miRNAs are genome-encoded small RNAs that canonically repress translation of target mRNAs, which they recognize by base-pairing. In contrast, the non-canonical function of miRNAs in translational activation in G0-phase cells has been reported. Our knowledge of miRNA-mediated translational activation is still limited; in particular, it is unclear whether miRNA-mediated translational activation affects endogenous mRNAs significantly. The main aim of my research will be the genome-wide evaluation of miRNA-mediated translational activation in endogenous mRNAs. As quiescent and post-mitotic cells are pervasive in living organisms, our results will reveal important aspects of miRNA biology that have been overlooked in standard culture systems.
I will address endogenous activation targets using ribosome profiling. Ribosome profiling is a method based on deep sequencing of RNA fragments protected by ribosomes that measures the translation of each mRNA quantitatively. I will introduce miRNAs into G0-phase HeLa cells, synchronized by serum-starvation, and analyze how miRNA-mediated translational activation impacts endogenous mRNAs, how miRNAs activate translation, and how differently miRNAs recognize the target mRNAs.
One of my previous reports has suggested that the poly(A) tail of target mRNAs might be a key factor for miRNA-mediated translational activation. I will address the impact of polyadenylation by RNA-Seq analysis of poly(A)+ and poly(A)- fractions of rRNA-depleted RNA and high resolution poly(A) tail assay (Hire-PAT). This result will provide insights for the function of poly(A) tail in miRNA-mediated translational activation.

Meiotic recombination has two key roles, it creates genetic diversity and promotes accurate segregation of chromosomes during meiotic cell division. Despite a plethora of knowledge about the genetic control of this process in lower eukaryotes, revealed primarily through forward genetic screens, our understanding of it in higher eukaryotes is lacking. This is due to the rapid divergence of meiotic proteins through evolution, which has limited the standard reverse genetic approaches. I will exploit our current knowledge of meiotic recombination in higher organisms and the recent advances in genomics, to conduct an ENU mutagenesis genome wide screen in mice for players of meiotic recombination and chromosome structure. As a readout for my screen, I will use behaviour of thoroughly described and robust indicators of double strand break repair and chromosome structure, namely phosphorylated histone H2AX called gamma-H2AX and synaptonemal complex protein SYCP3. This will not only allow me to identify proteins directly involved in the formation and repair of double strand breaks, but will also uncover components of the meiotic protein scaffold and those involved in sex chromosome inactivation. Subsequent characterisation of the candidates identified from my screen will allow me to bridge the gap and enhance our understanding of several aspects of the mammalian system, from DNA repair, to meiotic chromatin organisation, to conservation of the underlying strategies of meiosis.

This project aims to decipher how circadian gene expression is regulated on the translational level. Circadian clocks coordinate the daily timing of most physiological processes. During the last decade numerous molecular studies have used genome-wide transcriptome analyses to define rhythmic gene expression. However, proteomic approaches have shown a surprisingly high discrepancy between the circadian transcriptome and proteome, pointing to additional levels of circadian regulation. Circadian translational control is an attractive hypothesis for how rhythmic protein accumulation might be achieved from non-circadian transcripts. However, such translational mechanisms are still poorly investigated in the circadian field.
We will implement a recently developed technique termed “ribosome-profiling” to determine circadian translation rates in mouse liver in a genome-wide and quantitative fashion. In particular, we aim to discover proteins that are rhythmically translated from stable transcripts throughout the day. Based on the sequence information of these transcripts we will make predictions about molecular mechanisms and interaction partners that are involved in circadian translation. Hypotheses generated by these approaches will be assessed using in vitro and in vivo systems. Finally, loss-of-function analyses in different mouse models will give information about the relevance of the uncovered mechanisms for liver physiology.

Recently, our laboratory has shown that cancer cells need both genetic mutations and epigenetic alterations for their continuous growth. This was revealed through a focused RNAi screen where all epigenetic regulators were systematically evaluated for their role in proliferation of acute myeloid leukemia (AML). I performed a new RNAi screen in acute lymphoblastic leukemia (ALL). Remarkably, this screen identified that TRIM33 (Tripartite motif-containing 33) is required for the growth of ALL but not for 10 other forms of cancer tested. TRIM33 harbors a PHD and a bromodomain which recognizes methylated and acetylated histones, respectively. While TRIM33 is uniquely required for growth of ALL cells, I found surprisingly that TRIM33 is expressed ubiquitously in many cell types. Therefore, TRIM33 promotes growth of ALL in a highly context-specific manner, which might be related to the underlying genetic/epigenetic cellular state. To understand the mechanism underlying this intriguing phenotype, I will establish a tractable mouse-model system in which we can manipulate cellular genetic/epigenetic contextual features to evaluate the key determinants of TRIM33-addiction. This powerful system will enable us a comprehensive evaluation of TRIM33 function in ALL, which will include: 1) identifying the target genes of TRIM33 2) revealing genome-wide distribution of TRIM33 3) purifyingTRIM33-containing complexes in multiple cell types, and 4) reconstituting TRIM33 functions in vitro. The central goal of this project will be to define the context-specific mechanism that accounts for the addiction of ALL cells to TRIM33, a novel therapeutic target in this untreatable disease.

The vulva of the Caenorhabditis elegans (C. elegans) is the egg-laying and copulatory organ of the hermaphrodite (or female) and connects the uterus to the outside of the nematode. Due to its simple cellular organization and accessibility in-vivo, the developing vulva has become a paradigm for organogenesis and the two main molecular pathways underlying vulval patterning are mapped out in great detail. However, a quantitative understanding of the interplay between environmental factors and these molecular pathways is still lacking. Therefore, our first aim is to identify specific environmental cues that induce classifiable and numerically significant phenotypic effects on vulval development for wild-type C. elegans strains. From prior work on Drosophila and male nematodes, for instance, we expect that heat shocks during early developmental stages exert a strong influence on vulva phenotypes. Next, we will apply the identified experimental protocols to the classical pathway mutant strains. We expect that the same types of environmental perturbation will have a different effect in these mutants. These data will now be fit to a very recently introduced quantitative model, which to establishes the genotype to phenotype map via only a few aggregate variables. These fits will yield a quantitative correspondence between signaling pathways and environmental perturbations and enable us to dissect effective interactions between mutant genes and environmental stresses. They will furthermore allow us to infer specific perturbations that should be applied to one mutant strain in order to induce phenotypes of another. This model prediction will finally be experimentally tested.

Meiosis, the key to sexual reproduction, is a special cell division process that mediates the production of haploid gametes from diploid parental cells. To this end, meiosis critically depends on the accurate segregation of homologous chromosomes. Errors in this process are a major cause of infertility, and can also result in developmental disorders such as Down syndrome. However, the mechanisms regulating homologous pairing and its stabilization by the formation of the synaptonemal complex and recombination still remain unclear. In the proposed project I will investigate the role of cytoskeletal forces in meiotic chromosome pairing and synapsis. I will take advantage of the wealth of tools available to study meiotic chromosomes in the nematode Caenorhabditis elegans. By generating transgenic nematodes expressing novel fluorescent reporters, I will directly visualize the forces acting on meiotic chromosomes through in vivo imaging and image analysis. Using these tools, I will investigate the roles of specific molecular components, including the microtubule motor dynein, and HORMA-domain proteins, a family of conserved proteins of the meiotic chromosome axis. These experiments will test the hypothesis that forces generated by motors acting on the microtubule cytoskeleton create tension between homologous chromosomes to ensure accurate pairing. I will also explore how physical forces are transduced through the intact nuclear envelope, and along the length of the chromosomes. My analysis will advance our understanding of the regulatory mechanisms that ensure faithful chromosome segregation and genome stability.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic neoplasm resulting from the malignant transformation of T-cell progenitor cells. Despite the improved cure rate for childhood T-ALL, up to 25% of patients will fail frontline therapy and their prognosis is dismal. This is mainly because most efforts in understanding T-ALL biology have focused on the protein-coding genome, which represents less than 2% of our genetic material. Consequently, the molecular mechanisms underlying the disease are poorly understood. Non-coding RNAs (ncRNAs) have been shown to regulate important biological processes that support normal cellular functions. Here, I propose to investigate the implication of long ncRNAs (lncRNAs) in the pathogenesis of leukemia. I will perform whole transcriptome sequencing studies in primary and relapsed patient samples to identify and comprehensively annotate T-ALL-specific lncRNAs. I will functionally characterize the identified lncRNAs by loss and gain of function approaches to assess their contribution in induction and maintenance of T-ALL in vitro and in mouse xenograft models. Furthermore, I will test the lncRNAs expression in response to T-ALL targeted drug treatments, which will identify lncRNAs modulated by specific oncogenes. The proposed study will uncover a novel resource of important prognostic markers and will contribute to the complete understanding of mechanisms driving pathogenesis in T-ALL.

Neuronal circuits in the brain are constantly modified by external sensory experience. Synapses, the smallest unit of information processing in the brain, are highly dynamic in function and structure. It is still unclear how sensory experience is coded by synaptic function and structure. New imaging techniques have enabled high resolution exploration of dendritic spines, specialized post synaptic sites, in the living brain. These studies have focused on long term spine structural dynamics at time scales ranging from hours to days and months. However, it is not known how ongoing sensory experience may be rapidly transformed to synaptic structural and functional dynamics in the brain. Therefore, our main research aim is to address how a specific sensory input might change the structure and function of single spines. We propose to examine this question by combining in-vivo 2 photon imaging along with FLIM measurements of protein dynamics in single spines during the presentation of specific sensory input. By use of an optical cranial window and viral expression of tagged molecular sensors, this experimental setup could be utilized to examine how specific sensory experience is transformed into rapid structural and functional plasticity of synapses deep in the brain. Furthermore, we propose to examine how rapid spine dynamics corresponds to long term alterations in spine structures, which is associated with learning and memory. This project may help to better understand how sensory experience and learning is represented in rapid synaptic dynamics, and how these early events can lead to long term synaptic plasticity, enabling the brain to create novel memories while stably maintaining them over time.

Many microorganisms produce antibiotic compounds that target essential components of prokaryotic and eukaryotic cells. Xenobiotic production provides a means to disable competing microbes or host defenses to infection. Animals encounter these toxins in their environment, and posses a range of mechanisms mitigate their effects. These detoxification responses are tightly regulated, but how this regulation is achieved is poorly understood. In C. elegans, disruption of core cellular processes by RNAi activates the same detoxification responses that occur upon exposure to xenobiotics. This indicates that surveillance of core cellular processes, rather than detection of xenobiotics themselves, serves to trigger detoxification. I aim to understand the mechanism of surveillance of core cellular processes, and how information from this surveillance is relayed to regulate detoxification responses. I will use large-scale mutagenesis screens to identify factors that regulate detoxification responses, and transcriptome sequencing to gain a genome-wide view of their regulatory outputs. Some xenobiotic detoxification responses are regulated by endocrine signals. To distinguish factors that act in surveillance and responding tissues, I will establish a system for tissue-specific induction of xenobiotic stress. This work will constitute the first systematic genetic and genomic analysis of detoxification regulation in any animal. Conserved factors identified in C. elegans promise new insights into human disease and drug responses mechanisms.

Cell competition is the mechanism by which suboptimal cells are removed from a growing tissue through apoptosis. It is driven by a local comparison of cell fitness among neighboring cells. In Drosophila, multiple pathways can modify cell fitness and trigger cell competition, yet they all converge into few downstream signals leading to “loser” fate commitment and cell elimination. Loser fate is controlled by the upregulation of the membrane protein isoforms Flower-lose and to a lesser extent by the downregulation of the ubiquitously expressed isoform Flower-ubi. Moreover, loser cells also express protective signals (including the secreted protein Sparc) that delay their elimination. Current knowledge suggests that loser cell elimination relies on the comparison of the relative concentration among neighboring cells of loser signals (Flower-lose) and the absolute concentration of protective inputs (Sparc). Yet the description of the process is still very qualitative and the dynamics of these components is unknown. I propose to quantitatively dissect the rules leading to loser cell fate commitment. I will first perform live imaging of cell competition in order to correlate the endogenous levels and the dynamics of Flower-lose and Sparc with cell elimination. I will then quantitatively manipulate these components by developing light and chemical inducible gene expression in cell culture and in vivo. This study should shed new light on the process of cell competition and pave the way for a quantitative approach of developmental decision.

Animals can adapt to environmental changes by modifying their innate preferences, generating learned behavior that matches their experiences. Many genes required for learning and memory have been found through candidate gene studies and classical genetic methods. In this study I propose an alternative approach to seek the genetic basis of learning and memory, using natural variation among wild-type C. elegans strains. I will seek two wild-type strains with variations in learning and memory capabilities and identify the underlying genetic differences using quantitative behavior assays and linkage-based mapping techniques. A microfluidic system will be designed to monitor animals’ behavior upon classical conditioning, after pairing the attractive odor propanol with a noxious acidic pH stress of HCl. Upon successful learning and memory acquisition the worms’ attraction to propanol, the conditioned stimulus, will be reduced. Learning and short- and long-term memory phenotypes will be quantified by measuring the reduction in attraction to propanol. I will seek two wild-type strains with natural variability in learning and memory capabilities, and employ Quantitative Trait Locus (QTL) and Chromosome Substitution Strain (CSS) mapping techniques to identify the genetic basis of their behavior variability. I will characterize the identified genes’ function in the context of learning and memory, and seek the neural circuitry underlying the learned preference behavior using established genetic and imaging tools. Combining my approach with new microfluidic technologies and quantitative measurements, I aim to identify relevant genes that were not identified in classical approaches.

Selective attention allows flexible tuning of behavior according to the current behavioral goal. Adaptive changes in the response properties of neurons in the auditory cortex accompany attentional shifts. Yet, the causal mechanisms that enable such attention-related cortical adaptation remain largely unknown. Commonly suggested mechanism speculates that ‘top-down’ inputs, allegedly from the prefrontal cortex, actively modulates the local excitatory-inhibitory balance at the targeted sensory neurons. Here we suggest testing this proposed mechanism using a novel behavioral paradigm and optogenetic stimulation of specific types of neurons. We will train rats to selectively attend to a cued frequency (CueF) while ignoring a distracting frequency (DstrF), an octave or more apart. The tonotopic organization of the auditory cortex will be exploited to allow separate perturbation of the local neural activity at the CueF and DstrF receptive felids. To this end, the light-sensitive cation channel Channelrhodopsin-2 (ChR2) will be introduced into layer 2/3 excitatory neurons by means of in-utero electroporation. This will allow stimulating excitatory microcircuit at either of the receptive fields (CueF/DstrF) and probing the effect of such stimulations on selective attention and on local neural activity. In addition, ‘top-down’ projection neurons to the auditory cortex will be retrogradely transduced with ChR2 to allow stimulating them from outside of the auditory cortex. This will allow probing the causal role different projections to the auditory cortex play at controlling neural mechanisms of selective attention.

Circadian rhythms are thought to have evolved to enable organisms to organise their activities according to the Earth’s predictable daily cycles but data revealing the benefits of circadian rhythms are scarce. More difficult still is explaining why parasites that exclusively live within the bodies of other organisms have a circadian rhythm. For example, the developmental rhythm of many malaria parasite species is coordinated; parasites invade host red blood cells, replicate, and then release their progeny in a timed, synchronized burst. Similarly, there is increasing evidence of circadian rhythms in the effectors and regulators of host immune responses, but it is not known whether this influences protection from disease. What is lacking is an explicit recognition that rhythms in immune defence and parasite development could provide an evolutionary advantage to hosts, parasites or both, as well as recognition that both parties may control each other’s rhythms. We will integrate different perspectives and methods from evolutionary ecology, chronobiology, parasitology, and immunology to investigate the causes and consequences of rhythms for hosts and parasites. Our project uses an established rodent-malaria model system. We will capitalise on the reciprocal feedback from integrating empirical and theoretical approaches to answer the following questions: (A) How are rhythms in parasite development initiated and maintained? Do parasites use their own time-keeping mechanisms to organise development or do they use cues from the host's circadian rhythms? (B) How do rhythms affect the ability of hosts to cope with infection? Do rhythms maximise immune defence or are they a constraint resulting from competing demands on the immune system? (C) How do rhythms affect the survival and transmission of parasites? Do developmental rhythms enable parasites to avoid host immune responses or facilitate the exploitation of circadian driven resource availability? (D) Are rhythms in parasite development and host immune responses adaptive for parasites, hosts, or neither? Does the evolution of parasite rhythms drive the evolution of host rhythms, or vice-versa? Thus our project breaks new ground by elucidating: the role of rhythms in infections, how rhythms may be exploited for clinical benefit, and the adaptive significance of rhythms for hosts and parasites.

MicroRNAs (miRNAs) are essential components of the genetic regulatory networks in plants and animals. Through inactivation of specific mRNAs, especially those encoding transcription factors, they modulate the homeostasis, development and responses to environmental stimuli of multicellular organisms. In order to accomplish its biological functions a spatial/temporal balance between a given miRNA and its targets has to be reached. Thus, a fine-regulation of the miRNA action is critical. In contrast to animals, where multiple regulatory factors modulating miRNA action have been identified, only a few have been found in plants. In this project, I propose two main strategies to elucidate the mechanism regulating miRNAs. First, I will focus on the regulation of miRNAs generation; I developed a novel screening system that allowed me to identify numerous mutants defective in miRNA activity. As part of my research project, I will characterize the isolated mutants, establish their mechanisms of action and explore their relationship with other transcriptional processes. The second strategy will focus on later events on the life of a miRNA. I will study, by genetic and biochemical approaches, the processes that determine the fate of a mature miRNA duplex, a critical regulatory step. The project will be conducted in an integrative way, using multidisciplinary approaches to study miRNA regulation and its relationships with other pathways. My goal is to elucidate the intricate mechanisms regulating the miRNA activity. This knowledge is fundamental to understand how miRNAs affects downstream biological processes in plants and potentially in other organisms.

The liver is strategically located at a crossroads of the systemic circulation, allowing it to carry out its metabolic functions in lipid, carbohydrate and protein generation and in the degradation of toxic or waste products. In homeostasis, the liver also serves as a central immunological shield between the gut and systemic circulation. Pathogens, toxins, and harmless food-derived antigens can enter the liver directly from the gut lumen, and be scanned by liver antigen-presenting cells (APCs) and lymphocytes. Despite such continuous exposure to microbial and non-self molecules, the liver appears to be an immune-privileged organ, which favors the induction of peripheral tolerance rather than immunity. Liver tolerance is achieved by the cooperative action of various tolerogenic liver-resident APCs populations and immune regulatory mediators, which are released locally from liver resident cells. Following liver injury, inflammation is a pathological hallmark feature of chronic hepatitis. Sustained inflammation then promotes liver fibrosis and, as an end stage, liver cirrhosis, autoimmune hepatitis or hepatocellular carcinoma. Although much progress has been made in understanding the role of the liver in immune tolerance, the precise mechanisms, and whether tolerance is maintained during chronic inflammation remain poorly understood. During my studies I will explore the hypothesis that the liver serves as a cardinal immunological organ, responsible for the induction of systemic immune tolerance to food and bacteria antigens. During chronic hepatitis, liver immune tolerance is compromised and systemic food intolerance, inflammation, and autoimmune diseases develop. To experimentally validate this hypothesis, I will use specific mouse models of liver inflammation and antigen presentation ablation and characterize the cellular and molecular mechanisms underlying liver induced tolerance and disease.

Pausing auf RNA polymerase (RNAP) II in the promoter proximal region of a large set of genes is the major rate-limiting step in human gene expression. The aim of this proposed research is to understand the mechanism by which promoter proximal pausing of RNAPII is controlled in human cells. I will achieve this goal by a combination of genome-wide and biochemical methods.
My research will focus on the role of phosphorylation of the RNAPII C-terminal domain (CTD) in the regulation of promoter proximal pausing. I will adapt nascent elongating transcript sequencing (NET-seq), a novel approach that allows RNAPII transcription to be studied genome-wide at single-nucleotide resolution in yeast, to human cells. I will use NET-seq to determine promoter proximal pausing sites of RNAPII across the entire human genome with single-nucleotide precision. I will make use of the high spatial resolution of human NET-seq data to extract the DNA sequences that underlie promoter proximal pausing sites and to reveal pausing motifs. By performing NET-seq with monoclonal antibodies directed against site-specific phospho-modifications of the CTD, I will determine the CTD phosphorylation state of transcriptionally engaged RNAPII at promoter proximal pausing sites. To establish the role of CTD phosphorylation in the regulation of promoter proximal pausing in vivo and to reveal transcription factors that are involved, I will disrupt CTD phosphorylation by CTD kinase depletion and through site-specific mutations of the CTD.
This new approach is critical to understand the way RNAPII CTD phosphorylation functions in the regulation of promoter proximal pausing and how its dysregulation causes cancer.

Intracellular recording of the neuronal transmembrane potential has been crucial to our understanding of the biophysical mechanisms underpinning neuronal computation. Existing intracellular recording techniques, however, do not permit long-term measurements of this potential, being limited by electrode-induced changes in neuronal function and the inherent instability of these recording devices. In practice, intracellular electrical recordings are generally limited to less than one hour, but many important processes occur on timescales of hours to days such as learning and plasticity. While in isolated cases repeated recordings from an identified neuron can be a work-around, this remains particularly challenging in vivo.
Here we propose to develop minimally-invasive intracellular recording electrodes for in vivo recordings by combining two complimentary technologies that have been separately developed by the laboratories of Andreas Schaefer (MPI f. medical research, Heidelberg, Germany) and Nick Melosh (Materials Science & Engineering, Stanford, USA). Schaefer group has described solid-conductor intracellular nananelectrodes (SCINEs) that can be inserted into neuronal membranes to record supra- and sub-threshold neuronal potentials without causing membrane damage or diluting cytoplasmic molecules. Unfortunately, these nanoelectrodes only insert into membranes with low probability and limited duration - both, presumably, due to an unstable membrane-electrode interaction. Melosh group has developed technology that allows inorganic nanostructures to insert and fuse spontaneously and stably into lipid bilayers. This is accomplished by creating a 2-10 nm wide hydrophobic band around the hydrophilic metallic electrode, mimicking the internal hydrophobic region of the lipid bilayer. Upon contact with a lipid membrane, this band fuses into the core of the bilayer, thus allowing the nanostructure to traverse the membrane without disrupting it and providing stable adhesion with the cell. Melosh group has verified integration of such “Stealth Probes” into membranes through atomic force microscopy and intracellular cyclic voltammetry.
Together, we plan to develop biomimetically patterned solid-conductor nanoelectrodes, “stealth SCINEs” (sSCINEs), for non-invasive intracellular recording, with Melosh group focusing on design, and testing of membrane-electrode interfaces, and Schaefer group focusing on assessing sSCINEs for physiology and developing in vivo sSCINE recording preparations. First applications will be mapping stability and plasticity of synaptic properties and substhreshold receptive fields in the mouse olfactory bulb — experiments currently impossible due to limited recording duration of current intracellular recording techniques and the high-dimensional input space.

The eukaryotic genome is spatially organized and highly dynamic. In yeast, several inducible genes are repositioned from the nuclear interior to the nuclear periphery upon activation, where they interact with nuclear pore complexes (NPCs). This interaction is thought to optimize transcription and DNA repair and promote mRNA quality control and export. Interestingly, gene-NPC targeting is accompanied by the formation of gene loops, which correlate with a “memory” of previous transcription. Many factors involved in looping and NPC-targeting also play multiple roles in gene expression, which complicates the interpretation of reverse genetic approaches. An important challenge in the field is to establish direct cause-effect relationships between the three-dimensional folding and positioning of a gene and its activity. To approach this problem, I propose to engineer synthetic, inducible gene loops by using novel optogenetic tools in S. cerevisiae. This will allow to determine whether artificially induced gene looping and NPC-tethering can recapitulate NPC function in terms of transcriptional activation and memory, and facilitation of mRNA export. Additionally, I aim to test whether the synthetic system can bypass the requirement for essential components of the natural NPC-associated gene expression machinery. These experiments will create a powerful basis to address a number of key questions in the field.

Metastasis of any organ-specific cancer type has a poor prognosis and is the cause of 90% of cancer-related deaths. MicroRNAs (miRs), small non-coding RNAs regulating gene expression, are increasingly implicated in cancer progression, including metastasis.
A recent study by Dr. Hanahan’s group (Olson et al, 2009) examined the dynamic expression of miRs during different stages of tumour progression in a mouse model of pancreatic neuroendocrine tumour (PNET), and unveiled unique miR signatures in each stage; suggesting a putative role in cancer progression. One of the miR signatures was detected in a subset of primary PNET and shared common miRs with the liver bona fide metastasis miR signature. The former one, referred to as met-like primary (MLP) signature, suggests that a subset of primary tumours can diverge and acquire enhanced metastatic abilities. Preliminary data suggest that miRs -214, -137, -449, and -199 of the MLP signature might act in a synergistic way to promote metastasis.
The focus of the proposed fellowship is to characterize the synergistic effect of the aforementioned miRs. The contribution of each miR to the synergistic effect will first be determined, and subsequently the critical time-frame/stage for their role during the metastatic process will be elucidated. Furthermore, subsequent experiments will aim to identify target genes and cellular processes altered by miRs. Finally, this study will also examine the prognostic value of the miRs in human PNET cancer.
Understanding the role of miRs in PNET metastasis can provide guidance for future experiments as well as the design of novel therapeutic approaches targeting tumour metastasis.

Do cells think? We imagine that thinking is the product of complex nervous systems. Studies by our team and others, has demonstrated that seemingly simple microbes, such as brewers yeast, display signs of having memory and consequently the rudiments of intelligent behavior. Yeasts grown on a particular sugar typically activate genes that code for enzymes to digest that sugar. We found if yeasts are grown for a long time on glucose, they appear to maintain activation of the genes required to digest glucose, and consequently take much longer to switch on the genes required for growth on an alternative sugar once the glucose is used up. This history-dependent behavior can be interpreted as a rational decision because, for yeast, the constant availability of glucose in the past appears to imply that glucose is likely to become available again in the future. In other words, yeast cells somehow "remember" how long they have been consuming a certain sugar, and they adapt their behavior accordingly. These preliminary results show that cells and thus the biochemical machinery – the genes and proteins – that generate their behavior are influenced by the past. We will set up experiments to first quantify history-dependent behavior at the single-cell level and second to identify genes and specific molecular processes involved in memory. We will specifically focus on epigenetic mechanisms, those that do not involve changes to the genes themselves but to changes in the structure of DNA and the inheritance of particular proteins from cell to cell. Further, to identify why history-dependent behavior may have evolved, we will determine environments (sequential appearances of different sugars) that favor and environments that disfavor history-dependent behavior. Our research will not only reveal new insight into microbial physiology, but may also establish general principles into how previous experience shapes future choices for cells. Our efforts may open up new avenues of research, such as whether complex nervous systems may use similar strategies to store information or whether cancer cells “learn” differently from normal cells, perhaps in a way that could be harnessed to selectively kill these cells.