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Research: thoughts on a personal experience.

Dany Morissetdany.morisset@nib.sihttp://www.nib.si/en/

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Contents:• Slovenia

• The National Institute of Biology

• The Department of Biotechnology and Systems Biology

- Introduction

- GMO (Genetically Modified Organism)

• Personal career and some thoughts

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Shanghai

Slovenia

Where is Slovenia?

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Slovenia

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ItalyItaly

GermanyGermany

Venezzia Venezzia (Venice)(Venice)

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GENERAL INFORMATION

Area: ..........................20,273 km2 (80km x 260 km) 7,037km2 for Shanghai (city)Forest: .......................10,124 km2 (49.9%) Vineyards: ..................216 km2 Length of coast:..........46.6 km Capital: ......................LjubljanaPopulation: ................2,023,000 (2008) 18,450,000 in Shanghai (2007)Language: .................Slovene Currency: ..................Euro, 1 January 2007Education: .................Universities in Ljubljana, Nova Gorica and Maribor cca 90,000 students

Slovenia (Slovenija)

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Triglav, the highest Slovenian mountain

Bled, jewel of the Julian Alps

Ljubljana, the capital city Rafting on the Soča River

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Piran, Tartini’s coastal town

Piran bay

Julian Alps

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Postojna Cave Lipica horses

Proteus anguinus or human fishKarstic land in Škocjan park

10Vineyard from Štajerska Region

Slovenian specialities

Slovenian wine producing

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National Institute of BiologyNational Institute of Biology

at the Biological Centre at the Biological Centre in in LjubljanaLjubljana

ItalyItaly

GermanyGermany

Venezzia Venezzia (Venice)(Venice)

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NIB is the leading institution for basic and applied research in biological sciences in Slovenia…

and…

the third largest institution in the field of natural sciences in Slovenia!

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Mission

NIB is a public research institute, established by the

Republic of Slovenia with the mission:

- to create new (basic) knowledge on the field of

biology and related natural sciences;

- to transfer new knowledge into use for improvement

of the quality of life (to enrich the life on Earth).

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Staff

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69%

31%FEMALEASSISTANTS &DOCTORALASSISTANTS

MALE ASSISTANTS& DOCTORALASSISTANTS

75%

25%

FEMALEPOSTDOCTORALASSISTANTS

MALEPOSTDOCTORALASSISTANTS

67%

33%

FEMALE RESEARCHFELLOWS

MALE RESEARCHFELLOWS

50%50%

FEMALE SENIORRESEARCHFELLOWS

MALE SENIORRESEARCHFELLOWS

53%

47%FEMALE RESEARCHCOUNSELLORS

MALE RESEARCHCOUNSELLORS

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National Institute of Biology

• Departments– Department of Biotechnology

and Systems Biology– Department of Freshwater and

Terrestrial Ecosystems Research– Department of Entomology– Marine Biology Station– Department of Genetic

Toxicology and Cancer Biology

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Department of Biotechnology and Systems Biology

37 employees 8 senior researchers, 6 postdocs, 8 technical assistants, 5 research assistants, 8 PhD students, 2 administrative assistants

Pr. Dr Maja Ravnikar

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- Horizontal project organisation:- OMICS (Kristina Gruden)- MICROBIOLOGY (Maja Ravnikar)- GENETICALY MODIFIED ORGANISMS (Jana Žel)

- Infrastructure (Maruša Pompe Novak)- Quality Assurance and lab safety issues (Marjana Camloh)- Human resources and pedagogical activities (Maja Kovač, Marina Dermastia)- Bio-security (Mojca Milavec)

Activities

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Projects• Cca 55 project per year on 1,2 or 3 years

basis • Ratio between basic and applied research:

60% / 40 %• Applied research:

– Research for companies (LEK, BIA Separations, Krka...)

– Detection of pathogens (bacteria, phytoplasma, viruses)

– Detection of genetically modified plants in food, feed and seed

• Every year, application for 30 – 40 projects

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International cooperation

• 4 EU projects

• 13 bilateral projects

• 5 COST

• 2 Twining

• 2 ERA-NET

• 2 Technology Platforms

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OMICS:Systems biology approaches

Methodologies

– Transcriptomics: DNA microarrays and quantitative real time PCR

– Proteomics

– In-situ hybridization

– Biochemical and cytological research

– Bioinformatics and biostatistics

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• Plant-pathogen/pest interactions– Potato – Virus PVY– Potato – Colorado Potato Beetle– Grapevine – Phytoplasma

• Cell therapy of cancer

• Projects with pharmaceutical industry:– Drug discovery, drug production

Biological problems::

OMICS:Systems biology approaches

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MICROBIOLOGY

• New findings of plant pathogens, emerging diseases, biosecurity

• Study of biology of plant pathogens – biodiversity• Development of diagnostic methods

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Real time RT- PCR:PVYNTN - distinction of strains CSNV, TSWV, INSVToMV (CIM), CaMV (CIM),PePMV - distinction of strains, Rotaviruses – water samplesXylophyllus ampelinus,Eam, Phytoplasmas

Microarray technology, ring trial

New Methods:

Development of sensitive methods for plant pathogen detection

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Concentration and purification of plant viruses

- Using CIM® chromatographic supports, quick and efficient method for virus concentration in irrigation water was elaborated

- Studies of virus purification: purification step for ToMV is finished in one day

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- Transgenic plants: Development and research of plants resistant to pathogens and pests- Research and development, projects- Development of methods for routine detection of GMOs- Policy advisors, quality system, Working Groups

Genetically Modified Organisms (GMOs):

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• Selection of promising genes from transient expression• “omics” studies for stable transformation

Transgenic plants

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• Co-Extra EU project– Traceability and coexistence of GMOs– Integrated project, 54 partners, 24Mo Euros– Objectives:

• Fill-in the gap between the set legal framework and its practical implementation

– Works:• Critical point of GMO quantification (extraction, DNA

quantification…)

• Automation set-up, machine robustness

• Gene variability, new reference genes and materials, new chemicals

• New detection methods (microarray, real-time detection)

Research and development, projects

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GMOs diagnostics• Development and validation of analytical

methods in GMO-testing• Introduction and validation of analytical methods• Routine diagnostics (private, inspectorate...)• Follow-up of results in proficiency tests• Participation to international studies• Respect of QA (ISO 17025)

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National Reference laboratory• Cooperation with different ministries in Slovenia

– Routine analysis– Sc. and Tec. experts, Slovenian representative– Training– Communication

• Cooperation with standardization bodies (CEN, Slo)• Cooperation with CRL (JRC, Ispra, Italy)

– Validations of methods– Consultant

• Cooperation with ENGL– Member of Steering Committee– Cooperation in WG

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Studies, PhD

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Studies, PhD

• B.sc. Biochemistry

• M.sc. Biochemistry

• Pre-PhD Biotechnology, opt. Biochemistry

• PhD microbiology & Biochemistry

• Postdoc microbiology & Biochemistry

Poitiers University, France

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“Structure/function relationships of an anti-listerial bacteriocin, mesentericin Y105”

• Microbiology, Biochemistry and Molecular biology. Food safety orientated

• Purely fundamental research but partially financed by private company (Danisco, ex Rhodia Food, Lactic ferment company)

Opportunity to give lectures in University (Freshmen, Sophomores, Juniors): Biophysics, Biochemistry, Microbiology and molecular Biology

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First postdoc

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First postdocPostdoctoral studies in University Laval and Hospital Laval, Quebec city, Qc, Canada

“Role of the new WAP protein, HE4”

• Use of previous knowledge: cloning, biochemistry, microbiology

• New knowledge: cell biology, enzymology, yeast, cancer/inflammation processes

• Purely fundamental, public funding

• University + hospital

• First experience abroad

New experience in different domain, flexibility, living abroad

Loss of contact with France, less opportunities to come back, need for more applied research

Neutrophil inhibitorsPDE4 inhibitors

Alveolarrepair Mucoregulators

Immunosuppressants?

Protease inhibitors AAT, SLPI, pre-elafin, TIMPs

Mediator antagonistsLTB4 inhibitors

Chemokine antagonists

Quit smoking

Emphysema Mucushypersecretion

PROTEASESNeutrophil elastaseCathepsinsMMPs

Neutrophil

Alveolar macrophage

CD8+

lymphocyteChemotactic

mediators(IL-8, LTB4)

MCP-1?

I nflammatory response pathway (and/or others?)

Receptor

I nflammation(SLPI , pre-elafi n, HE4HE4)

I nvasion(SLPI , pre-elafi n)

Cell proliferation(SLPI , ps20, HE4HE4)

Cell death / apoptosisAngiogenesis(ps20)

+/- ?

I nflammatory response pathway (and/or others?)

Receptor

I nflammation(SLPI , pre-elafi n, HE4HE4)

I nvasion(SLPI , pre-elafi n)

Cell proliferation(SLPI , ps20, HE4HE4)

Cell death / apoptosisAngiogenesis(ps20)

+/- ?

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Actual work• Started 2005.

• “Development of alternative DNA-amplification system to PCR technology”

– Within CoExtra project

– Broad view on different DNA-amplification systems

– Adapt ideas to GMO context

– First “real” applied project (milestones, deliverables, deadlines): project manager

– Wild card for the development

Real motivation

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Traceability issues • Alternative real-time DNA amplifications, real-time NAIMA• Improving microarrays in GMO detection: Target amplification

using NAIMA, signal amplification dendrimersMorisset et al., Nucl. Acids Res. August 2008.

Morisset et al., 2008. Eur. Food Res. Tech.. 227: 1287-1297

WP5 & & meeting11-12 MayLjubljana

ssRNA/ssDNA

second universal primer

Reverse Transcription

RNAse H activities

T7-universal primer

RNA (-)

100-1000 copies

NAIMA multiplex amplification

Primer extension

3’

5’

Transcription

ss DNA

T7-RNA polymerase promoter sequence + cap sequence

Second universal sequence

3’5’

3’5’

3’

3’

3’

3’

3’

3’

3’

RNA pol

5’3’

ds DNA

5’3’

WP5 & & meeting11-12 MayLjubljana

ssRNA/ssDNA

second universal primersecond universal primer

Reverse TranscriptionReverse Transcription

RNAse H activitiesRNAse H activities

T7-universal primerT7-universal primer

RNA (-)

100-1000 copies

NAIMA multiplex amplification

Primer extensionPrimer extension

3’3’

5’5’

TranscriptionTranscription

ss DNA

T7-RNA polymerase promoter sequence + cap sequence

Second universal sequence

T7-RNA polymerase promoter sequence + cap sequence

Second universal sequence

3’5’3’5’3’5’

3’5’3’5’

3’3’

3’3’

3’3’

3’3’

3’3’

3’3’

3’3’

RNA pol

5’3’

ds DNA

RNA polRNA pol

5’3’

ds DNA

5’3’

5’3’3’

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• Added new responsibilities: “responsible analyst for routine GMO detection”– Work in a Quality system (ISO 9001/17025)– Few research per se– Developing new methods (new markers)– Introduction methods validated in ring-trial– Prepare and control in-house validation– Follow-up lab’s performances (studies, PT)– Follow-up of GMO’s novelty– Advise authorities

Actual work

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Relation with authorities– Opinions for legislation

purposes

– Official control support (recommendations, opinions..)

– Public presentations on GMOs (like for Farmers Unions...)

– Scientific and technical assistance to the competent authorities

NATIONAL INSTITUTE OF

BIOLOGY

Ministry of Health

Ministry of Environment and Spatial Planning

Ministry of Agriculture,Forestry and Food

Inspectorate for Agriculture, Forestry and

Food

Inspectorate for the Environment and Spatial

PlanningHealth Inspectorate

Ministry of Defence and Slovenian Research Agency

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• Development of novel approaches to GMO field– How to simplify routine analysis (automation, mixes,

procedure)– How to decrease cost (screening, GMO track)– How to handle unapproved GMOs

• Participation to several WG/works– ENGL– JRC– CEN– CoExtra

• Participation to congresses/lectures/(review)

Actual work

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National Reference laboratory

• Cooperation with CRL (JRC, Ispra, Italy)– Validations of methods (17 until now)– Other (Working Groups for quality assurance

of laboratories coop. in validations…)• NIB is ENGL member since 2004

– Member of Steering Committee– Cooperation in WG:

• Working group on GMO analytes• Working group of unknown GMOs

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Conclusion

• Free choice of career:– There is no intelligent design (!)– Use previous knowledge for new topics– Possibility to quit “wet” science

• Applied/fundamental: make your choice– Pressure is different– Length/aim are different

• Need for motivation: – short/long projects– are you made for science?

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Conclusion• Industry/public:

– Applied research also in public/ Commercial aspects (patents/ commercialisation/spin-off)

– Industry’s choice vs search for funding (but SME also look for funding)

– One project/ work on different projects at one time

• Network: extremely important– Collaboration on papers (better success)– Ease application for projects– Trust– Career opportunities

• Salary/perception: don’t dream!