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Research group of
Functional Genomics and Proteomics
http://bio.kuleuven.be/df/ls/people
Input
Environ
mental
stimuli
Output
Fysiological
processes
Behaviour
Processing ?
Basics of wiring, neuronal circuits: understanding
intercellular signalling
small model organisms
Neuropeptides, neuronal communication
and neuroplasticity
Humane hersenen
~100 miljard neuronen C. elegans
302 neurons
Mechanisms of biological processes and behaviours conserved during evolution.
200 neuropeptides
• Learning and memory
• Ageing
• Feeding behaviour • Reproduction
• Sleep
• Addiction
Extensive genetic toolkit: knockouts, cell-
specific knockouts, overexpression, cell
specific rescues
• Small (1 mm) freeliving soil nematode
• Feeds on bacteria
• 959 transparant cells
• Easy to cultivate
• Genome sequenced
At the level of the genotype Big ideas from small brains
C. elegans TOPICS
BRAIN CONTROL: what and how do we learn/forget? * * *
- neuropeptides and learning & memory
- sleep wakefulness mechanism
NEURODEGENERATION: *
why don’t we know why? (Huntington, Alzheimer Disease)
AGEING: WHO WANTS TO LIVE FOREVER? * * *
neuron-specific -omics in long-lived worms-
WEIGHT CONTROL * *
- why and how do we gain weight?
REPRODUCTION
what is happening in puberty?
Basics of wiring, neuronal circuits,
understanding intercellular
signalling
oxytocin trust
bonding Love hormone
Nieuwsblad november 2012:
Oxytocin ('the Love Hormone') houdt
uw man weg van andere vrouwen.
Ron Paul Won't Use Brain Chemical
Oxytocin to Influence Voters, Obama and
Romney Will
Nematocin mutants can’t associate sensory stimuli with presence/absence of food. They can’t learn from earlier experiences.
Nematocin: Pavlov’s dog revisited
Wormen leren op... | Facebook
www.facebook.com/faculteitwetenschappen/
Wormen leren op dezelfde manier als mensen! Tot die bevinding
kwam Isabel Beets, doctoraatsstudente in de on
Wormen leren op dezelfde manier als mensen... Tof. - Topsy
In het nieuws – KU Leuven DE MORGEN. Wormen kunnen net als
mensen verbanden leggen tussen prikkels uit hun omgeving en zo
Worm leert zoals mens | Eos Wetenschap - P1
29 okt. 2012 – Wormen kunnen net als mensen verbanden leggen
tussen prikkels uit hun omgeving en zo leren uit hun ervaringen.
Wormen leren uit hun fouten - Wetenschap - Nieuws -
Knack.bewww.knack.be ›
Signaalmolecule om te leren ontdekt bij de worm –
Wetenschapsforum www.wetenschapsforum.nl › ... ›
Wormen leren op dezelfde manier als mensen - HLN.be
www.hln.be/... 26 okt. 2012 – Wormen kunnen net als mensen
verbanden leggen tussen prikkels uit hun omgeving en zo leren uit
opgedane ..., lees meer op Kanaal Planet.
Twitter: houbi@houbi27 oktober
„Wormenlerenopdezelfdemanieralsmensen”leesikinDM. Maar
is dat dan met of zonder iPads in de klas?
oxytocine
Molecular pathways
• molecular signals used by
VP/OT during learning ?
• up- and downstream players ?
Neuronal circuit dynamics
• neuronal circuit behind
vasopressin/oxytocin effects on
learning ?
• changes in cellular response upon
learning?
• evolutionary conserved circuit?
calcium imaging optogenetics
BRAIN CONTROL neuropeptides and learning and short-term memory
The behaviour of the worm can be genetically manipulated by light by making transgene worms in which specific neurons express light-sensitive ion channels, Channelrhodopsine-2 (ChR2, depolarisation, activation) and Halorhodopsine.
Photoactivation of defined neurons in the head let the worms crawl in the shape of a triangle
From genetics to behaviour: Understanding experience-based learning behaviour
Optogenetic tools.
BRAIN CONTROL neuropeptides and long-term memory
Info: Isabel Beets, Liesbet Temmerman
Is vasopressin/oxytocin involved in longterm learning & memory?
Are there gender-specific differences?
- Set up longterm memory test (spaced training)
- identify cells involved via GFP reporter constructs and confocal microscopy
- identify downstream players via single-cell RNA sequencing
Build model for learning & memory
Alzheimer’s disease • World’s most common neurodegenerative disease • Dementia: dramatic emotional and financial consequences • Amyloid-β and Tau known toxic aggregates in brain
Techniques
• Single copy insertion @ known place in
genome (MosSCI)
• Microinjection
• Confocal microscopy
• Behavioural/learning assay
• Molecular genetics
Aim • Generate humanized C. elegans Alzheimer
models • Measure toxic effects of Amyloid-β and Tau • Analyse learning/forgetting behaviour,
movement
Neurodegeneration: why don’t we know why?
Everyone ages.
Why?
Naakte molrat 28 jaar
Huismuis 4 jaar
AGEING: WHO WANTS TO LIVE FOREVER? * * *
Ageing
Before 1990:
ageing = random accumulation of
damage
The “rateofliving”and oxidative
damage theory
Antioxidants have no
lifespan extending effect
Ageing 1993:
Knocking out one gene doubles life span in
Caenorhabditis elegans
Ageing is a biological proces contralled by
signalling molecules and pathways!
Uit Kenyon et al., 1993
Ageing: Who wants to live forever? Neuron-specific -omics in long-lived worms
Techniques:
Many life-extending interventions available in C. elegans
insulin pathway mutations, dietary restriction, life-extending→
compounds (metformin, spermidine,…)
Topic 1: Neuronal control of longevity: Neurons play a central role in the endocrine regulation of aging which neuronal proteomic & peptidomic changes allow
animals to live longer?
Proteomics & Peptidomics Fluorescent labelling
of neurons
Info: Wouter De Haes, Liesbet Temmerman
Timing lifespan
Contact: [email protected]
Topic 2: Cognitive decline in ageing
• Ageing leads to cognitive decline; process is poorly understood
• The applicant will profile the age-related decline of cognition in C. elegans and identify the molecular mechanisms involved.
• Techniques: Associative learning assays, RNAi
• Several naturally occurring compounds can increase lifespan • The applicant will screen promising compounds from
medicinal herbs on their ability to increase lifespan in C. elegans.
• Techniques: Lifespan assays, RNAi,…
Topic 3: Effect of medicinal plant extracts on ageing
Ageing: Who wants to live forever?
All animals sleep
What is the molecular mechanism? Is it evolutionarily conserved?
BRAIN CONTROL: SLEEP-WAKEFULNESS MECHANISMS
IN C. ELEGANS
NLP-22
Induction of sleep and
molting
GNRR-
3
In vivo localisation
Overexpression , rescue and double mutants
(molecular biology, PCR, cloning...)
sleeping worm movie.mov
Assesing sleep
behavior
Feeding behaviour and metabolism
- Hart en vaataandoeningen - Diabetes - Hoge bloeddruk - Galblaas dysfunctie - Ademhalingsproblemen - Sommige vormen
van kanker
1.6 miljard met overgewicht
400 miljoen obese
1/3 in US
1/7 in België
Onderzoekers hopen met de toepassing van genomics-technieken de sleutelprocessen in kaart te brengen die leiden tot een verstoorde stofwisseling.
Curing obesity: unravelling the CK-signalling system
Signaling pathways regulating food intake and fat storage are highly conserved in C. elegans
Molecular techniques
- (qRT)-PCR
- receptor de-orphanization
- in vivo localisation, cell specific rescue
- in vivo calcium imaging
Phenotyping
- Food intake
- Fat content
- Live motility tracking
CCK/CK Aim: molecular and genetic
dissection of CK-pathway
controlling satiety and fat storage
Contact: [email protected]
Restoring (ab)normal feeding behavior in Caenorhabditis elegans through genetic engineering
Supervisor: Lise Peeters ([email protected])
blanco DM Nlp-12 Ckr-2 Eat-2 N2
Feeding
assay
Techniques • generation of transgenic lines by MosSci-biotic
• Localisation of receptor by fosmid recombineering and
modified pSM vectors
• Starvation and & refeeding experiments, real-time PCR
Mutagenesis
GFP fluorescent reporter worms for oocyte maturation
Fluorescent worms
worm sorter
Non fluorescent worms
Sequencing van genoom en vergelijken met wildtype
Molecular genetic deciphering of the reproductive pathway in the model organism Caenorhabditis elegans
Aim: identify genes that influence oocyte maturation
1) FORWARD GENETICS
TARGET PHENOTYPING
2) defects with respect to r
egg-laying behavior and
development
3) qRT-PCR analysis of the effects on steroidogenesis
1) one-step WGS and SNP
mapping,
genetic rescues
vit-2::gfp
reporter
EMS
mutagenesis mutant identification
2) REVERSE GENETICS
fundamental insights in genetics of
reproduction
RNAi of candidate genes
TECHNIQUES
Molecular genetic deciphering of the reproductive pathway in themodel organism Caenorhabditis elegans
Supervisor: Liesbeth Van Rompay ([email protected])
TECHNOLOGY DEVELOPMENT
2D-DIGE
Differential
proteomics
Microarray-
analysis
Transcript-
omics
Mass
spectrometry
Peptidomics
RNA
interference
Gene Silencing
Next
generation
sequencing
Phenotypic plasticity in locusts:
epigenetic regulation
Elucidation of locust phase polyphenism with gel based proteomics.
2 phenotypes
1 Genome
Epigenetic component?
DNA
Methyl-Cytidine
Differential DNA methylation?
Differential PROTEOME?
Workflow:
Tissue dissection
Protein extraction
Differential analysis: 2D-DIGE
Protein identification
- MALDI TOF/TOF mass spectrometry
- Bioinformatics de novo protein identification
Brain
More info?
Towards a novel insect pest control strategy with endogenic parasites
Endogenic
gut parasites
Isolation and
characterisation
of the locust-parasite
mutualism
Genome sequencing project
of the parasite
Modification of the transgenic
parasites
-Fluorescent tags
- study of infection dynamics
Modification of the parasite
which is lethal for the host
More info:
Creativity
Genetic
engineering
Proteome analysis of FFPE colon tumor tissue ([email protected])
Seperation of proteins
FFPE tissue Protein extraction Proteomics Mass spectrometry
Formalin Fixed Paraffin Embedded tissue
PROBLEM:
Formalin-induced protein cross-links pose challenges for
proteome analyses of FFPE tissue
OBJECTIVES:
• Develop a reliable proteomics workflow enabling the analysis of the FFPE tissue
proteome
• Identify prognostic biomarker candidates for colorectal cancer
TECHNIQUES:
• Proteomics:
Gel Enhanced Liquid Chromatography, gel free approach, 2D gel electrophoresis
• Mass spectrometry
Aphid = agricultural pest, easy to breed, endosymbionts, diverse
host-pathogen interactions, reproductive plasticity, viviparous ...
epigenetic control
microRNA control
.
. International context: Erasmus .
Summertime: asexual
reproducing females
LD SD
Photoperiod shortening
Fall: switch towards sexual reproducing females
.
ERASMUS FRANCE: APHID PHENOTYPIC PLASTICITY
EPIGENETIC AND MICRO-RNA CONTROL
OVERVIEW ALL TOPICS & CONTACTS
BRAIN CONTROL: neuropeptides and learning&memory (2) Isabel Beets
sleep and wakefulness Lotte Frooninckx NEURODEGENERATION: why don’t we know why? (Huntington or Alzheimer Disease)
Roel Van Assche WHO WANTS TO LIVE FOREVER? neuron-specific -omics in long-lived worms
Wouter De Haes Cognitive decline in ageing
Liesbet Temmerman Effect of medicinal plant extracts on ageing
WEIGHT GAIN: role of cholecystokinin (curing obesitas) Kevin Van Calster restoring (ab)normal feeding behaviour by genetic engineering
Lise Peeters MOLECULAR GENETICS OF REPRODUCTION: forward and reverse genetics
Liesbeth Van Rompay LOCUST PEST CONTROL: from harmless parasite to Trojan horse
Epigenetic cause of solitary-migratory behavior switches in locusts?
Bart Boerjan CLINICAL: proteome of FPPE tissue Valerie Broecks ERASMUS: Université de Rennes: Aphid phenotypic plasticity: epigenetics and microRNA’s
info: website http://bio.kuleuven.be/df/ls/ [email protected] or [email protected]