<p> Bronchopulmonary dysplasia (BPD) is a serious respiratory complication for the preterm newborn characterized clinically by prolonged respiratory distress and histologically by alveolar simplification and decreased pulmonary vasculature. The development of BPD is well linked to oxidative stress suffered by the newborn as a result of a preterm fetal-neonatal transition, supplemental oxygen, infection, increased inflammation, and mechanical ventilation. Damage suffered by oxidative stress may be through direct mechanisms or through alteration of redox&not;sensitive pathways involved in cell death, cell survival, differentiation, and proliferation. Redox&not;sensitive modifications regulating protein function and redox-sensitive pathways have mainly been ascribed to oxidative modification of cysteine thiols. As their modification is critical for protein function, maintenance of the thiol redox status is crucial. Thioredoxin-1 (Trx1) functions in maintenance of thiol redox homeostasis, and its redox activity is intimately linked to antioxidant, cytoprotection, proliferation responses, and cytoprotection. While Trx1 targets of redox regulation have been identified, we hypothesize that additional protein may be redox regulated and that Trx1 target profiles may change during oxidative stress. Therefore a novel immunoprecipitation approach, identified as the substrate trap approach, was developed to identify Trx1 targets. The following demonstrates the use of the substrate trap approach for identification of Trx1 redox targets and further application of the approach to identify alterations in target profiles in response to oxidative stress. Use of nuclear targeted substrate trap was successfully employed to enrich from nuclear Trx1 targets. As a final component the characterization of the Trx1 system in mouse from late embryonic development through the first week of life animals were exposed to room air or hyperoxia (model of BPD). Characterization indicates impairment of the Trx1 system in response to hyperoxic injury. As Trx1 is known to regulate proliferation, cell death, survival, differentiation pathways, impairment of the Trx1 system during early neonatal development may potentiate hyperoxic injury and alterations in lung development. Better understanding of Trx1 interactions occur through the substrate trap in a physiological model of BPD will help elucidate redox-signaling pathways involved in BPD pathogenesis.</p>

<p> Manipulating the phenylpropanoid (Pp) pathway has been of great focus to bio-engineers as this pathway is responsible for production of many compounds that are important to human health for their known antioxidant, anti-viral, anti-inflammatory, anti-allergenic and vasodilatory properties. The secondary by products of the Pp pathway are important for the physiological well-being of the plant as it contributes to plant&rsquo;s ability to tolerate changing environment. Plant bio-engineering, involves manipulating gene expression of proteins that regulate functional proteins which are known to attribute to stress tolerance. Our research focused on one such regulatory protein called the 14-3-3 lambda (14-3-3&lambda;) protein and its effects on anthocyanin production in two different plants: a plant model <i>Arabidopsis thaliana </i> (<i>A. thaliana, Columbia-0</i>), and a naturally drought tolerant resurrection plant <i>Selaginella lepidophylla</i> (<i> S. lepi</i>). Due to their structural characteristics the family of 14-3- 3 proteins bind to many different client proteins and hence can function as signaling factors in eukaryotes. Anthocyanins are anti-oxidants produced in plants that alter plants physiology to resist stress. The goal of this study was to establish which nodes in the anthocyanin synthesis pathways are influenced by 14-3-3&lambda; in both <i>A. thaliana</i> and <i>S. lepi </i>. Data from this study established the steps in the Anthocyanin pathway that 14-3-3&lambda; affects to alter anthocyanin production during normal hydration and drought stress states. Based on our published studies and experimental data we have identified that the 14-3-3&lambda; isoform is playing a significant role in the anthocyanin pathway during drought stress. Using a reverse genetics approach, the amounts of secondary anthocyanin metabolites produced in a 14-3-3&lambda; knockout mutant were compared to the wild-type <i> A. thaliana</i> during normal hydration and drought conditions. Analytical techniques such as high performance liquid chromatography (HPLC) and liquid chromatography-Mass Spectrometry (LC-MS/MS) in combination with open access databases were used for metabolite profiling. The metabolite profile lead to candidate metabolites that differed between the drought-treated and hydrated groups in the knockout mutants and wild-type. Identification of these metabolites determined the nodes of Pp pathway that were affected by 14-3-3&lambda;, namely the enzymes chalcone synthase and chalcone isomerase. These findings in <i> A. thaliana</i> were expanded in the naturally drought resistant plant <i> S. lepi</i> using similar analytical approaches employed in <i> A. thaliana</i>. The results proved that 14-3-3&lambda; affects biosynthesis of anthocyanin during drought stress in <i>A. thaliana</i> and <i> S. lepi</i> in a similar manner, hence suggesting a similar role of 14-3-3&lambda; in the production of anthocyanins in both the plants.</p><p>

<p> The eukaryotic cytoskeleton is composed of varying proteinaceous filaments and is responsible for intracellular transport, cell proliferation, cell morphogenesis, and cell motility. Microtubules are one of three cytoskeletal components and have a unique polymer structure. The hollow cylinders undergo rapid polymerization and depolymerization events (<i>i.e.</i> dynamic instability) to promote assembly at the leading edge of the cell and disassembly in the rear of the cell to drive the cell front forward and facilitate directional migration. High-resolution light microscopy and automated tracking allow visualization and quantification of microtubule dynamics (<i>i.e.</i> growth speeds and growth lifetimes) during time-lapse imaging. These techniques were used to understand how the physical environment influences molecular control of endothelial cell morphology. The ultimate goal of this work is to test hypotheses relevant to vascular development and diseases associated with endothelial cell angiogenesis &ndash; defined as the development of new blood vessels from pre-existing vessels. Angiogenesis is of particular relevance because it is a commonality underlying many diseases affecting over one billion people worldwide, including all cancers, cardiovascular disease, blindness, arthritis, and Alzheimer's disease.</p><p>

<p> Genome instability is a hallmark of human cancers. Patients with Bloom&rsquo;s syndrome, a rare chromosome breakage syndrome caused by inactivation of the RecQ helicase BLM, result in phenotypes associated with accelerated aging and develop cancer at a very young age. Patients with Bloom&rsquo;s syndrome exhibit hyper-recombination, but the role of BLM and increased genomic instability is not fully characterized. Sgs1, the only member of the RecQ family of DNA helicases in <i>Saccharomyces cerevisiae,</i> is known to act both in early and late stages of homology-dependent repair of DNA damage. Exo1, a 5'&ndash;3' exonuclease, first discovered to play a role in mismatch repair has been shown to participate in parallel to Sgs1 in processing the ends of DNA double-strand breaks, an early step of homology-mediated repair. Here we have characterized the genetic interaction of <i>SGS1</i> and <i> EXO1</i> with other repair factors in homology-mediated repair as well as DNA damage checkpoints, and characterize the role of post-translational modifications, and protein-protein interactions in regulating their function in response to DNA damage. In <i>S. cerevisiae</i> cells lacking Sgs1, spontaneous translocations arise by homologous recombination in small regions of homology between three non-allelic, but related sequences in the genes <i>CAN1, LYP1,</i> and <i>ALP1.</i> We have found that these translocation events are inhibited if cells lack Mec1/ATR kinase while Tel1/ATM acts as a suppressor, and that they are dependent on Rad59, a protein known to function as one of two sub-pathways of Rad52 homology-directed repair.</p><p> Through a candidate screen of other DNA metabolic factors, we identified Exo1 as a strong suppressor of chromosomal rearrangements in the <i> sgs1&Delta;</i> mutant. The Exo1 enzymatic domain is located in the N-terminus while the C-terminus harbors mismatch repair protein binding sites as well as phosphorylation sites known to modulate its enzymatic function at uncapped telomeres. We have determined that the C-terminus is dispensable for Exo1&rsquo;s roles in resistance to DNA-damaging agents and suppressing mutations and chromosomal rearrangements. Exo1 has been identified as a component of the error-free DNA damage tolerance pathway of template switching. Exo1 promotes template switching by extending the single strand gap behind stalled replication forks. Here, we show that the dysregulation of the phosphorylation of the C-terminus of Exo1 is detrimental in cells under replication stress whereas loss of Exo1 suppresses under the same conditions, suggesting that Exo1 function is tightly regulated by both phosphorylation and dephosphorylation and is important in properly modulating the DNA damage response at stalled forks.</p><p> It has previously been shown that the strand exchange factor Rad51 binds to the C-terminus of Sgs1 although the significance of this physical interaction has yet to be determined. To elucidate the function of the physical interaction of Sgs1 and Rad51, we have generated a separation of function allele of <i> SGS1</i> with a single amino acid change <i>(sgs1-FD)</i> that ablates the physical interaction with Rad51. Alone, the loss of the interaction of Sgs1 and Rad51 in our <i>sgs1-FD</i> mutant did not cause any of the defects in response to DNA damaging agents or genome rearrangements that are observed in the <i>sgs1</i> deletion mutant. However, when we assessed the <i>sgs1-FD</i> mutant in combination with the loss of Sae2, Mre11, Exo1, Srs2, Rrm3, and Pol32 we observed genetic interactions that distinguish the <i>sgs1-FD</i> mutant from the <i>sgs1 </i> deletion mutant. Negative and positive genetic interactions with <i> SAE2, MRE11, EXO1, SRS2, RRM3,</i> and <i>POL32</i> suggest the role of the physical interaction of Sgs1 and Rad51 is in promoting homology-mediated repair possibly by competing with single-strand binding protein RPA for single-stranded DNA to promote Rad51 filament formation.</p><p> Together, these studies characterize additional roles for domains of Sgs1 and Exo1 that are not entirely understood as well as their roles in combination with DNA damage checkpoints, and repair pathways that are necessary for maintaining genome stability.</p><p>

Breast cancer is the most frequent tumor type among women. Heightened susceptibility of the breast to tumor development has been associated with early menarche, nulliparity, exposures to ionizing radiation, and family history, but the underlying molecular mechanisms are poorly understood. Unfortunately, the etiology of breast cancer is complex and is complicated by the fact that it is a heterogeneous disease. The p53 tumor suppressor gene was altered in a large proportion of these spontaneous breast tumors implicating its involvement in the progression of breast cancer development. The aim of this dissertation was to determine the regulation of p53 in the normal mammary gland and whether it is involved in suppressing the development of mammary tumors. To evaluate the effect of p53 on mammary tumor formation, the first component of this work involved the characterization of BALB/c- p53-deficient mice. BALB/c-p53+/− and p53−/− mice were examined for tumor spectrum and mammary abnormalities. Mammary transplants were performed to evaluate the role of p53 in tumor suppression in the mammary gland. This work demonstrated that p53 is critical in suppressing mammary tumorigenesis in the mammary gland as BALB/c mice deficient in p53 readily develop mammary carcinomas. The second element of this project examined the expression, localization and activity of p53 in normal mammary tissues. Since the mammary gland is a tissue that is sensitive and responsive to local and systemic hormones, the last chapter of this dissertation focused on the hormonal effects on p53 activity. Results from these experiments demonstrated that p53 was expressed at high levels localized to the cytoplasm of the ductal epithelium of the quiescent mammary gland. P53 was not responsive to radiation-induced DNA damage suggesting its function is compromised in the nulliparous mammary gland. Further experiments demonstrated that the functional state of wild type p53 in the mammary epithelium could be regulated by hormonal stimuli.

The development and maintenance of multicellular organisms depends fundamentally on cell division, a series of events largely mediated by the mitotic spindle. Errors in spindle formation and/or function are often associated with severe consequences, most notably cancer. In order to elucidate the cause of such errors and the potential for therapeutic intervention, it is imperative to attain a clear understanding of how cell division normally operates. In this regard, this dissertation focuses on the activity of two microtubule-based motor proteins, Eg5 and dynein, prior to and immediately following nuclear envelope breakdown during mitosis. I show that prophase microtubules are remarkably more dynamic than their metaphase counterparts, moving both toward and away from centrosomes across a wide distribution of rates. Inhibition of Eg5, dynein and Kif2a revealed that a subset of this motion is consistent with microtubule flux, a well-established phenomenon temporally limited to metaphase and anaphase spindles by the preceding literature. My data indicates that flux is operational throughout all of mitosis, possibly functioning at early stages to collect centrosomal components. Immediately following prophase, cells begin assembling bipolar spindles. While the establishment of spindle bipolarity fails in the physical or functional absence of Eg5, I show that co-inhibition of dynein restores a cell's ability to organize microtubules into a bipolar structure. Despite inhibition of both Eg5 and dynein, these spindles are morphologically and functionally equivalent to controls. Together, these data suggest that Eg5 and dynein share an antagonistic relationship and that a balance of forces, rather than a definitive set of players, is important for spindle assembly and function. To determine how Eg5- and dynein-mediated forces functionally coordinate to bring about antagonism during spindle assembly, I utilize a nocodazole washout assay. I show, via in vivo imaging and in silico modeling, that spindle collapse in the absence of functional Eg5 requires dynein activity and an initial intercentrosomal distance of less than 5.5μm. These data are consistent with a model in which dynein antagonizes Eg5 by crosslinking and sliding antiparallel microtubules, a novel role for dynein within the framework of spindle assembly.

Chloroplast biogenesis relies on the import of thousands of nuclear encoded proteins into the organelle and proper sorting to their sub-organellar compartment. The majority of nucleus-encoded chloroplast proteins are synthesized in the cytoplasm and imported into the organelle via the Toc-Tic translocation systems of the chloroplast envelope. In many cases, these proteins are further targeted to subcompartments of the organelle (e.g. the thylakoid membrane and lumen or inner envelope membrane) by additional targeting systems that function downstream of the import apparatus. The inner envelope membrane (IEM) plays key roles in controlling metabolite transport between the organelle and cytoplasm, and is the major site of lipid and membrane biogenesis within the organelle. In contrast to the protein import and thylakoid targeting systems, our knowledge of the pathways and molecular mechanisms of protein targeting and integration at the IEM are very limited. Previous reports have led to the conclusion that IEM proteins are transferred to the IEM during protein import via a stop-transfer mechanism. Recent studies have shown that at least two components of the Tic machinery (AtTic40 and AtTic110) are completely imported into the stroma and then re-inserted into the IEM in a post-import mechanism. This led me to investigate the mechanisms and pathways involved in the integration of chloroplast IEM proteins in more detail. I selected candidates (AtTic40 for post-import and IEP37 for stop-transfer) that are predicted to have only one membrane-spanning helix and adopt the same IEM topology to facilitate my analysis. My studies confirm the existence of both stop-transfer and post-import mechanisms of IEM protein targeting. Furthermore, I conclude that the IEP37 transmembrane domain (TMD) is a stop-transfer signal and is able of diverting AtTic40 to this pathway in the absence of AtTic40 IEM targeting information. Moreover, the IEP37 TMD also functions as a topology determinant. I also show that the AtTic40 targeting signals are context dependent, with evidence that in the absence of specific information in the appropriate context, the AtTic40 TMD behaves as a stop-transfer signal. This is an indication that the stop-transfer pathway is the default mechanism of protein insertion in the IEM.

U14 small nucleolar RNA (snoRNA) is required for processing of 18S ribosomal RNA. It was hypothesized that U14 might base pair with 18S RNA through two highly conserved U14 sequence elements known as domains A and B. Using Saccharomyces cerevisiae as the experimental system, I showed that: (1) the domain A and B elements are functionally interdependent, and (2) single-point mutations in domain A combined with complete substitution of domain B causes lethality while either mutation alone does not. Direct interaction of U14 with 18S RNA was shown by demonstrating that a lethal mutation in U14 domain A can be suppressed with a mutation which restores complementarity in the corresponding region of 18S RNA. Y-domain in yeast U14 was postulated to serve as a recognition element for vital intermolecular or intramolecular interactions. Consistent with this assumption, mutations in several conserved nucleotides of the loop cause growth defects. In contrast, alterations to the stem have little or no effect. Using a lethal mutation in the loop, three different intragenic suppressor mutations were mapped to three positions adjacent to the primary mutation, and are predicted to influence the structure of the loop. An extragenic suppressors (UF1) able to rescue a cold-sensitive mutation in the loop encodes an essential putative ATP-dependent RNA helicase. Loss of UF1 gene expression caused a reduction in 18S rRNA production, without affecting accumulation of 25S rRNA or U14 snoRNA. Pulse-chase analysis showed that depletion of UF1 protein impaired pre-18S rRNA processing. Finally, an effort was made to define minimum pre-rRNA substrates that can be used to produce functional 18S and 25S rRNAs in vivo. The rDNA operon was split either between the 18S RNA and 5.8S/25S coding units, or between the 18S/5.8S RNA and 25S RNA coding units. The test fragments were expressed from GAL7 promoters. The results showed that functional rRNAs could be produced in trans, but only when the operon was divided between the 18S RNA and 5.8S/25S RNA coding sequence.

Cytokinesis, the process of physically separating cells for division, requires the precise orchestration of numerous physical, mechanical, chemical and biological processes. For these processes to function well, complex coordination of various proteins, with crosstalk between them, either as signaling molecules or as just plain structural components that contribute to the physical separation must exist. Actin, a structural polymer and myosin, a motor are two proteins that contribute to this process significantly. Both proteins are assembled in the contractile ring and together are responsible for the process of constriction. A thorough understanding of the behavior of these proteins, in the contractile ring as well as outside in a cell undergoing cytokinesis is therefore important to prevent possible defects that might lead to deleterious diseases. In this dissertation research, a combination of techniques are made use of, that involve live imaging of fluorescently labeled proteins in cells undergoing cytokinesis along with the use of drugs that either disrupt the structure (integrity) or function of cytokinetic proteins. I generated two LLCPK1 (pig epithelial cell lines); one that stably expresses GFP-actin and the other that stably expresses Tandemn Dimer RFP-myosin regulatory light chain (TDRFP-MRLC). Live imaging and analysis of cells expressing GFP-actin shows that actin in the contractile is highly dynamic and need to be dynamic. Evidence is presented for new roles of Myosin II, in addition to generating the force for cytokinesis. Myosin not only contributes to disassembly of actin in the contractile ring but is also required to maintain actin in the equatorial region. Live imaging of the cell lines that expresses TDRFP-MRLC or GFP-actin helped in the better understanding of the role of microtubules in simultaneously regulating actin and myosin dynamics, not only in the contractile ring to allow ingression, but also in preventing contractile activity outside in the contractile ring. Cytokinesis involves other proteins besides actin and myosin, which help in their recruitment, assembly, ingression and subsequent disassembly. Decreasing the accumulation of actin in the contractile ring, by treatment with Latrunculin B facilitated the examination of spatial and temporal events involved in building the ring. Actin, myosin and other proteins organized as nodes that coalesce during ingression, similar to the fission yeast. We conclude that this mode of cytokinesis a highly conserved feature of cytokinesis.

The ultimate means of limiting the influence of an individual cell on the physiology of a multicellular organism is to induce the death of that cell. Apoptosis is a genetically regulated form of cell death that removes cells that are malfunctioning, unnecessary or damaged. During development, cells are produced in excess and those that are not optimal in form, location or function are removed via apoptosis. In the adult organism, apoptosis allows for the turnover of cells that have carried out specialized functions and maintains tissue homeostasis. Negative selection is the developmental process by which immature T cells that have inappropriate reactivity to self antigen are induced to undergo apoptosis. During work in the lab confirming the requirement for the orphan nuclear hormone receptor, Nur77, for thymocyte apoptosis, an upregulation of the early growth response 1 gene (egr-1) was observed. This thesis investigates the requirement for transcriptional activation mediated by EGR-1 during the apoptosis of DO11.10, a cell line model of thymocyte negative selection. A dominant negative form of EGR-1, WT1EGR1, was expressed in DO11.10. The ability of these transfectants to undergo apoptosis in response to a variety of stimuli was measured. Another important function of apoptosis is to limit the life span of activated immune cells. The inception of the second part of this work was the clinical observation that exposure of non-healing wounds to hyperbaric oxygen (HBO), 100% oxygen at elevated atmospheric pressures, aids in the healing of these wounds. The hypothesis tested here is that HBO enhances the apoptosis of immune cells. Such an enhancement would promote the resolution of chronic inflammation and aid in wound healing. It is demonstrated that HBO enhances apoptosis of immune cells in response to stimuli relevant to both the regulation of the immune system and the application of HBO as an adjuvant to anti-cancer therapy. This study provides a new approach for studying the role of oxygen and its derivatives in apoptosis. The findings also support the continued investigation of expanding the clinical application of HBO.