Scientia 2014

Vol. 1 | Spring 2014

Letter from

the Editor The first issue has arrived! After many months of editing, sorting

through articles, formatting, and marketing, the premier issue is at

last ready. Scientia is presented to you by BURST (Baylor Under-

graduate Research in Science and Technology), Baylor’s premier

research organization for undergraduates. We started off as a small

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we have grown into something much more than we imagined.

I am proud to say that Scientia is a journal for undergraduates by

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upon which undergraduate research in the sciences can be recog-

nized. In this issue you will find articles about organic chemistry

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thank Dean Nordt for his enthusiasm and outstanding support of

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cause by holding this journal in your hands, you are helping BURST

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Sincerely,

Kelli Hicks

Editor In Chief, Scientia

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form upon which undergraduates of

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research in the areas of biological

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Editorial Board

Editor-in-Chief: Kelli Hicks ‘14

Assistant Editor: Mallory Myers ‘16

Student Editors

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Jimmy Kuhn ‘14

Savan Patel ‘15

Sai Konde ‘17

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Special Thanks

Dean Lee Nordt

URSA Steering Committee

1

Scientia | Baylor Undergraduate Journal of Science and Technology 2


Characterization of CD200

Expression after Nervous System Injuries ..................4

Richa Manglorkar ‘15

The Preparation of Certain Phenylazopurines................. 16

Hayden Jefferies ‘15

Gene Therapy and Parkinson’s Disease: A Review............... 24

Dustin Buller ‘14

Effect of Regular Strength Tylenol© on

Simocephalus serrulatus Population Growth . ................36

Asha Scott ‘16, Andrea Bodale ‘16, Marisa Pinson ‘16

Gender Differences In Neurobiological Processing. . ..............40

Krystal Miller ‘15

Table of Contents

3

Scientia | Baylor Undergraduate Journal of Science and Technology

After injury to the nervous system, the body must resolve its inflammatory response in order to promote neuronal repair and regeneration. CD200/C200R is a ligand-receptor pairing that is found throughout the body, including the central nerv-ous system (CNS). In the context of the CNS, CD200 is primarily expressed on neurons which help maintain microglia, the pri-mary expressers of the CD200 receptor (CD200R), in an anti-inflammatory state. In order to better understand the role of CD200 in regulating inflammation, this study utilized several types of nervous system injuries in a C57/Bl6 mouse model and characterized variations in CD200 expression post-injury. Immunocytochemistry was used to identify the extent and in-tensity of CD200 expression in three sciatic nerve cut mice (7 days post injury (dpi)), three sciatic nerve cut mice (14 dpi), three T9 dorsal hemi-section mice (7 dpi), and three dorsal rhi-zotomy mice (7 dpi). We hypothesized that CD200 expression would decrease in regions of microglial activation following lesion and loss of sensory or motor axon degeneration. Based on the preliminary results from a representative sample per group, microglia and astrocytes are activated but CD200 ex-pression does not significantly change in the spinal cord dorsal horn after sciatic nerve cuts, dorsal rhizotomy, or caudal to the dorsal hemi-section injuries. Based on a very limited change in CD200 expression post inju-ry, we concluded that CD200 is not exclusively on sensory ax-ons, and that the ligand-receptor complex may not play a prin-ciple role in post-injury inflammation within certain types of nervous system injuries.

Richa Manglorkar, Bryan Brautigam, Lyn Jakeman

SUCCESS Program; Department of Biology, Baylor University; Bio-medical Sciences Graduate Program; Center for Brain and Spinal Cord Repair; Department of Physiology and Cell Biology; The Ohio State University Wexner Medical Center, Columbus, OH 43210

4


Introduction

5

CD200 and CD200R have been found to play various roles in the body such as in viral infections, cardiovascular interactions, cancerous tumor cells, and the central nervous system (Holmannova et al. 2012). CD200 is a glyco-protein ligand found on the surface of neurons. The corresponding receptor CD200R is primarily found on microglia, which are the immune defense cells of the central nervous system. When CD200 binds to CD200R it has an immu-nosuppressant effect on microglia. Therefore, in homeostatic conditions, CD200 will bind to CD200R and microglia will not activate any immune re-sponse. However, studies show that CD200 gene-targeted mice that are CD200-/- have an increased microglial immune response, characterized by the microglia taking on a more phagocytic, macrophage-like morphology. Micro-glia activation can also induce an inflammatory response which creates a mi-croenvironment that is not conducive for neuronal regeneration (Hoek 2000).

In terms of central nervous system injury, when neuronal regeneration is inhibited by factors such as inflammation, the brain and spinal cord are un-able to heal properly. Therefore, if a pathway that suppresses nervous system inflammation can be identified, potential therapies could target a specific in-flammatory mechanism. Previous work by Bryan Brautigam in Dr. Jakeman’s lab showed that the distribution of CD200 is similar to that of sensory axons and that CD200 expression decreases at the site of microglial activation fol-lowing spinal cord crush or contusion injuries. This study characterizes the expression of CD200 in the spinal cords of mice with three different types of nervous system injuries.

Based on the types of injuries and known anatomical features of the nervous system, each injury in this study had a predicted outcome in terms of axon degeneration and CD200 expression. The sciatic nerve cut injury would cut the dorsal sensory axons from the dorsal root ganglion where the sensory neuron cell body resides. However, the sensory axons located in the spinal cord would still be attached to the cell body. The cut would also cut ventral motor axons, which are responsible for transporting electoral impulses to muscle fibers, from the motor neuron cell body. Therefore, CD200 expression should decrease in the dorsal horn and possibly the ventral horn as well. The microglial activation for the sciatic nerve cut should move more dorsally in the 14 dpi animals compared to the 7 dpi animals. The dorsal rhizotomy would cut the dorsal sensory axons from the dorsal root ganglion where the sensory neuron cell body resides, which would cause degeneration of sensory axons in the spinal cord. Therefore, CD200 expression should decrease in the dorsal horn, but have no change in the ventral horn because the motor neu-rons will still be intact. The dorsal hemisection would cut the corticospinal tract caudal to the injury site causing degeneration. The dorsal section of the spinal cord rostral to the injury site would also degenerate. Therefore, CD200 expression should decrease in the corticospinal tract caudal to the injury site. CD200 expression should also decrease in dorsal axons rostral to the injury site. Portions of these results have been included in a Master’s Thesis by Bry-an Brautigam (Brautigam 2013).


Materials and

Methods

Surgery There were four total surgery groups with n=3 per group (Figure 1). Group one was a sciatic nerve cut (7 dpi, n=3), group two was a T9 dorsal hemi-section (7 dpi, n=3), and group three was a L5 dorsal rhizotomy (7 dpi, n=3). All three of these groups were perfused seven days post-injury. Group four was also given a sciatic nerve cut (14 dpi, n=3) in the same manner as group one. All mice were anesthetized with ketamine and xylazine based on their weight. Post-surgery all animals were ad libitum food and water throughout the recovery process. All groups were given daily checks until perfusion day.

Perfusion The mice were given a lethal dose of ketamine (120mg/kg) and xylazine (14mg/kg) before perfusion. 0.1M Phosphate-Buffered Saline (PBS) was pumped through the animal and then the solution was changed to 4% para-formaldehyde (PFA) in 0.1M PBS.

Figure 1: Shows a cross section of the spinal cord and the three types of surgery per-formed on the mice. (Aldoskogius et al. 1998)

Dissection The entire central nervous system (CNS) was removed during this process and all animals with sciatic nerve cuts had their sciatic nerves removed as well. Tis-sue samples were post-fixed in 4% PFA for two hours at 4ºC. After two hours, the solution was changed to 1M PB and left at 4ºC overnight. The next day, the solution was changed to 30% sucrose solution in preparation for blocking and freezing.

Tissue Processing Using a Cryostat, tissue sample blocks were cut in 10μm-thick cross sections and placed on slides in order to perform immunohistochemistry stains.

Figure 1—Injury Models

6


Blocking The central nervous system was cut into separate segments using the schemat-ic above. The spinal cord segments were placed into blocks with Tissue-Tek O.C.T. Compound to prepare for Cryostat cutting. Each blue square represents a segment of tissue that was cut and frozen in a Cryostat cutting solution. Block A was the brain, which was not placed in Tissue-Tek O.C.T. Compound and was instead frozen with dry ice. Block G was the sciatic nerves collected from six sciatic nerve injury animals. This study examined block E in the lumbar enlargement area based on the prediction that there should be a reaction in that area for all three types of nervous system injuries performed on the mice. Block F in the sacral region was also examined for caudal reactions based on the dorsal rhizotomy and dorsal hemi-section. This study focuses on block E and F.

Immunohistochemistry The CD200/GFAP stain was performed using a 1% blokhen/0.1% FG/0.1% Tx-100 in PBS blocker solution. The primary antibodies were 1:2000 rat anti-CD200 and 1:1500 rabbit anti-GFAP. The secondary antibodies were 1:200 goat anti-rabbit 488 and 1:200 goat anti-rat 546. DAPI was at a concentration of 1.5μL in 50mL. The Iba-1/DAB stain was performed using 6% H2O2 in methanol solution and the block was a 4% BSA/0.3% Tx-100/in 0.1M PBS solution. The primary was antibody 1:1000 rabbit anti-iba-1.The slides incubated with Elite-ABC and DAB (3, 3'-Diaminobenzidine) solution. The dehydration step consisted of: two minutes in 70% ethanol twice, two minutes in 95% ethanol twice, and three minutes in 100% ethanol twice. The clearing step consisted of four minutes of Histoclear three times. The EC/CV stain used acetone for 5 minutes and the Eriochrome Cyanine solu-tion for 30 minutes. Next was 5% Iron Alum for approximately 5 minutes and then Borax-Ferricyanide for 2 minutes. Afterwards the slides were placed in 80% alcohol and 1% Cresyl Violet for 10 minutes and then followed by a series of rinses in the following solutions: 95% alcohol/0.1% acetic acid, 100% alco-hol, Histoclear. The CGRP/DAB stain was performed using a 5% normal goat serum in PBS block, 20% H2O2 in methanol, and a primary antibody 1:20,000 rabbit anti-CGRP overnight. The secondary antibody was 1:2000 biotinylated goat anti- rabbit. The slides incubated with Elite-ABC and DAB (3, 3'-Diaminobenzidine) solution. The dehydration step consisted of: two minutes in 70% ethanol twice, two minutes in 95% ethanol twice, and three minutes in 100% ethanol twice. The clearing step consisted of four minutes of Histoclear three times.

7


Materials and

Methods

(cont.)

Results

Immunohistochemistry Definitions

Quantification Quantification was done using the Image J program. After an image was se-lected, the color channels were split in order to reduce background signal. Then a consistent threshold was chosen and the dorsal horn region was quan-tified using percent area.

Using EC/CV staining, sections of the spinal cord were identified based on their corresponding vertebral levels. In the 7dpi sciatic nerve cut mice (Figure 2A-2D), the site of injury was found at L5, which confirms that the sciatic nerve was properly cut since the sciatic nerve joins the spinal cord at L5 (Figure 2A). Another cross-section was stained with iba-1/DAB (Figure 2B). A clustering of activated microglia can be seen in the sensory neurons within the dorsal horn and the motor neurons within the ventral horn. Next, CD200 and GFAP expres-sion were examined in section caudal to the confirmed microglial activation. Visually and quantitatively, there was little to no difference in the expression of the CD200 ligand in both the dorsal and ventral horn on the injured right-side when compared to the uninjured left-side (Figure 2C and Figure 6). However, there is a slight increase in GFAP expression in the outer most part of the dor-sal horn and the middle of the ventral horn on the injured side of the spinal cord which indicates astrocyte activation (Figure 2D). In all comparative anal-yses, the left side of the cross section was used as the control. Next, the 14dpi sciatic nerve cut animals (Figure 2E-2H) were analyzed using the same stains as the 7dpi sciatic nerve cut animals. The EC/CV stain revealed that the injury occurred in the L4/L5 region as expected (Figure 2E). The Iba-1/DAB stain shows a large activation of microglia in the ventral horn and a slightly more diffuse microglial activation in the dorsal horn (Figure2F). The difference in microglia activation could be due to the time period difference after which the 7 dpi and 14 dpi animals were euthanized. The 14dpi animals had more time to resolve their injuries, which could account for the subsiding

8

Name Stain

CD200 CD200 ligand—found on the membrane of neurons

GFAP—Glial Fibrillary Acidic Protein Astrocytes—increases expression with astrocyte activation

Iba-1 Microglia—primary antibody

DAB—3, 3'-Diaminobenzidine Chromagen substrate which produces a black color

EC—Eriochrome Cyanine Histological stain for lipids to see Myelin

CV—Cresyl Violet Neurons

CGRP—Calcitonin Gene-Related Peptide Sensory axons—found in the dorsal horns


Figure 2—Sciatic Nerve Cut

microglia activation. There is no change in CD200 expression in areas of micro-glial activation in comparison to the uninjured side of the animal (Figure 2G). There is an increase in astrocyte activation in both the dorsal and ventral horn as indicated by the GFAP expression (Figure 2H).

Figure 2: This figure shows the cross-sectional area of a mouse with a sciatic nerve inju-ry that was euthanized 7 dpi (A-D) and another mouse that was euthanized 14 dpi (E-H). (A) EC/CV stain of the L5 vertebral level. (B) iba-1/DAB stain with white arrows that indicate microglial activation in the right dorsal and ventral horn. (C) CD200 stain with white arrows indicating where microglial activation occurred and where CD200 expres-sions did not change in comparison to the left uninjured side. (D) GFAP stain with white arrows indicating a slight up regulation in GFAP signifying astrocyte activation. (E) EC/CV stain of the L4/L5 vertebral level. (F) iba-1/DAB staining with white arrows that indi-cate microglial activation in the right dorsal and ventral horn. (G) CD200 stain with white arrows indicating where microglial activation occurred and where CD200 expressions did not change in comparison to the left uninjured side. (H) GFAP stain with white arrows indicating a slight up regulation in GFAP signifying astrocyte activation (UI = side of the animal that remained uninjured, I = injured side of the animal).

9


Results

(cont.)

After a dorsal rhizotomy, the distal portions of sensory axons that are no longer attached to the cell body begin to show signs of Wallerian degenera-tion. Sensory axons are found only on the dorsal horns of the spinal cord. This degeneration can be observed using a CGRP/DAB stain that stains function-ing sensory axons black. An absence of this stain indicates that the sensory axons have degenerated, confirming the success of a proper dorsal rhizoto-my. Figure 3 shows a 7 dpi sciatic nerve cut animal that has functioning sen-sory axons on both sides in the dorsal horns (Figure 3). However, the 7 dpi dorsal rhizotomy animal is missing functioning sensory axons on the injured right side. This validates a proper rhizotomy and confirms the injury site. Fur-thermore, the CGRP stain also confirms that the sciatic nerve cut was properly performed and that the cell body was still attached to the distal sensory ax-ons.

Figure 3: This figure shows the difference between two mice, one of which has both sets of dorsal sensory neurons intact and another that has degeneration of sensory axons on the right side. The 7 dpi dorsal rhizotomy animal does not have any staining on the right side were the nerve cut took place, confirming the injury site. The dorsal rhizotomy animals were compared to sciatic nerve cut animals since the sciatic nerve cut animals had their sensory axons intact.

By implementing both EC/CV and Iba-1/DAB on spinal cord samples collected from the dorsal rhizotomy animal, the stains revealed that the injury occurred in the L4/L5 vertebral level of the animal (Figure 4A). The Iba-1/DAB stain showed a clustering of activated microglia in the top of the right dorsal horn, which is where sensory axons are located (Figure 4B). However, unlike the sci-atic nerve cut animal, there is no microglia activation in the ventral horn. CD200 expression does not significantly change when compared to the unin-jured left side of the animal (Figure 4C). However, a slight decrease on the in-jured right side has been observed in some dorsal rhizotomy cross sections. There is little to no change in GFAP expression in comparing the left and right sides of the animal (Figure 4D).

Figure 3—CGRP

10


Figure 4: This figure shows the cross-sectional area of a mouse with a dorsal rhizotomy injury at L5 that was euthanized at 7 dpi (A-D) and another mouse with a dorsal rhizot-omy injury at S3 that was euthanized at 7 dpi (E-H). (A) EC/CV stain of the L5 vertebral level. (B) iba-1/DAB staining with the white arrow indicating microglial activation in a very localized region of the right dorsal horn. (C) CD200 stain with white arrows indicat-ing where microglial activation occurred and where CD200 expressions did not change in comparison to the left uninjured side. (D) GFAP stain with the white arrow indicating no change in GFAP expression in the area of microglial activation. (E) EC/CV stain of the S3 vertebral level. (F) iba-1/DAB staining with the white arrow indicating microglial acti-vation in a very localized region of the right dorsal horn. (G) CD200 stain with white ar-rows indicating where microglial activation occurred and where CD200 expressions did not change in comparison to the left uninjured side. (H) GFAP stain with the white ar-row indicating an increase in astrocyte activation/GFAP expression in the area of micro-glial activation (UI = side of the animal that remained uninjured I = injured side of the animal).

Figure 4—7dpi Dorsal Rhizotomy L5 and S3

11


Results

(cont.)

Dorsal roots leave the spinal cord and form the peripheral nervous system. Roots in the cervical and thoracic portion of the spinal cord leave parallel to their corresponding vertebral level. However, more caudal roots such as the lumbar and sacral slope downwards as they exit the spinal cord and are no longer parallel. Therefore, when the dorsal rhizotomy was performed at the L4/L5 level, the root that was cut could have led to a more caudal level of the spinal cord. Figures 4E-4H show sections of the sacral 3 level of a dorsal rhi-zotomy animal, which was confirmed by EC/CV staining (Figure 4E). The Iba-1/DAB stain revealed that there was microglial activation in the right dorsal horn, as was hypothesized previously (Figure 4F). There is no significant change in CD200 expression; there is, however, a significant astrocyte reac-tion (Figure 4G, 4H). The cross sections below were taken caudal to the site of the T9 dorsal hemi-section. However, there is no evidence of a large microglial reaction as ex-pected in the dorsal region of the spinal cord, specifically the corticospinal tract. This could indicate that the axons severed at the T9 level of the spinal cord have not degenerated as far as the lumbar levels by 7 dpi (Figure 5A). There was no change in CD200 expression, but a mild increase in GFAP in the corticospinal tract of the lumbar region of these mice (Figure 5C, 5D).

Figure 5: This figure shows the cross-sectional area of a mouse with a dorsal hemi-section injury that was euthanized 7 dpi. (A) EC/CV stain of the L4/L5 vertebral level. (B) iba-1/DAB staining indicating microglial activation (C) CD200 expressions did not change in comparison to the ventral uninjured site. (D) GFAP stain indicating a slight astrocyte reaction in the corticospinal tract region. (UI = side of the animal that re-mained uninjured I = injured side of the animal)

Figure 5—7dpi Dorsal Hemi-section

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Figure 6 demonstrates a relationship between microglia and CD200 expres-sion. The significant increase in microglia activation in the right dorsal horn confirms that the area that is being quantified is the injury site. Furthermore, if CD200 expression were affected, it would happen in that particular region. Both sciatic nerve cuts and two dorsal rhizotomy sites have increased micro-glial activity. However, the dorsal hemi-section did not have increased micro-glial activity because the precise injury site was not located. After confirming an increase in microglia, Figure 6 shows that CD200 expression does not change in the sciatic nerve cut animals. Interestingly, there is a decrease in CD200 expression in the L5 dorsal rhizotomy and an increase in CD200 ex-pression in the S3 dorsal rhizotomy. This could be due a variation in individu-al animals or a difference in injury sites, which would have to be investigated further. The dorsal hemi-section serves as a negative control that indicates no change in CD200 expression within the dorsal horns.

Figure 6: This figure shows quantification of both the Iba-1/DAB stain for microglia and CD200. This graph only quantifies expression found in the dorsal horn and compares the left uninjured side to the right injured side. (SN 7dpi = sciatic nerve cut 7 days post injury, SN 14dpi = sciatic nerve cut 14 days post injury, DR L5 = dorsal rhizotomy at the L5 verte-bral level, DR S3 = dorsal rhizotomy at the S3 vertebral level, HEMI = T9 dorsal hemi-section).

Figure 6—Quantified Microglia and CD200 Expression

Discussion The working hypothesis was that after injury, when axons expressing CD200 were severed, microglia activation occurred because CD200 was down regu-lated. Our hypothesis also stated that the down-regulation of CD200 would cause CD200R to remain unbound and contribute significantly to the result-ing inflammatory response. Based on the results in Figure 6, that hypothesis was disproven. Iba-1/DAB enabled identification of areas with microglia acti-vation surrounding the injured sensory afferents and motor neurons. Howev-

13


Discussion

(cont.)

er, a section only 180μm away did not show a down regulation of CD200 when compared to the uninjured side of the animal. This suggests that CD200 expres-sion does not directly cause the observed microglia activation. Furthermore, the GFAP stain reveals that there is slight astrocyte activation in areas correspond-ing to microglial activation. This confirms the site of injury on account of the fact that after injury, astrocytes surrounding injured axons are also activated. This same analysis holds true in both the 7dpi sciatic nerve cut mice and the 14dpi sciatic nerve cut mice. After using CGRP to confirm the site of the dorsal rhizotomy and Iba-1/DAB to confirm areas of microglia activation, CD200 does not change its level of ex-pression in the dorsal rhizotomy injury site. There is, however, an increase in GFAP expression and astrocyte activation. Therefore it can be deduced that ac-tivation in both microglia and astrocytes does not necessarily indicate a down regulation in CD200 expression. It can be deduced that because a loss of CGRP staining did not affect CD200 levels, that CD200 is not found on the axons of dorsal horn sensory neurons. Although there is probably inflammation post in-jury, the inflammation does not seem suppressed by a pathway that involves CD200. The T9 dorsal hemi-section did not cause changes in CD200 expression, but fur-ther exploration in more rostral sites reveals changes that are evident as far away as the lumbar spinal cord. A protein kinase C- γ (PKC-γ) stain revealed that the corticospinal tract was cut caudal to the injury site (data not shown). This study indicates that an injury at the T9 site did not manifest caudally in the lower lumbar region, except for some possible localized astrocyte activation. The results suggest that CD200 is not restricted to sensory axons, and also indi-cates that CD200/CD200R is not the only mechanism at play in the inflammato-ry response to axotomy. Preliminary results from our lab indicate that CD200 may be down regulated after contusion injuries; however this decreased expres-sion can be attributed to neuronal degeneration since CD200 is located on neu-ronal cell membranes. Future studies would include more types of injuries in order to determine if the CD200 anti-inflammatory pathway responsiveness is dependent on various in-jury models. Furthermore, expression of CD200R on microglia would also have to be investigated since there is an obvious microglial reaction post-injury in both the sciatic nerve cuts and dorsal rhizotomy. Furthermore, because the dor-sal rhizotomy animals did have conflicting variations in CD200 expression, a larger study would be prudent for that injury model. Future studies would also include a CD200Fc drug that mimics the interaction of CD200 with its receptor. Binding a functionally similar ligand to CD200R would induce the anti-inflammatory response in the nervous system after particular injuries. The ago-nist would induce the CD200/CD200R even if CD200 was down regulated and could possibly give damaged neurons the proper microenvironment to pro-mote regeneration. A CD200Fc drug study in vivo would also help to determine if this anti-inflammatory mechanism is dependent on external factors, and whether or not an anti-inflammatory response can be maintained after various types of injury.

14


References

Acknowledgments I would like to thank Feng Yin and Kent Williams for their technical assis-tance and discussions of the project. Also, I would like to thank the SUC-CESS program and my SUCCESS peers for their training and advice. This work was supported by NIH grant NS043246.

Aldoskogius, Hakan, and E. N. Kozlova. "Central Neuron-Glial and Glial-Glial In-teractions Following Axon Injury." Progress in Neurobiology 55 (1998): 1-26. Print. Brautigam, Bryan. “Examining the Regulation of Inflammation through CD200 and CD200R Following Spinal Cord Injury”. Diss. The Ohio State University, 2013. Print. Hoek, R. M., S. R. Ruuls, C. A. Murphy, G. J. Wright, R. Goddard, S. M. Zurawski, B. Bloom, M. E. Homola, W. J. Streit, M. H. Brown, A. N. Barclay, and J. D. Sedgwick. "Down-regulation of the Macrophage Lineage through Interaction with OX2 (CD200)." Science 290 (2000): 1768-71. Print. Holmannova, D., M. Kolackova, K. Kondelkova, P. Kunes, J. Krejsek, and C. Andrys. "CD200/CD200R Paired Potent Inhibitory Molecules Regulating Immune and Inflamma-tory Responses; Part I: CD200/CD200R Structure, Activation, and Function." Acta Medi-ca (Hradec Kralove) 55 (2012): 12-7. Print.

15


The focus of this project was to synthesize a number of diazo-nium salts and couple the products with theophylline. Aniline along with other aniline derivatives were diazotized and cou-pled with theophylline to yield typical azopurines. In a similar reaction, guanine gave a product whose structure was undeter-mined. When N,N-dimethylaniline was diazotized, a precipi-tate resulted, in a manner unlike other aniline derivatives. Six (6) aniline derivatives were diazotized and subsequently cou-pled with theophylline. Guanine was employed in one of the coupling reactions. Two of the coupled products were reduced to give products in low yields. Insoluble diazotized dialkylami-noanilines were observed and set aside for further study.

Hayden Jefferies, Dr. Jesse W. Jones

Department of Chemistry and Biochemistry, Baylor University

16


Introduction

17

Since ancient times, dyes have played major roles in human civiliza-tions and have been important characteristics in culture and everyday life. Ev-idence of dying processes is abundant throughout many major civilizations. Today, dyes are used in nearly every commercial product and are largely used in the textile, food, and paint industries.

Among the different classes of dyes, the azo dye class is indisputably one of the most important classes. Modern azo dyes are engineered to be re-sistant to acids and fading. About half of the dyes used in industry are azo dyes.

Azo dyes are typically red, orange, or yellow. An azo compound is characterized by any molecule of general formula R-N=N-R. Azo dyes are molecules that have a diazo functional group between two aromatic rings. The nature of the aromatic substituents on both sides of the azo group con-trols the colors of the azo compounds; as well as the water-solubility of the dyes and how well they bind to a particular fabric.

Azo dyes can be formed by diazotization of an aromatic amine, such as aniline, followed by the coupling of the diazotized salt with another aro-matic compound such as a purine like Theophylline.

Diazonium salts, or diazonium compounds, are a group of organic compounds sharing a common functional group R-N2+ X- where R can be an alkyl or aryl group and X is an inorganic or organic anion such as a halogen. Diazonium salts are often intermediates in the organic synthesis of azo dyes.

Materials and

Methods

Preparation of 4-Chlorobenzenediazonium Chloride (HJ57A)

p-Chloroaniline (I: Y=p-chloro; well ground, 10 g/0.078 mol) was dissolved in 100 ml of 5% hydrochloric acid and stirred for 5 minutes. Sodium Nitrite (5.4 g/0.078 mol) in 25 ml water was added to the solution at 0-10o and allowed to stand at room temperature for 15 minutes.

Preparation of 8-(4-chlorophenylazo)theophylline (HJ57B)

The 4-chlorobenzenediazonium chloride solution was added in small aliquots to a solution of theophylline (21.7 g/0.12 mol) previously dissolved in an aque-ous solution of potassium hydroxide (100 ml at 7%). The mixture turned a vis-cous orange color following the addition. Water (100 ml) was added and the mixture was allowed to continue to stir. After 55 minutes, hydrochloric acid (20 ml; 5%) was added to adjust the pH to approximately 5. As the acid was added, the mixture changed dark yellow. The resulting slurry was filtered, washed with water (30 ml) and dried at 85o for 15 hours. The light orange pre-cipitate weighed 29 g when dry.

Preparation of 8-Aminotheophylline (HJ65)

The light orange 8-(4-chlorophenylazo)theophylline (IV: Y=p-chloro; 10 g) was dissolved into potassium hydroxide (200 ml; 10%) at 70-80o and stirred (30


minutes after the final addition). The mixture changed dark orange/red. So-dium hydrosulfite (50 g) was then added to the mixture at 70-80o in small portions and stirred for 30 minutes after final addition. Additional potassi-um hydroxide (5 g) was added followed by the addition of sodium hy-drosulfite (7 g). The mixture was filtered while still warm and the filtrate was acidified and became peach-colored. After being allowed to stand in the refrigerator overnight, the mixture was filtered and the precipitate was al-lowed to stand and dry overnight. The precipitate weighed 2.5 g.

Preparation of 4-(diethylamino)benzenediazonium Chloride (HJ59A

N,N-Diethylaniline (I: Y=diethylamino; 5 g/0.033 mol) was dissolved in hy-drochloric acid (50 ml; 5%). A solution of sodium nitrite (2.3 g/0.033 mol) and water (10 ml) was added to the solution drop-wise at 0-10o and stirred for 5 minutes. The solution was allowed to stand at room temperature for 15 minutes. The solution changed from dark red to a dark green color im-mediately upon mixing.

Preparation of 8-(4-diethylaminophenylazo)theophylline (HJ59)

4-(diethylamino)benzenediazonium chloride (prepared from 5 g N,N-diethylaniline (II, Y=4-diethylamino) and excess sodium nitrite) was added in small aliquots to a solution of theophylline (9.2 g/0.05 mol) and potassium hydroxide (50 ml; 5%). The mixture changed light green upon addition. Af-ter 15 minutes of stirring, hydrochloric acid (30 ml; 5%) was added to adjust the pH to approximately 5. As the acid was added, the mixture changed dark green. The resulting slurry was filtered, washed with water (30 ml) and dried at 85o 30 minutes. The light green precipitate weighed 9 g after dry-ing.

The precipitate (9 g) was dissolved in potassium hydroxide (5%; 200 ml or approximately 20 ml per gram) and concentrated hydrochloric acid was added until the pH was approximately 5. When the pH turned neutral, the mixture changed from dark green to light green. The mixture was filtered and the light green precipitate was dried in the oven at 85o to yield 9 g of product.

Preparation of 8-aminotheophylline (HJ68)

The 8-(4-diethylaminophenylazo)theophylline precipitate (IV: Y=4-diethylamino; 5 g) was dissolved into potassium hydroxide (100 ml; 10%) at 70-80o and stirred (30 minutes after the final addition). The mixture changed

18


dark green. Sodium hydrosulfite (32 g) was then added to the mixture at 70-80o in small portions and stirred for 30 minutes after final addition. The mixture was allowed to stand at 10-15o for 30 minutes before being filtered and then acidified. The precipitate was dried to air overnight to yield 4 grams of prod-uct.

Diazotization of N,N-Dimethylaniline (HJ63)

N,N-Dimethylaniline (I: y=4-dimethylamino) (5 g/.04 mol) was dissolved in concentrated hydrochloric acid (10 ml) and stirred. Sodium nitrite (2.5 g/.04 mol) was dissolved in water (10 ml) and added to the N,N-dimethylaniline so-lution in small aliquots at 0-10o. As the sodium nitrite was added, the mixture changed to dark red, bubbles formed, and finally changed a bright orange col-or. The mixture was allowed to stir 15 minutes after final addition then stand at room temperature for 5 minutes before the orange precipitate was filtered and washed with water (30 ml). The precipitate weighed 4 g.

Diazotization of N,N-diethylaniline in Acid Media (HJ66)

N,N-Diethylaniline (I: y=4-diethylamino; 5 g/0.033 mol) was dissolved in hydro-chloric acid (50 ml; 5%) and stirred for 10 minutes. Sodium nitrite (2.3 g/0.033 mol) dissolved in water (10 ml) was then added to the solution drop-wise at 0-10o and allowed to stir at room temperature for 15 minutes. The solution changed from light red to a dark red color immediately upon mixing and then changed to a dark green color after sitting for 15 minutes.

Preparation of 8-(4-N,N-Diethylaminophenylazo)guanine (HJ66)

4-(N,N-diethylamino)benzenediazonium chloride (prepared from 5 g N,N-diethylaniline (I; Y=diethylamino) and excess sodium nitrite) was added in small aliquots to a solution of guanine (5 g/.033 mol) and potassium hydroxide (25 ml; 10%) at room temperature. The mixture changed dark green upon ad-dition. After 40 minutes of stirring, concentrated hydrochloric acid (5 ml) was added to adjust the pH to 7. The resulting slurry was filtered, washed with wa-ter (30 ml) and allowed to stand for 30 minutes.

The precipitate was dissolved in potassium hydroxide (30 ml; 10%) and con-centrated hydrochloric acid (1 ml) was added until pH 7 was achieved. The mix-ture was stirred 20 minutes before it was filtered and the precipitate was al-lowed to stand overnight. The precipitate weighed 9 g.

Preparation of 3-Chlorobenzenediazonium Chloride (HJ69A)

3-Chloroaniline (I: y=3-chloro; 5 g; 0.039 mol) was dissolved in hydrochloric acid (50 ml; 5%) and stirred for 5 minutes. A solution of sodium nitrite (2.7 g/0.040 mol) and water (10 ml) was added to this solution in small portions at

19


Materials and

Methods

(cont.)

0-10o and allowed to stand at room temperature for 15 minutes. The solu-tion changed light orange immediately upon adding the last amount of so-dium nitrite.

Preparation of 8-(3-Chlorophenylazo)theophylline (HJ69B)

3-Chlorobenzenediazonium chloride (II: Y=3-chloro) prepared from 5 g 3-chloroaniline and excess sodium nitrite) was added in small aliquots to a solution of theophylline (6.3 g/0.035 mol) and potassium hydroxide (50 ml; 5%). After 30 minutes of stirring, 75 ml KOH 5% was added. Hydrochloric acid (4 ml; 5%) was added to adjust the pH to approximately 5. The result-ing mixture was filtered, washed with water (60 ml) and dried to air. The dark red precipitate weighed 8.5 g when dry.

Preparation of o-Methoxybenzenediazonium Chloride (HJ70A)

o-Anisidine (I; Y=o-methoxy; 5 g; 0.039 mol) was dissolved in hydrochloric acid (50 ml; 5%) and stirred for 5 minutes. A solution of sodium nitrite (2.7 g/0.040 mol) and water (10 ml) was then added to the solution in small por-tions at 0-10o and allowed to stand at room temperature for 15 minutes. A thick, dark red sludge precipitated at the bottom of the beaker while the mixture was light orange colored and was very insoluble. Additional water (25 ml) was added and the mixture was stirred until mostly dissolved.

Preparation of 8-o-Methoxyphenylazotheophylline (HJ70B)

o-Methoxybenzenediazonium chloride (y=o-methoxy) prepared from o-anisidine (5 g) and excess sodium nitrite was added in small aliquots to a solution of theophylline (6.3 g/0.035 mol) and potassium hydroxide (50 ml; 10%) and stirred for 30 minutes. Hydrochloric acid (5 ml; 5%) was added to adjust the pH to approximately 5. The resulting mixture was filtered, washed with water (30 ml) and dried to air. The dark blue-black precipitate weighed 1 g.

Preparation of 4-Methoxybenzenediazonium Chloride (HJ71)

p-Anisidine (I: Y=p-methoxy; 5 g; 0.039 mol) was dissolved in hydrochloric acid (50 ml; 5%) and stirred for 5 minutes. A solution of sodium nitrite (2.7 g/0.040 mol) and water (10 ml) was added to the solution in small portions at 0-10o and allowed to stand at room temperature for 15 minutes. The col-or changed from dark violet to brownish-red and was very insoluble. Water (25 ml) was added and the mixture was stirred until mostly dissolved.

20


Reaction of Theophylline with 4-Methoxybenzenediazonium Chloride (HJ71)

4-Methoxybenzenediazonium chloride (II: Y=4-methoxy; prepared from 5 g p-anisidine and excess sodium nitrite) was added in small aliquots to a solution of theophylline (III; 6.3 g/0.035 mol) and potassium hydroxide (50 ml; 10%) and stirred for 30 minutes during which the mixture turned brown. Hydrochloric acid (1 ml; 5%) was added to adjust the pH to approximately 5. The resulting mixture was filtered, washed with water (30 ml) and dried to air. The dark violet precipitate weighed 6.5 g.

Preparation of benzenediazonium chloride (HJ72A)

Aniline (I: Y=null; 5 g; 0.039 mol) was mixed with hydrochloric acid (50 ml; 5%) and stirred for 5 minutes. A solution of sodium nitrite (2.7 g/0.040 mol) and water (10 ml) was added to the mixture in small portions at 0-10o and allowed to stand at room temperature for 15 minutes. The color changed yellow and was very thick and insoluble. Water (25 ml) was added and it was stirred until mostly dissolved.

Preparation of 8-phenylazotheophylline from aniline (HJ72B)

Benzenediazonium chloride (I: Y=null; prepared from 5 g aniline and excess sodium nitrite) was added in small aliquots to a solution of theophylline (III; 6.3 g/0.035 mol) and potassium hydroxide (50 ml; 10%) and stirred for 30 minutes during which the mixture turned golden brown. Hydrochloric acid (3 ml; 5%) was added to adjust the pH to approximately 5. The resulting mixture was fil-tered, washed with water (30 ml) and dried to air. The red precipitate weighed 10 g when dry.

Results and

Discussion

Aniline (I, Y=null) was diazotized with sodium nitrite in acid at 5o C to yield benzene diazonium (II, Y=null). The diazonium salt was coupled with theophyl-line (III) in base to give compound IV (Y=null).

p-Chloroaniline (I, Y=p-chloro) was diazotized in the same manner to produce compound II (Y=p-chloro). The diazonium salt was coupled with theophylline (III) to yield compound IV (Y=p-chloro). Compound IV was reduced using sodi-um hydrosulfite and yielded compounds V and II (Y=p-chloro). This reaction yielded a small amount of precipitate and it is suspected that the sodium hy-drosulfite was spent.

3-Chloroaniline (I, Y=3-chloro) was diazotized with sodium nitrite in acid at 5o C to yield compound II (Y=3-chloro). The diazonium salt was coupled with the-ophylline (III) to yield compound IV (Y=3-chloro).

o-Anisidine (I, Y=o-methoxy) was diazotized with sodium nitrite in acid at 5o to yield compound II (y=o-methoxy). The diazonium salt was coupled with theo-phylline (III) to yield compound IV (Y=o-methoxy).

21


References

Acknowledgments

"SYNTHESIS OF AN AZO DYE FOR INCORPORATION INTO CRYSTALS."Http://depts.washington.edu/chemcrs/bulkdisk/chem242a_spr10/info_Azo%20Dye%20Lab.pdf. University of Washington, n.d. Web. 24 July 2013.

European Ban on Certain Azo Dyes, Dr. A. Püntener and Dr. C. Page, Quality and Environ-ment, TFL

IUPAC, Compendium of Chemical Terminology, 2nd ed. (the "Gold Book") (1997). Online corrected version: (2009) "azo compounds".

I would like to acknowledge Dr. Jesse Jones of Baylor University for the op-portunity to work alongside him, as well as his incredible creativity, guidance, and patience with me as we worked together to complete this project. I would also like to thank Baylor University for the resources and facilities that made the project a success.

p-Anisidine (I, Y=p-methoxy) was diazotized in the same manner to yield com-pound II (Y=p-methoxy). The diazonium salt was coupled with theophylline (III) to yield compound IV (Y=p-methoxy).

N,N-Diethylaniline (VII) was diazotized with sodium nitrite in acid at 5o C to produce compound VIII. Compound VIII was coupled with theophylline (III) to yield compound IV (Y= diethylamino). Compound IV was reduced with sodium hydrosulfite to yield compounds V and II (Y=diethylamino). It was again sus-pected that the sodium hydrosulfite used to reduce compound IV was spent.

N,N-Diethylaniline (VII: Y=diethylamino) was diazotized with sodium nitrite in acid at 5o C to yield compound VIII. Compound VIII (: Y=diethylamino) was coupled with guanine (IX) to yield compound X which was subsequently re-duced with sodium hydrosulfite.

N,N-Dimethylaniline (I, Y=dimethylamino) was diazotized with sodium nitrite in acid at 5o C. The resulting precipitate was isolated for further study.

22

http://en.wikipedia.org/wiki/International_Union_of_Pure_and_Applied_Chemistry

http://en.wikipedia.org/wiki/Compendium_of_Chemical_Terminology

http://goldbook.iupac.org/A00560.html


Scheme 1

Scheme 2

23


In this review, a historical perspective on the development of gene therapy in relation to Parkinson’s disease is provided by communicating the findings of work done in years ranging from 2000-2012. This will be done the five following steps: 1) by first exploring gene therapy, its various types, and their ad-vantages and disadvantages vis a vis the treatment of human diseases, 2) by providing an example of an effective use of gene therapy, 3) by identifying two relevant genes identified as disease-causing when mutated, 4) by discussing the pathology of Parkinson’s disease in relation to these two genes, and 5) by finally discussing the potential of gene therapy in relation to Parkinson’s disease, specifically with regard to these two genes.

Dustin Buller, Devan Jonklaas

Department of Chemistry and Biochemistry, Baylor University

24

Vol. 1 | Spring 2014 25

Gene therapy works – simply summarized – by placing therapeutic DNA, which encodes for a desired product, in a delivery vehicle – a vector – which is specific for certain cell types in a target organism. This is typically done in a virus, by replacing the viral genome with a cassette containing the thera-peutic DNA. The vector is then delivered to its specific target cells, enters them, and releases its DNA. The vector genome is then expressed to desired products. This process is shown below in Figure 1.

Gene therapy made its clinical debut almost twenty years ago, when a retro-viral vector was used to deliver lymphocytes to an immunocompromised young girl. Though successful, this implementation raised many concerns about the safety of gene therapies. Only recently have advances in immunol-ogy and virology led to in vivo, clinical techniques which can be considered safe enough for use in conjunction with diseases of various tissues and or-gans.1

A large part of the improved safety and efficiency of gene therapy as a clini-cal technique has been the development of a streamlined generic strategy for vector engineering and transduction. These processes are depicted in Figure 2 and Figure 3. Although the examples shown depict processes in-volving a viral vector, the same general processes can be employed for the synthesis of any vector, simply by replacing the viral packaging DNA with packaging instructions for some other carrying and delivery mechanism.

Figure 1. A depiction of the process of gene therapy using an adenovirus vector

(available from NIH, public domain)


In the generic vector engineering process, a wild-type virus is first selected for its clinically relevant target-cell specificity. Its DNA is then extracted and the genes in the viral DNA essential for viral replication are replaced with the thera-peutic cassette – the DNA fragment which will be expressed as desired prod-ucts. The resulting DNA (containing the cassette) is called the vector DNA. The viral replication DNA is then placed in what is called the helper DNA, which can be contained in a plasmid or a helper virus. Both the helper DNA and vector DNA are inserted into a packaging cell, which uses the replication genes in the

helper DNA to replicate the vector DNA, which then encodes for its own packaging, finally resulting in a viral vector, specific to target cell types and con-taining a therapeutic cas-sette.2

Transduction occurs when the viral vector reaches its target cell. The vector en-ters the cell, typically through a receptor-mediated process (this is how vectors are cell-specific). Inside the cyto-sol, the vector’s viral coat-ing is broken down by degradative enzymes and

the vector DNA enters the cell nucleus. Through varied processes, depending on the type of vector, the vector DNA either becomes integrated into the cell genome or remains in the nucleus as a plasmid. In either case, the vector’s re-sultant host-cell dsDNA is translated and expressed into desired therapeutic proteins.2

Thus far when viral vectors have been used there are five types of viruses which have advanced at least to the stage of clinical trials as viral vectors for gene therapy. These are the retrovirus, the lentivirus, the adenovirus, the adeno-associated virus (AAV) and the herpes simplex virus (HSV).2

Retroviruses have been used extensively due to their efficient integration into

Figure 2. A generic vector engineering process2

26


the chromatin of target cells, and the ease of modifying their cell affinity. Retrovirus target cells are dictated al-most entirely by the glyco-proteins present on the ret-rovirus’ viral envelope, due to the glycoproteins’ inter-actions with target-cell re-ceptors. It has been shown that glycoproteins can be efficiently substituted during vector engineering for those of a different virus via a pro-cess called pseudotyping. In this way, retroviruses can be

easily engineered to target specific host cells. However, retroviruses tend to establish chronic infection that can develop into malignancy and immunodefi-ciency.2

Lentiviruses are a subset of retroviruses which are transported by active rather than passive transport across nuclear membranes. This mode of nuclear en-trance allows for the benefits of both being able to target non-dividing cells, and of being able to engineer lentiviral self-inactivating vectors. Additionally, lentiviruses have been found to be easily derived from non-primate animal lentiviruses, resulting in a smaller immune response to the non-human vec-tors.2

Adenoviruses were one of the first viruses used for vectors due to their early effective gene transfer in the respiratory system. Similarly to lentiviruses, they have often been used in non-human forms in order to skirt immunological dif-ficulties. However, due to their complexity, highly-controlled vector engineer-ing via the effective removal of the entirety of the viral genome is quite diffi-cult.2

AAVs are named as such because they normally require an adenovirus helper in order to function as vectors. Little is known about the AAV transduction mechanism, providing a significant barrier in effective use, yet since AAVs lack actual virus coding sequences, they are not treated by the human body with viral immunological responses. Thus, provided more is discovered about the

Figure 3. A generic transduction process2

27


specifics of their mechanistic functions, AAVs likely promise to serve as safe and effective vectors in the near future.2 HSV and related viruses are currently the most successful and effective vectors for gene transfer in vivo in humans, primarily due to their ability to persist in humans in a non-potent, disease-free state following transduction. This has been shown to hold true even in immunocompromised hosts. The primary diffi-culty of HSV and related viruses exists in their inability to specifically target cer-tain host cell-types due to a complex cell entry mechanism involving multiple different viral envelope glycoproteins.2

Generally, the primary problem with viral vectors in general is the tendency of these vectors to elicit immune system responses, however minimal, from the human body. These responses are dangerous in that their interference with vec-tor function can have serious DNA-altering side effects which result in diseases such as various malignancies. Thus, there has been significant research done in the development of non-viral nanoparticles as DNA delivery vehicles.3

The most significant non-viral means of DNA transport for gene therapy on which successful work has been done is the concept of DNA condensation – the use of DNA condensing agents in order to condense DNA down into tiny, pri-marily-DNA nanoparticles. In this way, the DNA effectively is the vector. It trans-ports itself, introducing no harmful secondary compounds as in the case of viral vectors. A diagram of this process is shown below in Figure 4.

Figure 4. DNA condensation and uptake in mammalian cells.4

28


In the presence of polycation condensing agents, DNA is easily compacted into smaller, super-ordered structures including spheroids, rods, and toroids. The interactions of these DNA-polycation structures with anionic membrane glycoprotein receptors allow easy entrance into the cell via endocytosis. The cationic condensation agents conveniently induce an increased pH in their cy-tosolic carrier vesicles and thus prevent lysosomes from degrading the vesicles prematurely. However, the eventual high-pH-induced proton influx in the car-rier vesicles causes a destabilization in the vesicle membrane and the release of the DNA complex. The complex is allowed to enter the nucleus via the nu-clear pore, separates from the polycation vehicle, and decondenses.4

The appeal of this mechanism of gene delivery comes in both the avoidance of immune responses and in a lack of damaging delivery byproducts, yet there is significant progress to be made before DNA condensation provides a pre-dictable and efficient mechanism for gene delivery. The unpredictability of the method comes with the lack of an obvious structure-activity relationship be-tween condensation agents and resultant DNA nano-particle shape. Since the resultant post-condensation shape of the DNA determines the efficiency of DNA transfection, this also means that there has not been found a correlation between condensation agent structure and transfection efficiency. Thus, it is impossible, or at least very difficult, to develop a pipeline-like way of develop-ing an ideal condensation agent.4

One very interesting thing to note (and the reason for the emphasis on viral vectors and endocytosis-mediated condensation for gene therapy) is the en-tirely ineffective mobility of unpackaged DNA throughout cells, both in the cytosol and more drastically in the nucleus. Lukacs et al. report relative diffu-sion constants (as compared with water) of less than 0.19 in the cytoplasm for DNA fragments of only 100 base pairs, decreasing six-fold to 0.032 for frag-ments of 500 base pairs. They report relative nucleic DNA diffusion constants consistently below 0.025 for DNA ranging from 20-5000 base pairs in size. This data, shown in Figure 5 below, indicates that gene delivery via DNA itself is highly ineffective. It can then be concluded that packaging and transport mechanisms such as viral vectors and the endocytosis-induced vesicles of DNA condensation are crucial to effective gene delivery, and also that the de-velopment of other, more effective packaging and transport mechanisms is important, if not invaluable.5

29


The use of viral vector delivered gene therapy has been shown to be quite suc-cessful in both animal and clinical trials by many groups for illnesses affecting various tissues. A study conducted by Morishita et. al. well illustrates the long-term effects of successful gene therapy. In their study, human insulin vector was administered via a HVJ-liposome complex to diabetic mice. For up to 14 days on average following a single transfection, the mice were shown to have in-creased insulin levels (Figure 6), decreased plasma glucose levels (Figure 7), and even after three months following a single transfection significantly de-creased BUN and creatinine levels were observed (Figure 8). This indicates much improved insulin production and resulting metabolic functions due to the expression of the vector genome.6

Figure 5. Relative DNA mobility vs. DNA size in both the cytoplasm and the nucleus5

Figure 6. Plasma insulin concentration in diabetic mice for 14 days following a

single transfection of human insulin vector6

Figure 7. Plasma glucose concentra-tion in diabetic mice for 18 days fol-

lowing a single transfection of human insulin vector6

30


It has been shown that the onset of Parkinson’s Disease (PD) is typically caused by the mutation of one or more of its five primary genetic risk factors: SNCA (which encodes for α-synuclein), MAPT (which encodes for microtubule associated protein tau), GBA (which encodes for the lysosomal enzyme gluco-cerebrosidease), LRRK2 (which encodes for leucine-rich repeat kinase 2), and OMI/HTRA2 (which encodes for an unnamed serine protease that is known to cleave beta-amyloid). Of these five genomes whose mutations are shown to correlate most strongly with the presence of PD, it is useful to focus primarily on SNCA (and thus the protein α-synuclein) and LRRK2 (and thus the leucine-rich repeat kinase 2), as the majority of gene therapy work has been done in relation to these two targets.7

α-Synuclein is a highly charged protein commonly found in abundance around synaptic vesicles whose specific function are currently not well known. It has been shown in many genetic studies that genetic mutations and alterations at the SCNA gene encoding for α-synuclein have a higher correlation to the symptoms of PD than any other genetic mutation. SCNA mutation has been shown to lead to the formation of Lewy bodies – a major component of the pathology of several neurodegenerative diseases including PD – via the path-way shown in Figure 9. 8

As shown above, mutant α-synuclein has been shown to form irregular β-

sheets which lead to aggregation, protofibril formation, and ultimately the creation of a fibrillar α-synuclein polymer in a pathway analogous but not identical to the formation of amyloid plaques in Alzheimer’s disease. The other suggested α-syn pathology is via small oligomers, which have been shown to

Figure 8. BUN (d) and creatinine (e) concentrations on average for 3 months follow-

ing a single transfection of human insulin vector6

31


bear significant toxicity on their own right. Regardless of the pathological path-way, a mutation of SCNA leading to mutant α-synuclein or wild-type α-

synuclein in excessive amounts plays a large part in PD. Thus, SCNA is a viable target for gene therapy. 8

The mechanism of LRRK2 pathology is not very well known. It has, however, been shown that LRRK2 kinase activity is required for neuronal toxicity, and suggested that LRRK2 inhibition is a viable treatment for PD. This implies that the engineering of a vector gene that encodes for either less active LRRK2 or non-toxic LRRK2 inhibitors could provide a viable treatment via gene thera-py.9,10

α-Synuclein in particular has shown incredible promise as a future target for gene therapy treatment of PD. Figure 10 shows the fibrillation activity of wild-type α-syn and three PD-linked mutant α-synucleins vs. time when treated with a fibrillation-retarded α-synuclein mutant as done by Koo and colleagues at Sejong University. It was observed that the fibrillation-retarded “β-sheet breaker peptides,” as Koo calls them, were very effective in inhibiting the fibril for-mation of all α-synucleins tested in vitro. This provides a promising avenue for PD treatment via gene therapy, awaiting only the development of a viable DNA sequence (and accompanying viral vector) encoding for a fibrillation-retarded α-synuclein mutant.11

Figure 9. The pathways of α-synuclein inclusive pathology8

32


Ultimately, few in vivo experiments have been performed on the gene therapy treatment of Parkinson’s disease due to the very recent discovery that genetic treatment was a relevant approach to treating the disease. However, it has now been shown that gene therapy has potential to be a highly effective treat-ment for PD due to the reported use of mutant proteins and kinases as effec-tive treatments in vitro. Future work should focus on the development of safe and viable delivery mechanisms including viral and non-viral vectors contain-ing PD-treatment-relevant mutant genes, and on the in vivo testing of the effi-cacy of these vectors. Ideally this would progress to clinical trials for gene therapy mediated treatments for Parkinson’s disease, providing the first ever viable treatments for the debilitating neurodegenerative disease.

Figure 10. Prevention of α-synuclein fibrillation by fibrillation-retarded mutants11

33


(1) Björklund, T.; Kirik, D. Scientific rationale for the development of gene therapy strategies for Parkinson’s disease. Biochimica et Biophysica Acta (BBA) - Molec-ular Basis of Disease 2009, 1792, 703–713.

(2) Kay, M. A.; Glorioso, J. C.; Naldini, L. Viral vectors for gene therapy: the art of turning infectious agents into vehicles of therapeutics. Nat Med 2001, 7, 33–40.

(3) Ferrari, M. Nanovector therapeutics. Current Opinion in Chemical Biology 2005, 9, 343–346.

(4) Vijayanathan, V.; Thomas, T.; Thomas, T. J. DNA Nanoparticles and Development of DNA Delivery Vehicles for Gene Therapy†. Biochemistry 2002, 41, 14085–14094.

(5) Lukacs, G. L.; Haggie, P.; Seksek, O.; Lechardeur, D.; Freedman, N.; Verkman, A. S. Size-dependent DNA Mobility in Cytoplasm and Nucleus. J. Biol. Chem. 2000, 275, 1625–1629.

(6) Morishita, R.; Gibbons, G. H.; Kaneda, Y.; Ogihara, T.; Dzau, V. J. Systemic Admin-istration of HVJ Viral Coat–Liposome Complex Containing Human Insulin Vector Decreases Glucose Level in Diabetic Mouse: A Model of Gene Therapy. Bio-chemical and Biophysical Research Communications 2000, 273, 666–674.

(7) Bras, J. M.; Singleton, A. Genetic susceptibility in Parkinson’s disease. Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease 2009, 1792, 597–603.

(8) Waxman, E. A.; Giasson, B. I. Molecular mechanisms of α-synuclein neurodegen-eration. Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease 2009, 1792, 616–624.

(9) Greggio, E.; Zambrano, I.; Kaganovich, A.; Beilina, A.; Taymans, J.-M.; Daniëls, V.; Lewis, P.; Jain, S.; Ding, J.; Syed, A.; Thomas, K. J.; Baekelandt, V.; Cookson, M. R. The Parkinson Disease-associated Leucine-rich Repeat Kinase 2 (LRRK2) Is a Di-mer That Undergoes Intramolecular Autophosphorylation. J. Biol. Chem. 2008, 283, 16906–16914.

(10) Löw, K.; Aebischer, P. Use of viral vectors to create animal models for Parkin-son’s disease. Neurobiology of Disease 2012, 48, 189–201.

(11) Koo, H.-J.; Choi, M. Y.; Im, H. Aggregation-defective α-synuclein mutants inhibit the fibrillation of Parkinson’s disease-linked α-synuclein variants. Biochemical and Biophysical Research Communications 2009, 386, 165–169.

References

34


35


The purpose of this experiment is to test the effect of aceta-minophen on Simocephalus serrulatus offspring survivability. Four one-week trials were run in which there were four con-tainers for each of the four different concentrations of dis-solved acetaminophen: 0 ng/L, 225 ng/L, 450 ng/L, and 900 ng/L. The hypothesis stated the concentration of 900 ng/L would have the most detrimental impact on S. serrulatus off-spring. The results of this experiment show there is no signifi-cant difference in offspring survivability between the control group and the different treatment groups.

Asha Scott, Andrea Bodale, Marisa Pinson, Dr. Marty Harvill

Department of Biology, Baylor University,

36


Introduction

37

Materials and

Methods

The presence of pharmaceuticals in the environment is growing at an alarm-ing rate. The variety of pharmaceuticals in the market is increasing rapidly, and with the presence of both parent chemicals and subsequent metabolites, pharmaceuticals are increasing at an even higher rate in the environment (Guillén, et al., 2012). There has been a call to address the issue of potential environmental impact since these pharmaceuticals that are entering the envi-ronment have been engineered to work at such miniscule levels, meaning that even at low concentrations, pharmaceuticals in the environment have the potential to have a large impact (Arnold, et al., 2013).

This study is focused on the effects of acetaminophen on organisms in water-ways in the United States. Acetaminophen is one of the most commonly used household drugs. It has been shown through an FDA supported study that between 2-5% of acetaminophen taken orally passes through the body un-metabolized and is excreted unchanged in the urine (McNeill Consumer Healthcare, 2002). One study that collected samples from streams across the United States found the median concentration of acetaminophen to be 110 ng/L with a maximum concentration of 10,000 ng/L in one instance (Kolpin, et al., 2002). Since so many organisms are dependent upon the success of inver-tebrates at the bottom of the food chain, such organisms are often used as indicators of environmental quality. Of the several organisms that were tested by Kim et al. (2012), the particular species of Daphnia studied—Daphnia magna— was deemed the most acetaminophen-sensitive organism. In the present study, the toxicity of acetaminophen will be tested by using Simo-cephalus serrulatus, a species of Daphnia indigenous to the Waco Wetlands. This preliminary study evaluated the effect of acetaminophen at the concen-trations of 225 ng/L, 450 ng/L, and 900 ng/L on the survivability of on S. ser-rulatus offspring.

The Lake Waco Wetlands allows water to pass through different cells before it flows back out to the Bosque River. The Lake Waco Wetlands are made up of four cells, which are shallow basins of water separated by raised banks. Water becomes progressively more pure as it passes from cell to cell through pipes. The last cell, Cell 4, should contain the least amount of acetamino-phen. Sixteen mesocosms were set up using clear plastic Sterilite 5.7L con-tainers. Four containers were designated for each of the three different con-centrations to be tested and another four were designated as the 0 ng/L ac-etaminophen control. The tested concentrations were 225ng/L, 450 ng/L, and 900 ng/L. The containers were filled with 1 liter of Cell 4 Waco Wetland water that had been filtered through 180 micron-mesh. Five lab-grown S. serrulatus were placed into each mesocosm. Stock solution was made using 4.0 mg/L of powdered acetaminophen tablets and D.I. water. This stock solution was then used to make the different concentrations of acetaminophen to be tested. Required amounts of stock solution were calculated and pipetted into the


Results

Discussion

different mesocosms to achieve the desired concentrations. No stock solution was added to the four control mesocosms. Fluorescent lights were hung 36.8 cm above the mesocosms and set with a timer to a 12-12 day-night cy-cle. Each S. serrulatus trial was then left alone for a one week interval, at which point the S. serrulatus in the differing concentrations of the current trial were counted and the data for each mesocosm was recorded. After counting, the experiment was re-set three more times, resulting in a total of four trials.

Figure 2 shows that the population means are not statistically significantly dif-ferent. This is supported by a p-value that shows little deviation from the mean. The long lines on either end of the boxes in the Box-and-Whiskers plot repre-sent the maximum and minimum populations that were counted for each con-centration. The means are represented by where the two colors meet. The pop-ulation means for the differing concentrations for the offspring survivability of

Figure 1: Experimental setup

Figure 2 shows the mean number of S. serrulatus per concentration. Using ANOVA, no statistical difference among treatments and control group were found (p-value = 0.5877>0.05).

38


References

Acknowledgments

S. serrulatus do not greatly differ. The hypothesis was not supported since there was no significant difference between the population means for each concentration. The data gathered in this ex-periment proved that the 112 ng/L concentration of acetaminophen that was found in naturally flowing lake water is not detrimental to the pop-ulation of S. serrulatus (Fig. 3) since the concen-trations that were tested, which were more than twice the amount of the naturally occurring con-centration, had no damaging effect on the S. ser-rulatus population. This means that there is no need to worry about the current levels of dis-solved acetaminophen in the water systems in which acetaminophen is present in levels equal to or less than 900ng/L. This encompasses the majority of U.S. streams which had a median concentration of 110ng/L (Kolpin, et al., 2002).

A special thanks to Dr. Marty Harvill, Nora Schell, Baoqing Ding , The College of Arts and Sciences, Department of Biology, Lake Waco Wetlands.

Arnold, K.E.; Boxall, A.B.A.; Brown, A.R.; Cuthbert, R.J.; Gaw, S.; Hutchinson, T.H.; Jobling, S.; Madden, J.C.; Metcalfe, C.D.; Naidoo, V.; Shore, R.F.; Smits, J.E.; Taggart, M.A.; and Helen M. Thompson. (2013) “Assessing the exposure risk and impacts of pharmaceuticals in the environment on individuals and ecosystems.” Biology Letters, 9: 20130492.

Guillén, D.; Ginebreda, A.; Farré, M.; Darbra, R.M.; Petrovic, M.; Gros M.; and Barceló, D. (2012) “Prioritization of chemicals in the aquatic environ-ment based on risk assessment: Analytical, modeling and regulatory perspective.” Science Of The Total Environment, 440, pp. 236–252.

Kolpin, D.; Furlong, E.; Meyer, M.; Thurman, E.M.; Zaugg, S.; Barber, L.; and Buxton, H. (2002) "Pharmaceuticals, Hormones, and Other Organic Wastewater Contaminants in U.S. Streams, 1999-2000: A National Re-connaissance." USGS Staff -- Published Research. Paper 68.

McNeill Consumer Healthcare. 2002. "Overview of Acetaminophen Metabo-lism and Toxicology." Response to Docket No. 1977N-0094L.Vol 1, pg. 101

Kim, P.; Park, Y.; Ji, K.; Seo, J.; Lee, S.; Choi, K.; Kho, Y.; Park, J.; and Choi, K. (2012) "Effect of chronic exposure to acetaminophen and lincomycin on Japanese medaka (Oryzias latipes) and freshwater cladocerans Daphnia magna and Moina macrocopa, and potential mechanisms of endocrine disruption." Chemosphere. Volume 89, Issue 1, pg. 10–18.

Figure 3: Simocepha-lus serrulatus

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The prevalent view on gender differences in neurobiologi-cal processing is males are generally unilateral whereas females are bilateral. In both pain and language processing females demonstrated dual hemispheric activity while males favored one hemisphere. This may have connections to the hunter-gatherer time in human history, when men’s and women’s individual tasks exerted different evolutionary pressures on the way each gender's brain functioned. Hormones such as estrogen and testosterone influence cognitive activity as well. An in depth review shows that these theories, in addition to other suggested explanations, sup-port the view that males are unilateral and females are bilateral.

Krystal Miller, Renee Michalski

Department of Psychology and Neuroscience, Baylor University

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Vol. 1 | Spring 2014 41

The generally accepted finding of many studies on the difference between male and female brains is that males tend to express unilateral processing while females demonstrate bilateral processing. Several aspects, such as pain and language, have been studied in order to determine how men and wom-en differ in hemispheric preference. Possible explanations for these differ-ences in the tendency to be unilateralized or bilateralized include origins in the past human activities of hunting and gathering, hormonal influences on brain development and function, and an array of more abstract theories such as physiological gender identity (masculine or feminine) and sexual orienta-tion.

General Functional Organization Based On Hemispheres

The Left Hemisphere

The left hemisphere is known for its role in language and calculations. Damage to the left side from strokes, tumors, or injuries may impair reading, writing, speaking, arithmetic, reasoning, and understanding (Myers, 2013, p. 76). The frontal lobe on the left is more active in individuals who are happy, enthusiastic and positive (Myers, 2013, p. 467). Stimulation of the insula area in the left hemisphere produces parasympathetic mediated calming effects of lowing heart rate and blood pressure (Wasan, Anderson & Giddon, 2010). The left half contains the two structures well known for speech production (Broca's Area) and language comprehension (Wernicke's Area). Damage to Broca's Area will result in the lack of verbal production while damage to Wer-nicke's Area will result in the production of senseless or meaningless words and sentences (Myers, 2013, p. 365). Belin et al. (1998) investigated the ad-vantage of the left hemisphere during language tasks using rapid formant transitions (very quick frequency changes of spectral peaks of about 40 msec) and extended formant transition stimuli (similar to rapid formant tran-sitions but at a duration of 200 msec). Both the rapid and extended formant transition stimuli induced bilateral activation in the left superior temporal gyri. However, the rapid formant transition stimuli showed a primarily left-biased cortical pattern, activating an area almost double in size in the left hemisphere than in the right. Conversely, the extended formant transition stimuli activated only the auditory cortices, and the pattern was almost sym-metrical. This suggests a left-biased asymmetry of auditory activation.

The Right Hemisphere

The right hemisphere is known for its role in perceptual, creative, and artistic tasks. While the left may be involved with language production, the right hemisphere excels in making inferences, in modulating speech to make meanings clear, and in the orchestration of our sense of self (Myers, 2013, p. 79-80). The right hemisphere is dominant in spatial and targeting tasks as well. Activation of the right prefrontal cortex is associated with negative emotions such as disgust and depression and is seen in individuals with ‘negative’ personalities (Myers, 2013, p. 467). The right hemisphere is also


more involved than the left in the processing of pain (Coghill, Gilron & Iadaro-la in Wasan, et al, 2010) and in attending to pain (Wasan, et al, 2010); stimula-tion of the insula in the right hemisphere produces arousing effects of raising heart rate and blood pressure via the sympathetic nervous system. Damage to the right half of the brain will result in emotional problems such as trouble characterizing emotions in faces, matching emotional expressions, and group-ing pictorially presented and written emotional scenes.

Corpus Callosum Connection

The left and right halves of the brain are connected by a band of axon fibers called the corpus callosum. The corpus callosum transfers motor, senso-ry, and cognitive information between the brain hemispheres. Severing this band will disrupt the relaying of information between the left and right hemi-spheres. This has been a very effective treatment for patients with epilepsy as the abnormal brain activity cannot bounce back and forth between the two halves, thus ending the epileptic episodes (Myers, 2013). The corpus callosum in females is larger in cross section and more densely packed with fibers than in the male, making the hemispheres in females more “extensively intercon-nected” (Ardekani, Figarsky, & Sidtis, 2012).

Organizational Comparison

Language Processing

Females demonstrate a more bilateralized neurological process for lan-guage. Baxter et al. (2003) found that males and females show a difference in fMRI activation patterns during a semantic processing language task. An ex-ample of such a task is hearing a general classification (ex: birds) and deciding if the specific classification (ex: flamingo) match. The word pairs may be cor-rect (ex: vegetables/celery) or incorrect (ex: beverages/lemon) and the partici-pant must notify only the correct pairs. Females demonstrated activation on both their left and right hemispheres evenly, with significant activation in the left and right superior temporal gyri and predominant left inferior frontal gy-rus activation. Males demonstrate a more unilateralized neurobiological pro-cess. During the semantic language processing task, fMRI activation patterns showed increased left hemisphere activity in males, (diffusely in the left inferi-or frontal gyrus, the left superior temporal gyrus, and the cingulate areas), while the right hemisphere was generally inactive (Baxter et al. 2003). Shaywitz et al. (1995) used echo-planar functional magnetic resonance imaging to study neural activity during orthographic, phonological, and semantic language tasks. They found that females exhibited a more even distribution between the left and the right hemisphere. Females also employed the inferior frontal area bilaterally as well. The amount of neural activity in the male brain detected by the echo-planar functional magnetic resonance imaging was almost double in the left hemisphere when compared to the right during orthographic, phono-logical, and semantic language tasks (Shaywitz et al., 1995). This information reveals that males process language in their left hemisphere while the right

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http://en.wikipedia.org/wiki/Facial_expressions


hemisphere remains uninvolved.

During rhyming tasks, males were lateralized to the left inferior frontal area, which once again shows males using one specific hemisphere (Shaywitz et al., 1995). As the left hemisphere is specialized for language with Broca’s and Wernickie’s areas both present in that hemisphere, men represent the ‘typical’ brain function for language, which is lateralization on the left.

Pain Processing

Gender differences are found in pain processing as well. Wasan, Ander-son, and Giddon (2010) collected data on patients with chronic left-sided, right-sided, or bilateral spinal pain. In the females, there was essentially no differ-ence in the effect of left-sided pain versus right-sided pain on mood or enjoy-ment of life, anxiety, depression, or total negative affect. Their results indicate that despite females reporting more average pain, females did not have the connection between pain and psychiatric symptoms found in males. This demonstrates that females were not adversely affected by pain symptoms on either side of the body.

Wasan et al. (2010) also discovered that for male patients with chronic spinal pain on the left side of their body, interference with mood or enjoyment of life, anxiety, depression, and total negative affect were more severe than males with right-sided pain. Pain from the left side of the body is processed by the right side of the brain. As the right hemisphere is more involved than the left in negative emotions (Myers, 2013), the processing of pain (Coghill, Gilron & Iadarola in Wasan, et al, 2010) and in attending to pain (Wasan, et al, 2010), these findings are not surprising. However, this finding was seen only in males; despite females reporting similar levels of pain. Their results indicate that for males, left-sided pain resulted in more distress, depression, and psychological health interference. These findings provide another correlate with right-hemisphere dominance in males. There is a very real application for this knowledge in the world of medical assessment and treatment. Men with left-sided pain are more likely to display negative psychological symptoms such as depression and may benefit from psychiatric, pharmacological or behavioral interventions for these symptoms.

Functional Connectivity

A study done by Tomasi and Volkow (2012) explored the laterality pat-terns of short-range and long-range connectivity of brain areas using function-al connectivity density mapping (FCDM) with special computing and graphing methods to analyze gender differences in MRIs from a large public database. Short-range connections were thought to underlie specific functional speciali-zation while long-range connectivity represented functional integration. High levels of connectivity are thought to increase efficiency of neural processing. Females showed greater left lateralization of long-range connectivity in the in-ferior frontal cortex areas, whereas males had a higher density of long-range fibers on the right in the superior temporal cortex. This would seem to indi-

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cate that females integrate information more on the left and males more on the right. Of interest here are the findings on short-range connectivity fibers. Although the differences were limited to small brain areas, the density of short-range fibers was different from left to right in the males, but not in the fe-males. This polarity of short-range functional connectivity in males would sug-gest that males localize some functions in one hemisphere, whereas females tend to use both hemispheres.

Tomasi and Volkow's (2012) study found a greater lateralization of functional connectivity in males than females, which included a greater density of short-range fibers on the right in the inferior frontal and parietal cortices as well as the superior temporal cortex. Density of long-range connectivity fibers was also higher on the right in the superior temporal cortex for males. Short-range fibers mediate specific functions while long-range fibers allow for inte-gration. These results demonstrate a consistent favoring of the right hemi-sphere for specific functions in males and right lateralization of integrative tasks occurring in the superior temporal cortex. Since these data were ob-tained in the resting state, the results would seems to indicate that the male brain is generally formulated for processing on the right side, which confirms the suggestion of a gender dimorphism in neurological lateralization.

Facial Processing In determining whether a face presented on a computer screen was

male or female, females had a tendency to be 10 ms faster than males. Proverbio, Mazzara, Riva, and Manfredi (2012) found asymmetric inter-hemispheric transfer times in males with faster latencies in right to left hemi-sphere transfer (with a speed of 170 ms) and slower latencies in left to right hemisphere transfer (with a speed of 185 ms). This demonstrates a difference for males between the left and right transfer not seen in females. Analysis of brain wave-evoked potentials during this task demonstrated the time for inter-hemispheric transfer of visual information was symmetric in females with their right hemisphere to left hemisphere time essentially equal to their left hemi-sphere to right hemisphere time, demonstrating a greater hemispheric transfer in females, consistent with anatomical findings mentioned earlier showing more fibers in the female corpus callosum (Proverbio, Mazzara, Riva, and Manfredi, 2012).

Emotional Processing

Emotional intelligence is defined as problem solving with and about emotions including the ability to perceive, use, understand, and manage emo-tions on the self and others, according to Mayer, Salovey, Caruso, and Si-tarenios (2003). Their study investigated a possible connection between emo-tional intelligence and a right hemispheric dominance for facial and emotional processing. First, as expected, they confirmed a right hemispheric dominance for detection of emotional facial expression in both males and females. When asked to detect emotion on a chimeric face (one that was half neutral and half happy), females showed more of a right hemisphere dominance. The bias

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score was (-.34) for females, compared to a (-.23) male score, which shows that females were more lateralized than males for this task. Also, scores for the emotional intelligence tests showed a trend toward higher females' scores.

The Castro-Schilo and Kee (2010) study investigated connections be-tween emotional intelligence and a right hemispheric dominance for facial and emotional processing. While both males and females were lateralized to the right hemisphere for facial emotion detection, males were slightly less lateral-ized than females for detection of emotion in chimeric faces. This disputes the theory that males are more unilateral. They did note however, that males with a higher emotional intelligence score showed a greater dominance in the right hemisphere during facial emotion tasks. This may be due to different strategies used by the different genders. A global processing is a right hemisphere spe-cialization and local processing is a left hemisphere. Therefore, a global strate-gy may be the characteristic of a male's approach to emotional intelligence ra-ther than a female's possible localized approach.

Theories & Explanations

The Hunter-Gatherer Hypothesis

The ‘Hunter-Gatherer’ hypothesis has been used to explain differences in performance and processing between males and females. Males typically perform better in spatial tasks while females are better at object recall (Kimura, 1996). This separation of skills has theorized links to a past age during which the divisions of labor for men and women were significantly different, with men being hunters and women being foragers. The tasks at which our ances-tors excelled determined the evolutionary path for our modern day labor divi-sions. As described in 1992 by Silverman & Eals (in Eals & Silverman, 1994, p. 88), hunter, land-trekking men would use different spatial skills for tracking and killing animals than gatherer, homemaking women would use in foraging for edible plants. The hunter would need to orient himself in space and main-tain that orientation while moving about. These skills would pay forward to map reading and maze learning. His spatial skills would extend to accurate placement of deadly force for both food procurement and defense, useful in military applications today. The forager would need to locate certain objects among a group of objects and may make cognitive maps ‘incidentally,' that is, while moving about or engaging in other tasks. This serves for remembering the placement of objects.

When tested on their memory of object presence and location on-paper, females were better than men at remembering the location of objects and their performance on memory for the actual object, although it did not meet statistical significance. The ‘in-person’ version of the test included direct-ly instructing subjects to remember the objects in the ‘directed’ condition, and simply allowing them to sit in the room but later asking them to recall the ob-jects and their locations in the ‘incidental’ condition. Both common (paper, pen, ruler) and uncommon (teapot) objects were used. Women scored higher

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in directed and incidental conditions of common object memory and location. For identification of uncommon objects, men scored higher in the directed con-dition while women did better in the incidental condition. The authors suggest that the woman’s attentional style was more attuned to the environment and developed in response to demands of caretaking of both children and home, as well as foraging. Indeed, their skill for remembering locations of objects makes women better at using physical landmarks as guidance along a route from place to place. (Eals and Silverman, 1994). In contrast, men prefer using north, south, east, west and distance information for guidance Brown (2013)..

Another skill related to foraging is location-based inhibition-of-return which is thought to facilitate foraging by orienting attention toward novel loca-tions compared to recently inspected ones. From an evolutionary perspective, if inhibition-of-return facilitates successful foraging, then women might be ex-pected to exhibit greater location-based inhibition-of-return than men. When Brown (2013) tested this hypothesis, he found evidence of greater inhibition-of-return for women than men and greater inhibition-of-return to the higher spa-tial frequency target. The spatial frequency of the target selects for a specific visual pathway through the thalamus, with low spatial frequency traveling the magnocellular pathway which preferentially processes motion and orientation, and high frequency stimuli traveling through the parvocellular pathway, which carries form, color and fine detail information (Pinel, 1997). The results support the position that women have an object-based attention orientation and men have a location-based attention orientation. This view of perceptual/attentional processing differences between men and women provides a helpful framework from which to view sex differences in spatial abilities and others. Women are quicker and more accurate when recognizing objects compared to men. This may be taken as proof of the theory that women became bilateralized through the evolution of strengths related to past obligations as forgers and gatherers for their tribes and families.

Psychological Masculinity or Femininity

Psychology may play a role in this as Bourne and Maxwell (2010) re-vealed when they tested for psychological gender identity in their study of per-ception of emotion in chimeric faces. Participants were shown chimeric faces comprised of vertically split faces of which one half was neutral and the other expressed an emotion from the basic six being tested: anger, disgust, fear, hap-piness, sadness, and surprise. These faces were presented in pairs, one on top of the other, with one face showing the emotion on the left side and the other showing the emotion on the right side. The participants were asked to decide which face they believe looked more emotive by pressing either an upper but-ton for the upper face or a lower button for the lower face. Psychological gen-der-identity was assessed by a scale of twenty masculine, twenty feminine, and twenty neutral items. Right hemisphere dominance was seen as would be ex-pected in an emotional-processing task. The overall statistics were significant for happiness, sadness, and surprise. Biological males showed more lateraliza-

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tion than females for anger, happiness, sadness and surprise. Interestingly, psy-chological masculinity was a significant predictor for all six emotions. The high-er the masculinity, the more lateralization to the right hemisphere for the pro-cessing of both negative and positive emotions. So again we see that a more masculine mind will be more unilateral when processing faces and their emo-tions.

Hormonal Influence

Sexual hormones also influence organization in the brain. Lust et al. (2011) studied the correlation of prenatal testosterone and neural lateralization using a database of testosterone levels assessed from amniotic fluid samples collected between the 16th and 18th weeks of pregnancy. At six years of age, the children were observed during tests for handedness that consisted of ten tasks requiring motor movement (drawing, tooth brushing, throwing far, ham-mering, stirring, erasing, opening a lid, cutting with scissors, grasping a glass of lemonade, and turning the hands of a clock). Lateralization of language was assessed using a dichotic listening task. High prenatal testosterone was associ-ated with strong language lateralization to the left hemisphere. While testos-terone levels did not predict right or left-handedness per se, high testosterone levels were associated with low strength of handedness. These results imply a differential effect of prenatal testosterone on language lateralization and handedness. Lust et al. (2011) suggests that prenatal testosterone is the most important factor, rather than sex, in determining lateralization.

Kimura (1996) investigated the role of hormones on neurobiological processing and found that androgens seems to enhance male spatial function as seen when females born with congenital adrenal hyperplasia exhibit in-creased spatial ability when compared to their unaffected sisters. Testosterone also plays a role in spatial ability with the optimal levels being higher in wom-en and lower in men. Men have higher testosterone levels in the morning and during autumn, both times having the worst spatial performance for men, compared to spring and evening times, while non-spatial tasks showed no sig-nificant performance difference. Women performed better on spatial tasks during low-estrogen phases of their menstrual cycle and better on verbal and fine manual skills during high-estrogen phases. This research leads to the con-clusion that there must be early organization of the neurocognitive systems due to some hormonal influences causing sex differences.

Developmental Differences

There may be a developmental growth aspect to the differences in brain processing. Brain development during the primary years of life are character-ized by regressive processes such as pruning of the axons, dendrites, and loss of synapses leading to less gray matter; and progressive processes such as the myelination of neurons leading to an increase in white matter. Using a high-resolution magnetic resonance imaging for volumetric analysis, De Bellis et al. (2001) investigated the correlations of age, gender, cerebral gray and white

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matter, and area of the corpus callosum in a group of males and females, rang-ing from age 6 to 17. The results showed that young females go through signif-icant developmental changes but at a slower rate than their male counterparts. Males between ages 6 and 18 exhibited a 19.1% decrease in gray matter vol-ume in comparison to females, who exhibited a 4.7% reduction. However, males showed a 45.1% increase in white matter and a 58.5% increase in corpus callo-sum area, while females had a 17.1% increase in white matter volume and a 27.4% increase in corpus callosum area. Intracranial and cerebral volumes did not significantly increase with age, while cerebral gray matter exhibited the ex-pected decrease and white matter and corpus callosum area showed the ex-pected increase with age. Males, respectively, had larger intracranial volumes and cerebral volumes than females with an 11% and 12% difference. Males lose gray matter more quickly than females and increase their white matter and cor-pus callosum area more quickly than females, leading to females keeping their synapses for a longer period of time, therefore being able to use them, whiles males do not and cannot. This is a relevant distinction between the maturing male and female minds.

Sexual Orientation

Sexual orientation has also been suggested as a possible source of di-chotomy between male and female processing. Doreen (1996) discovered that sexual orientation seems to have no affect on paper-and-pencil spatial tasks, but when given the finger dexterity task that favors women, homosexual men performed equivalently to heterosexual males. However, heterosexual males performed better on targeting tasks and made fewer errors than homosexual men who performed relatively similar to heterosexual women. This refutes the previously accepted explanation that men perform better at targeting because of their physical or structural advantages. What seemed to be homosexual men as intermediates between heterosexual men and women has been proposed, by Hall and Kimura (1995), to be homosexual men exhibiting either male-typical or female-typical patterns, depending on the task at hand.

Conclusion

The research done to investigate the lateralization of neurological pro-cesses has been extensive and enlightening. Literature reviewed here has sup-ported the notion that despite inherent functional differences between the right and left hemispheres, men tend to be more lateralized in mental func-tioning, while women show more bilateral processing. This is true for lan-guage, where men demonstrate more lateralized neurological activity in the left hemisphere while women demonstrate use of both hemispheres. Analysis of fiber connectivity shows a large density of short-range fibers in the right hemisphere of the male brain; thus the male brain is formulated to process individual functions on the right. Indeed men show a quicker transfer of infor-mation from the right to the left hemisphere than in the other direction, sug-gesting quicker information processing on the right, resulting in readiness for transfer. A right-sided dominance would explain men’s excellent spatial skills.

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Men suffer from this weighty use of the right hemisphere when experiencing pain from the left side of the body, as left-side body pain has been shown to trigger depression and negative affect in males, but not in females. On the oth-er hand, women process language bilaterally and do not have a hemispheric concentration of short-range, specific-function fibers on either side. Support-ing the idea of bilateral functioning in females is a corpus callosum with a larg-er diameter and greater fiber density, which supports large amounts of infor-mation transfer equally in both directions.

Several theories have been put forth to explain these differences in functioning between the male and female brain. Evolutionarily speaking, these differences may have risen from a division of labor where spatial abilities fa-vored the hunting male, and near-range object location aided the forager, child-caring female. Differences have been tied to increased testosterone pre-natally and, developmentally in childhood and puberty, and in everyday/time of day functioning. More so than genetic sex, psychological gender-identity is associated with male and female brain functioning.

Gallagher (2010) calls attention to the idea that current neuroscience research is establishing that the left and right sides of our bodies and minds are not segregated, mechanical entities, but are, instead, extensions of neuro-logical networks that aid in defining ourselves in regards to gender and emo-tion. With this in mind, we may soon see our medical staff categorizing pa-tients based on left/right sided pain as a tool for both diagnosing and treating, especially if those patients are male. Or we may soon use the gender differ-ences in language, spatial skills, and memory to better assign employees to tasks that will allow them to take advantage of their evolutionary skills. This research could have applications in many areas that may allow our society to further advance medicine and our understanding of the genders.

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Ardekani, B. A., Figarsky, K., & Sidtis, J. J. (2012) Sexual dimorphism in the human corpus cal-losum: An MRI study using the OASIS brain database. Cerebral CORTEX, 23(10), 2514-2520. doi:10.1093/cercor/bhs253

Baxter, L. C., Saykin, A. J., Flashman, L. A., Johnson, S. C., Guerin, S. J., Babcock, D. R., & Wishart, H. A. (2003). Sex differences in semantic language processing: A functional MRI study. Brain and Language, 84, 264-272. Belin, P., Zilbovicius, M., Crozier, S., Thivard, L., Fontaine, A., Masure, M. C., & Samson, Y.

(1998). Lateralization of speech and auditory temporal processing. Journal of Cogni-tive Neuroscience, 10, 536-540.

Bourne, V. J. & Maxwell, A. D. (2010). Examining the sex difference in lateralisa tion for processing facial emotions: Does biological sex or psychological gender identity matter? Neuropsychologia, 48, 1289-1294. Brown, J. M. (2013). A sex difference in location-based inhibition-of-return. Personality and

Individual Differences, 54, 721-725. Castro-Schilo, L. & Kee, D. W. (2010). Gender differences in the relationship between emo-

tional intelligence and right hemisphere lateralization for facial processing. Brain and Cognition, 73, 62-67.

De Bellis, M. D., Keshavan, M. S., Beers, S. R., Hall, J., Frustaci, K., Masalehdan, A., ... Boring, A. M. (2001). Sex differences in brain maturation during childhood and adolescence. Cerebral Cortex Jun, 11(6), 552-557.

Eals, M. & Silverman, I. (1994). The hunter-gatherer theory of spatial sex differences: Proxi-mate factors mediating the female advantage in recall of objects arrays. Memory And Cognition, 88-96.

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Richa Manglorkar is a Junior Biology major from Flower Mound, Texas. Her future plans are to attend medical school in the fall of 2015. Hayden Jefferies is a Junior Biology major from Spring, Texas. His future plans are to continue conducting research and to attend medical school to become a surgeon. Dustin Buller is a Senior Biochemistry major, minoring in Computer Science, from Fort Worth, Texas. His future plans are to do mission work for a year and then attend medical school. Asha Scott is a Nursing (B.S.N.) major from Houston, Texas, who plans on graduating in December 2016 and entering the workforce as a Pediatric or Labor & Delivery nurse. She is considering doing research in one of the afore-mentioned fields.

Andrea Bodale is a Biology Premedical major from Phoenix, Arizona. After she graduates in May 2016, Andrea plans on attending medical school, earning her doctor of medicine degree, and opening her own private practice.

Marisa Pinson is from Beaumont, Texas, and is a Biochemistry B.S. major ex-pecting to graduate in May 2016. She plans on entering an MD/PhD program after college and continuing on to research medicine.

Krystal Miller is a Junior Neuroscience Major from Keller, Texas. Her future plans are to attend medical school and to become a pediatric neuropa-thologist at the Children’s Medical Center in Dallas specializing in neurologi-cal disorders.

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