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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
91

Mitochondrial inheritance and cell cycle regulation in Saccharomyces cerevisiae

Crider, David Garry January 2012 (has links)
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.
92

Characterization of Gf a Drosophila trimeric G protein alpha subunit

Quibria, Naureen January 2012 (has links)
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.
93

Obesity and Aggressive Prostate Cancer: Bias and Biology

McBride, Russell Bailey January 2012 (has links)
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.
94

The Role of Hiwi in Stem Cell Maintenance and Sarcomagenesis

Siddiqi, Sara January 2012 (has links)
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.
95

Alcohol alters the expression of Soluble N-Ethylmaleimide-Sensitive Factor Attachment Protein Receptors (SNAREs) and spontaneous γ-Aminobutyric Acid (GABA) release...

Varodayan, Florence Prabha January 2013 (has links)
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.
96

The Intrinsic Caspase Death Pathway in Stroke Neurodegeneration

Nsikan, Akpan E. January 2013 (has links)
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.
97

The Intrinsic Caspase Death Pathway in Stroke Neurodegeneration

Akpan, Nsikan January 2013 (has links)
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.
98

Microfluidic Selection and Applications of Aptamers

Hilton, John January 2013 (has links)
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.
99

Elucidating the Biological Function of PWWP-Domain Containing Protein Complexes

Reddy, Bharat January 2013 (has links)
In eukaryotes, nuclear DNA is folded with histone proteins in the form of chromatin, and this structure plays a critical role in multiple biological processes, including development, DNA damage repair, and aging. Post-translational modifications of histones, such as acetylation and methylation, are essential regulators of chromatin structure and function. Consequently, misregulation of these post-translational modifications has causal roles in numerous diseases, including multiple types of cancer. However, the mechanisms that direct the localization of histone-modifying enzymes and regulate their activities are not fully understood. This thesis focuses on the characterization of a class of proteins containing the PWWP domain. This domain is often present in chromatin proteins, and it is predicted to recognize methylated histones based on structural analysis. Here, we have demonstrated that the PWWP domain proteins in fission yeast bind to methylated histones. Additionally, we have shown that proteins with this domain form complexes with diverse histone modifying activities to regulate multiple cellular processes. Methylation of histone H4 lysine 20 (H4K20me) is essential for the activation of a DNA damage checkpoint, which blocks the progression of cell cycle to allow sufficient time for DNA damage repair. In fission yeast, only the enzyme Set9 catalyzes H4K20me, and the mechanisms that underlie the regulation of this protein are poorly characterized. Here we showed that Set9 forms a stable complex with the PWWP domain containing protein Pdp1. The PWWP domain of Pdp1 binds to H4K20me, demonstrating that the PWWP domain constitutes a novel methyl-lysine recognition motif. Moreover, the binding of PWWP domain to methylated H4K20 plays a critical role in regulating Set9 activity, thus facilitating higher degrees of H4K20 methylation. Histone H3K9 methylation is critical for heterochromatin assembly in diverse organisms. The RNAi pathway is required for the formation of pericentric heterochromatin, although the exact role that RNAi plays in heterochromatin assembly remains a topic of significant debate. We discovered that a separate PWWP domain protein, Pdp3, forms a stable complex with the H3K14 histone acetyltransferase Mst2. Interestingly eliminating the enzymatic activity of the Pdp3-Mst2 complex obviates the requirement for the RNAi machinery in pericentric heterochromatin functions. Furthermore we demonstrated that one function of RNAi during heterochromatin assembly is to exclude the Pdp3-Mst2 complex, thus maintaining low levels of RNA polymerase II localization to pericentric regions in order to retain the parental histone modification patterns for its passage through generations. Altogether, my results have firmly demonstrated that the PWWP domain is a novel class of methyl-lysine binding motifs. Moreover, in fission yeast the PWWP domain proteins form stable complexes with other chromatin proteins to regulate diverse cellular processes.
100

Analysis of The RING Domain And BRCT Repeats of BRCA1

Reid, Latarsha January 2011 (has links)
Mutations within BRCA1 often contribute to breast cancer susceptibility. Many of these mutations cluster within two highly conserved regions that may be important for BRCA1's functions: the RING domain and the BRCT repeats. BRCA1's RING domain has E3 ubiquitin ligase activity, which is greatly enhanced when it forms a heterodimer with Bard1. This region is of particular interest because it displays the only known enzymatic activity of BRCA1. The BRCT repeats have phosphopeptide binding activity, which is necessary for BRCA1's interaction with DNA repair proteins BACH1, ABRAXAS, and CtIP. To test the importance of these two domains we generated cell lines and mouse models with point mutations that either eliminate the E3 ligase activity of Brca1 while maintaining its interaction with Bard1 (I26A) or eliminate its phosphopeptide binding activity (M1717R). We found that the E3 ubiquitin ligase activity of Brca1 is dispensable for its role in cell viability, embryonic development, double strand break repair, and tumor suppression. Interestingly, we were unsuccessful at generating homozygous BRCT mutant ES cells and homozygous M1717R Brca1 MEFs displayed a proliferation defect, spontaneous chromosomal aberrations, and centrosomal amplification. Our data shows that the BRCT repeats are crucial for BRCA1's role in DNA repair because BRCT mutant MEFs do not recruit Brca1 or Rad51 to IR induced DNA damage sites, they have a defect in homology directed repair, and M1717R Brca1 is not hyperphosphorylated in response to DNA damage in these cells. We were able to generate homozygous M1717R Brca1 mice, but with a very low frequency (<1%). All Brca1M17171R/M1717R mice produced thus far have been males and they are sterile. Our analysis indicates that the fertility defect is not due to a defect in meiosis. Introduction of the M1717R mutation in a conditional mammary or pancreatic tumor model also reduces the tumor latency to the same degree as the introduction of a Brca1 null mutation. Therefore our data shows that BRCA1 carries out the majority of its functions through its BRCT repeats.

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