<p> Arteriogenesis, the expansion of collateral arteries, is vital process for compensatory blood flow to tissues in response to vascular occlusions. In patients with peripheral arterial disease (PAD), arteriogenesis is crucial for overcoming limb ischemia, but for many, further treatment is required. In the United States alone, approximately 80,000 individuals lose limbs to this disease each year. Thus, it is critical to understand the mechanisms regulating arteriogenesis. Indirect evidence suggests that mitogen-activated protein kinase kinases 1 and 2 (MEK1 and 2) are involved in arteriogenesis, but this has not been directly tested. To do this, we used a mouse model of hindlimb ischemia, femoral artery ligation (FAL), treated with a specific allosteric MEK1 and 2 inhibitor (PD0325901). Whereas control mice showed increased myogenesis, angiogenesis, and arteriogenesis, mice dosed daily with PD0325901 failed to recover. In order to examine for any temporal regulation, mice were treated days 8 to 28 post surgery. Interestingly, mean arterial luminal area increased. In a complimentary experiment, mice were treated out to 7 days post surgery, treatment was stopped, and tissues were collected at day 28 post surgery. Although muscle tissue had recovered by this time, mean arterial luminal area remained low relative to controls, suggesting a critical window of MEK1 and 2 signaling being necessary for recovery. Surprisingly, results from MEK2^{&ndash;/&ndash;} mice also fail to undergo arteriogenesis after surgery, indicating this effect may be specific to MEK2 alone. BrDU-injected mice co-stained with either CD31 or &alpha;SMA show that loss of MEK2 predominantly affects endothelial cells within the arteries. Lastly, equivalent results are shown in mice lacking tumor endothelial marker 8 (TEM8). These results indicate MEK2 activity is required for arteriogenesis, and show the first known physiological role for TEM8. In addition, the results have implications in the current use of MEK1 and 2 inhibitors for anti-cancer therapy, as these drugs may affect remodeling arteries. Our results also have potential implications for future therapies for PAD, as MEK2 activation after blockage could stimulate arterial growth, preventing the need for amputation.</p><p>

<p> Meiosis is a highly conserved specialized cell division that occurs in many organisms, including budding yeast and mammals. Meiosis divides the chromosome number of the cell in half to create gametes for sexual reproduction. A single round of chromosome duplication is followed by two rounds of chromosome segregation, Meiosis I (homologs segregate) and Meiosis II (sister chromatids segregate). Proper segregation at Meiosis I requires that homologs are connected by both crossovers and sister chromatid cohesion. Crossovers are formed by the repair of double strand breaks (DSBs) preferentially by the homolog. The choice of repair template is determined at the time of strand invasion, which is mediated by two recombinases, Rad51 and the meiosis-specific Dmc1. Rad51 is necessary for Dmc1 to function properly but its strand exchange activity is inhibited both by Dmc1 and Mek1, a meiosis-specific kinase, which is activated by DSBs. Mek1 suppresses interaction between Rad51 and its accessory factor Rad54 in two ways. First, phosphorylation of Rad54 lowers its affinity for Rad51. Second, phosphorylation stabilizes Hed1, a meiosis-specific protein that binds to Rad51 and excludes Rad54. Although <i>RAD54</i> is not required for wild-type levels of interhomolog recombination, <i> rad54</i>&Delta; diploids exhibit decreased sporulation and spore viability, indicating the presence of unrepaired DSBs. My thesis tested the idea that Mek1 kinase activity is down-regulated after interhomolog recombination to allow Rad51-mediated repair of any remaining DSBs. </p><p> Meiotic recombination occurs in the context of a proteinaecous structure called the synaptonemal complex (SC). The SC is formed when sister chromatids condense along protein cores called axial elements (AEs) comprised of the meiosis-specific proteins, Hop1, Red1 and Rec8. AEs are brought together by interhomolog recombination, which creates stable connections and the gluing together of the AEs by the insertion of the transverse filament protein, Zip1, in a process called synapsis. Pachynema is the stage of meiotic prophase in which chromosomes are fully synapsed and where interhomolog recombination has proceeded to the double Holliday junction (dHJ) stage. </p><p> Meiotic progression requires transcription factor <i>NDT80</i>, a middle meiosis transcription factor required to express >200 genes, including the polo-like kinase, CDC5 (required for Holliday junction resolution and SC disassembly) and <i>CLB1</i> (required for meiotic progression). Diploids deleted for <i>NDT80</i> arrest in pachynema with unresolved dHJs. I used an inducible version of <i>NDT80</i> (<i>NDT80-IN </i>) to separate prophase into two phases: pre-<i>NDT80</i>, when interhomolog recombination occurs and post-<i>NDT80</i>, when it is proposed that inactivation of Mek1 allows intersister recombination to repair residual DSBs. <i>RAD54</i> is sufficient to function after interhomolog recombination, as inducing both <i>RAD54</i> and <i>NDT80</i> simultaneously rescues the spore inviability defects observed in <i>NDT80-IN rad54&Delta;</i> diploids. Using an antibody specific for phosphorylated Hed1 as an indicator of Mek1 kinase activity, I showed that Mek1 is constitutively active in <i>ndt80</i>-arrested cells and that induction of <i>NDT80</i> is sufficient to abolish Mek1 activity. Furthermore, inactivation of Mek1 by Ndt80 can occur in the absence of interhomolog strand invasion and synapsis. Mek1 inactivation correlates with the appearance of <i>CDC5</i> and the degradation of Red1. My work demonstrates that the sole target of <i>NDT80</i> responsible for inactivating Mek1 is <i>CDC5</i>. </p><p> Unrepaired DSBs trigger the meiotic recombination checkpoint resulting in prophase arrest, which requires Mek1 and works by sequestering Ndt80 in the cytoplasm. Mek1 also delays meiotic progression in wild-type cells, likely through inactivation of Ndt80. My work shows that Ndt80 in turn negatively regulates Mek1. Based on my observations, as well as published work showing that synapsis results in the removal of Mek1 from chromosomes, I propose that recombination and meiotic progression are coordinated by regulation of Mek1. </p>

<p> Colorectal cancer (CRC) is the third most commonly diagnosed cancer and the third leading cause of cancer death in the United States. Twin cohort studies indicate that inherited susceptibility accounts for approximately 35% of all CRC cases, but only 5-6% of CRC cases can be attributed to known functional mutations. We were the first to identify a germline mutation in Transforming Growth Factor Beta Receptor 1 (<i>TGFBR1</i>) that is also somatically acquired in tumors, a 9 bp in frame deletion within exon 1 (rs11466445), which results in a receptor with decreased TGF-&beta; signaling properties. The observed association between this hypomorphic variant and cancer risk led us to hypothesize that constitutively decreased TGF-&beta; signaling may contribute to the development of CRC. </p><p> In this dissertation, we developed a novel mouse model of <i>Tgfbr1 </i> haploinsufficiency (<i>Tgfbr1</i>^+/&minus;) and found that <i>Tgfbr1</i>^+/&minus; mice were twice as likely as <i>Tgfbr1</i>^+/+ mice to develop CRC. We subsequently identified two human haplotypes associated with constitutively decreased <i>TGFBR1</i> expression and CRC risk and found that decreased <i> TGFBR1</i> expression is strongly associated with three SNPs: rs7034462, rs11466445 and rs11568785. Further examination of <i>TGFBR1</i> haplotype tagging SNPs suggests that the <i>TGFBR1</i> rs7034462-TT is a novel moderate penetrance risk genotype, which has high penetrance among African Americans, the ethnic group with the highest risk for CRC. Our results provide strong support for the novel notion that rs7034462-TT is a potentially clinically relevant CRC susceptibility genotype that may identify individuals at high risk of dying from CRC.</p>

<p> The integrity of the mechanisms that control gene transcription during development and in post-natal life is essential to maintain tissue homeostasis and impede the development of genetic diseases such as cancer. Inheritance of a defective hyperpatahyroidism 2 (<i>HRPT2</i>) allele, an essential regulator of gene transcription, predisposes individuals to a constellation of symptoms ranging from endocrine abnormalities to parathyroid adenomas and jaw bone tumors (HPT-JT). In order to elucidate the function of the <i> HRPT2</i> gene and the pathogenesis that results upon spontaneous inactivation in familial cases of parathyroid cancer and HPT-JT, mouse models were generated that allow for deletion of <i>Hrpt2</i> within different stages and tissues during development. We have used the <i>Hrpt2</i> flox mouse model to delete <i>Hrpt2</i> in mesenchymal progenitor cells as well as committed, terminally differentiated osteoblasts and osteocytes. Whereas loss of <i>Hrpt2</i> in mesenchymal progenitors was embryonic lethal, genetic deletion of <i>Hrpt2</i> in mature bone forming cells led to increased bone mass and bone strength. However, <i>Hrpt2 </i> conditional knockout bones had increased cortical porosity and osteocyte apoptosis associated with increased osteoblast specific gene expression. This work suggests that <i>Hrpt2</i> is required for cell proliferation and differentiation and acts as a transcriptional repressor in terminally differentiated cell types.</p><p> Control of gene transcription defines cell identity and fate. Ascorbic acid (AA, also known as vitamin C) is an essential vitamin for humans and is well known for its role in collagen synthesis. AA acts as a cofactor for TET enzymes, which hydroxylate methylated cytosines. Here, we characterize how 7 days of AA treatment causes changes in gene transcription, 5-hydroxymethylcytosine deposition, and the active chromatin marks H3K4me3 and H3k27ac in MC3T3-E1 murine pre-osteoblasts cells, initiating cell differentiation and expression of the osteoblast phenotype. Though 5hmC deposition was not specific for only highly expressed genes, it was highly enriched at transcriptional start sites and CpG islands. While H3K4me3 was mostly unchanged, H3K27ac was predictive of driving gene expression. This work suggests that AA causes dramatic changes to the epigenome through epigenetic modifiers to impact cell differentiation. </p><p>

Breast cancer is the most common tumor among women with inherited mutations in the p53 gene (Li-Fraumeni syndrome). The tumors represent the basal-like subtype which has been suggested to originate from mammary stem/progenitor cells. In mouse mammary epithelium, mammosphere-forming potential was increased with decreased dosage of the gene encoding the p53 tumor suppressor protein (Trp53). Limiting dilution transplantation also showed a 3.3-fold increase in the frequency of long-term regenerative mammary stem cells in Trp53-/- mice. The repression of mammospheres by p53 was apparent despite the absence of apoptotic responses to radiation indicating a dissociation of these two activities of p53. The effects of p53 on progenitor cells were also observed in TM40A cells using both mammosphere-forming assays and the DsRed-let7c-sensor. The frequency of long-term label-retaining epithelial cells (LRECs) was decreased in Trp53-/- mammary glands indicating that asymmetric segregation of DNA is diminished and contributes to the expansion of the mammary stem cells. Treatment with an inhibitor of γ-secretase (DAPT) reduced the number of Trp53-/- mammospheres to the level found in Trp53+/+ cells. These results demonstrate that basal levels of p53 restrict mammary stem/progenitor cells. Notch is a target of γ-secretase suggesting that the Notch pathway is a therapeutic target to prevent expansion of this vulnerable pool of cells. The expansion of p53-deficient mammary stem/progenitor cells can also be reversed after the expression of C-terminal p53, suggesting that the C-terminal domains of p53 may be responsible for the regulation of mammary stem/progenitor cells self-renewal. In parous mammary gland, increased p53 responsiveness sensitized mammary stem/progenitor cells to ionizing radiation without affecting the self-renewal of these cells, which may be responsible for the parity-induced protection against breast cancer.

The Drosophila morgue gene was identified as a regulator of programmed cell death and protein ubiquitination. It has been shown to enhance programmed cell death via promoting the turnover of DIAP1, a conserved anti-apoptotic protein. Morgue protein contains a zinc finger motif, an F box domain and a ubiquitin E2 conjugase variant domain with a Cysteine to Glycine substitution at the catalytic site. This unique domain/motif architecture suggests that Morgue may have very distinctive activities. However, how and what each domain/motif contributes to Morgue function remains unexplored. My dissertation project focused on a study of Morgue protein evolution and function using a combination of bioinformatics, genetics and biochemical methods. The results suggest that Morgue exhibits widespread but restricted phylogenetic distribution among invertebrate metazoans; the study of Morgue's origin provides an example of how multi-domain proteins may evolve. Results of functional studies revealed that over-expression of Morgue can induce a homozygous lethal phenotype that is independent of either F-box or the Glycine in the UEV domain. In addition, co-immunoprecipitation experiments have shown that Morgue associates with SkpA and Lys48 linked polyubiquitin chains, indicating that Morgue might be a multi-functional protein in PCD and ubiquitination.

The alleles cse1-1 and cse2-l were cold-sensitive yeast mutants that display increased mitotic chromosome nondisjunction phenotypes. Two high dosage suppressors, SCM1 and SCM2, were isolated which suppress the cold-sensitive phenotypes of cse1-1 and cse2-1 cells, respectively. DNA sequence analysis revealed that SCM1 is identical to SRP1, which was previously isolated as an extragenic suppressor of an RNA polymerase I mutant. SRP1 encodes a 67 kD protein containing an arm (armadillo) motif that localizes to the periphery of the nucleus. Mutational analysis indicates that SRP1 is essential for viability and that the C-terminus is important for function. Genetic analysis indicates that SRP1 and CSE1 functionally interact in chromosome transmission. In addition, a putative mouse homolog of SRP1 called MSG1 was isolated, which also contains an arm motif in the central domain. Overexpression of MSG1 suppresses the cold-sensitive phenotype of cse1-1 cells suggesting a possible functional similarity between SRP1 and MSG1. In addition to the cold-sensitive and chromosome missegregation phenotypes, cse2-1 also causes temperature sensitivity. Overexpression of SCM2 only suppresses the cold-sensitive phenotype. SCM2 encodes a 65 kD protein that is highly homologous to known amino acid permeases. Disruption of SCM2 causes slow growth. The scm2$\Delta$ trp1-$\Delta$101 double mutant cells exhibit a synthetic cold-sensitive phenotype and grow much more slowly at the permissive temperature than cells with either a scm2$\Delta$ or trp1-$\Delta$101 single mutation. Amino acid uptake assays revealed a decreased accumulation of tryptophan in the scm$\Delta$ cells indicating that SCM2 encodes a tryptophan permease. A temperature-sensitive allele of phosphoinositide-specific phospholipase C gene in yeast, plc1-1, causes a 9-fold increase in chromosome missegregation. The temperature sensitivity and the chromosome missegregation phenotypes of plc1-1 cells were both partially suppressed by calcium. The mutant plc1-1 allele was cloned and found to encode a single amino acid substitution in the protein. The yeast PLC1 protein contains a putative EF-hand calcium binding domain and the enzymatic activity of PLC1 is dependent upon calcium. Five EF-hand mutants have been generated. Three of these mutants exhibit a temperature-sensitive phenotype that can be suppressed by calcium. The effects of the plc1 mutations on chromosome segregation and cell lysis were also investigated.