The integrity of stratified epithelial sheets in skin is dependent on intercellular adhesion, which is in part mediated by adherens junctions. These junctions are dynamic, allowing for tissue remodeling during morphogenesis and tissue repair following injury. Wound healing is an orchestrated process that involves movement of epidermal cells to close the gap, inflammatory responses to prevent infection at the injured site, and hyperproliferation to replenish lost cells. Targeted loss of the adherens junction protein alpha-catenin in skin results in enhanced inflammatory, hyperproliferative, and migratory behaviors, changes in epithelial cell morphology, and development of squamous cell carcinoma in situ. The molecular roots of these phenotypes appear to be intrinsic to the epidermal keratinocyte. I have developed an approach to dissect the mechanisms underlying the complex phenotypes arising from alpha-catenin loss in skin. I devised a series of RNAi screens in wild type and alpha-catenin mutant keratinocytes to assay how the genes that are differentially expressed in these cells contribute to each phenotype. I then propose functional analyses of my identified factors to address the phenotypic consequence of their loss in skin, the nature of their interaction with alpha-catenin, and their function in wound repair and squamous cell carcinoma tumor development and progression.

A challenging frontier in neuroscience is to understand the molecular pathways that regulate the organization, function and plasticity of circuitry mediating specific behavioural responses to environmental stimuli. Using pheromone-initiated behaviours as a model, the present study proposes a methodology to investigate the molecular pathways regulating the organization and function of the female versus male pheromone-mediating circuitry that leads to sexual versus aggressive behaviours, respectively, in adult males. Transgenic mice will be generated in which neurons activated in response to female versus male pheromones can be recognized by their expression of a fluorescent reporter. Using established single-cell gene expression profiling techniques, I propose to isolate and analyze pheromone receptor-expressing neurons in the vomeronasal organ and within higher order central nervous system (CNS) structures to address two fundamental questions: (1) What is the combinatorial pheromone receptor code that mediates processing of female versus male pheromones? (2) What are the distinct molecular pathways that regulate the organization and function of higher order circuitry processing female versus male pheromone information? This study is anticipated to significantly advance our understanding of the molecular logic that underlies female versus male pheromone-initiated behaviours in adult males. Further, the project proposes a general methodology for the investigation of molecules regulating the organization, function and plasticity of circuitry in the adult CNS.

Splicing is a fundamental gene regulation process that removes the non-coding regions and joins the coding regions of pre-mRNA to produce proper mature mRNA for protein translation. Most of the eukaryotic genes display alternative splicing patterns that allow the production of multiple proteins from the same pre-mRNA, significantly enriching the complexity of the proteome. Even though the chemistry of the splicing reaction is known, comparatively little is known about the splicing code, namely how splice sites are chosen and splicing decisions are made by the cellular splicing machinery. There is evidence that in mammals constitutive and alternative splicing events are regulated by trans-acting proteins that are recruited by cis-acting elements along the RNA sequence such as exonic and intronic splicing enhancers as well as exonic and intronic splicing silencers. Although substantial effort has been recently put into the identification and characterization of cis-acting elements that regulate splicing, a detailed and comprehensive study of trans-acting proteins recruited by the intronic/exonic splicing enhancers/silencers in mammalian cells remains to be explored. Therefore, this project aims to establish functional relationships between the known cis-acting elements and trans-acting factors, study the biological function of the splicing isoforms of the trans-acting factors and discover novel trans-acting factors by an RNA interference medium-throughput screen. Since many genetic diseases arise from mutations in cis-acting elements, a comprehensive library of the associated trans-acting factors might point to novel therapeutic targets.

Mutations in Pkd1 and Pkd2 genes, encoding polycystin-1 (PC-1, a membrane receptor) and polycystin-2 (PC2, an ion channel) proteins respectively, are implicated in the development of skeletal diseases. The PC1 and PC2 complex is believed to form a mechanosensitive receptor-ion channel complex that is involved in flow detection in kidney epithelial cells and is essential to normal kidney physiology. Although recent evidence suggests the presence of PC1 and PC2 in osteocytes (the mechaonsensor in bone) may be important for their physiology, their role in the mechanosensitivity of these cells remains unknown. To fully address the functional role of the ciliary PCs in the mechanosensitivity of osteocyes, our research plan consists of three main steps, divided as follows: 1). Mechanosensitivity of the primary cilium in OB/OC cells. 2). Role of PCs in the mechanosensitivity of OB/OC cells. 3) Link between PC-stimulation and cellular activation of OB/OC cells. We believe these approaches will not only lead to a better understanding of the molecular mechanisms underlying the mechanosensitivity of bone cells, but will also shed light on mechanisms involved in the pathogenesis of skeletal diseases.

Cell to cell variability in gene expression and protein levels has recently become a subject of intense study. However, it is still unclear whether such variability has any bearing on cell fate in mammalian cells. This question is especially relevant to cell death, which has a central role in tumor suppression, the immune response, and host-pathogen interactions. Is cell death a deterministic process, in which genetically identical cells in a homogenous environment behave the same? Or is there an element of chance, where cells under the same conditions behave differently? To try to answer this question, I propose to challenge macrophage cells with diverse pro-apoptotic factors, alone and in combination with TNF, a pro-survival factor in macrophages. I will also examine variability in cell death after infection with macrophage tropic HIV. I will use time-lapse microscopy to determine whether the probability of cell death during a given time interval is the same or different between cells, and whether the extent of variability depends on the pathway involved. If significant variability in cell fate does exist, I will investigate how different cell fates are correlated to different temporal activation dynamics of the NF-kappaB pathway, a central pro-survival pathway in many cell types. Understanding variability in cell death may lead to better understanding of processes where cell death plays a key role, such as HIV infection, cancer therapy, and the immune response. This systems biology based understanding of immune cell physiology will be a new direction for me, but my background in systems biology and live cell imaging gives me the tools to carry out this project.