Transformation of Ideas into Testable Hypotheses and Aims

Jason G. Umans, MD, PhD GHUCCTS

Georgetown – Howard Universities Center for Clinical and Translational Science

Georgetown University | Howard University MedStar Health Research Institute | Oak Ridge National Laboratory | Washington DC Veteran’s Administration Medical Center

Hypothesis

• A supposition made on the basis of limited evidence as a starting point for further investigation.

• A statement that explains or makes generalizations about a set of facts or principles, usually forming a basis for possible experiments to confirm its viability. – Greek hupothesis foundation, supposition, under-

placing.

Pediatric Origins of Scientific Method

• Underlying knowledge or observations • Ask questions • Create hypotheses, make predictions • Test hypotheses (experiments or other approach) • Evaluate evidence, draw tentative conclusions • Iterate or move on

• Eg., Peek-a-boo

Hypotheses-Research-Grants

• Why do we favor hypothesis-based research? – Clarity – Benchmarks for progress – Basis for aims – Basis for experiments – Basis for replication, extension, alternative testing

Quantitative Research Paradigms

• Reasoning from general to specific • Explain (causal) relationships between

(objectively and reproducibly measurable) variables

• Predictions are specific, unique, and falsifiable (with available methods) – Rejection of hypothesis leads to alternative

explanation(s)

Getting to a Question • Know everything about your field.

– Read everything (old and new) – Know how others are thinking about or

approaching research in your field-go to meetings, lectures

– Find the voids (that really matter) – Are there tools to ask questions in ways that were

not possible in the past? – Are there any missing data between the literature

and the ability to ask an important new question?

• Hypothesis must build on available knowledge to advance the field (in important way) – Must lead to specific predictions (usually from

comparisons, raising issue of appropriate controls) – Must be measurable – Must drive the research rather than follow the

available technology – Useful to avoid descriptive or data-gathering

exercises – Useful to tie together aims and experiments

Hypothesis and Specific Aims • These better be perfect! • Role of (short) introduction stating overall

objectives of project and long term goal • Review current state of the art,

limitations/gaps (and their consequences) • Your contributions and ability to move field

forward • New world view and how to advance the field

– This is nice place for an overarching hypothesis

Hypotheses in the Specific Aims • 2-4 Specific Aims (in a 5y grant) • Must be complementary and not depend on

prior aims, results, or untested methods • Should each relate to the overall hypothesis

and, if possible, to a narrower hypothesis • One approach is to start with tag-line, then

hypothesis, experimental approach, payoff and context

• You are allowed ONE descriptive aim – Don’t force an hypothesis when it isn’t

Where to start?

• Writing: – Title* – Specific Aims (with hypotheses)* – Research Strategy – Innovation – Abstract* *these are the roughly 1.5 pages plus 81 characters that everyone on study section will read

Where to start?

• Thinking: – A critical idea (is it worth 5 years of your life and

much of the NIH’s money; will you be able to convince all of the stakeholders of this?)

– The idea must be • Original • Important • Add important new knowledge

Specific Aim: Introductory paragraph

Statement of central hypothesis What existing knowledge gap will these studies fill?

Why is this problem important? Why am I studying this problem?

Although SP-C is the first surfactant component to appear during lung development, the function of SP-C is the least understood of the surfactant proteins. Several lines of evidence strongly support the hypothesis that SP-C is important for surfactant function and turnover: Replacement surfactants with recombinant SP-C as the sole protein component have excellent surface properties in vitro and in vivo; SP-C facilitates the uptake of lipids by Type II cells; and, most importantly, several term human infants with normal levels of SP-A and SP-B but undetectable SP-C protein rapidly developed severe RDS and ultimately succumbed to the disease. The overall goal of this project is to assess the role of SP-C in surfactant homeostasis (see Fig.1). SP-C function(s) will be identified by systematically altering the structure of the molecule and evaluating changes in surfactant assembly, secretion, biophysical activity, recycling and catabolism. The central hypothesis of this proposal is that the extramembrane domains of SP-C are important for intracellular trafficking, packaging of lamellar body phospholipids and/or secretion whereas the transmembrane domain of SP-C is essential for the biophysical properties of the peptide and/or uptake of alveolar phospholipids.

Tim Weaver (Cincinnati ) was kind enough to put this grant on the web

Why is this experiment important? How will the hypothesis be tested?

Clear statement of hypothesis or goal Descriptive title for each specific aim

Structure of a specific aim

Specific Aim 1. Identification of Motif(s) Required for Intracellular Trafficking of SP-C. This aim will test the hypothesis that the sorting determinant(s) which directs SP-C to the regulated secretory pathway is located at the NH2-terminal end of the mature peptide. Deletion constructs of SP-C will be cloned in frame with green fluorescent protein (GFP) and transiently transfected into PC-12 cells. Intracellular trafficking of SP-C GFP will be monitored by confocal microscopy. These studies will identify the peptide domain(s) which is essential for trafficking of SP-C to dense core granules of the regulated secretory pathway.

Structure of a specific aim

Specific Aim 2. Identification of Motif(s) Required for Secretion of SP-C. SP-C is a transmembrane protein which is secreted into the airway. This aim will test the hypothesis that inward vesiculation of the SP-C proprotein in multivesicular bodies is essential for secretion of SP-C and that the NH2- and/or COOH-terminal propeptide is required for inward vesiculation. Deletion constructs of the SP-C proprotein will be transiently transfected into isolated Type II epithelial cells and the kinetics of SP-C secretion assessed by immunoprecipitation. Inward vesiculation will be assessed by immunogold labeling of ultrathin cryosections. These studies will provide insight into the mechanism whereby an integral membrane protein can be secreted.

Structure of a specific aim

Specific Aim 3. The Effect of Palmitoylation on SP-C Trafficking and Surfactant Homeostasis. This aim will test the hypothesis that palmitoylation of Cys5-Cys6 and/or Lys11 is important for packaging of surfactant phospholipids in lamellar bodies; we will also test the hypothesis that palmitoylation alters SP-C mediated uptake of surfactant phospholipids from the alveolus. The three amino acids in SP-C known to be palmitoylated will be individually or combinatorially mutated to prevent acylation. Peptide trafficking, lipid packaging, and secretion of non-palmitoylated SP-C will be evaluated in transiently transfected Type II epithelial cells. In order to assess the importance of SP-C palmitoylation for surfactant homeostasis, palmitoylation mutants will be expressed in SP-C knockout mice. The pathophysiology associated with the loss of SP-C palmitoylation will provide new insight into the physiologic importance of this post-translational modification for surfactant homeostasis.

Structure of a specific aim

Specific Aim 4. The Role of the Transmembrane Domain in Surfactant Homeostasis. This aim will test the hypothesis that the amino acid composition, and in particular the valine content, of the membrane spanning domain is critical to the function of SP-C. SP-C constructs in which the transmembrane has been selectively mutated will be expressed in SP-C knockout mice. Mutations resulting in perturbation of surfactant homeostasis will provide new insight into the function of SP-C and the structural basis for this function.

Hypothesis formulation

• It is not enough to formulate the hypothesis- you must rigorously test it – Design experiments that establish cause-effect – Experiments that establish a correlation do not

rigorously test the hypothesis

Reality intrudes. Anticipate it….

• Hypotheses are frequently wrong – Structure your experiment so that a negative

outcome is informative – Propose an alternative hypothesis (usually in

section on anticipated results/interpretation following each experiment in Methods)

Example hypotheses

• We will test the hypothesis that EGF treatment is correlated with SP-C expression

• We will test the hypothesis that expression of SP-C is developmentally and spatially regulated

• We will test the hypothesis that annexin I mediates lamellar body formation and surfactant recycling in alveolar type II cells and the annexin I-mediated processes are under growth factor and cellular calcium control

Potential problem areas • How many specific aims (too many/few) • Integration of specific aims (do they make a cohesive whole) • Contingent aims • Relationship of specific aims to central hypothesis • Goals vs hypotheses • Depth vs breadth • Novelty • Lack of hypotheses • Alternative hypotheses • Organization • Physiological/biological/clinical relevance/importance

Now, to writing the grant Starting with the hypotheses

• Most R01 applications set out to test an hypothesis, rather than search for a problem or simply collect information.

• Your hypothesis triggers everything you plan to do. Conceptually, think of it as your destination, determining the course of your research and the terminal point of all its pathways.

• Choose a hypothesis that is well-focused and testable, and that your experimental results will be able to prove or disprove. Reviewers must believe that your hypothesis is sound and important so your research will be able to make a high impact on its field.

A few more examples: (Specific) Winners

From Jacques Banchereau, Ph.D., Baylor University: • Systemic lupus erythmatosus patients with active disease display

considerable alterations in their CD8+ T cell compartments, including effector CD28+ CTLs and suppressor CD28- subsets. We surmise that an excess of killer cells in lupus results in the characteristic tissue damage and explains an excess of dying cells that are considered as key factors in this disease.

From Volker Briken, Ph.D., University of Maryland College Park: • This proposal seeks to test the hypothesis that the capacity of

Mycobacterium tuberculosis to inhibit infection-induced apoptosis of macrophages is a major pathway of the bacteria to avoid the host’s innate and adaptive immune response.

And (Poorly-focused) Losers

• Understanding the strategies of Escherichia coli to subvert host cells will allow for improved ways of preventing and treating E. coli -related diseases.

• Rheumatoid arthritis patients with active disease show many alterations in their immune profile.

• A wide range of molecules can inhibit HIV infection.

Transition from Hypotheses to Aims • For your aims, think high level. Ask what two or three objectives

you could achieve within the time you are planning to request. Make each aim an achievable objective, not a best effort, one with clear endpoints peer reviewers can easily assess.

• Limiting your application to few Specific Aims keeps you clear of the typical new applicant trap of being overly ambitious. Like your topic, your Specific Aims should build on your previous experience.

• My Specific Aims can test my hypothesis. • They are doable within the grant period I am requesting. • The aims are concrete and well-focused. • I can define endpoints my peer reviewers will be able to assess.

Transition from Aims to Strategy

• Start planning (not writing!) the strategy by sketching out experiments to conclusively accomplish each aim, including alternatives for if you 1) have an exciting finding or 2) get a “negative” result

• Make sure all the experiments (and alternatives) track with the specific aims and link back to hypotheses.

• It's a good idea to create a flowchart and timeline for planning purposes -- you can also include it in the application if you wish.

Did I plan adequately? • After finalizing this planning work, i.e., before writing,

check that: • Each aim links to overarching and/or specific hypothesis • Experiments are appropriate an sufficient to fully

address each aim. • I created a flow diagram that shows the branching of the

possible pathways I could take. • I planned those experiments too. • My team and I are qualified to execute the experiments. • I or they have the required resources to do the work.

• Think of the one-page Specific Aims as a capsule of your Research Plan. All your reviewers read your; excite them about your project!

• Write a narrative. Use at least half the page to present and provide the context for your planned research. A good way to start is with a sentence that states your project’s overarching goals. Then:

• Describe the significance of your research. • Give your rationale for choosing the project — in some cases, you

may want to explain why you did not take an alternative route. • Briefly describe your aims and show how they build on your

preliminary studies and your previous research. • To target a study section with broad expertise, summarize the

status of research in your field and explain how your project fits in. • State your hypothesis.

Many also used their aims to:

• State the technologies they plan to use. • Note their expertise to do a specific task or that of

collaborators. • Describe past accomplishments related to the project. • Describe preliminary studies and new, key findings in the field. • Explain their area’s biology. • Show how the aims relate to each other.

• Depending on your situation, decide which items are important

for you. For example, it’s a good idea for a new investigator to highlight preliminary data or qualifications.

• Many people use bold or italics to emphasize items they want to bring to the reviewers’ attention, such as the hypothesis or rationale.

Elements of Style • List your aims. After the narrative, enter your

aims as bold bullets, or stand-alone or run-on headers (as in List your aims. at left).

• State your plans using strong verbs like identify, define, quantify, establish, determine.

• Describe each aim in one to three sentences. • Consider including bullets under each aim to

refine your objectives.

Did you get it right? • I kept to the one-page limit. • Each of my aims is a narrowly focused, concrete, and achieveable. • My aims highlight the significance of the research to science and health. • My text states how my work is innovative. • I describe the biology to the extent needed for my reviewers. • I give a rationale for choosing the topic and approach. • I tie the project to my preliminary data and other new findings in the field. • I explicitly state my hypothesis and why testing it is important. • My aims (and the experiments they lead to) will test my hypothesis and are logical. • I can design and complete the experiments that will fulfill each aim. • I use language that an educated non-expert can understand, to the extent possible. • Used bullets, bolding, or headers so reviewers can easily spot aims (and other key items).

• For each element listed above, analyze your text and revise it until your Specific Aims hit

all the key points you’d like to make. • After the aims, some people add a closing paragraph, emphasizing the significance of the

work, their collaborators, or whatever else they want to focus reviewers’ attention on.

Links to Web Resources

• http://funding.niaid.nih.gov/researchfunding/grant/pages/appsamples.aspx

• http://funding.niaid.nih.gov/researchfunding/gra

nt/pages/applying.aspx • http://www.nlm.nih.gov/ep/Tutorial.html • http://www.cc.nih.gov/training/resources/grant_

writing.html