<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> The genetic code is highly conserved across all domains of life, enabling horizontal gene transfer (HGT) between organisms and across ecosystems via horizontally-transferred genetic elements such as viruses and plasmids. While HGT increases genetic diversity, it poses a risk to engineered biological systems by introducing new genes that destabilize engineered functions or allowing the expression of engineered genes in wild organisms with unknown effects. A model organism engineered with an alternative genetic code may provide new insight into the origins of the genetic code while also providing a stable chassis for engineered biological systems.</p><p> The Isaacs Lab recently developed an <i>Escherichia coli</i> strain lacking both UAG stop codons and Release Factor 1, resulting in the first genomically recoded organism (GRO) with an unassigned codon in its genetic code. Here, we demonstrate that this alternative genetic code lacking UAG codon assignment confers resistance to multiple viruses (&lambda;, M13, PI, MS2) at titers up to 10¹¹ PFU/mL and impairs conjugative plasmid function (F and RK2) up to 10⁵-fold. Propagating viruses on a mixed microbial community containing standard and alternative genetic codes also reduced viral population fitness and prompted viral adaptation to the alternative genetic code. In investigating the molecular mechanism underlying the resistance to viruses and conjugative plasmids, we found that UAG-ending genes elicit ribosomal stalling and the tmRNAmediated ribosomal rescue response, resulting in degradation of UAG-ending proteins and suggesting that genomic recoding may be a broadly applicable strategy to impair horizontal gene transfer into other organisms.</p><p> To prevent the expression of engineered genes in wild organisms, we reassigned the UAG codon in the GRO to a sense codon incorporating the non-standard amino acid <i>4</i>-acetylphenylalanine (pAcF) through the introduction of an orthogonal translation system (OTS). We then created a library of UAG-containing variants and assessed escape of UAG-containing genes from the GRO into wild-type organisms for both a non-selective green fluorescent protein (GFP) and selective chloramphenicol acetyltransferase (CAT) gene. While 1 UAG codon impaired the expression of GFP in wild-type organisms, at least 2 UAG codons were required in CAT to consistently prevent escaped expression in wild-type organisms with a standard genetic code. Additionally, sequencing revealed that wild-type organisms enabled expression of CAT by mutating UAG codons to UGG coding for tryptophan or CAG coding for glutamine. By placing UAG at sites in proteins that cannot tolerate a tryptophan or glutamine substitution, we can create UAG-containing genes further isolated from expression in wild organisms.</p><p> As biotechnology increasingly targets open-environment applications such as bioremediation or disease treatment in humans, we require methods to stabilize and control the genetic information that we encode in engineered biological systems. Because alternative genetic codes can both confer resistance to horizontal gene transfer into an engineered system and restrict expression of engineered genes in wild-type organisms, genomic recoding of organisms to contain alternative genetic codes is a promising path towards increasing the stability and safety of engineered biological systems. However, open-environment applications will expose engineered biological systems to new stresses not represented in the laboratory environment, and further work is required to validate these methods will be robust in conditions of limiting nutrients or other cellular stresses. Additionally, while we have demonstrated genetic isolation of the GRO with respect to genes both entering and leaving the cell, we cannot currently have both properties simultaneously because UAG is the sole open codon. We envision that current research into further codon reassignments, including the reassignment of sense codons, will pave the way for alternate genetic codes with multiple codon reassignments. By expanding recoding efforts to multiple species, we envision the development of synthetic microbial communities with alternate genetic codes that are genetically isolated and robust to perturbation by HGT.</p>

<p> Despite being comprised of networks with extensive positive feedback, the brain is able to prevent runaway activity. Neural networks are remarkably good at maintaining an activity setpoint while still permitting learning-related or developmental plasticity. To accomplish the delicate balance between change and stability, neural networks employ a group of homeostatic negative feedback mechanisms. This suite of homeostatic mechanisms sense and adjust neuronal excitability to keep firing rates within some target range. To date, the most well described manner in which neurons homeostatically regulate their excitability is through adjustment of excitatory or inhibitory synaptic weights, or by modulating their intrinsic excitability. It is perplexing why the neuron should have several means to accomplish the same outcome. Experiments demonstrating the collaborative or solo induction of homeostatic mechanisms have provided only limited insight into how homeostatic signaling pathways are organized to generate and maintain firing rate set-points (FRSP).</p><p> In order for neurons to maintain a FRSP, deviations from this value must modulate an internal signal that subsequently triggers homeostatic mechanisms to restore excitability to its set-point. The CaMKIV pathway is a calcium-dependent signaling element that plays a crucial role in regulating excitatory synaptic strength. The CaMKIV cascade is highly sensitive to activity and can modulate transcription, making it an ideal candidate to integrate incoming activity and modulate the excitability of neurons. Therefore, the major aim of this thesis was to characterize the role of CaMKIV in inducing multiple forms of homeostatic plasticity in tandem. Here we leverage our expertise in measuring homeostasis in neocortical neurons <i>in vitro</i> to determine how manipulating the activation state of nuclear CaMKIV affects neuronal excitability. </p><p> We found that excitatory synaptic scaling and intrinsic plasticity were bidirectionally induced by manipulating CaMKIV activity even without any perturbations to network activity. In contrast, CaMKIV had no impact on inhibitory synaptic weights. Additionally, we found that CaMKIV activity bidirectionally regulated spontaneous firing rates. Taken together, our data suggests that CaMKIV activity is used by the neuron to monitor the firing set point and gate homeostatic mechanisms to correct for drift from this target. The data presented in this thesis contribute that excitatory synaptic scaling and intrinsic excitability are tightly coordinated through bidirectional changes in the same signaling pathway, while inhibitory synaptic scaling is sensed and regulated through an independent signaling mechanism. This body of work contributes to a better understanding of neuronal homeostasis and will hopefully help us determine how malfunctions in homeostatic plasticity contributes to neurological and neurodevelopmental disorders.</p><p>

<p> Treatment for prostate cancer patients who experience recurrent disease involves androgen deprivation therapy (ADT) as prostate tumors are primarily driven by activation of the androgen receptor (AR). However, most patients on this therapy relapse within a few years after which this treatment fails to extend survival and progresses to castration resistant prostate cancer (CRPC). Treatment for CRPC often involve inhibitors of the AR itself, however, patients on these treatments often fail as well. The main cause for the failure of many therapies is acquired resistance to treatment; therefore, there is an urgent need to better understand this resistance for improved disease management. Protein translation plays an important role in altering signaling pathways by modifying protein expression levels, and offer promising targets for preventing acquired resistance. mTOR (mechanistic target of rapamycin) is a key regulator of protein translation in humans, and multiple mTOR inhibitors have been developed over the years and used in many diseases as treatment, including prostate cancer. EIF4E is a key component of the translational mechanism in eukaryotic organisms and its phosphorylation has been implicated in resistance to several therapies in many cancer types. EIF4E is involved in both cap-dependent and &ndash;independent translation, however, mTOR regulates only cap-dependent translation. Here I demonstrate using my data from <i>in vitro</i> studies as well as human-derived tumor-xenograft models that phosphorylation of eIF4E at Ser209 plays a significant role in the resistance of prostate tumors to AR and mTOR inhibition, by changing the mechanism of protein translation from cap-dependent to cap-independent to maintain tumor cellular proliferation, growth and survival. In recent years, many clinical trials used combinations of mTOR and AR inhibitors in patients with CRPC who have failed first line therapy; many of these studies fail especially if they are conducted in patients who had been pre-treated with an AR inhibitor; whereas others partially succeed if they are used in untreated patients. The overall goal of my thesis is to study the role of eIF4E phosphorylation in the development of resistance to mTOR and AR inhibitors in prostate cancer. My data points to AR as a suppressor of eIF4E phosphorylation, therefore explaining why prior treatment with the AR inhibitor bicalutamide made patients resistant to a combination of bicalutamide with the mTOR inhibitor RAD001. Furthermore, our results show that the receptor tyrosine-protein kinase ErbB3 negatively regulates phosphorylation of eIF4E, and high levels of ErbB3 may be an indicative of tumors that would respond to the combination therapy. Taken together, our studies demonstrate the mechanisms by which prostate cancer acquires resistance to mTOR and AR inhibition and explain some of the responsible proteins and pathways that are involved in this process. We also indicate promising biomarkers for evaluation of therapy effectiveness with this combination in prostate cancer patients.</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>

<p> Neural stem progenitor cells (NSPCs) in the mammalian brain contribute to life-long neurogenesis and brain health. Adult mammalian neurogenesis primarily occurs in the subventricular zone (SVZ) and the subgranular zone (SGZ) of the dentate gyrus. Epigenetic repression is a crucial regulator of cell fate specification during adult neurogenesis. How epigenetic repression impacts adult neurogenesis and how epigenetic dysregulation may impact neoplasia or tumorigenesis remains poorly understood. Examination of epigenetic regulation in the adult mammalian brain is complicated by the heterogeneous nature of neurogenic niches and by the highly orchestrated fate specification processes within neural stem progenitor cells involving myriad intrinsic and extrinsic factors. To overcome these challenges, we utilized a cross-species approach. To model histone modifications as they exist <i>in vivo</i> for epigenetic profiling, we isolated neural stem progenitor cells from the adult SVZ and SGZ of non-human primate baboon brains. To determine cellular and molecular changes within the adult SVZ and SGZ following loss of epigenetic repression, we utilized multiple mouse models, including conditional <i> Ezh2</i> and <i>Suv4-20h1</i> knockouts. To model the non-cell type specific effects common to small molecule screening and brain chemotherapeutic agents, induction of conditional knockout utilized a recombinant Cre protein. Finally, to model epigenetic mechanisms during SVZ-associated glioblastoma (GBM) tumorigenesis, we conducted comparative analysis between healthy NSPCs and GBM specimens from humans. The convergence of baboon, mouse and human models of adult neurogenesis revealed that epigenetic repression is a critical mechanism regulating proper neural cell fate and that epigenetic dysregulation may be a driver of GBM.</p><p>

The Notch and TGF13 signaling pathways have been shown to play important roles in regulating endothelial-to-mesenchymal transition (EndMT) during cardiac morphogenesis. EndMT is the process by which endocardial cells of the atrioventricular canal and the outflow tract repress endothelial cell phenotype and upregulate mesenchymal cell phenotype. EndMT is initiated by inductive signals emanating from the overlying myocardium and inter-endothelial signals and generate the cells that form the heart valves and atrioventricular septum. The Notch and TGFf3 pathway are thought to act in parallel to modulate endothelial phenotype and promote EndMT. Vascular endothelial (VE) cadherin is a key regulator of cardiac endothelial cell phenotype and must be downregulated during EndMT. Accordingly, VE-cadherin expression remains stabilized in the atrioventricular canal and outflow tract of Notchl-deficient mouse embryos, while activation of the Notch or TGFP pathways results in decreased VE-cadherin expression in endothelial cells. However, the downstream target gene(s) that are involved in regulating endothelial cell phenotype and VE-cadherin expression remain largely unknown. In this thesis the transcriptional repressor Slug is demonstrated to be expressed by the mesenchymal cells and a subset of endocardial cells of the atrioventricular canal and outflowtract during cardiac morphogenesis. Slug is demonstrated to be required for cardiac development through its role in regulating EndMT in the cardiac cushion. Data presented in Chapter 6 further suggests that Slug-deficiency in the mouse is compensated for by a increase in Snail expression after embryonic day (E) 9.5, which restores EndMT in the cardiac cushions. Additionally, the Notch pathway, via CSL, directly binds and regulates expression of the Slug promoter, while a close Slug family member, Snail is regulated by the TGFB pathway in endothelial cells. While Notch does not directly regulate Snail expression, Notch and TGFB act synergistically to regulate Snail expression in endothelial cells. It is further demonstrated that Slug is required for Notch mediated EndMT, binds to and represses the VE-cadherin promoter, and induces a motile phenotype. Collectively the data demonstrate that Notch signaling directly regulates Slug, but not Snail, expression and that the combined expression of Slug and Snail are required for cardiac cushion morphogenesis. / Medicine, Faculty of / Medicine, Department of / Experimental Medicine, Division of / Graduate

Fusion genes caused by chromosomal rearrangements are a common and important feature in haematological malignancies, but have until recently been seen as unimportant in epithelial cancers. The discovery of recurrent fusion genes in prostate and lung cancer suggests that fusion genes may play an important role in epithelial carcinogenesis, and that they have been previously under-reported due to the difficulties of cytogenetic analysis of solid tumours. In particular, breast cancers often have complex, highly rearranged karyotypes which have proved difficult to analyse using classical cytogenetic techniques. The aim of this project was to search for fusion genes in breast cancer by using high-resolution mapping of chromosome rearrangements in breast cancer cell lines. Mapping the chromosome rearrangements was initially done using high-resolution DNA microarrays and fluorescence in-situ hybridisation, but moved to high-throughput sequencing as it became available. Interesting candidate genes identified from the mapped chromosome rearrangements were investigated on a larger set of cell lines and primary tumours. The complete karyotypes of two breast cancer cell lines were constructed using a combination of microarrays, fluorescence microscopy, and high-throughput sequencing. A number of potential fusion genes were identified in these two cell lines. Although no expressed fusion genes were found, the complete karyotypes gave insight into the number and mechanisms of chromosome rearrangement in breast cancer, and identified interesting candidate genes which may be of importance in tumourigenesis. Two genes which were fused in other breast cancer cell lines, BCAS3 and ODZ4, were disrupted by chromosome rearrangements and identified as interesting candidate genes in tumorigenesis. A bioinformatic pipeline to process high-throughput sequencing data was set up and validated, and shown to more accurately predict fusion genes than other methods, and can be used to investigate further cell lines and tumours for recurrent fusion genes. The pipeline was used to analyse data from 3 other breast cancer cell lines and predict chromosomal rearrangements and fusion genes, several of which were found to be expressed. Of the fusions predicted in the cell line ZR-75-30, 7 expressed fusion genes were identified, and may have functional significance in breast cancer.

Under suitable conditions an unfolded protein molecule refolds spontaneously into a precise three-dimensional shape (known as its conformation), which is fixed by the chemical structure of the molecule. Can the relationship between the shape and chemical sequence of a protein ever be fully understood? I still cannot answer this question, which has troubled me for several years. It may seam surprising that one can work on a problem that could be insoluble. My reasons are as follows: Firstly, the folding problem is the most fundamental problem of theoretical molecular biology. Life is ordered in space and time: a body is an ordered aggregate of cells; a cell is an ordered aggregate of macromolecules; and a macromolecule is an ordered aggregate of atoms. The building blocks of living matter are highly ordered protein molecules, which also use their precise shapes to catalyse the biochemical reactions that make life dynamic. Proteins are to life sciences what the atom is to physics and chemistry. When physical laws determine how the thousands of atoms of a protein fold from a random coil into a precise three dimensional arrangement, dead matter comes to life. The complex order that is found in proteins is unknown in physics or chemistry; it is as if a motor car assembled itself when all the pieces were joined in a line and shaken about. Using electronic computers it may be possible to mimic nature and calculate how the amino acid sequence determines the folded shape of a protein. Apart from many practical uses, a general solution to the folding problem would be fundamentally important.