Heat Shock Protein- 90 (HSP90) is an essential molecular chaperone. HSP90 relies on its intrinsic ATPase activity as well as interactions with co-chaperone proteins to chaperone its clients. HSP90 is also an extracellular protein, performing both a signaling and chaperoning role. Extracellular client, matrix metalloproteinase-2 (MMP2) relies on HSP90 for its stability. MMP2 mediates extracellular matrix remodeling through its gelatinolytic activity. MMP2 activity is also tightly regulated by its endogenous inhibitor, the Tissue Inhibitor of Metalloproteinase-2 (TIMP2). At present how HSP90 performs its chaperoning role in the extracellular matrix is uncertain. In this thesis, I describe that TIMP2 acts as the first bona fide extracellular co-chaperone of eHSP90, and show that TIMP2 is a stress inducible protein. I describe how TIMP2 directly interacts with HSP90, and how TIMP2 decelerates the HSP90 ATPase cycle. TIMP2 also sensitizes HSP90 to both ATP and N-terminal pharmaceuticals. Overall, TIMP2 acts as both a scaffold and a disruptor of the client/chaperone relationship between MMP2 and HSP90, performing both a HSP90 co-chaperone and MMP2 inhibitor role, non-mutually exclusively. The activatory co-chaperone AHA1 competes with TIMP2 for HSP90 binding. TIMP2 and AHA1 are able to form two independent ternary complexes with MMP2 and HSP90; as a result, the TIMP2 complex is MMP2 proteolytically inactive and the AHA1, active. This competition is further described in vivo where it can be inhibited by both _AHA1 antibodies and TIMP2 AHA1 antibodies and TIMP2 exogenous protein treatments, whilst induced following AHA1 protein and _AHA1 antibodies and TIMP2 TIMP2 antibody treatments. Finally, the role of phos-Y-TIMP2 was examined in relation to its interaction with HSP90. To address this, a novel methodology to purify hTIMP2 from E.Coli without previously necessary refolding strategies in a scale-able manner suitable for therapeutic TIMP2 treatments, was developed. Wild type recombinant human TIMP2 and phospho-mutants Y90E, Y90F, and TE (Y62E, Y90E, Y165E) were purified, were inhibitory towards MMP2, and modulated TIMP2 interaction with HSP90. Taken together, I demonstrate how extracellular HSP90 is regulated by co-chaperones to facilitate the chaperoning of pro-invasive client, MMP2. It further shows ways in which we can manipulate this system to promote an inactive MMP2 protease, a key strategy in cancer therapeutics.

The complex organization and trajectory of development of the central nervous system (CNS) depends not only on cell-cell interactions but also on interactions of cells with the extracellular environment. The extracellular environment in the CNS contains an organized network of proteins, glycans and glycoproteins and comprises what is called the CNS extracellular matrix (ECM). Components of this ECM are dynamically regulated not just during development but are also involved in various neurobiological processes in the adult. They are implicated in normal nervous system physiology as well as in nervous system disease and pathology. A complete understanding of the CNS therefore, requires an understanding of the composition, structure and function of the neural ECM also. Our laboratory focuses on a specialized substructure of the neural ECM, called perineuronal nets (PNNs). PNNs are conspicuous structures within the neural ECM and are thought to be key regulators of neuronal plasticity. Traditionally their role has been demonstrated in regulating forms of plasticity seen during development, but recent work has implicated PNNs in other forms of learning and plasticity across various brain regions as well. Interest in these structures has been further peaked by studies linking their alterations to a variety of neurological and neuropsychiatric diseases such as Alzheimer's disease and schizophrenia. However, despite this growing interest in PNNs, the mechanisms by which they modulate neural functions are poorly understood. The limited mechanistic understanding of PNN function is derived primarily from the fact that there are no existing models, tools or techniques that specifically target them without also disrupting the surrounding neural ECM. Therefore, a rigorous investigation of PNN function to date has not been possible. Our inability to specifically target PNNs is driven by an incomplete of understanding of their molecular composition and structure. While PNNs comprise of many known ECM components which are broadly expressed in the CNS ECM, PNNs appear clearly distinct from their surroundings. They are highly ordered and stable and show a regular organization and geometry not present in the diffuse neural ECM. The exact molecular mechanisms by which various PNN components come together and aggregate on the cell surface to form these structures however, is not iii clearly known. The primary goal of our laboratory has therefore been to determine the molecular structure, composition and formation of PNNs. Through our work described in chapters 2 and 3 of this thesis, we were able to develop a new model of PNN structure. In our proposed model, PNN components are bound to the cell surface by two distinct types of interactions, one dependent on the classical HA scaffold of PNNs and the other mediated by a complex formed by the ECM glycoprotein tenascin-R (TNR) and the chondroitin sulfate proteoglycan receptor protein tyrosine phosphatase zeta (RPTPz). Our work also allowed us to provide evidence that PNN components are immobilized on the neuronal surface by a GPI-linked mechanism. We identify the GPI-linked protein contactin-1 (CNTN1) as a key receptor molecule for PNNs. To our knowledge, this is the first ever identified receptor for PNNs. We feel that our findings presented here give us important insight into PNN structure and represent the initial important steps in understanding the formation of this unique ECM subcompartment.

In1899, psychiatrist Emil Krapaelin introduced a separation between schizophrenia (SZ) and bipolar disorder (BD), formerly “dementia praecox” and“manic-depressive disorder”, which came to be known as the Krapaelinian dichotomy, and has prevailed over the past century (Kraepelin, 1904). Although Emil Krapaelin postulated that these are distinct entities, multiple converging lines of evidence suggest that SZ and BD have a shared etiology: (1) first-degree relatives of a SZ-affected individual are at higher risk for BD than the general population, and vice versa (Lichtenstein et al., 2009), (2) recent work from genome-wide association studies (GWAS) and rare variant studies revealed that SZ and BD share common risk genes, suggesting that these disorders share a set of molecular substrates, and (3) second-generation antipsychotics exhibit effectiveness in ameliorating psychosis and mania (Buckley, 2008). SZ and BD are highly heritable mental illnesses with a lifetime prevalence near 1%. Onset typically occurs in late adolescence to early adulthood. Their etiology is complex and multi-factorial. SZ and BD are among the leading causes of disability around the globe(Global Burden of Disease Study 2013 Collaborators, 2015). There isa constellation of symptoms related toSZ, including hallucinations (e.g., auditory, olfactory, visual), delusions (e.g., persecutions, grandiosity), thought disturbances, affective flattening, and anhedonia. SZ and BD have clinical resemblances like psychosis, though this is more widely recognized as a hallmark of SZ. The core feature of BD is extreme changes in mood ranging from periods of mania followed by severe depression, which is also referred to as “switching”. Drugs for treating SZ and BD have changed very little over the past 50 years, and those that are used today are not always effective and can elicit severe side effects. SZ and BD research is evolving rapidly but our understanding of these disorders is still in its infancy. One of the major advances in the field has been the “big data” revolution. Technological advances have been a critical driving force of this revolution, including emergence of DNA microarray chips for high-throughput genome-wide genotyping and gene expression profiling. These technologies became widely adopted in psychiatry and led to a proliferation of genome-and transcriptome-wide studies in psychiatry to aid in the discovery of novel genes and pathways related to mental illness. Despite SZ and BD having a strong genetic basis, identifying susceptibility genes was a significant challenge. Combining data across laboratories became a fundamental strategy to overcome inherent weaknesses with statistical power and methodological biases, which has proven be to a fruitful strategy for GWAS (Cross Disorder Group of the Psychiatric Genomics Consortium, 2013; Ripke et al., 2014; Ruderfer et al., 2013; Sklar et al., 2011). Yet, a robust methodological and statistical framework for analyzing combined collections of gene expression data has been lacking in psychiatry. Microarray studies of SZ and BD suffered from low statistical power and drawbacks that affected their reproducibility (Draghici, Khatri, Eklund, & Szallasi, 2006; Evans, Watson, & Akil, 2003). Combining gene expression data from numerous sources and addressing methodological issues may help to uncover reliable molecular associations. Even though the relevance of gene expression to physiology is not always clear, gene expression abnormalities in mental illness can provide fundamental insight into gene regulatory networks in brain and peripheral tissues, and provide a framework for interpreting genomics data. Integrating findings between GWAS and gene expression studies has the potential to elucidate the etiological overlap of SZ and BD. Moreover, gene expression signatures of mental illnesses may have biomarker utility and set up a foundation for identifying better drug targets. Data sharing is now a common place. Although microarrays are gradually being replaced by more sensitive and precise technologies such as next-generation sequencing, data harmonizing will be a pervasive issue unless dealt with now.In this dissertation, I present two review papers describing the current state of SZ and BD genetics research followed by three primary research studies that I performed to answer these prevailing questions: (1) what are the genes, pathways, and regulatory elements that relate to risk for SZ and BD, and are these similar or different across disorders? (2) what genes and pathways are abnormally expressed in SZ and BD, and might these differences converge with genomic evidence? (3) can differences between SZ and BD reflected in gene expression profiles offer biomarker utility and a basis for developing disorder-specific classifiers?My primary hypothesis for this work is SZ and BD exhibit overlapping abnormalities across pathways related to neurodevelopment, neurotransmission, and immunity/cellular response to stressors; furthermore, these abnormalities are relevant for pathophysiology. My dissertation work encapsulates the development of methodologies and computational tools to analyze large “poly-omics” data sets, i.e., jointly analyzing genomic, epigenomic, and transcriptomic data to identify abnormalities gene expression regulation and molecular substrates that are common between and unique to SZ and BD. My work uncovered convergent evidence of dysregulation among genes, pathways, and regulatory molecules associated with SZ and BD. Major outcomes of this thesis may help to lay the groundwork for causal inference of the effect of genetic variants on cellular phenotypes, biological sub-typing of mental illness through gene expression profiling, and rational drug design.

Biosensor development continues to be driven by the growing need to accurately detect and monitor analytes with many biotechnology, clinical, agriculture, and military applications. With their well-established capacity for molecular recognition, proteins are the go-to choice of binding elements in many conventional sensor designs. Switchable proteins offer the potential of integrating analyte binding and signal transduction within a single molecule, thus reducing the need for complex and expensive detection equipment and opening the door to miniaturization and in vivo applications. The principal challenge is that the majority of natural binding proteins do not undergo a large-scale change in conformation upon target binding. This work describes two complementary protein design strategies for the rational conversion of ordinary binding proteins into ligand induced conformational switches for biosensing purposes. In the first approach, we applied the Alternate Frame Folding (AFF) mechanism to the human sulfiredoxin (hSrx) and the fibronectin (FN3) monobody scaffold towards the creation of an ATP biosensor and a customizable biosensor platform, respectively. In a second novel approach, the Protein Fragment Exchange (FREX) mechanism was demonstrated in a proof-of principle study that converts the FN3 scaffold into a biosensor, capable of genetic encoding and application in mammalian cells. While these designs were based on well established principles of protein folding and thermodynamics, the results obtained from these studies also offer important insights regarding protein sequence-structure-function relationships.

The structural maintenance of chromosome (SMC) proteins form the core of Cohesin, Condensin and the Smc5/6 complex, which are essential for organization and metabolism of chromosomes during the cell cycle. The Smc5/6 complex is implicated a role in non-essential homologous recombination-mediated (HR) DNA repair. Inactivating Smc5/6 (via temperature-sensitivity mutant) in the S phase, but not in the G2 phase, causes mitotic failure. Hence, we hypothesize that the Smc5/6 complex has an major role in DNA replication. We analyzed the genome-wide DNA replication temporal program in different genetic backgrounds. Mapping replication fork-associated ssDNA in WT and rad53 cells in the presence of replication stress allowed us to separate early and late replication origins. Using this method, we revealed a strain background difference of origin usage in A364a and W303. We then studied the genome-wide replication dynamics in a hypomorphic mutant, smc6-P4, using a density transfer coupled with microarray method. Overall replication dynamics of the mutant are similar to that of the WT cells with exceptions in the early S phase. However, we captured a difference in the replication profile of an early S phase sample in the mutant, prompting the hypothesis that the mutant incorporates ribonucleotides and/or accumulates single-stranded DNA gaps during replication. We then tested whether we can exacerbate the DNA replication stress hypersensitivity of the smc6 mutant by inhibiting ribonucleotides excision repair (RER) pathway. Contrary to our expectation, impairment of ribonucleotide excision repair, as well as virtually all other DNA repair pathways, alleviated smc6 mutant’s hypersensitivity to induced replication stress. We propose that nucleotide incision with impaired Smc5/6 complex has a more disastrous outcome than the damage itself. We found that the smc6 mutant has a significant increase in ssDNA level under replication stress but inhibiting RER pathway decreases it. This result suggests that Smc5/6 complex prevents unsupervised ssDNA formation during DNA replication as a result of damage incision repair. We reason that excess ssDNA in the Smc5/6 defective cells may subsequently engage in toxic recombination. Our study thus provides novel perspectives for the role of the Smc5/ 6 complex during DNA replication.

Humanaromatase(AROM) catalyzes theconversion of androgens to estrogensand is a major breast cancer drug target. Structural investigation has provided insights intothe active siteandaromatization mechanism.Utilization of the structural data has permitted rational design of a series of novel steroidal inhibitors. Investigation ofthe roles of key amino acids is facilitated by a recombinant AROM identical in crystal structureto the placental AROM.We use mutagenesis, chromatography, ultracentrifugation, spectrophotometry, enzyme kinetics, and X-ray crystallography to probe the roles of critical residues and the molecular basis of oligomerization. Furthermore, weevaluate the potencies of novel inhibitors and determine the structural basis of inhibition andselectivity. A critical active site residue D309 withan elevated pKa remainsprotonated at neutral pHand facilitatessubstrate binding and catalysis.The “gatekeeper” R192, linked to D309 via a watermolecule, is postulated to have a role in proton relay and substrate selectivity. D309N and R192Q mutants are virtually inactive supporting thehypothesis that both play keyrolesin aromatization.AROM oligomerization is driven bytheD-E loop of one moleculeand heme-proximal region of another via hydrogen bonding, electrostatic interactions between E181 and K440, and shape complementarity.Del7, generated by deletionof 7 residues in the D-E loop, experiences 65% reductionin activitydue to the loss of oligomer formation. Mutants Del4, E181A, and E181K exhibit normal enzymatic activity,and maintain some oligomeric interactions. The heme-proximal interface is also the putative coupling site of the reductasethatsupplieselectronsfor aromatization. The siteis larger than the active site, and at least twice aslarge asother P450s.MutantsK440Qand Y361Fof this region are virtuallyinactive.Collectivelythe results suggestfunctional significanceof oligomerization. Several newly designedAIs are superiortoexemestane, the steroidal AI currently used as a drug, in inhibition and anti-proliferation assays. The C6β-(pent-2-yn-1-yloxy) side chains ofthe most potent compoundspenetrate the access channelunique to AROM and havethe sameconformation asin the enzyme-free state.Astructural-based approachcan improve drug efficacy by improving specificity and selectivity, and reducing sideeffects.

Myeloproliferative neoplasms (MPNs) are a class of clonally-derived hematologic malignancies characterized by uncontrolled proliferation of myeloid lineage cells. Theyare classified into Philadelphiachromosome-positive (Ph+)MPNs, consisting of chronic myelogenous leukemia (CML), and Philadelphiachromosome-negative (Ph-)MPNs, consisting of polycythemia vera (PV), essential thrombocythemia (ET), primary myelofibrosis(PMF).The JAK2V617F mutation is the most common abnormality in Ph-MPNs, occurring in ~95% of PV patients, 55% of ET patients and 65% of PMF patients. JAK2V617F mutation results in constitutive activation of the JAK2 tyrosine kinase. Deletion of chromosome 20q (20q-) is a common chromosomal abnormality in myeloid neoplasms, including about 24%of MFcases. The 20q-lesion can coexist with JAK2V617Fmutation in MPN and MDS/MPN. The PTPN1gene is located on human chromosome 20q, within the commonly deleted region. PTPN1 is a tyrosine phosphatase and a known negative regulator of JAK-STAT signaling. The role of PTPN1 loss in the pathogenesis of MPNs and the mechanism by which loss of PTPN1 might contribute to various MPN phenotypes remains elusive. The goalsof this dissertation were to determine the effects of PTPN1 deficiency alone and in JAK2V617F-induced MPNs in vivo. To determine the mechanism by which PTPN1 mediates its tumor suppressor function using hematopoietic cells andassessment of PTPN1 status in 20q-MPN patients.Using conditional knock-out PTPN1 mouse model, we show that deletion of PTPN1 causes an MPN-like phenotype, characterized by increased WBC and NE counts and splenomegaly, compared to control mice. We also show that loss of PTPN1 causesfibrosis in older mice. PTPN1 knockdown significantly increased cell proliferation and activation of JAK2, MAPK and AKT signaling, whereas over expression in JAK2V617F-expressing cells attenuated cytokine-independent cell proliferation and signaling. Our data revealed a cooperative effect between PTPN1 deficiency and JAK2V617F expression in mice as shown by enhanced severity of theMPN phenotype and transformation to MF. Cell autonomous BMT revealed that the effects of PTPN1 deficiency are cell autonomous.Taken together, our results suggest a novel tumor suppressor function for PTPN1 in MPNs.

The response of memory B cells to challenge infection is fundamental to longterm protection against pathogens. Following challenge, memory B cells can rapidly differentiate into antibody-secreting cells (ASCs) to produce a secondary antibody response. Memory B cells have also been shown to re-enter into germinal centers and undergo additional rounds of affinity maturation. Both the isotype of the B cell and the signals that generated the B cell have been proposed to modulate how memory B cells respond. Initial studies proposed BCR-intrinsic factors are responsible for the differentiation of memory cells. IgM memory cells undergo differentiation in GCs following antigen challenge, while IgG memory cells rapidly differentiate into ASCs. Other studies found no link-between BCR isotype and differentiation. We investigated the differentiation of T-bet+ CD11c+ IgM memory B cells following challenge infection. IgM memory cells differentiated into IgM-producing plasmablasts. Other IgM memory B cells entered germinal centers, underwent class switching, and became switched memory cells. Yet other donor cells were maintained as IgM memory cells. The IgM memory cells also retained their multi-lineage potential following serial transfer. The kinetics of the IgM memory response mimicked the kinetics of the primary response. Thus, IgM memory cells can differentiate into all effector B cell lineages, and undergo self-renewal, properties that are characteristic of stem cells; however, differentiation occurs with the same kinetics of the primary response. We propose that memory B cells have varying degrees of stem cell likeness. IgM memory stem cells retain the most differentiating capacity but respond to challenge similarly to naïve cells, while IgG effector memory cells are primed to rapidly differentiate into IgG ASCs.

Eye formation begins with the specification of the eye field from the anteriorneural plate during the transition from gastrulation to neuralization. The morphogeneticgradients formed by BMP and Activin/Nodal ligands organize the body axis andregulate intracellular signals to bias the cell lineages.Therefore, to understand theextracellular signaling mechanisms governing eye field specification, we looked closerat the mechanisms responsible for neuralization. This project takes advantage of thedevelopmental accessibility of theXenopus laevisembryo to investigate the role ofBMP and Activin/Nodal signaling pathways during early eye development.Nogginis a secreted BMP antagonist and is sufficient to induce formation of ectopic retinaltissue. My results presented in Chapter 2 show that intracellular repression of BMP-Smad1/5/8 and Activin/Nodal-Smad2/3 signaling was sufficient to replicate the retina-promoting activity of Noggin and efficiently drive pluripotentXenopusprimitive ectodermto a retinal progenitor cell fate. Furthermore, my work presented in Chapter 3 suggeststhat repression of BMP and Activin/Nodal in the anterior neural plate allows the anteriorneural patterning transcription factor, Otx2, to directly activate expression of the earliesteye field transcription factor,tbx3. This study elucidates the initial step by which theanterior neural plate is patterned to specify the eye field. We next translated our findings to mouse embryonic stem cell culture (Chapter 4). We observed that mouse embryonic stemcells can be efficiently directed towards retinal progenitor cells by first converting themto an early primitive ectoderm-like state, followed by simultaneous repression of BMPand Activin/Nodal and activation of FGF signaling. After differentiation, we observedthat photoreceptor genesis is synchronized, resulting in 96% of aggregates expressingmarkers for cone photoreceptors after 9 days. This differentiation protocol is more efficientand quicker than previously published mouse stem cell protocols. Optimizing treatmentconditions resulted in up to 62% of cells expressing the cone/rod photoreceptor marker,CRX. This study was the first to show that photoreceptor-like cells can be generated frommouse pluripotent stem cells as efficiently as published human pluripotent stem cell reports,but with a shorter culture period. These studies will provide the basis of future work todetermine the underlying molecular mechanism driving retinal differentiation.

Sarcomeres are the most basic unit of muscle cells. Formins are actin regulatory proteins that are important for actin filament polymerization and nucleation, and might be responsible for the actin filamentassembly in sarcomeres. In Caenorhabditis elegans, two formins (CYK-1 and FHOD-1) were found in the body wall muscle (BWM), specifically in the Z-line of sarcomeres. Previously, BWM were analyzed in null cyk-1(ok2300) mutant worms, derived from heterozygous parents, and fhod-1(tm2363) mutantworms, andwere found to have smaller muscles than wild-type worms. Yet, there was still functional CYK-1 present in the worms due to inherited maternal CYK-1. To eliminate this potential source of CYK-1, cyk-1(or596ts) temperature sensitive mutant worms were used to allow for all CYK-1 to be non-functional at 26°C. The focus of this study was to understand the importance of cyk-1on muscle development in C. eleganslarvae. Wild-type, fhod-1(tm2363), cyk-1(or596ts), and fhod-1(tm2363);cyk-1(or596ts) double mutant wormsat L1 larval stage were observed for worm shape and muscle abnormalities. Abnormal worm shapes were observed in fhod-1, cyk-1, and more commonly in fhod-1;cyk-1mutant worms at permissive and restrictive temperatures. Abnormal muscle was observed in both permissive and restrictive temperatures forfhod-1, cyk-1, and fhod-1;cyk-1mutant worms that had abnormal worm shapes, while all worms that had normal body shape usually had normal muscle at both temperatures. Worms were alsotested to determine long-termeffects of cyk-1and fhod-1mutations on muscle development. Worms were held at permissive or restrictive temperatures for various times. Fhod-1and cyk-1mutant worms showed reduced muscle size compared to wild-type, while fhod-1;cyk-1mutant worms displayed a more severelyreduced muscle size. Next, cyk-1orfhod-1were tested to see whether they can rescue muscle cell size after mosaic expressionin a cyk-1(-) or fhod-1(-) mutant worm background. Fhod-1(+) muscle cells showeda significant increase in muscle cell size compared to fhod-1(-) cells, while cyk-1(+) muscle cells showeda non-significant increase in muscle size compared to cyk-1(+/-) cells. This shows cell-autonomousexpressionoffhod-1influencesmuscle growth, while cyk-1expression in another organ might influence muscle development.