Genetic information can be epigenetically modified independently of the DNA sequence itself. These chromatin-based regulatory mechanisms play a fundamental role in many biological processes, for example differentiation and development. Despite recent advances in mapping epigenetic states genome-wide, a mechanistic understanding of how they affect gene expression and the transcription machinery is missing. During Dosage compensation (DC), the expression of the X chromosome is equalized between females (XX) and males (XY). As DC operates independently of the DNA sequence, it provides an ideal system to study transcriptional regulation by epigenetic mechanisms. In Drosophila, DC operates on the male X by histone acetylation and recruitment of the MSL complex. I plan to address, how the MSL complex affects transcription by studying the biochemical and biophysical properties of a fully recombinant complex in the context of designer chromatin. For this, I will take advantage of recently developed techniques to produce homogenously modified histones. I then plan to establish a highly purified in vitro transcription system to analyze, whether and how histone modifications and the MSL complex affect transcription. Third, I would like to study whether co-transcriptional processes such as RNA processing are enhanced for X-linked genes as part of the DC process in vivo. Here, I foresee to discover potentially novel crosstalks between the DC machinery, transcription and chromatin. Altogether, these studies will elucidate the mechanistic basis of modulated expression during DC and also provide important progress in understanding how the epigenome affects the transcription machinery.

Decisions can arise from multiple systems in the brain, which can be dissociated behaviorally, neurally, and computationally. Much progress has been made in our understanding of the “Pavlovian” and “habit” systems, but the neural mechanisms underlying the “planning” system remain elusive. An emerging model system for the study of planning at the neurobiological level is the recording and manipulation of “place cells” in the hippocampus of rats solving navigation problems. Sequences of place cell activity can be decoded to reveal signatures of a planning process: possible trajectories are represented serially at decision points and following reward receipt, and include constructed, never-experienced trajectories. However, computational work clearly shows that serial, brute-force search will fail in complex environments. Accordingly, many efficient algorithms for approximate search have been developed in artificial intelligence (AI) and reinforcement learning (RL), but the algorithms used by the brain to plan in complex environments are unknown.
We propose to elucidate the neural mechanisms of planning in complex environments through the recording, manipulation, and simulation of hippocampal activity, afforded by interdependent approaches rooted in neurobiology, engineering, and computational modeling. We will record place cell activity on-line during planning and off-line during consolidation periods, on tasks sufficiently complex to require approximate planning methods, and that satisfy established behavioral criteria for planning. AI and RL-derived simulations will be applied to these tasks to generate contrasting predictions about the content and frequency of hippocampal sequences, and provide a computational basis for targeted manipulations. To test the impact of hippocampal sequences on associated circuits and behavior, we will develop real-time methods for sequence decoding, enabling the disruption and reinforcement of specific sequences with electrical and optogenetic tools. The combination of these approaches permits the disentangling of interactions between fast, “on-line” planning and the slower “structure learning” of appropriate task representations. The proposed work will reveal the real-time dynamics and associated computational principles that underlie navigational planning in complex environment, and uniquely links the neural circuit, representational and computational levels of a model planning system.

The overall goal of this proposal is to study the relationship between characteristics of functional brain networks and behavior. We will use a combination of imaging and graph theoretical approaches to create and analyze functional brain networks generated during memory formation and retrieval followed by targeted optogenetics to manipulate identified network hubs. At the behavioral level we will use fear conditioning to generate memories. To permanently tag neurons activated during memory acquisition we will use the recently developed TRAP (targeted recombination in active populations) mouse line in which expression of Cre recombinase is controlled by the neuronal activity regulated c-fos promoter. We will tag neurons active during fear memory acquisition with a genetically encoded fluorescent marker. After memory retrieval brains, will be processed for imaging using the CLARITY technique. Functional networks will be built by examining the co-variation of the number of neurons expressing the fluorescent marker (acquisition) or elevated levels of activity regulated genes such as c-fos (retrieval) throughout the brain. Graph theoretical analysis will be used to identify brain regions with high interconnectivity (i.e, hubs). We will interrogate the network using targeted optogenetics in which adeno associated viruses expressing inhibitory opsins in a Cre recombination dependent manner are infused into hub brain regions. We will examine the effect of inhibiting hubs during testing on memory retrieval and the resulting functional network. In this way we will be able to draw causal inferences between characteristics of functional brain networks and memory expression.

During the past 40 years, behavioral studies have confirmed the presence of magnetoreception in all major groups of Vertebrates, including fish, amphibians, reptiles, birds, and mammals, as well as in insects, crustaceans, and in a variety of microorganisms including the magnetotactic bacteria and protists. Electrophysiological studies in birds and fish indicate that the ophthalmic branch of the trigeminal nerve is one of the conduits of magnetic field information to the brain, implying that this pathway evolved before the last common ancestor of Vertebrates; more recent work suggests the vestibular system in birds might also be involved. Other work suggests magnetoreception might also arise in the retina, perhaps through optical effects on a protein like cryptochrome.
Despite this widespread presence in animals, the question of whether or not humans possess a remnant of these ancient magnetosensory systems has remained an extraordinarily controversial issue. Humans possess both biological magnetite, as well as cryptochromes, arguing that magnetoreception is a physical possibility. However, reports of magnetic fields influencing human orientation, made by a British group 30 years ago, failed the ‘acid test’ of Science when the initial orientation effects could not be repeated, thereby precluding tests of the magnetic influence. In contrast, more recent and as yet preliminary results from psychological experiments by the Kirschvink group support the hypothesis that some human subjects may have a subconscious remnant of this magnetic sensory system. Using our combined geophysical, psychological, and engineering skills, our goals for this HFSP project are to attempt verification, improve the power of the procedures, map the neural circuits underlying the sensitivity, and ultimately develop behavioral-neural techniques to bring human magnetoreception into conscious awareness. If successful, these tools could also be applied to test biophysical hypotheses for the underlying transduction mechanism(s). As a significant byproduct, the psychophysical and engineering techniques developed will lead to more interactive BMI (Brain Machine Interface). It will be applicable to any other cases where a human brain has potentially plastic sensory abilities but not revealed yet.

Cell size is one of the most fundamental aspects that define the properties of a cell. During cell division, daughter cell size is determined by the position of the mitotic spindle, which is controlled by dynein-based cortical forces that pull on the spindle. During my HFSP fellowship, I identified cortical dynein regulatory systems that ensure equal-sized cell division in HeLa cells. However, the biological significance of equal-sized cell division has not been studied. For example, maintenance of cell size may play an important role in stem cell self-renewal.

To precisely control spindle position in a variety of cell types, I will develop a light-inducible system in which cortical dynein regulators are targeted to locally illuminated regions to manipulate spindle position in a spatially and temporally controlled manner. This optogenetic approach represents a powerful tool that will allow me both to understand the molecular functions of targeted proteins, and to manipulate spindle position to generate unequal-sized divisions in cell types that normally divide symmetrically.

Using the optogenetic system in parallel with RNAi, I will analyze the significance of symmetric cell division in differentiated cells and embryonic stem cells.

In this proposal, I will:

1. Develop an optogenetic-based approach to manipulate spindle position

2. Define the roles of symmetric cell division in HeLa and non-transformed Rpe1 cells

3. Analyze the mechanisms and roles of symmetric cell division in self-renewing ES cells.

The support of the CDA is critical for allowing me to establish my independent research and will enable me to assemble a team to conduct these experiments.

In all domains of life ribosomes catalyze protein synthesis. To ensure that these large molecular machines are correctly assembled, a plethora of protein factors are deployed in eukaryotes, which facilitate the assembly pathway in which 4 ribosomal RNAs and 79 proteins are integrated into a fully functional ribosome. Defects in ribosome assembly are associated with several serious human diseases, which underline the central importance of this process.

The initial transcription of pre-ribosomal RNA (35S pre-rRNA in yeast, containing 18S, 5.8S and 25S rRNA) is followed by a hierarchical assembly of a giant complex termed the Small Subunit Processome (the 2.2 Megadalton SSU Processome) from several multi-protein sub-complexes. This complex is unique to eukaryotes and most of its constituents are essential proteins. While many of the protein components of the SSU Processome have been identified, our understanding of this essential complex is currently limited by a lack of structural and mechanistic insights.

This proposal aims at the structural and mechanistic characterization of the SSU Processome. To unravel its functional architecture an integrated structure-function approach will be applied, which combines yeast genetics, novel biochemical tools as well as complementary structural studies using X-ray crystallography and cryo-electron microscopy. These approaches will be used synergistically to elucidate the inner workings of the SSU Processome during eukaryotic ribosome assembly.

Proliferating cells must grow and duplicate their contents before dividing. The cell cycle comprises the set of regulatory interactions that protect against improper division by imposing a strict order on the processes leading up to cytokinesis. A key aspect of this control is the coordination of overall growth with division to maintain a consistent cell size across populations and generations. In S. cerevisiae this is achieved by delaying progression through the cell cycle in G1 phase until the cell has reached a minimum size. Although genetic screens and in-depth studies have identified a set of proteins affecting cell size regulation, the molecular mechanism that allows cells to measure their size is still obscure. One major candidate for a size sensor is the unstable G1 cyclin Cln3, which triggers irreversible commitment to cell division. Since Cln3 concentration is thought to remain constant through G1, we hypothesize that cells may titrate the number of Cln3 molecules, which increases proportionally with cell size, against their DNA, which remains constant in G1. Such a mechanism could result in an enzyme activity proportional to the number of Cln3 molecules rather than to their concentration. Here, we propose to test this hypothesis both in vitro and in vivo. First, we will determine if the phosphorylation of key Cln3-Cdk1 substrates is affected by the presence of DNA in vitro. Second, using single cell quantitative microscopy,we will confirm the relevance of DNA-dependent phosphorylation to size control in vivo. Demonstrating that DNA dependence of Cln3-Cdk1 substrate phosphorylation is required for size control would directly support the titration model of cell size control and thereby solve a longstanding question in biology.

Synthetic biology offers the possibility of new therapies based on functional cellular implants to cure intractable diseases, such as immune diseases and metabolic diseases. For example, the need for diabetics to inject insulin after every meal could potentially be avoided by introducing functional cellular implants that rapidly release insulin on demand. This would dramatically improve patients’ quality of life. However, it is difficult to rapidly control protein secretion in response to stimulation because current artificial cellular functions rely mainly on slow transcriptional control of transgenes within the cells.
My proposed project aims to create new cellular functions capable of rapidly modulating biological systems by secreting effector molecules outside the cells in response to external stimuli. It is established that a well-regulated exocytosis pathway exists in a wide variety of cells and can be artificially reconstructed in artificial liposomes. Therefore the project will focus on creating new, rapidly responsive cellular functions by artificially modulating the exocytosis pathway in living cells.
I intend to validate this design approach for innovative future cell-based therapies by constructing a new therapeutic model. Specifically, I propose to construct a model system for treating rejection after transplantation without the need for administration of immunosuppressive drugs, by incorporating artificial cellular functions into the surface layer of the transplant. These functions will serve to sense contact of active T cells and to rapidly secrete T-cell-blocking effector molecules only in the immediate vicinity of the transplant.

Misfolding diseases arise as a consequence of accumulation of undesired proteins in the cell, which is due to failure of protein quality control system. The ubiquitin proteasome system, involved in this control, degrades a large number of cellular proteins and dysfunction of the proteasome has been associated with a number of misfolding diseases. Thus, rescuing the defects resulting from proteasome inhibition could be of a therapeutic use. The aim of this project is to identify strategies to rescue from proteasome impairment. Recent unpublished work in the host laboratory has led to the identification of a potent and previously unidentified suppressor of proteasome dysfunction in yeast. Since the function of the proteasome is evolutionary conserved it is likely that a protein of similar function exists in mammals. The first goal of my project is to identify this homologue. In addition, the host lab has recently discovered that cells can survive with undegraded proteins that accumulate as a result of proteasome inhibition when supplemented with amino acids. I will determine whether amino acid supplementation can rescue deleterious consequences of protein misfolding in an in vivo model of a misfolding disease. In summary, this research can lead to better understanding of proteasome assembly and function as well as highly impact misfolding diseases field.

Combine the well established RNA switch in the laboratory of Dr. Hirohide Saito at Kyoto University with cytotoxic supramolecular nanofibers from the laboratory of Dr. Bing Xu at Brandeis University, the objective of the proposed research is to design a two-component device for selectively elimination of cancer cells based on the recognition of over-expressed RNA binding proteins (RBPs) in cancer cells and the degradation controlled cytotoxicity of molecular nanofibers. To achieve the objective, the proposed research will design RNA switches that response to RBPs, such as Sam68, eIF4E, La, or HuR (L. Wurth, Comp. Funct.Genom., 2012), that are over-expressed in cancer cells. The recognition of RBPs in cancer cells prohibits the expression of downstream mRNA that codes for esterase. On the contrary, low expression of RBPs in normal cells allows the expression of esterase. Therefore, the toxic nanofibers, which can be degraded into nontoxic fragments by the esterase, will only induce cytotoxicity in cancer cells, while leaving the normal cells unharmed. The proposed research innovatively combines synthetic biology with supramolecular chemistry, by which both precise recognition and elimination of cancer cells can be achieved. Importantly, the two-component device unlikely would induce adverse side effect on normal cells and tissues, as the device, by itself, is nontoxic and toxicity appears only under the recognition of cancer marker RBPs. The high tolerance of the device by normal cells and tissue not only may lead to a new approach for cancer therapy, but also will contribute to the research in iPS cells for the removal of cancer cells from iPS cell cultures.

The origin and maintenance of new species is one of the key questions in evolutionary biology. Yet, little is known about the genetic and epigenetic basis of speciation, especially outside of model organisms. In conventional models of speciation, the genetic architecture of diverging species will become so different that genes or genomic regions are incompatible if brought together in hybrids. A recent idea is that a broad variety of epigenetic mechanisms, like methylation, may also play a role in the hybrid phenotype. This project will investigate the role of methylation and gene expression in hybrid incompatibilities. For this, I will use the recently speciated Formica rufa group ants and their hybrids. First, I will study gene expression and methylation patterns genome-wide by RNA-seq and whole genome bisulfite sequencing and test if gene expression and methylation are disrupted in hybrids. Second, I will test the hypothesis that genomic regions implicated in hybrid incompatibilities are associated with divergent or disrupted methylation and/or expression patterns. The novelty of this project lies in combining genome-wide data on genetic incompatibilities, methylation and expression. This project will generate new data on the epigenetic basis of speciation, a promising research area where only a few studies exist at present. Furthermore, the project will widen my scientific and analysis skills by learning me to master genome-wide sequence data. The results will shed important insight into genetic and epigenetic basis of speciation, how methylation coordinates gene expression and the mechanisms that underlie hybrid dysfunction and novelty.

Cell surface receptors, transporters, and ion channels underlie communication between the intracellular and extracellular environments. Cell surface receptors exist in several stable states and likely sample multiple conformations during transitions between these states. Yet, fundamental mechanisms relating these conformational changes to protein function remain poorly understood. Single molecule studies have revealed dynamics of purified proteins, transplanted out of their cellular environment, but existing single molecule methods lack the sensitivity to examine protein dynamics in live cells. Moreover, many important membrane proteins cannot be purified and reconstituted, making them impossible to study with existing single molecule methods. The proposed research will extend single-molecule measurements to these previously inaccessible proteins. Our studies will utilize plasmonic nanostructures that will enable spatial isolation of proteins within nanoscale ‘chambers’, by growing cells on arrays of these tiny apertures. While the optical confinement of single receptors in these apertures will virtually eliminate background fluorescence, the plasmonic antenna will enhance the fluorescence emission of dyes and increase photostability. These cooperative features will significantly improve detection efficiency and allow for the resolution of single protein dynamics in live cells.
We will couple this innovative live cell, single molecule, imaging system with patch clamp electrophysiology that will enable precise control of voltage-gated ion channel gating. This will allow us to examine conformational dynamics of receptors at sub-millisecond time scales using single-molecule FRET. We will apply this new technique to measure conformational changes in Kv1.2 voltage gated potassium channels which are essential electrical regulators in the nervous system. Applied changes in membrane voltage trigger structural rearrangements within these ion channels, culminating in opening of the channel pore. The proposed studies will allow us to detect these conformational changes in single ion channels, and directly correlate conformational changes to receptor function. This collaborative and multidisciplinary project has the potential to establish a novel platform for a broad range of single-molecule applications in live cells.

Although conserved ageing pathways have been studied in invertebrate model systems, the links between them are still poorly described. Medical interventions for human ageing are likely to use adult stem cells (ASCs) as therapeutic agents. Thus, it would seem timely to utilize model systems that have ASCs to understand how ASCs respond to one of the key drivers of ageing, oxidative stress. DNA replication causes the chromosomes to shorten as cells divide unless telomerase activity counteracts this process. Also, as animals increase in chronological age, oxidative damage would contribute to telomere shortening that slows down tissue’s homeostatic turnover. However, evidences have suggested other telomere-independent functions of telomerase that arise during oxidative stress to protect cells from oxidative damage. I aim to investigate how ASCs in ageing and non-ageing flatworms cope with oxidative stress and regulate reactive oxygen species (ROS) homeostasis. Telomere maintenance and telomerase activity are adapted to asexual reproduction, such that they allow the avoidance of ASCs senescence by telomere shortening. I will use flatworms with sexual and asexual biotypes to understand how they avoid ageing when challenged by ROS. I will study how telomerase activity is regulated, how this affects stem cell maintenance and whether telomerase exerts protective functions on mitochondria to prevent mitochondria dysfunction during oxidative stress. This study will yield unprecedented insights into biological immortality, regenerative capacity and ageing, making use of powerful flatworm models to understand the links between oxidative stress and telomerase in the context of ASCs.

Our present concept of CRL function derives mainly from scrutinized SCF and APC/C complexes that drive cell cycle progression by targeted proteolysis of their substrates. However, accumulating evidence highlights vital roles for CRL3 and CRL4 in orchestrating mitotic events. To further illuminate the function of these CRLs during cell division we need to (i) identify important adaptors and their critical substrates, (ii) decipher the biological output of individual ubiquitination reactions as well as (iii) extend our knowledge of general CRL enzymology and regulation. As of yet the identification of substrates for specific E3 ligases has been technically challenging. This primarily reflects short substrate residence times, rapid turnover of ubiquitinated substrates destined for degradation and often low cellular abundance. Here, I will pursue a novel strategy of a single molecule TIRF microscopy-based flow-cell assay tailored to overcome these limitations. In line with our research priorities this assay supports fast compositional probing of novel CRL complexes and ultrasensitive detection of prevalently transient and rare physiological E3 ligase–substrate encounters from cell extracts. On top of that, I will exploit this single-molecule imaging technique to recapitulate and quantitatively analyze mechanistic aspects of dynamic E3 ligase function. A list of novel putative mitotic Cul4 substrate specific adaptors that emerged from in silico predictions and high-content RNAi screening previously conducted in our lab provides the biological foundation for this study that includes a comprehensive analysis of novel CRL4 complexes in mammalian cells.

Throughout the animal kingdom, social interactions such as parental and mating behavior, territorial defense and predator avoidance are triggered by specific sensory stimuli. In rodents, chemosensory systems provide the main source of information for social interactions. Decades of research has showed that a vast range of animal cues trigger innate social behaviors through the vomeronasal pathway. But how are those cues encoded in the brain to generate specific behavioral responses is still poorly understood. In my proposed work, I firstly plan to dissect the neural coding of critical parameters relevant to social behaviors in awake animals using microendoscopy-based calcium imaging. I will examine the representation of species- and sex- related natural stimuli in the medial amygdala (MeA), a central node of the vomeronasal sensory-motor transformation. Transgenic lines expressing light-activated opsins (ChR2 or C1V1) driven by specific vomeronasal organ receptors (VRs) as well as natural stimuli will be used to uncover the MeA-receptor maps corresponding to discrete vomeronasal inputs. Furthermore, the basic principles of MeA organization will be examined in genetically identified neuronal populations, using markers such as the enzyme aromatase (Ar) and the thyrotropin-releasing hormone (TRH). Finally, taking advantage of chronic imaging by microendoscopy, I seek to find the mechanisms of both instinctive and learned social behavioral changes triggered by past experience. For this purpose, I will observe the representation of both natural pheromones and single receptor population inputs in the MeA during experience-dependent as well as learning-involved social behaviors.

Pharmaceutical drugs represent one of the most powerful methods available for manipulating important aspects of our physiology. As such, their development has been a big driver for the advances in healthcare over the last century. Drugs have been used not only to treat diseases directly, but also as an experimental tool for researchers trying to understand how the body works.
Although the many benefits of pharmaceutical drugs are well known, so are their limitations. Drugs commonly have ‘side effects’ on aspects of our physiology that were previously healthy and, over repeated application of a drug, people often become desensitized, meaning that they require higher and higher doses to achieve the desired effects. One can view both of these limitations as a consequence of the failure of drugs to recapitulate the ‘natural’ modulation of physiology that generally keeps us healthy. Natural checks and balances on cell physiology act by regulating the activity of specific proteins in specific cells in a particular part of the body, while drugs often are much less selective in the nature and location of their activity. Similarly, while the activity of a natural process is modulated in ‘real time’, drug concentrations build up slowly and hang around for a long time. It is, of course, not time to throw the pharmaceutical baby out with the bath water, nevertheless alternative methods of manipulating physiology would be most welcome.
As many as a third of currently prescribed drugs target a specific mode of communication between cells that originates with a large family of proteins called G protein-coupled receptors (GPCRs). We propose developing a group of modified GPCRs that will be light sensitive. These proteins will turn ordinary cells into photoreceptors, allowing us to control their physiology simply by shining light on them. Because only those cells containing these engineered proteins will respond to light, and because its easy to switch light on and off rapidly (or to leave the light on for hours/days), this approach will allow scientists to approach a more ‘natural’ modulation of cell physiology. In the first instance these proteins will be available for researchers to use in cell cultures and/or animals to gain a better understanding of how the body works. Ultimately, we hope that they will be adapted for treating diseases in people.

Myeloid cell infiltration occurs at early stages of melanoma and is correlated with tumor development. Populations of myeloid cells have been proposed to regulate tumor development and maintenance in different manners. In the proposed studies I will identify the mechanisms underlying myeloid cell infiltration into melanoma tumors and elucidate how infiltrating myeloid cells affect tumor cells and vice versa. The zebrafish is unique for high resolution in-vivo imaging of the activity of the myeloid and tumor cells, and transgenic and gene inactivation technology can be used to probe function. A BRAF;p53 model of melanoma has been created by the Zon laboratory, and a specific transgenic using the crestin promoter reports melanomas at the 4 cell stage of tumorigenesis. We plan to watch the myeloid cells migrate to the tumor cells for the first time and describe the populations. Myeloid-expressed GFP transgenic fish will be used to follow the populations. RNA-seq of the infiltrating myeloid and tumor cells, compared to human primary melanoma samples, will define potential chemotactic molecules and genetic alterations in the cells following myeloid and tumor cell contact. I will use a unique plasmid system to express transgenes of the chemotactic factors in the tumors or to inactivate pathways in myeloid cells. This will provide the first genetic evidence for myeloid cell-tumor interactions, and may lead to new therapies directed at these mechanisms.

The function of human body to a large extent depends on direct cell-to-cell communication mediated by gap junctions composed of intercellular channels. They permit the exchange of ions and small molecules between cells. Calcium homeostasis modulator 1 (CALHM1) is a recently characterized gap junction-like ion channel involved in the Alzheimer’s disease (AD) and in taste-stimuli-promoted neurotransmitter ATP release from taste sensing cells. CALHMs 2–3 are associated with neurodegenerative Creutzfeldt-Jakob’s disease.

The molecular architecture of CALHMs remains poorly understood. No three-dimensional structures of CALHMs are available and only one structure of gap junction protein is known. This limits the mechanistic understanding of ion and ATP recognition, translocation across the membrane, and release by CALHMs (as well as by most large pore channels). Thus the signal transduction underlying the CALHM-associated neurodegenerative diseases is poorly understood.

To decipher permeation and regulatory mechanism of CALHM, I will integrate biophysical, electrophysiological, cell-based analyses to investigate the structure and function of (1) human CALHM cytoplasmic domains, (2) the full length CALHMs, and (3) CALHMs in complex with ligands such as ATP, calcium and cobalt (blocker of the channel) ions and antibodies. These studies will reveal the overall architecture of at least one CALHM alone and in complex with its ligands, provide atomic level understanding of how CALHM may function in healthy individuals and patients. Structure of CALHM-ligand complexes may also provide a platform for the rational design of therapeutically valuable molecules for clinical interventions.

Over the past tens of thousands of years, humans have expanded into a hugely diverse range of environments. This expansion has been accompanied by dramatic phenotypic changes. As a result of this, many traits vary substantially across modern human populations, often with important medical implications. Understanding which traits have been under selection, and which genomic loci have responded to this selection is important both for our understanding of human history, and for our knowledge of the genetic architecture underlying phenotypic variation in modern humans. One exciting new source of information about this evolution comes from recently developed technology to extract DNA sequence data from ancient samples. Though this data is relatively easy to analyze, it cannot be generated in large quantities, which limits it usefulness. Conversely, data from modern humans is easy to obtain, but statistically difficult to analyze. This project aims to tackle the challenging problem of developing methods that can combine both of these sources of information in order to take advantage of both of their strengths. This will allow us to answer questions both about the role of selection in the evolution of specific genomic loci, and about the relative importance of different types of selection. In particular, we will be able to test the hypothesis that polygenic selection, or weak selection on large numbers of loci, is responsible for much of the recent phenotypic evolution of humans.

To determine the neurons and mechanisms a fly employs to integrate blends of male pheromone and food odor, I will proceed in four steps. 1)I will build a reference library of flies’ individual behavioral responses to blends of male pheromone and food odors. 2)I will identify the activated receptor neurons using loss of function mutants, and resolve their downstream neurons based on literature and recent mapping data of the lateral horn neurons from the Jefferis group. The lateral horn is the most promising site of integration. 3) I will identify the integrator neurons by recording from the downstream neurons using electrophysiology and calcium imaging. If the level of neuronal activity depends on the presence of both food odor and male pheromone, then the targeted neurons are likely the integrators. I will backtrace signals to confirm that the identified, and not other upstream neurons, are the integrators. I will also verify that stimulating their presynamptic inputs is sufficient to trigger behavioral integration. 4)I will reversibly inactivate the neurons to eliminate the reponses to blends, which tests whether the neurons are necessary. I will stimulate the neurons to recapitulate behavioral integration, which tests whether the neurons are sufficient. Last, I will modulate the excitability of the integrator(s) and measure input levels. If required input levels change coordinately with changes in excitability, then activity within the integrators is critical for behavioral integration. Together the results will advance our understanding on how behavioral choices are elicited at the neuronal level.