<p> Cancer encompasses a broad range of complex malignancies characterized by diverse sites of origin, genetic landscapes, and disease progressions. This diversity dictates the manner in which these cancer cases are clinically managed, as well as the overall prognosis. Despite these differences, most cancers exhibit universal hallmarks that contribute to tumor growth and cell survival. As such, these hallmarks are the focus of many research efforts and represent high priority targets for anticancer therapy. While some novel treatment regimens targeting these features have provided good outcomes, effective therapeutics for aggressive cancers are still needed. </p><p> To that end, I used patient- derived xenografts (PDX) of colorectal cancer (CRC) and an established cell line of advanced prostate cancer (CaP) to target oncogenic drivers, specifically mTOR and MEK, and altered metabolism for decreased tumor cell growth. My work defined genomic landscapes in CRC for which molecularly targeted agents (against MEK alone and in combination with mTOR) were most effective. My research also revealed the glycolytic nature of CaP which, when inhibited, upregulated autophagy. Combined inhibition of glycolysis and autophagy reduced CaP cell viability, thereby supporting autophagy&rsquo;s role in cell survival during times of metabolic stress. </p><p> Chapter 1 provides an introduction on the hallmarks and history of cancer to frame the work described in Chapters 2 and 3. Chapter 2 presents the development of clinically relevant PDX models of CRC, and their sensitivity to targeted therapies. Chapter 3 describes the metabolic characteristics of CaP and the effects of inhibiting glycolysis. Chapter 4 contains unpublished data that interrogates the role of autophagy in CaP. Finally, chapter 5 presents conclusions from this work and future research directions.</p><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> <i>Wolbachia</i> are gram-negative, obligate, intracellular bacteria infecting a majority of insect species and filarial nematodes. In both insects and nematodes <i>Wolbachia</i> are primarily transmitted through the female germ line. <i>Wolbachia</i> carried by filarial nematodes are the cause of the neglected diseases African river blindness and lymphatic filariasis afflicting millions worldwide. In order to combat these diseases, we created a <i>Wolbachia</i>-infected Drosophila cell line that enabled high throughput screening for novel potent anti-<i> Wolbachia</i> compounds. Of the 36,231 compounds screened in house, 8 compounds dramatically reduced <i>Wolbachia</i> titer both in the cell and nematode based screen. Significantly, we discovered that the albendazole metabolite, albendazole sulfone, reduces <i>Wolbachia</i> titer in Drosophila melanogaster and the filarial nematode <i>Brugia malayi </i> perhaps by directly targeting <i>Wolbachia</i> FtsZ. Using the <i>Wolbachia</i>-infected cell line, we discovered that in addition to vertical germ line transmission, <i>Wolbachia</i> are efficiently transmitted horizontally via cell-to-cell transmission. We show that horizontal transfer is independent of cell-to-cell contact, can efficiently take place within hours, and uses both host cell phagocytic and clathrin/dynamin-dependent endocytic machinery. Modifications to our high-throughput screen in combination with genome-wide RNA interference (RNAi) identified host factors that influence <i> Wolbachia</i> titer. When these host factors were tested in <i> Drosophila melanogaster in vivo</i> we found that maintenance of <i>Wolbachia</i> titer relies on an intact host Endoplasmic Reticulum (ER) associated degradation (ERAD) system. These data, in combination with electron microscopy studies, demonstrated that <i>Wolbachia</i> is intimately associated with the host ER and suggested a previously unsuspected mechanism for the potent ability of <i>Wolbachia</i> to prevent RNA virus replication. To examine the impact of nutritional on <i>Wolbachia </i> titer, Drosophila were fed sucrose- and yeast-enriched diets. These conditions resulted in increased and decreased <i>Wolbachia</i> titer in Drosophila oogenesis, respectively, and that somatic TOR and insulin signaling mediate the response of the yeast-enriched diet on <i>Wolbachia </i>. Taken together, these studies provide initial insights into the molecular and cellular interactions between <i>Wolbachia</i> and its insect and nematode hosts.</p><p>

<p> Progression through the mammalian cell cycle is a tightly regulated process that allows cells to replicate their genomes and divide properly. In growth factor-deprived conditions or in response to stress, the cell will exit the cell cycle either reversibly through quiescence, or permanently via senescence. Studies have shown that the SIN3 family of proteins plays a crucial role in these cell cycle exit processes. SIN3 proteins are highly conserved, and exist in mammals as two family members: SIN3A and SIN3B, which function as flexible scaffolding proteins to assemble co-repressor complexes. Our laboratory has recently implicated SIN3B as a critical mediator of each of these cell cycle exit processes. However, its mechanism of action and the consequences of its disruption pertaining to cancer progression have not been comprehensively elucidated. Here we demonstrate that SIN3B is required for the induction of senescence in a mouse model of prostate cancer, and thus prevents the progression to aggressive and invasive carcinoma. In addition, through interaction analysis, we uncovered a novel and robust association between SIN3B and the DREAM complex. The DREAM complex, comprised of p107/p130, E2F4/5, DP1 and the MuvB core complex, is responsible for the repression of hundreds of cell cycle-related transcripts during quiescence. We determined that the deletion of <i>SIN3B</i> resulted in the derepression of DREAM target genes during quiescence, but was not sufficient to allow quiescent cells to resume proliferation. However, the ectopic expression of APC/C^CDH1 inhibitor EMI1 was sufficient for <i>SIN3B</i> deleted cells, but not wild-type cells, to reenter the cell cycle. These studies demonstrate a critical role for SIN3B in the senescence and quiescence programs, and provide important mechanistic insight into the molecular pathways that exquisitely regulate cell cycle exit.</p><p>

<p> Present day genomic technologies are evolving at an unprecedented rate, allowing interrogation of cellular activities with increasing breadth and depth. However, we know very little about how the genome functions and what the identified genes do. The lack of functional annotations of genes greatly limits the post-analytical interpretation of new high throughput genomic datasets. For plant biologists, the problem is much severe. Less than 50% of all the identified genes in the model plant <i>Arabidopsis thaliana,</i> and only about 20% of all genes in the crop model <i>Oryza sativa</i> have some aspects of their functions assigned. Therefore, there is an urgent need to develop innovative methods to predict and expand on the currently available functional annotations of plant genes. With open-access catching the &lsquo;pulse&rsquo; of modern day molecular research, an integration of the copious amount of transcriptome datasets allows rapid prediction of gene functions in specific biological contexts, which provide added evidence over traditional homology-based functional inference. The main goal of this dissertation was to develop data analysis strategies and tools broadly applicable in systems biology research. </p><p> Two user friendly interactive web applications are presented: The Rice Regulatory Network (RRN) captures an abiotic-stress conditioned gene regulatory network designed to facilitate the identification of transcription factor targets during induction of various environmental stresses. The <i>Arabidopsis </i> Seed Active Network (SANe) is a transcriptional regulatory network that encapsulates various aspects of seed formation, including embryogenesis, endosperm development and seed-coat formation. Further, an edge-set enrichment analysis algorithm is proposed that uses network density as a parameter to estimate the gain or loss in correlation of pathways between two conditionally independent coexpression networks.</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>

We consider the problem of decontaminating a network where all nodes are infected by a virus. The decontamination strategy is performed using a Cellular Automata (CA) model in which each node of the network is represented by the automata cell and thus, the network host status is also mapped to the CA state (contaminated, decontaminating, decontaminated). All hosts are assumed to be initially contaminated and the status of each cell is synchronously updated according to a set of local rules, based on the state of its neighbourhood. Our goal is to find the set of local rules that will accomplish the decontamination in an optimal way. The metrics used to define optimality is the minimization of three metrics: the maximum number of decontaminating cells at each step, the required value of the immunity time of each cell and the number of steps to complete the sanitization algorithm. In our research, we explore the designing of these local decontamination rules by refining the concept of the neighbourhood radius of CA with the addition of two new dimensions: Visibility Hop and Contamination Distance. Additionally, a research tool that help us manage our study have been developed.

The aim of the present study was to determine how lymphokines could exert their biological action on the skin during the course of the delayed hypersensitivity reactions. Four groups of experiments were conducted to investigate: (1) The production and the separation of lymphokines, (2) the protease activity of lymphokines and the effect of lymphokines on the kinin-forming system, (3) the effect of lymphokines on mast cells and platelets, and (4) the effect of enzyme-treated lymphokines on the skin inflammatory reaction. Guinea pig lymph node lymphocytes, stimulated by either the specific antigen DNP-BGG or by concanavalin A, were used to generate lymphokines. Parameters for testing lymphokine activities were those of migration inhibitory factor (MIF) and skin reactive factor (SRF). Separation of MIF and SRF from the lymphokine preparation by gel filtration, electrophoresis and fractional precipitation with ammonium sulphate was unsuccessful, which indicated that the physical properties of MIF and SRF were similar. Lymphokine preparations contained little or no neutral and acidic protease activities. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

<p> This study examined various hybrid peptides that possess both collagen [(POG)_n] and cell penetrating peptides (CPP) [(RRG)_n or R_n] sequences. The hybrid peptides were able to fold into triple helical conformation when the surrounding temperature was lower than their transition temperature (T_m) which resulted in cellular internalization. The peptide that lacked collagen [(POG)_n] domain failed to penetrate the cell. The hybrid peptide under study, FL7V1, was shown to have the ideal T_m (17.3&deg;C) for the potential purpose as a drug carrier. <i>In vitro</i> study of FL6V1 with temperature gradient showed cellular internalization at low temperatures (10&deg;C-20&deg;C) while no uptake was achieved at high temperatures (24&deg;C-32&deg;C). <i> In vivo</i> study of FL7V1 with <i>P. leidyi</i> corresponded with the results of <i>in vitro</i> study at constant and gradient temperature.</p>