<p> Remodeling of the mouse mammary gland is a highly coordinated process that occurs after the removal of suckling pups from the mother. Involution, or shrinking of the mammary gland, after removal of the pups has been linked to apoptotic events within the mouse mammary tissue during forced weaning. Several transcription factors are hypothesized to be involved in this process. A transcription factor known as GATA-3, which was first identified in the thymus, is also important for maintenance of various tissue types within the mouse mammary gland; its loss leads to epithelial cell detachment and eventual death. Another transcription factor known as fetal zinc liver finger protein 1, or Fliz1, has been found to regulate GATA-3 in T-cells. This interaction had not been elucidated during involution in mouse mammary tissue. I hypothesized that Fliz1 is expressed at heightened levels during mouse mammary gland involution following forced weaning of pups, and that this expression correlates with a decrease in GATA-3 levels, with increased expression of the pro-apoptotic protein BAD. Using qRT-PCR, immunoblotting and immunohistochemistry I have shown that Fliz1 is indeed expressed in involuting mouse mammary gland tissue as well as several other tissue types. However, levels of Fliz1 remain fairly constant during involution. The findings also show that Cathepsin L, a known apoptotic marker for mammary gland involution, is substantially up-regulated during the process of mammary gland involution in the mouse. The study also revealed that GATA-3 levels as hypothesized decrease substantially during the process of mouse mammary gland involution, indicating that GATA-3 is required for maintenance of the mouse mammary gland.</p>

<p> In the presence of arachidonic acid (AA), <i>Saccharomyces cerevisiae </i> produces prostaglandin E₂ (PGE₂). <i> S. cerevisiae</i> and its metabolites may be consumed in products manufactured using the yeast (e.g. beer). Neutrophils are immune cells present in the gastrointestinal (GI) tract during inflammation. As a lipid-signaling molecule, PGE₂ can potentially modify neutrophil functions and exacerbate pre-existing inflammation. As neutrophil migration is a hallmark of inflammation, we investigated the impact of PGE₂ on neutrophil chemotaxis. Chemotaxis assays were performed on neutrophils isolated from human whole blood using the chemotactic agents f-Met-Leu-Phe (fMLP) or interleukin-8 (IL-8). Neutrophil chemotaxis was concentration dependent as it was enhanced 3.5-fold at low concentrations of PGE₂ (0.1 nM-10 nM) and reduced 3.0-fold at higher concentrations of PGE₂ (100 nM).</p><p> The biochemical pathway utilized by <i>S. cerevisiae</i> to produce PGE₂ is unknown. Identifying enzymes that metabolize AA may direct approaches to reduce the impact that yeast PGE₂ may have on neutrophils. <i>S. cerevisiae</i> does not have genes homologous to those involved in mammalian AA metabolism. We employed RNAseq transcriptome sequencing to study the lipid biosynthetic pathway in <i>S. cerevisiae </i> and observed 1248 genes upregulated in yeast that were cultured in the presence of AA relative to yeast that were cultured without AA. Notably, genes that mediate beta-oxidation of fatty acids (<i>Pot1, Pox1, Faa1 and Faa2</i>) were upregulated up to 2.3-fold.</p><p> The results demonstrate that low concentrations of PGE₂ enhance neutrophil chemotaxis that is mediated by fMLP or IL-8, suggesting that PGE₂ may aid in recruiting neutrophils from regions that are distant to a site of inflammation. Once a higher concentration of PGE₂ is encountered by neutrophils, neutrophils may halt their migration and engage effector functions such as phagocytosis and superoxide production. Increased expression of genes involved with fatty acid metabolism points to enzymes that may utilize AA to produce PGE₂ in <i>S. cerevisiae</i>. Experiments testing PGE₂ levels in knock-out strains of yeast will identify genes involved in PGE₂ production. Results of this study have implications to reduce potential off-target effects caused by yeast PGE₂ in consumables.</p>

<p> Small non-coding RNAs (sRNAs) are being increasingly recognized as critical regulators of a wide variety of processes in bacteria. To investigate the contribution of unknown sRNAs to virulence gene regulation in <i>Vibrio cholerae,</i> we undertook a screen to identify previously uncharacterized sRNAs under the control of the major virulence gene activator in <i> V. cholerae,</i> ToxT. Using a combination of direct sRNA cloning and sequencing together with a genome-wide ToxT <i>in vitro</i> binding assay, we identified 18 putative ToxT-regulated sRNAs. Two of these ToxT regulated sRNAs were located within the <i>Vibrio</i> Pathogenicity Island-1 (VPI-1), the genetic element that encodes ToxT and the Toxin Co-regulated Pilus (TCP). We verified regulation of these sRNAs by ToxT and showed that deletion of one of them, now designated <i>tarB</i>, caused a variable colonization phenotype when competed against the parental strain in an infant mouse model of <i>V. cholerae</i> infection. Infections progressing for 18 hours or less showed the &Delta;<i>tarB</i> strain was out-competed by the wild type strain, while those carried out longer, showed &Delta;<i> tarB</i> out-competing the wild type. Additionally, if inoculated from a resource poor environment the &Delta;<i>tarB</i> strain also showed decreased colonization relative to wild type. Using a bioinformatic approach, we identified that <i>tarB</i>-mediated regulation of the gene <i> tcpF</i> was primarily responsible for the <i>tarB</i> mutant's <i> in vivo</i> colonization phenotype. Further investigation of genes regulated by <i>tarB</i> using genome-wide transcriptional profiling of a <i> tarB</i> over-expressing strain revealed that <i>tarB</i> also directly regulates genes involved in iron and amino acid uptake. We determined that <i>tarB</i> has a repressive effect on many genes within the VPI-1, but has an activating effect on <i>tcpP/tcpH,</i> encoding regulators upstream of ToxT. Taken together, the data suggest that <i> tarB</i> plays an important role in regulating virulence and metabolic genes early after <i>V. cholerae</i> infection, but that this repressive effect on virulence genes later in infection may lead to reduced replication <i> in vivo.</i></p>

Previously, we have demonstrated that a DNA helicase-like protein, termed RTEL (regulator of telomere length) is essential for the maintenance of genomic stability. RTEL deficiency induced telomere loss and genomic instability, leading to embryonic lethality. However, the role of RTEL in these biological pathways is largely unknown. To uncover RTEL’s function(s), we applied several approaches to identify the proteins that could interact with RTEL. Proliferating Cell Nuclear Antigen (PCNA), the key regulator of the replication fork, was found to be a strong candidate. In this study, we have demonstrated the interaction between RTEL and PCNA. Further characterization of the interaction between RTEL and PCNA revealed that the interaction is important for maintaining genomic stability. Due to the essential role of PCNA in nucleic acid metabolism as a component of the replication and repair machinery, its interaction with RTEL could be the key to the role of RTEL in the maintenance of genomic stability and mouse development. Along with a bioinformatics approach, we have employed several biochemical approaches to identify the interaction of PCNA with RTEL. Using co-immunoprecipitation, we have demonstrated that RTEL can specifically interact with PCNA. A PCR-based mutagenesis method was used to mutate the PCNA-interacting motif (PIP) in RTEL. Further we have demonstrated that several key amino acids in the PIP motif are responsible for mediating RTEL/PCNA interaction by using co-immunoprecipitation and immunofluorescence studies. Using a gene-targeting approach, we have specifically knocked-in a mutant RTEL with a mutation in PIP motif into mouse genome. Thus we have developed a transgenic mouse model to study the significance of the interaction between RTEL/PCNA in vivo. This study not only validated the interaction of RTEL with PCNA, via the PIP box, but also generated the RTEL PIP mutant alleles for further functional analysis by transgenic approaches. We have employed biochemical and cytogenetic studies to characterize the phenotypes in RtelI1169A/I1169A mouse. This is the first direct genetic approach to address whether PCNA is an important downstream mediator of RTEL’s function in the regulation of genomic integrity

<p> Blast injury-induced neurotrauma (BINT) is steadily increasing in prevalence due to escalated terror activity and constitutes the signature injury associated with current military conflicts. BINT produces significant neurological deficiencies and there is a growing concern that the injury may produce long-term consequences that affect the resilience and the performance of soldiers. One of the potential consequences is an increased susceptibility to Parkinson's disease (PD). A vital goal aimed at curtailing the post-deployment long-term consequences of blast injury-induced neurotrauma is to further our knowledge of pathogenic mechanisms responsible for the escalation of post injury diseases. <i> The purpose of this project is to investigate the molecular mechanism underlying the susceptibility of PD in post-blast rats.</i> We have identified acrolein, a highly reactive aldehyde that persists days to weeks following brain-injury and perpetuates oxidative insult, as a potential therapeutic target to curtail chemically mediated damage, a common feature of BINT and PD. <b>Our hypothesis is that acrolein is a key pathological factor linking BINT and the development of PD in our rat model.</b></p>

<p> Doc induces cell death by inhibiting translation; however, the mechanism of Doc-induced cell death and the cellular target of the toxin were unknown. One theory suggested that Doc inhibits translation elongation by binding directly to the 30S ribosomal subunit. Later evidence showed catalytic activity in distant homologs of Doc. These homologs contain a Fic-domain that has been shown to modify target GTPases by AMPylation and phosphocholination. Therefore, [³⁵S] &ndash; Met, &alpha;[³²P] &ndash; ATP, and &gamma;[³²P] &ndash; ATP were used in conjunction with an S30 extract to confirm that Doc inhibits translation, to assess the mechanism of modification, and to identify the modified target. The results showed that Doc is an enzyme that inhibits translation and phosphorylates a protein target.</p>

The Hippo pathway plays a key role in controlling organ growth and size. In mammals, core pathway components include the Lats1/2 and Mst1/2 kinases, which phosphorylate the transcriptional regulators, Taz and Yap. To identify novel upstream pathway regulators high throughput protein-protein interaction screens, called LUMIER (LUminescence-based Mammalian IntERactome) were performed together with a functional screen using a luciferase reporter that examines Hippo pathway responses. The screens revealed 1103 protein-protein interactions and 227 transcriptional regulators, which were particularly enriched in cytoskeletal regulators. A subset of these hits including BTK, Dvl1, Dvl2, Dvl3, Ing2, Magi2, Mark4 and Trip6 were validated by manual LUMIER assays and co-immunoprecipitation (Co-IP). Of particular interest was the microtubule dynamics regulatory protein MARK4. Loss of Mark4 prevents Taz activity demonstrating its role as a potential negative regulator of the Hippo pathway. Further studies could help decipher mechanisms of how Mark4 and the other cytoskeletal hits act to modulate the Hippo pathway.

Myeloproliferative neoplasms (MPNs) are a group of disorders characterized by an excess production of a specific, fully functional blood cell type. Many cases involve deregulation of a protein tyrosine kinase. JAK2 is one such kinase, involved in a subset of MPNs. JAK2-selective inhibitors are currently being evaluated in clinical trials. In order to identify inhibitor-resistant JAK2 mutations before they appear in the clinic, we utilized TEL-JAK2 to conduct an in vitro random mutagenesis screen for JAK2 alleles resistant to JAK Inhibitor-I. Isolated mutations were evaluated for their ability to sustain cellular growth, stimulate downstream signalling pathways, and phosphorylate a novel JAK2 substrate in the presence of inhibitor. When testing the panel of mutations in the context of the Jak2 V617F allele, we observed that a subset of mutations conferred resistance to inhibitor. These results demonstrate that small-molecule inhibitors select for JAK2 inhibitor-resistant alleles. Chronic myeloid leukemia is an MPN characterized by the presence of the BCR-ABL fusion gene. We determined that a specific cohort bearing deletions near the ABL gene, which is associated with poor prognosis, do not suffer from genomic instability. We also examined the role of a putative tumour suppressor gene EXOSC2 as an explanation for the reduced survival time, and suggest it may have a role in disease progression.

The Hippo pathway plays a key role in controlling organ growth and size. In mammals, core pathway components include the Lats1/2 and Mst1/2 kinases, which phosphorylate the transcriptional regulators, Taz and Yap. To identify novel upstream pathway regulators high throughput protein-protein interaction screens, called LUMIER (LUminescence-based Mammalian IntERactome) were performed together with a functional screen using a luciferase reporter that examines Hippo pathway responses. The screens revealed 1103 protein-protein interactions and 227 transcriptional regulators, which were particularly enriched in cytoskeletal regulators. A subset of these hits including BTK, Dvl1, Dvl2, Dvl3, Ing2, Magi2, Mark4 and Trip6 were validated by manual LUMIER assays and co-immunoprecipitation (Co-IP). Of particular interest was the microtubule dynamics regulatory protein MARK4. Loss of Mark4 prevents Taz activity demonstrating its role as a potential negative regulator of the Hippo pathway. Further studies could help decipher mechanisms of how Mark4 and the other cytoskeletal hits act to modulate the Hippo pathway.

<p>Saccharomyces cerevisiae cells exposed to a variety of physiological stresses transiently delay bud emergence or bud growth. To maintain coordination between bud formation and the cell cycle in such circumstances, the morphogenesis checkpoint delays nuclear division via the mitosis-inhibitory Wee1-family kinase, Swe1p. Swe1p is degraded during G2 in unstressed cells, but is stabilized and accumulates following stress. Degradation of Swe1p is preceded by its recruitment to the septin scaffold at the mother-bud neck, mediated by the Swe1p-binding protein Hsl7p. Following osmotic shock or actin depolymerization, Swe1p is stabilized, and previous studies suggested that this was because Hsl7p was no longer recruited to the septin scaffold following stress. However, we now show that Hsl7p is in fact recruited to the septin scaffold in stressed cells. Using a CDK mutant that is immune to checkpoint-mediated inhibition, we show that Swe1p stabilization following stress is an indirect effect of CDK inhibition. These findings demonstrate the physiological importance of a positive feedback loop in which Swe1p activity inhibits the CDK, which then ceases to target Swe1p for degradation. They also highlight the difficulty in disentangling direct checkpoint pathways from the effects of positive feedback loops active at the G2/M transition.</p> / Dissertation