No description available.

<p>After finishing a human genome reference sequence in 2002, the genomics community has</p><p>turned to the task of interpreting it. A primary focus is to identify and characterize not only</p><p>protein-coding genes, but all functional elements in the genome. The effort has identified</p><p>millions of regulatory elements across species and in hundreds of human cell-types. Nearly</p><p>all identified regulatory elements are found in non-coding DNA, hypothesizing a function</p><p>for previously unannotated sequence. The ability to identify regulatory DNA genome-wide</p><p>provides a new opportunity to understand gene regulation and to ask fundamental questions</p><p>in diverse areas of biology.</p><p>One such area is the aim to understand the molecular basis for phenotypic differences</p><p>between humans and other primates. These phenotypic differences are partially driven</p><p>by mutations in non-coding regulatory DNA that alter gene expression. This hypothesis</p><p>has been supported by differential gene expression analyses in general, but we have not</p><p>yet identified specific regulatory variants responsible for differences in transcription and</p><p>phenotype. I have worked to identify regulatory differences in the same cell-type isolated</p><p>from human, chimpanzee, and macaque. Most regulatory elements were conserved among</p><p>all three species, as expected based on their central role in regulating transcription. How-</p><p>ever, several hundred regulatory elements were gained or lost on the lineages leading to</p><p>modern human and chimpanzee. Species-specific regulatory elements are enriched near</p><p>differentially expressed genes, are positively correlated with increased transcription, show</p><p>evidence of branch-specific positive selection, and overlap with active chromatin marks.</p><p>ivSpecies-specific sequence differences in transcription factor motifs found within this regu-</p><p>latory DNA are linked with species-specific changes in chromatin accessibility. Together,</p><p>these indicate that species-specific regulatory elements contribute to transcriptional and</p><p>phenotypic differences among primate species.</p><p>Another fundamental function of regulatory elements is to define different cell-types in</p><p>multicellular organisms. Regulatory elements recruit transcription factors that modulate</p><p>gene expression distinctly across cell-types. In a study of 112 human cell-types, I classified</p><p>regulatory elements into clusters based on regulatory signal tissue specificity. I then used</p><p>these to uncover distinct associations between regulatory elements and promoters, CpG-</p><p>islands, conserved elements, and transcription factor motif enrichment. Motif analysis</p><p>identified known and novel transcription factor binding motifs in cell-type-specific and</p><p>ubiquitous regulatory elements. I also developed a classifier that accurately predicts cell-</p><p>type lineage based on only 43 regulatory elements and evaluated the tissue of origin for</p><p>cancer cell-types. By correlating regulatory signal and gene expression, I predicted target</p><p>genes for more than 500k regulatory elements. Finally, I introduced a web resource to</p><p>enable researchers to explore these regulatory patterns and better understand how expression</p><p>is modulated within and across human cell-types.</p><p>Regulation of gene expression is fundamental to life. This dissertation uses identified</p><p>regulatory DNA to better understand regulatory systems. In the context of either evolution-</p><p>ary or developmental biology, understanding how differences in regulatory DNA contribute</p><p>to phenotype will be central to completely understanding human biology.</p> / Dissertation

During vertebrate oocyte maturation, the chromosomes progress to and arrest at metaphase of meiosis II in preparation for fertilization. This process includes emission of the first polar body. The second polar body is emitted after fertilization. A number of proteins are accumulated during oocyte maturation. Inhibition of this de novo translation does not appear to affect the progression of meiosis during oocyte maturation. The role of these pools of proteins has yet to be elucidated. Curiously, several of the upregulated proteins are key players in mitosis, including INCENP, a subunit of the chromosome passenger complex implicated in chromosome segregation and cytokinesis. During early stages of development in Xenopus laevis, the embryo cycles through mitosis, also known as embryo cleavage, every 30min with little to no time for transcription/translation. Our goal is to determine if the de novo translation of these mitotic proteins during oocyte maturation has a role in early embryogenesis. We used morpholino oligonucleotides antisense to INCENP mRNA (INCENPmorpho) to inhibit de novo translation during oocyte maturation. Using confocal imaging and the host transfer technique, these injected oocytes were matured, fertilized and assessed for developmental competency. INCENPmorpho and a control morpholino (ctrlmorpho) had no discernable effect on 1st or 2nd polar body emission. Whereas ctrlmorpho embryos developed normally, INCENPmorpho embryos did not cleave. Thus, de novo translation of INCENP during oocyte maturation is necessary for embryogenesis. Specifically, accumulation of INCENP and other mitotic proteins during oocyte maturation may be a common strategy in this species to prepare for the rapid and synchronous mitoses during early embryogenesis.

Understanding the specific mechanisms regulating chemotherapeutic drug anti-cancer activities will uncover novel strategies to enhance the efficacy of these drugs in clinical settings. Activating Transcription Factor 3 (ATF3) is a stress inducible gene whose expression has been associated with survival outcomes in cancer models. This study characterizes the chemotherapeutic drugs, cisplatin and Histone Deacetylase Inhibitor (HDACi), M344 as novel inducers of ATF3 expression. Cisplatin is a DNA damaging agent widely used in various tumour types including lung, head and neck, and ovarian carcinomas. The HDAC inhibitor, SAHA, has recently been approved as a single agent in the treatment of subcutaneous T-cell lymphoma and HDACis themselves show potential for synergistic anti-cancer effects when used in combination with established chemotherapeutic drugs, including cisplatin. This study evaluates the mechanisms by which cisplatin and HDACi induce ATF3, as well as the role ATF3 plays as a mediator of cisplatin-induced cytotoxicity and the enhanced cytotoxicity between HDACi and cisplatin in combination. In this study, we demonstrate that cytotoxic doses of cisplatin and carboplatin consistently induced ATF3 expression in a panel of human tumour derived cell lines. Characterization of this induction revealed a p53, BRCA1, and integrated stress response (ISR) independent mechanism, all previously implicated in stress mediated ATF3 induction. Analysis of MAPKinase pathway involvement in ATF3 induction by cisplatin revealed a MAPKinase dependent mechanism. Cisplatin treatment, in combination with specific inhibitors to each MAPKinase pathway (JNK, ERK and p38) resulted in decreased ATF3 induction at the protein level. MAPKinase pathway inhibition led to decreased ATF3 mRNA expression and a reduction in the cytotoxic effects of cisplatin as measured by MTT cell viability assay. In A549 lung carcinoma cells, targeting ATF3 with specific shRNAs also attenuated the cytotoxic effects of cisplatin. Similarly, ATF3 -/- MEFs were shown to be less sensitive to cisplatin induced cytotoxicity as compared with ATF3+/+ MEFs. Taken together, we identified cisplatin as a MAPKinase pathway dependent inducer of ATF3 whose expression regulates in part cisplatin’s cytotoxic effects. Furthermore, we demonstrated that the HDAC inhibitor M344 was also an inducer of ATF3 expression at the protein and mRNA level in the same human derived cancer cell lines. Combination treatment with M344 and cisplatin lead to increased induction of ATF3 compared with cisplatin alone. Utilizing the MTT cell viability assay, M344 treatment was also shown to enhance the cytotoxic effects of cisplatin in these cancer cell lines. Unlike cisplatin, the mechanism of ATF3 induction by M344 was found to be independent of MAPKinase pathways. Utilizing ATF4 heterozygote (+/-) and knock out (-/-) mouse embryonic fibroblast (MEF) M334 induction of ATF3 was shown to depend on the presence of ATF4, a known regulator of ATF3 expression as part of the ISR pathway. HDACi treatment did not affect the level of histone acetylation associated with the ATF3 promoter as determined through Chromatin immunoprecipitation (ChIP) analysis, suggesting that ATF3 induction was not a direct effect of HDACi mediated histone acetylation. We also demonstrated that ATF3 regulates the enhanced cytotoxicity of M344 in combination with cisplatin as evidenced by attenuation of cytotoxicity in shRNAs targeting ATF3 expressing cells. This study identifies the pro-apoptotic factor, ATF3 as a novel target of M344, as well as a mediator of the co-operative effects of cisplatin and M344 induced tumour cell cytotoxicity.

The inhibitor of apoptosis (IAP) proteins traditionally regulate programmed cell death by binding to and inhibiting caspases. Recent studies have uncovered a variety of alternate cellular roles for several IAP family members. The cellular inhibitor of apoptosis 1 (cIAP1) protein, for instance, regulates different axes of the NF-κB signalling pathway. Given the extensive functions of NF-κB signalling in muscle differentiation and regeneration, I asked if cIAP1 also plays critical roles in skeletal muscle myogenesis. In a primary myoblast cell-culture system, genetic and pharmacological approaches revealed that loss of cIAP1 dramatically increases the fusion of myoblasts into myotubes. NF-κB signalling occurs along a classical and an alternative pathway, both of which are highly active in cIAP1-/- myoblasts. Suppression of the alternative pathway attenuates myotube fusion in wildtype and cIAP1-/- myoblasts. Conversely, constitutive activation of the alternative pathway increases myoblast fusion in wildtype myoblasts. cIAP1-/- mice have greater muscle weight and size than wildtypes, as well as an increased number of muscle stem cells. These results identify cIAP1 as a regulator of myogenesis through its modulation of classical and alternative NF-κB signalling pathways. Loss of the structural protein dystrophin in the mdx mouse model of Duchenne muscular dystrophy leads to chronic degeneration of skeletal muscle. The muscle pathology is strongly influenced by NF-κB signaling. Given the roles demonstrated for cIAP1 in cell culture and in vivo, I asked whether loss of cIAP1 would influence muscle pathology in the mdx mouse. To address this question, double-mutant mice were bred lacking both cIAP1 and dystrophin (cIAP1-/-;mdx). Histological analyses revealed that double-mutant mice exhibited reduced indications of damage on several measures, as compared to single-mutant (cIAP1+/+;mdx) controls. Unexpectedly, these reductions were seen in the “slow-twitch” soleus muscle but not in the “fast-twitch” extensor digitorum longus (EDL) muscle. The improvements in pathology of double-mutant solei were associated with reductions in muscle infiltration by CD68-expressing macrophages. Finally, the double-mutant mice exhibited improved endurance and resistance to damage during treadmill-running exercise. Taken together, these results suggest that loss of cIAP1, through its multiple regulatory functions, acts to improve myogenesis and increase muscle resistance to damage.

Platinum-based chemotherapies are effective agents in the treatment of a wide variety of human cancers. However, patients with recurrent disease can become resistant to platinum-based chemotherapy, leading to low overall survival rates. Activating transcription factor 3 (ATF3) is a stress-inducible gene that is a regulator of cisplatin-induced cytotoxicity. ATF3 protein expression was upregulated after cytotoxic doses of cisplatin treatment in a panel of cell lines. A chromatin immunoprecipitation assay showed that upon treatment with cisplatin, ATF3 directly bound to the CHOP gene promoter and this correlated with an increase in CHOP protein expression. In a 1200 compound library screen performed on cancer cell lines, disulfiram, a dithiocarbamate drug, was identified as an enhancer of the cytotoxic effects of cisplatin. This increased cytotoxic action was likely due to disulfiram and cisplatin’s ability to induce ATF3 independently through two separate mechanisms, namely the MAPK and integrated stress pathways. Furthermore, ATF3 protein and mRNA levels were variable amongst human ovarian and lung cancer tissues, suggesting the potential for basal expression of ATF3 to be predictive of cisplatin treatment response. Thus, understanding ATF3’s role in cisplatin-induced cytotoxicity will lead to novel therapeutic approaches that could improve this drug’s efficacy.

Background: Escherichia coli (E.coli) O86a:K61 belongs to the Enteropathogenic E. coli (EPEC) group of pathogens. Acute gastroenteritis affects 2-4 billion people annually and EPEC is associated with 10-40% of hospitalized diarrhea cases globally. Coagulation Factor (F) V circulates as an inactive procofactor (Mr 330kDa) which upon thrombin activation to the active cofactor, FVa, functions in prothombinase to accelerate prothrombin to thrombin conversion by 300,000-fold. The ability of E.coli O86a:K61 to cause intestinal hemorrhage is of interest because previous research demonstrated that during E.coli O86a:K61 sepsis in baboons, a dose-dependent inactivation of FV was observed as the bacterial dose increased. These results suggested a secreted E.coli protease may have mediated this effect on FV. This research has focused on the purification, identification, and characterization of a secreted E. coli O86a:K61 protease that inactivates FV. The final partially-purified protease inactivated FV to a 250kDa product by immunoblotting, and possessed a 900-fold increase in specific activity versus FV in human plasma compared to the culture supernatant. At least 3 proteins were observed upon SDS-PAGE. Proteolytic inactivation of FV was activated by up to 500-fold with β-mercaptoethanol and 2-fold with 1M urea. The protease was heat stable retaining all of its activity versus FV after 1h at 70°C or 80°C, and partial activity (50%) at 95°C. Proteolysis of FV was blocked by 90% with alpha-1-protease inhibitor; however, the protease was resistant to 1.5 mM PMSF, and unaffected by E64, or iodoacetamide. FV is a major regulator of the coagulation process and its inactivation by the secreted E.coli protease would be expected to result in a net bleeding tendency which may contribute to the mucosal hemorrhage observed in humans with associated hemorrhagic colitis. Proteolytic inactivation of FV is predicted to result in decreased bacterial containment by host fibrin thereby increasing pathogen survival and growth. FV inactivation by the secreted E.coli protease may be part of a novel pathogenic virulence mechanism that deregulates the blood coagulation process to enhance bacterial infectivity and transmission. / UOIT

The receptor activator of nuclear factor-kB (RANK) and its ligand, RANKL has have been implicated as an important link between breast cancer and metastasis to bone because of their ability to activate intracellular signal cascades leading to altered cancer cell behaviour and bone breakdown. The JAK/STAT5a cell signaling pathway is also crucial to breast biology and is involved in transcriptional regulation of many genes. The objective of this study is to determine if RANKL mRNA expression is regulated through the JAK/STAT5a pathway by stimulating human breast cancer cell lines, MCF-7 and MDA-MB-231, with prolactin (PRL), epidermal growth factor (EGF) and heregulin-beta1 (HRG-1), all known to activate STAT5a and play a role in breast cancer progression. This study shows that RANKL expression is upregulated by PRL, EGF and HRG-1, and that PRL and HRG-1 regulate transcription through the JAK/STAT5a pathway. / UOIT

No description available.

Modern communication systems require high performance radio frequency (RF) and microwave circuits and devices. This is becoming increasingly challenging to realize in the content of cost/size constraints. Integrated circuits (ICs) satisfy the cost/size requirement, but performance is often sacri¯ced. For instance, high quality factor (Q factor) passive components are difficult to achieve in standard silicon-based IC processes.<p>In recent years, microelectromechanical systems (MEMS) devices have been receiving increasing attention as a possible replacement for various on-chip passive elements, offering potential improvement in performance while maintaining high levels of integration. Variable capacitors (varactor) are common elements used in various applications. One of the MEMS variable capacitors that has been recently developed is built using deep X-ray lithography (as part of the LIGA process). This type of capacitor exhibits high quality factor at microwave frequencies.<p>The complementary metal oxide semiconductor (CMOS) technology dominates the silicon IC process. CMOS becomes increasingly popular for RF applications due to its advantages in level of integration, cost and power consumption. This research demonstrates a CMOS voltage-controlled oscillator (VCO) design which is used to investigate methods, advantages and problems in integrating LIGA-MEMS devices to CMOS RF circuits, and to evaluate the performance of the LIGA-MEMS variable capacitor in comparison with the conventional on-chip CMOS varactor. The VCO was designed and fabricated using TSMC 0.18 micron CMOS technology. The core of the VCO, including transistors, resistors, and on-chip inductors was designed to connect to either an on-chip CMOS varactor or an off-chip LIGA-MEMS capacitor to oscillate between 2.6 GHz and 2.7 GHz. Oscillator phase noise analysis is used to compare the performance between the two capacitors. The fabricated VCO occupied an area of 1 mm^2.<p>This initial attempt at VCO fabrication did not produce a functional VCO, so the performance of the capacitors with the fabricated VCO could not be tested. However, the simulation results show that with this LIGA-MEMS capacitor, a 6.4 dB of phase noise improvement at 300 kHz offset from the carrier is possible in a CMOS-based VCO design.