The production of retroviral enzymes requires a translational recoding event which subverts normal decoding, either by direct suppression of termination with the insertion of an amino acid at a stop codon (readthrough), or by an alteration of the reading frame of the mRNA (frameshift). It has been determined that retroviral readthrough and frameshift require cis-acting factors in the mRNA to stimulate recoding on the eukaryotic ribosome. Here we investigate the affects of trans acting factors on recoding, primarily in the context of the MoMLV gag-pol junction. We report the effects of a host protein, Large Ribosomal Protein Four (RPL4), on the efficiency of recoding. Using a dual luciferase reporter assay, we show that transfection of cells with an RPL4 cDNA expression construct enhances recoding efficiency in a dose-dependent manner. The increase in the frequency of recoding can be more than 2-fold, adequate to disrupt normal viral production. This effect is cell line specific, and appears to be distinct to RPL4 among ribosomal proteins. The RPL4 increase occurs with both retroviral readthrough and frameshift sequences, and even at other viral readthrough regions that do not involve RNA secondary structures. We show that RPL4 effects are negated by release factor over-expression, and that RPL4 will increase readthrough above the levels of a hyperactive mutant and in addition to G418. When cotransfected with Moloney murine leukemia provirus, the RPL4-mediated increase in readthrough reduces the amount of virus released. We also examined the effects of aminoglycoside drugs and the small molecule PTC124 on readthrough of the MoMLV gag-pol junction. We show that G418, paromomycin and PTC124 increase readthrough of our MoMLV reporter in a dose dependent manner in 293A cells. These drugs reduce viral replication, as measured by a recombinant transducing virus assay. We further examine G418 and paromomycin in an in-vitro system; readthrough is increased to higher levels than those seen in vivo. G418 displays deleterious effects on cell viability and overall translation. Paromomycin does not appear as toxic, suggesting differences in interactions by which these drugs enhance readthrough.

Movement and positional control of mitochondria and other organelles are coordinated with cell cycle progression in the budding yeast, Saccharomyces cerevisiae. Recent studies have revealed a checkpoint that inhibits cytokinesis when there are severe defects in mitochondrial inheritance. An established checkpoint signaling pathway, the mitotic exit network (MEN), participates in this process. Here, we describe mitochondrial motility during inheritance in budding yeast, emerging evidence for mitochondrial quality control during inheritance, and organelle inheritance checkpoints for mitochondria and other organelles.

In the morphogenesis of tissue development, how coordination of patterning and growth achieve the correct organ size and shape is a principal question in biology. Efficient orchestrating mechanisms are required to achieve this and cells have developed sophisticated systems for reception and interpretation of the multitude of extracellular stimuli to which they are exposed. Plasma membrane receptors play a key role in the transmission of such signals. G-protein coupled receptors (GPCRs) are the largest class of cell surface receptors that respond to an enormous diversity of extracellular stimuli, and are critical mediators of cellular signal transduction in eukaryotic organisms. Signaling through GPCRs has been well characterized in many biological contexts. While they are a major class of signal transducers, there are not many defined instances where GPCRs have been implicated in the process of development to date. The Drosophila wing provides an ideal model system to elucidate and address the role of GPCRs in development, as its growth is regulated by a small number of conserved signaling pathways. In my thesis work, I address the role of a trimeric G alpha protein in Drosophila, Gαf, and what part it may play in development. In particular, I explore the role of Gαf as an alpha subunit of a trimeric complex, to determine what heptahelical receptors might act as its cognate receptor.

Obesity is suspected to be a risk factor for aggressive PC due to its associations with altered circulating levels of metabolic and sex steroid hormones involved in prostate development as well as oncogenesis. However, the current observational evidence linking obesity to aggressive PC is inconsistent or conflicting, and there is growing concern that much of the heterogeneity across studies may be the result of obesity interfering with PC screening, diagnosis, and treatment. We performed a critical review of studies analyzing the association between anthropomorphic measures and overall PC risk, as well as risk of aggressive disease, and illustrate how unique aspects of PC diagnosis and treatment render its risk factor associations unusually susceptible to selection biases which are largely unabated by conventional statistical adjustment. Using a counterfactual framework to describe the selection processes that give rise to these biases, we demonstrate instances in which the use of marginal structural models (MSM) and inverse probability weighting (IPW) may be able to address such biases. Using data collected on a series of patients referred for prostate biopsy, and found to have PC, we examined the association between BMI, clinical and pathological characteristics. We found evidence of differential receipt of radical prostatectomy (RP) by BMI category, and history of obesity which, in the latter case, partially attenuated the association between obesity and high grade biopsy. After multivariate statistical adjustment and IPW, obesity was associated with increased odds of higher pathological grade and stage after RP, associations which were not apparent without the use of IPW. We also examined the association between one's exposure to history of obesity (measured at age 20, 40 and near the time of diagnosis), and found that men with a BMI ≥30 at all three measures had an increased odds of high pathologic stage (≥pT3), tumor volume >30mm3, and positive surgical margins, compared to never obese. In the multivariate models which did not use inverse probability weights, only the association between chronic obesity and high pathological grade reached statistical significance. These findings suggest that treatment selection factors caused a bias toward the null in our estimates of the associations between history of obesity and adverse tumor characteristics, and would have substantively altered the overall findings of the study. We then conducted multiplex immunoflorescence immunohistochemistry on tissue microarrays (TMA) made from representative cores of tumor tissue from RP specimens. Using a semi-automated, florescence microscopy and imaging technique, we measured nuclear expression or androgen receptor (AR), epithelial insulin like growth factor I receptor (IGF-IR), and proliferation marker Ki67, in 357 cases who received a RP. We then tested for associations between patient history of obesity and other demographic and clinical characteristics. Expression of AR and Ki67 were positively associated with tumor grade and stage, while Ki67 and IGF-IR were associated with tumor volume in excess of 30mm3. We also found an inverse association between IGF-IR and tumor grade. We did not, however, find that history of obesity was significantly associated with expression of any of the biomarkers. Thus we have found no evidence that the association between chronic obesity and aggressive disease is mediated by differential expression of androgen or IGF-I receptor, or greater tumor proliferation (Ki67). As researchers continue to understand the underlying causes of aggressive PC and pursue the goal of personalized medicine, studies such as these become increasingly important as they have the potential to reduce the biases inherent in these dataset and explore important interactions between risk factors, and tumor phenotypes that may point the way to new preventive and treatment.

This dissertation serves to analyze the molecular and cellular signaling mechanisms by which aldose reductase (AR) contributes to ischemia/reperfusion (I/R) injury, specifically in the myocardium. It provides a comprehensive overview of the pathophysiology associated with acute coronary syndromes, the mechanisms of normal myocardial substrate metabolism, and how metabolism is altered under ischemic conditions. Specific chapters in this dissertation aim to elucidate whether AR pathway mediated I/R injury is linked to changes in glycogen synthase kinase 3β (GSK3β) phosphorylation, and whether an increased flux via AR and resultant I/R injury is mediated by changes in autophagy.

Sarcomas are cancers of connective tissues, such as bone, adipose and cartilage, and are thought to arise from the aberrant development of the mesenchyme. As such, mesenchymal stem cells are thought to be the cell of origin for sarcomas. Genetic or epigenetic lesions at particular points during the differentiation of a mesenchymal stem cell into its terminal mesenchymal cell type are able to give rise to specific subtypes of sarcomas. Recently, a number of reports have identified elevated expression of the human Piwi homolog--called Hiwi--in a variety of human cancers, including gastric cancer, pancreatic cancer, gliomas and, most relevant for this dissertation, sarcomas. In sarcomas, Hiwi is highly expressed and elevated Hiwi prognosticates shorter patient survival. Hiwi is the human homolog of the Piwi family of proteins, which are members of the Paz-Piwi Doman (PPD) family. During normal development, Piwis are thought to maintain stem cells of the germline, and indeed their expression is limited to early development and to the adult germline. Piwis are thought to maintain stem cells in the germline with small RNA partners, called piwi-interacting RNAs (piRNAs). More specifically, Piwi/piRNA complexes in the germline are thought to maintain transposon silencing, and thus ensure genomic stability. A detailed mechanism by with Piwis suppress transposon migration in the germline remains an area of active investigation, but is thought to occur via DNA methylation of transposon regions. In this way, Piwis are critical for maintenance of genomic integrity of germline stem cells during normal development. Thus, the finding that Piwis are elevated in human cancers is directly in conflict with its known role in ensuring genomic stability during development. Piwi homologs are critical for maintenance of germline stem cells during development but aberrant Hiwi expression has also been identified in all cancers examined, including in sarcomas. A potential connection between mesenchymal stem cells, sarcomas and Hiwi remains unexplored. Moreover, the role of Hiwi in sarcomas is unknown. In the studies presented here, we demonstrate that over-expressing Hiwi in mesenchymal stem cells inhibits their differentiation in vitro and generates sarcomas in vivo. Secondly, transgenic mice expressing Hiwi (mesodermally-restricted) develop sarcomas. Conversely, inducible down-regulation of Hiwi in human sarcomas inhibits growth and re-establishes differentiation. These data reveal that Hiwi is directly tumorigenic. We have also identified the presence of piRNAs in our Hiwi-expressing models. We further show that DNA methylation correlates with Hiwi expression and that cyclin-dependent kinase inhibitor (CDKI) tumor suppressor genes are silenced upon Hiwi over-expression. Moreover, Hiwi's tumorigenic effects are reversible using DNA de-methylating agents. These studies reveal for the first time not only a novel oncogenic role for Hiwi as a driver of tumorigenesis, but also suggest that the use of epigenetic agents may be clinically beneficial for treatment of tumors that express Hiwi. Additionally, our data showing that Hiwi-associated DNA hyper-methylation with subsequent genetic and epigenetic changes favoring a tumorigenic state reconciles the conundrum of how Hiwi may act appropriately to promote genomic integrity during early development (via transposon silencing) and inappropriately in adult tissues with subsequent tumorigenesis.

Many synapses within the central nervous system are highly sensitive and responsive to ethanol. Although the regulation of postsynaptic receptors by alcohol is well studied, the mechanisms underlying the presynaptic effects of alcohol to alter neurotransmitter release remain relatively unexplored. This dissertation addresses whether alcohol-induced changes in transcriptional activity can promote synaptic vesicle fusion and therefore, neurotransmitter release. To identify a transcriptional pathway by which ethanol can regulate neurotransmitter release, we first investigated the effects of acute alcohol on the expression of genes encoding for synaptic vesicle fusion machinery proteins that form the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) complex. The proteins in this complex reside on the vesicle membrane (synaptotagmin 1 and synaptobrevin/vesicle-associated membrane protein, which is also known as VAMP) and the plasma membrane (syntaxin 1 and synaptosomal associated protein of 25 kDa, which is also known as SNAP-25), and their interactions within the SNARE complex trigger vesicle fusion and neurotransmitter release. We found that ethanol treatment of mouse cortical neurons increased the mRNA and protein expression levels of a subset of SNARE complex proteins, including synaptotagmin 1 (Syt1) and one of the isoforms of synaptobrevin, VAMP2, but not the other isoform, VAMP1. The gene induction of Syt1 and Vamp2 by alcohol occurs via activation of the transcription factor heat shock factor 1 (HSF1), while HSF1 transcriptional activity had no effect on Vamp1 mRNA levels. We then investigated whether ethanol altered neurotransmitter release in cortical neurons, using whole-cell voltage clamp electrophysiology. We found that alcohol increased gamma-aminobutyric acid (GABA) release via HSF1, but had no effect on glutamatergic synaptic vesicle fusion. Collectively, these data indicate that alcohol induction of HSF1 transcriptional activity triggers a specific coordinated adaptation in GABAergic presynaptic terminals that ultimately results in increased GABA release. This molecular mechanism could explain some of the transient changes in synaptic function that occur after alcohol exposure, and may underlie some of the enduring effects of chronic alcohol drinking on local circuitry.

Stroke has been a major source of morbidity and mortality for centuries. Eight-five percent of all strokes are ischemic in nature, meaning they are caused by the occlusion of a major cerebral artery. Despite extensive research to develop effective treatments for ischemic stroke, therapeutic options remain limited. Apoptosis (also termed "programmed cell death") is a process by which a stressed or damaged cell commits "suicide". In stroke, runaway apoptosis contributes to stroke neurodegeneration and neurological decline for days to weeks after disease onset. Cysteine-ASPartic proteASEs (caspases) are key mediators of apoptosis that are activated in distinct molecular pathways, but their impact in stroke is poorly defined. Direct evidence for caspase activation in stroke and the functional relevance of this activity has not been previously characterized. For this dissertation, we developed an unbiased technique for in vivo trapping of active caspases in rodent models of ischemic stroke. We isolated active caspase-9 as a principal contributor to ischemic neurodegeneration in rodents (Rattus norvegicus and Mus musculus). Caspase-9 is the initiator caspase for the intrinsic cell death pathway. Intranasal delivery of a novel, cell membrane-penetrating inhibitor for caspase-9 confirmed the pathogenic relevance of caspase-9 activity in stroke. Caspase-9 inhibition provided neurofunctional protection and established caspase-6 as its downstream target. Caspase-6 is an effector caspase and a member of the intrinsic death pathway that has never been implicated in stroke until now. Coincidentally, we discovered that caspase-6 is specifically activated within the axonal compartment. The temporal and spatial pattern of activation demonstrates that neuronal caspase-9 activity induces caspase-6 activation, which mediates axonal loss in the early stages of stroke (+/- 24 hours). We developed a novel inhibitor for caspase-6, based on a catalytically inactive clone, which demonstrated neuroprotective and axoprotective efficacy against ischemia. Collectively, these results assert that selective inhibition of caspase-9 and caspase-6 is an effective translational strategy for stroke. The impact of caspase activity is not restricted to neuronal death, as caspases can exacerbate inflammation and alter glial function. Thus, caspases are logical therapeutic targets for stroke. However, they have never been clinically evaluated due to a paucity of ideal drug candidates. This dissertation outlines fresh insights into the mechanisms of stroke neurodegeneration and offers novel caspase-based therapeutic strategies for clinical evaluation.

Stroke has been a major source of morbidity and mortality for centuries. Eight-five percent of all strokes are ischemic in nature, meaning they are caused by the occlusion of a major cerebral artery. Despite extensive research to develop effective treatments for ischemic stroke, therapeutic options remain limited. Apoptosis (also termed "programmed cell death") is a process by which a stressed or damaged cell commits "suicide". In stroke, runaway apoptosis contributes to stroke neurodegeneration and neurological decline for days to weeks after disease onset. Cysteine-ASPartic proteASEs (caspases) are key mediators of apoptosis that are activated in distinct molecular pathways, but their impact in stroke is poorly defined. Direct evidence for caspase activation in stroke and the functional relevance of this activity has not been previously characterized. For this dissertation, we developed an unbiased technique for in vivo trapping of active caspases in rodent models of ischemic stroke. We isolated active caspase-9 as a principal contributor to ischemic neurodegeneration in rodents (Rattus norvegicus & Mus musculus). Caspase-9 is the initiator caspase for the intrinsic cell death pathway. Intranasal delivery of a novel, cell membrane-penetrating inhibitor for caspase-9 confirmed the pathogenic relevance of caspase-9 activity in stroke. Caspase-9 inhibition provided neurofunctional protection and established caspase-6 as its downstream target. Caspase-6 is an effector caspase and a member of the intrinsic death pathway that has never been implicated in stroke until now. Coincidentally, we discovered that caspase-6 is specifically activated within the axonal compartment. The temporal and spatial pattern of activation demonstrates that neuronal caspase-9 activity induces caspase-6 activation, which mediates axonal loss in the early stages of stroke (<24 hours). We developed a novel inhibitor for caspase-6, based on a catalytically inactive clone, which demonstrated neuroprotective and axoprotective efficacy against ischemia. Collectively, these results assert that selective inhibition of caspase-9 and caspase-6 is an effective translational strategy for stroke. The impact of caspase activity is not restricted to neuronal death, as caspases can exacerbate inflammation and alter glial function. Thus, caspases are logical therapeutic targets for stroke. However, they have never been clinically evaluated due to a paucity of ideal drug candidates. This dissertation outlines fresh insights into the mechanisms of stroke neurodegeneration and offers novel caspase-based therapeutic strategies for clinical evaluation.

BioMEMS technology has the potential to increase the efficiency of conventional biological and medical protocols, by reducing their consumption of time and resources. Through more efficient surface-based chemical reactions and automation of tedious manual processes, orders of magnitude increases in efficiency across a number of metrics can be achieved by shifting conventional medical and biological protocols to the microscale domain. The SELEX process, by which aptamer sequences are selected via isolation from randomized libraries, is a time-consuming and resource-intensive protocol which is being performed with increasing frequency in both academic and private sector laboratories. Conventional approaches using macroscale technology cannot meet the current demand for selection of new aptamer sequences, as they require months of work and liters of expensive reagents. Microscale approaches to the SELEX process have been receiving attention in recent years due to their initial successes in reducing the time and reagents necessary to find aptamers. In particular, microscale "selection" or partitioning of weakly bound sequences from aptamer candidates, and on-chip integration of the protocol have separately been explored as approaches to scaling and improving SELEX. Initial results have shown that this technology can reduce resource requirements for SELEX by at least an order of magnitude. In this dissertation, a new approach to on-chip SELEX is developed which integrates highly efficient microfluidic selection and on-chip integration of the entire protocol. As a result, further reductions in processing time and reagent requirements can be realized. A demonstration of aptamer capabilities is first achieved via the development of a microfluidic aptasensor for cocaine, which utilizes aptamer-coated microbeads and fluorescent detection. Secondly, a technology necessary for on-chip integration of SELEX is developed: a novel bead-based polymerase chain reaction (PCR) protocol which vastly simplifies procedures for the capture and resuspension of ssDNA in solution. This protocol is then integrated on-chip with bead-based partitioning of weakly bound sequences to develop a microchip which performs temperature-specific isolation of aptamer sequences from a randomized library. Finally, this approach is further developed into a microfluidic SELEX chip which is capable of performing multiple rounds of temperature-specific SELEX. The novel bead-based protocol is shown to efficiently isolate target-binding sequences from a random library in a fraction of the time previously reported. As a result, this research provides a schematic for the development of highly efficient, integrated microfluidic SELEX devices. Such devices have the potential to impact a variety of fields including medical diagnostics, drug detection, and aptamer-based therapeutics.