Within the phosphatase field, identification of regulatory subunits and associated proteins has proven successful in determining the cellular role and potential substrates of phosphatases. This has been especially valuable for the PPP family of phosphatases due to its complex association with myriad of regulatory subunits, which dictate the activity, localization and substrate specificity of the phosphatases. To identify interactors of protein phosphatase 4 (PP4), I established a sensitive affinity purification coupled with mass spectrometry (AP-MS) approach to generate a high-density PP4 interaction network from the soluble fraction of human cell extracts. Our proteomic approach uncovered previously identified as well as new interactors; some may function as auxiliary regulators and others may serve as potential substrates. One of the interactor identified is a novel cytosolic PP4 regulatory subunit, which we termed PP4R4. PP4R4 displays weak homology to the PP2A A subunit of PP2Ac, but interacts specifically with PP4c and does not function as a scaffolding subunit to bridge other known regulatory subunits of PP4c. Remarkably, the PP4 interaction network revealed significant enrichment for proteins involved in transcription elongation and RNA processing. These interactors associate exclusively with PP4R3-PP4R2-PP4c holoenzyme. Consistent with this finding, PP4R3A possesses characteristics resembling that of splicing and transcription factors. We provided evidence suggesting that depletion of PP4c significantly reduced the transcription-elongation regulated genes, JUN and FOS, and altered the exon inclusion of selective genes. Our results define a high-density interaction network for the mammalian PP4 and uncover a potential role PP4 play in regulating the process of transcription elongation and alternative splicing.

Within the phosphatase field, identification of regulatory subunits and associated proteins has proven successful in determining the cellular role and potential substrates of phosphatases. This has been especially valuable for the PPP family of phosphatases due to its complex association with myriad of regulatory subunits, which dictate the activity, localization and substrate specificity of the phosphatases. To identify interactors of protein phosphatase 4 (PP4), I established a sensitive affinity purification coupled with mass spectrometry (AP-MS) approach to generate a high-density PP4 interaction network from the soluble fraction of human cell extracts. Our proteomic approach uncovered previously identified as well as new interactors; some may function as auxiliary regulators and others may serve as potential substrates. One of the interactor identified is a novel cytosolic PP4 regulatory subunit, which we termed PP4R4. PP4R4 displays weak homology to the PP2A A subunit of PP2Ac, but interacts specifically with PP4c and does not function as a scaffolding subunit to bridge other known regulatory subunits of PP4c. Remarkably, the PP4 interaction network revealed significant enrichment for proteins involved in transcription elongation and RNA processing. These interactors associate exclusively with PP4R3-PP4R2-PP4c holoenzyme. Consistent with this finding, PP4R3A possesses characteristics resembling that of splicing and transcription factors. We provided evidence suggesting that depletion of PP4c significantly reduced the transcription-elongation regulated genes, JUN and FOS, and altered the exon inclusion of selective genes. Our results define a high-density interaction network for the mammalian PP4 and uncover a potential role PP4 play in regulating the process of transcription elongation and alternative splicing.

Loss of the superficial zone of articular cartilage is an early change in osteoarthritis and with disease progression the deep zone (DZ) of cartilage shows progressive mineralization. To date, the mechanism(s) regulating post-natal articular cartilage mineralization is poorly understood. Previously, we have shown that inorganic polyphosphate inhibits mineralization of in vitro-formed DZ cartilage. We developed an indirect co-culture method to investigate the effect of superficial zone chondrocytes (SZC) on mineralization in DZ cartilage. Our findings suggest that SZC suppress mineralization by modulating polyphosphate levels in DZ cartilage via FGF-18. Furthermore, SZC promote glycosaminoglycan and collagen accumulation in the extracellular matrix of cartilage formed by DZ chondrocytes. This study provides insight into the interaction between chondrocyte subpopulations and possible mechanism(s) controlling post-natal articular cartilage mineralization. Moreover, the results of this study establish polyphosphate and FGF-18, separately or in combination, as therapeutic candidates for articular cartilage repair and osteoarthritis prevention.

Abstract The proglucagon‐derived peptides (PGDP) are expressed in islet alpha and gut enteroendocrine L cells. Although glucagon, glucagon‐like peptide‐1 (GLP‐1), and glucagon like peptide‐2 (GLP‐2) are derived from the same proglucagon gene, energy ingestion and nutrient assimilation represses proglucagon biosynthesis in the α‐cell, but stimulates the synthesis and secretion of GLP‐1 and GLP‐2 from the gut L cell. In the work presented in this thesis, I have identified novel G protein‐coupled receptors that stimulate GLP‐1 secretion and improve glucose homeostasis. G protein‐coupled receptor 119 (GPR119) is expressed in enteroendocrine cells and islets and is activated by nutrients (fatty acid derivatives) and small specific synthetic agonists. Activation of GPR119 enhances glucosestimulated insulin secretion from islet β‐cells and promotes incretin release from enteroendocrine cells in a cyclic AMP (cAMP)‐dependent manner. To determine the importance of gut hormones for the glucoregulatory actions of GPR119, I examined GPR119 activation in normal mice, isolated islets, and in mice with inactivation of gut hormone receptors. GPR119 activation directly stimulates insulin secretion from islets in vitro, yet requires intact incretin receptor signaling and enteral glucose exposure for optimal improvement of glucose tolerance in vivo. In contrast, activation of GPR119 inhibits gastric emptying independent of incretin receptors through GPR119‐dependent pathways. Another important feature of β‐cell GPCRs coupled to cAMP generation is their ability to protect the β‐cell from external injury. I have shown that mice lacking GPR119 (GPR119‐/‐) are more susceptible to streptozotocin (STZ)‐induced apoptosis while pharmacological activation of GPR119 failed to protect the β‐cell from STZ‐induced injury. Furthermore, GPR119‐/‐ mice iv display impaired incretin secretion and beta cell function when chronically fed a high fat (HF) diet. Conversely, abrogation of GPR119 signaling does not affect the beta‐cell adaptation (increased islet number and size) to the metabolic demand of high‐fat feeding. Mechanisms to increase β‐cell mass and function may be useful tools for the treatment of type 2 diabetes. GLP‐1 stimulates insulin biosynthesis, β‐cell proliferation and exerts antiapoptotic actions on β‐cells. To delineate novel mechanisms, important for the regulation of proglucagon gene expression and GLP‐1 secretion in the enteroendocrine L‐cell, I carried out a microarray‐based gene expression profiling and transcriptional networks analysis using RNA from murine gut GLUTag cells. To identify mechanisms unique to enteroendocrine L‐cells, I used the islet αTC1 cell line for comparative purposes. I identified a novel progesterone mediated signaling pathway involving activation of membrane GPCRs for the control of GLP‐1 secretion. In summary, these studies establish that GPR119 engages multiple complementary pathways for control of glucose homeostasis and suggest that endogenous GPR119 signaling plays a critical role in β‐cell adaptation to cytotoxic injury and nutrient excess. The studies provide evidence for a novel role for progesterone, regulating GLP‐1 secretion and controllingglucose homeostasis.

Abstract The proglucagon‐derived peptides (PGDP) are expressed in islet alpha and gut enteroendocrine L cells. Although glucagon, glucagon‐like peptide‐1 (GLP‐1), and glucagon like peptide‐2 (GLP‐2) are derived from the same proglucagon gene, energy ingestion and nutrient assimilation represses proglucagon biosynthesis in the α‐cell, but stimulates the synthesis and secretion of GLP‐1 and GLP‐2 from the gut L cell. In the work presented in this thesis, I have identified novel G protein‐coupled receptors that stimulate GLP‐1 secretion and improve glucose homeostasis. G protein‐coupled receptor 119 (GPR119) is expressed in enteroendocrine cells and islets and is activated by nutrients (fatty acid derivatives) and small specific synthetic agonists. Activation of GPR119 enhances glucosestimulated insulin secretion from islet β‐cells and promotes incretin release from enteroendocrine cells in a cyclic AMP (cAMP)‐dependent manner. To determine the importance of gut hormones for the glucoregulatory actions of GPR119, I examined GPR119 activation in normal mice, isolated islets, and in mice with inactivation of gut hormone receptors. GPR119 activation directly stimulates insulin secretion from islets in vitro, yet requires intact incretin receptor signaling and enteral glucose exposure for optimal improvement of glucose tolerance in vivo. In contrast, activation of GPR119 inhibits gastric emptying independent of incretin receptors through GPR119‐dependent pathways. Another important feature of β‐cell GPCRs coupled to cAMP generation is their ability to protect the β‐cell from external injury. I have shown that mice lacking GPR119 (GPR119‐/‐) are more susceptible to streptozotocin (STZ)‐induced apoptosis while pharmacological activation of GPR119 failed to protect the β‐cell from STZ‐induced injury. Furthermore, GPR119‐/‐ mice iv display impaired incretin secretion and beta cell function when chronically fed a high fat (HF) diet. Conversely, abrogation of GPR119 signaling does not affect the beta‐cell adaptation (increased islet number and size) to the metabolic demand of high‐fat feeding. Mechanisms to increase β‐cell mass and function may be useful tools for the treatment of type 2 diabetes. GLP‐1 stimulates insulin biosynthesis, β‐cell proliferation and exerts antiapoptotic actions on β‐cells. To delineate novel mechanisms, important for the regulation of proglucagon gene expression and GLP‐1 secretion in the enteroendocrine L‐cell, I carried out a microarray‐based gene expression profiling and transcriptional networks analysis using RNA from murine gut GLUTag cells. To identify mechanisms unique to enteroendocrine L‐cells, I used the islet αTC1 cell line for comparative purposes. I identified a novel progesterone mediated signaling pathway involving activation of membrane GPCRs for the control of GLP‐1 secretion. In summary, these studies establish that GPR119 engages multiple complementary pathways for control of glucose homeostasis and suggest that endogenous GPR119 signaling plays a critical role in β‐cell adaptation to cytotoxic injury and nutrient excess. The studies provide evidence for a novel role for progesterone, regulating GLP‐1 secretion and controllingglucose homeostasis.

Recent advances in the field of bionanotechnology have enabled researchers to design a variety of tools to detect, image and monitor biological process in cells. Despite this progress, the limited understanding of nanomaterial-cellular interactions has hindered the widespread use of these nanomaterials in biological systems. In this thesis, we examined the potential effects of metallic nanoparticle geometry on important cellular processes such as membrane trafficking, intracellular transport and subcellular signalling. We found that the size of nanoparticles plays an important role on their ability to interact with the cell surface receptors thus dictating their subsequent ability to activate intracellular signalling cascades. Interestingly, trafficking of these nanoparticles was dependent on their size due to biochemical and thermodynamical constraints. These findings suggest that nanomaterials actively interact with biological systems, thus, directly modulating vital cellular processes. In addition, by utilizing various physical and chemical properties of nanomaterials, we developed a novel class of hybrid nanoscaled carrier systems capable of delivering semiconductor quantum dots (QDs) into live cells without inducing membrane damage. Using biodegradable polymeric nanoparticles, bioconjugated QDs were encapsulated and delivered into trafficking vesicles of live cells. The environmentally sensitive surface charge of the polymeric nanoparticles exhibited positive zeta potential inside acidic endo-lysosomes, thus enabling their escape from the vesicular sequestration into the cytosol. Hydrolytic-induced degradation then releases the bioconjugate QDs for active labelling of subcellular structures for real-time studies. Unlike previously described intracellular QD delivery methods, the proposed system offers an efficient way to non-invasively deliver bioconjugated QDs without inducing cell damage, enabling researchers to accurately monitor cellular processes in real-time. The understanding of both physical and chemical properties of nanomaterials is crucial to the design of biocompatible nanosystems to study fundamental processes in biological systems. Here, we demonstrated that both the size and surface chemistry of nanoparticles can be modified to obtain desired biological responses. Future experimental efforts to study other physical and chemical properties could allow the development of more sophisticated and effective platforms for biological applications.

The Roles of Presenilin and FKBP14 in Drosophila Development and Notch Signalling; Diana L. van de Hoef, Department of Molecular Genetics, University of Toronto, 2008. The multimolecular gamma-secretase complex cleaves type 1 transmembrane proteins such as Notch and one of the genes targeted in Alzheimer’s disease known as APP. This complex comprises four components, known as anterior pharynx defective 1, presenilin enhancer 2, nicastrin and presenilin. Presenilin is an aspartyl protease that comprises the catalytic core of gamma-secretase, and mutated forms of presenilin cause early-onset familial Alzheimer’s disease. To further define the role of Drosophila Presenilin (Psn), I performed a genetic modifier screen to identify Psn-interacting genes. One of the genes that was identified, known as FKBP14, encodes a peptidyl-prolyl isomerase that may be involved in protein folding in the ER. I demonstrate that an immunosuppressant drug known as FK506, which binds FKBPs and abrogates their function, reduced Psn, anterior pharynx defective 1 and presenilin enhancer 2 protein levels in vivo. I also show that FKBP14 colocalized with anterior pharynx defective 1 and Psn in the ER, suggesting a role in gamma-secretase stability. Consistent with this, I demonstrate that FKBP14 binds with Psn and mediates Psn stability and Notch signalling in vivo. To further characterize the role of FKBP14 in development, I analyzed its expression pattern and phenotypes of an FKBP14 null mutant. I show that FKBP14 localized to embryonic hemocytes and larval tissues, in addition to being expressed in developing egg chambers. FKBP14 function is required during development, since FKBP14 null mutants are recessive lethal. These mutants exhibited defects in larval disc development that resulted in eye, wing and notum phenotypes reminiscent of Psn dominant-negative and Notch-dependent phenotypes. Furthermore, FKBP14 mutants displayed enhanced apoptosis in larval tissues, suggesting a possible involvement in apoptosis regulation. I then examined the effects of FKBP14 overexpression, and observed enhanced Psn protein levels in vivo. Interestingly, co-expression of FKBP14 and Psn resulted in synergistic bristle phenotypes, suggesting a role for FKBP14 function in the Notch signalling pathway. Consistent with this, FKBP14 mutants enhanced Notch loss-of-function phenotypes in the wing. Altogether, my data demonstrate an essential role for FKBP14 during development, particularly in Psn protein maintenance and Notch signalling.

The Epstein-Barr virus (EBV) infects over 90% of the human population and is associated with several human malignancies. The EBNA1 protein of EBV binds recognition sites in the latent origin of replication (oriP) and is important for the replication and segregation of EBV genomes in latently-infected cells. EBNA1 is also directly implicated in malignant transformation and immortalization of the host cell. EBNA1 does not have any known enzymatic activity and it employs cellular proteins to mediate its functions. One such protein is the ubiquitin specific protease, USP7, which is a key regulator of the p53 tumor suppressor. The aim of this thesis was to functionally characterize the interaction between EBNA1 and USP7. Here I show that USP7 promotes the DNA-binding activity of EBNA1 and is recruited along with an accessory protein, GMPS, to the oriP. The USP7-GMPS complex can deubiquitinate histone H2B and may enable epigenetic regulation of latent viral infection. Additionally, I present evidence for a direct role of EBNA1 in EBV-mediated carcinogenesis. EBNA1 prevents stabilization of p53 by USP7 and abrogates p53 activation by disrupting promyelocytic leukemia nuclear bodies (PML-NBs) that acetylate p53. This interferes with p53-activated gene expression and inhibits apoptosis. EBNA1-expressing cells also have impaired ability to repair DNA, but survive as well as or better than control cells. Thus EBNA1 creates a cellular environment conducive to transformation and immortalization. These studies have also allowed me to learn more about and expand on the known functions of USP7. I provide biochemical evidence suggesting that a P/A/ExxS motif is a preferred sequence for binding the USP7 N-terminal domain. Furthermore, I show USP7 is a negative regulator of PML proteins and PML-NBs and promotes p53 DNA-binding activity. Surprisingly, neither function required the deubiquitinase activity of USP7.

The Transforming Growth Factor beta (TGFb) superfamily of ligands are highly versatile, functioning throughout development and in adult organisms as diverse as worms and humans to regulate a myriad of biological activities. TGFb family members signal through their cognate serine/threonine kinase receptors to mediate the phosphorylation and activation of receptor-regulated Smads (R-Smads), that then complex with the common Smad (co-Smad/Smad4) to transduce TGFb signals from the membrane into the nucleus. This thesis recounts the first identification of a mammalian Smad-interacting transcription factor, Foxh1. Investigation of the Smad/Foxh1 DNA-binding complex, which mediates TGFb-dependent regulation of transcription from a Gsc enhancer, determined that both Smad and Foxh1 binding sites are required. These studies also defined the first known biological difference between the highly related R-Smads, Smad2 and Smad3. Specifically, it was shown that while both can similarly participate in Smad/Foxh1 DNA-binding complexes, Smad2 activates and Smad3 represses Foxh1-mediated TGFb-dependent transcription. A detailed analysis of the Gsc enhancer element subsequently defined the sequence req irements for a functional Smad/Foxh1 enhancer (SFE). This information was utilized to direct in silico, genome wide searches for genes harbouring evolutionarily conserved SFEs, which successfully expanded the repertoire of Smad/Foxh1 targets. Analysis of these targets revealed novel roles for Smad/Foxh1 signalling in forebrain development and retinoic acid production. Finally, the importance of Foxh1 to heart development was examined. The interaction between Foxh1 and the heart specific factor Nkx2-5 was characterized with respect to TGFb-dependent regulation of Mef2c expression via a compound Foxh/Nkx2-5 enhancer (FNE). Genome-wide searches for similar FNEs identified many potential Foxh1/Nkx2-5 targets, further analysis of which will provide greater insights into how Foxh1 functions in heart development. In summary, the work presented herein expands our understanding of the role of TGFb in development through the identification and characterization of Foxh1 and its genomic targets downstream of TGFb signalling. / PhD

In the budding yeast Saccharomyces cerevisiae, the G1-specific cyclin-dependent kinases (Cdks) Cln1-, Cln2-Cdc28 and Pcl1-, Pcl2-Pho85 are essential for ensuring that DNA replication and cell division are properly linked to cell polarity and bud morphogenesis. However, like most genes in S. cerevisiae, individual cyclin genes are not required for viability, and the phenotypes associated with deletion of any single cyclin gene tend to be subtle. My goal was to dissect the cellular roles of the G1 cyclins by systematically identifying their genetic interactions. To do this, I conducted Synthetic Genetic Array (SGA) screens using strains deleted for different combinations of cyclin genes. The results of screens with strains deleted for the G1 cyclin pairs, CLN1, CLN2, or PCL1, PCL2, confirmed a role for these cyclins in cell polarity regulation and identified novel G1 Cdk substrates, which I examined in more detail. One cell polarity regulator that showed an interesting pattern of genetic interactions with G1 cyclins was BNI1, which encodes a yeast formin protein. Overexpression of BNI1 caused an Synthetic Dosage Lethal interaction in the absence of both G1 cyclin pairs while its deletion caused synthetic lethality specifically in the absence of PCL1, PCL2. Consistent with these genetic interactions, phosphorylation of Bni1 was partially dependent on CLN1, CLN2. It has been proposed that Bni1 is regulated by intramolecular interactions. In an effort to discover how phosphorylation might affect Bni1 function, I developed assays to test for intramolecular interactions. In my experiments I found no evidence that Bni1 is regulated by intramolecular binding, as was proposed from parallels with its mouse homolog mDia1. I also found that deletion of BNI4, which encodes an adaptor protein that targets several proteins to the bud neck, results in severe growth defects in the absence of the Cdc28 cyclins Cln1 and Cln2, and overexpression of BNI4 was toxic in yeast cells lacking the Pho85 cyclins Pcl1 and Pcl2. I discovered that Bni4 was phosphorylated by Pcl1- and Pcl2-Pho85 in vitro and that phosphorylation of Bni4 was dependent on PCL1 and PCL2 in vivo. Further analysis showed that phosphorylation of Bni4 by Pcl-Pho85 is necessary for its localization to bud neck, and the bud neck structure can be disrupted by overexpressing BNI4 in pcl1pcl2 mutant cells. I propose that if Bni4 cannot be regulated by phosphorylation, it may titrate away an essential component that resides at the bud neck, thus causing catastrophic morphogenesis defects. The relationship between G1 Cdk activity and the polarity regulator Bni4 serves as a bridge to link the cell cycle machine to the regulation of cell.