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Characterization of Gf a Drosophila trimeric G protein alpha subunitQuibria, 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.
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Novel Platforms for Cardiovascular RepairMartens, Timothy January 2011 (has links)
Collectively the experiments discussed in this thesis represent an attempt to improve upon current cardiovascular cell therapy practices. The first chapter focuses on the regenerative efficacy of immunoselected mesenchymal progenitor cells in the setting of acute myocardial ischemia. Particular attention is paid to the mechanisms underlying the functional recovery attributed to administration of these cells as well as potential drawbacks to their delivery as a liquid suspension. Given the poor engraftment witnessed with this formulation, the second chapter focuses on the use of in situ polymerization of hydrogels to enhance immediate cell retention and survival. This approach is further developed in chapter three with particular attention paid to the constraints imposed by currently used percutaneous delivery catheters. The fourth and final chapter combines the use of hydrogels with a novel biomimetic scaffold in an attempt to further improve upon cell retention and survival as well as provide a post-implant environment that provides physical and biologic characteristics similar to native myocardium.
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Integrin-Linked Kinase, ECM Composition, and Substrate Rigidity Regulate Focal Adhesion - Actin Coupling, Modulating Survival, Proliferation and Migration: Towards a Biophysical Cancer BiomarkerChander, Ashok Coil January 2012 (has links)
The extracellular matrix (ECM) has been implicated in numerous physiological and pathogenic processes. Integrins are thought to be the primary receptors that cells use to transduce biochemical and physical signals from the ECM. Integrin - ligand binding is specific for ECM molecules and is regulated by specific protein-protein interactions that further regulate downstream cellular activity such as motility, survival, growth, and proliferation. Termed outside-in signaling, the engagement of integrins results in protein recruitment to sites of cell - ECM contacts known as focal adhesions. Focal adhesions (FAs) are central to cell spreading, motility, survival and growth and serve as both physical linkages between the ECM and cytoskeleton as well as signaling centers for a cell on 2D substrates. Termed focal adhesion-actin coupling, FAs physically link the cytoskeleton with the ECM via actin binding proteins and are involved in mechanically coupling the cell to the ECM. To date, FAs' signaling properties and FA- actin coupling have been unrelated and independent mechanisms. This study provides data that suggests the amount, or level, of focal adhesion coupling in addition to regulating traction force generation, motility events and the rigidity response, also regulates the amount of biochemical signaling towards survival, growth and proliferation. First, via a knockout cell line system I demonstrate that Integrin-Linked Kinase is involved in coupling Beta1 integrins to collagen and FAs. I then demonstrate that lack of coupling results in altered rigidity sensing, defects in spreading of the cytoplasm, lower force generation and collagen contraction, as well as altered localization and activation of MAP kinases. Specifically, when ILK null cells were plated on collagen coated glass they were unable to reinforce Beta1 integrin mediated interactions nor spread their cytoplasm or undergo contractile activity. In contrast, when ILK null cells were plated on fibronectin coated glass, ILK null cells progressed to the contractile phase of spreading and then retracted their adhesions, losing the ability to stabilize late stage Beta1 integrin mediated fibronectin interactions. Moreover, I demonstrate that actin retrograde flow regulates the localization and modification state of FA signaling molecules that regulate survival, growth, and proliferation. Secondly, via changing ECM composition and rigidity of the substrate, I demonstrate that the engagement of both Beta1 and Beta3 integrins via collagen type I and fibronectin increases focal adhesion size, focal adhesion-actin coupling, and activation of signaling molecules involved in translation, survival, growth, and proliferation. This investigation presents data that supports the idea that the degree of focal adhesion mediated ECM-cytoskeletal coupling correlates with the ability to activate signaling molecules and suggests a model in which focal adhesion-actin coupling regulates the localization and modification state of scaffold and signaling proteins that result in the modulation of survival, growth and proliferation. Finally, I propose the use of an experimentally derived metric to describe ECM-FA-actin coupling and present preliminary data that the proposed metric can also be used as a biomarker for specific disease states such as cancer.
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The Role of Hiwi in Stem Cell Maintenance and SarcomagenesisSiddiqi, 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.
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Mitochondrial Inheritance and Function in the Lifespan Control of Budding YeastMcFaline Figueroa, Jose Ricardo January 2012 (has links)
Mitochondria are essential organelles that cannot be synthesized de novo and must be inherited by daughter cells. During cell division, mitochondria align along the mother- daughter axis of the dividing cell, exhibit bidirectional poleward movement and are anchored at the cell poles. Mitochondria anchored at the bud tip and thus destined to be inherited by the daughter cell, show markers of increased fitness, lower superoxide burden and less oxidizing mitochondria, while less fit mitochondria are retained in the mother. In this work, the mechanism for anchorage of fit mitochondria to the bud tip and its effect on yeast lifespan determination are presented. Mitochondria at the bud tip are associated with cortical ER (cER) sheets underlying the plasma membrane. Mmr1p, a member of the DSL1 family of tethering proteins, mediates anchorage of mitochondria at the bud tip by binding to both mitochondria and cER at this site. A conserved protein phosphatase, Ptc1p, regulates mitochondrial anchorage by dephosphorylation of Mmr1p. Mitochondrial fitness decreases as a function of age, yet retention of less fit mitochondria occurs to the same extent in young and older cells. Disruption of mitochondrial anchorage at the bud tip by deletion of MMR1 results in a severe lifespan anomaly, such that some cells have drastically reduced lifespan and markers of aged cells, while others show increased lifespan and markers of young cells. Loss of anchorage also leads to defects in mitochondrial quality control during inheritance and mitochondrial fitness correlates to the aging phenotypes observed in mmr1-delta cells. These findings support the model that the mitochondrial inheritance machinery promotes retention of lower-functioning mitochondria in mother cells and that this process contributes to both mother- daughter age asymmetry and age-associated declines in cellular fitness.
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Analyzing Genomic Studies and a Screen for Genes that Suppress Information Loss During DNA Damage RepairPierce, Steven January 2013 (has links)
This thesis is concerned with the means by which cells preserve genetic information and, in particular, with the competition between different DNA damage responses. DNA is continuously damaged and imperfect repair can have extremely detrimental effects. Double strand breaks are the most severe form of damage and can be repaired in several different ways or countered by other cellular responses. DNA context is important; cell cycle, chromosomal structure, and sequence all can make DSBs more likely or more problematic to repair. Saccharomyces cerevisiae is very resilient to DSBs and primarily uses a process called homologous recombination to repair DNA damage. To further our understanding of how S. cerevisiae efficiently uses homologous recombination, and thereby minimizes genetic degradation, I performed a screen for genes affecting this process. >In devising this study, I set out to quickly quantify the contribution of every non-essential yeast gene to suppressing genetic rearrangements and deletions at a single locus. Before I began I did not fully appreciate how variable and contingent this type of recombination phenotype could be. Accounting for the complex and changing recombination baseline across many tests became a significant effort unto itself. The requirements of the experimental protocols precluded the use of traditional recombination rate calculation methods. Searching for the means to compare the utility of normalizations and to validate my results, I sought general approaches for analyzing genome wide screen data and coordinating interpretation with existing knowledge. It was advantageous during this study to develop novel analysis tools. The second chapter describes one of these tools we developed, a technique called CLIK (Cutoff Linked to Interaction Knowledge). CLIK uses preexisting biological information to evaluate screen performance and to empirically define a significance threshold. This technique was used to analyze the screen results described in chapter three. The screen in chapter three represents the primary work of this dissertation. Its purpose was to identify genes and biological processes important for the suppression of recombination between DNA tandem repeats in yeast. By searching for gene deletion strains that show an increase in non-conservative single strand annealing, I found that many genetic backgrounds could induce altered recombination frequencies, with genes involved in DNA repair, mitochondria structural and ribosomal, and chromatin remodeling genes being most important for minimizing the loss of genetic information by HR. In addition, I found that the remodeling complex INO80 subunits, ARP8 and IES5 are significant in suppressing SSA.
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Stressed Astrocytes: Insights on the Pathology of Alexander DiseaseGuilfoyle, Eileen M. January 2013 (has links)
Alexander disease (AxD) is a rare and fatal neurological disorder caused by mutations in the gene that encodes glial fibrillary acidic protein (GFAP), an intermediate filament protein found in astrocytes in the central nervous system. The clinical presentations of AxD are diverse, ranging from onset in infancy to onset in early adulthood, and include seizures, psychomotor retardation, ataxia, and a variety of neurological signs related to abnormal brain stem function. The defining neuropathological hallmark is the presence of cytoplasmic, proteinaceous inclusions called Rosenthal fibers in astrocytes. Although GFAP expression is astrocytic, AxD patients also show de/dysmyelination and variable amounts of neuronal loss, most severely in infantile-onset patients. Astrocytes undergo severe morphological changes, beyond that of typical reactive astrocytes, and develop several forms of cell stress. However, how stressed astrocytes cause the loss of myelin in this disease is unknown. In this work I have conducted a largely immunohistological investigation of AxD patient tissue, model mice, and primary astrocytes cultured from the AxD model mice, focusing on factors that might provide insight into the pathological manifestations of AxD and paying particular attention to those factors which might contribute to de/dysmyelination. To gain insight on the morphological transformation of astrocytes in AxD, I analyzed GFAP in the hippocampus of the most severely affected AxD mouse. Astrocytes in these mice lose their star-like shape, and become hypertrophic and often multinucleated. They accumulate large amounts of GFAP. Subsequent study of primary cultured astrocytes from AxD mice revealed that these cells have perinuclear inclusions of GFAP surrounded by displaced microtubules and displaced Golgi. I next investigated another mechanism of stress that may affect astrocyte function in AxD. Work in our lab and others' has demonstrated proteasomal inhibition in AxD astrocytes. Because the unfolded protein response in the endoplasmic reticulum (ER) can be enacted by proteasomal inhibition, I examined the immunohistochemical expression of two proteins commonly increased under conditions of ER stress. We found BIP/Grp78, an ER chaperone, increased in AxD patient astrocytes and model mice. Additionally, the CCAAT enhancer binding protein homologous protein (CHOP) was expressed by a small subset of astrocytes in the AxD mouse hippocampus, unveiling ER stress as a potential contributory factor in AxD pathology. Work in other labs has found iron in astrocytes in AxD model mice. To further elucidate mechanisms of cellular stress in AxD, I conducted an immunohistochemical analysis of iron and several regulatory proteins in AxD patients and found, by enhanced Perls' staining, Fe3+ in Rosenthal fibers and iron and ferritin accumulated in astrocytes. This finding is in marked contrast to what one sees in the normal CNS, with little staining of astrocytes, and easily detectable staining of oligodendrocytes. Finally, I examined the localization of the cell surface glycoprotein CD44, along with several related proteins, including its ligand hyaluronan. I found CD44 protein expression greatly increased in the white matter, cortex and hippocampus of AxD patients and in the hippocampus of AxD mice. Additionally, through use of a biotinylated hyaluronan binding protein, I found abnormally high levels of hyaluronan in the hippocampus of AxD mice in the same areas where increases in CD44 were found. Work elsewhere has found CD44 and hyaluronan in other disorders that affect myelination, and experiments have revealed an inhibitory effect of hyaluronan on oligodendrocyte development and myelination. The studies in this thesis contribute novel stressors to the list of those that impact astrocytes in AxD and, in particular, suggest the accumulation of iron in astrocytes as potentially important to the pathological manifestations of AxD. Additionally, my research has revealed dramatic increases in the expression of CD44 in AxD astrocytes which, in conjunction with widespread increases in hyaluronan, may be critical to understanding the mechanisms underlying the de/dysmyelination that occur in this disease.
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Notch deficiency leads to arteriovenous malformations and altered pericyte functionKolfer, Natalie January 2013 (has links)
During angiogenesis, nascent blood vessels sprout from pre-existing vasculature and recruit pericytes to induce maturation and vessel quiescence. Perictyes are associated with small vessels and capillaries where they share the basement membrane with the endothelium to provide vascular support. Pericytes are a critical component of the blood-brain barrier and regulate endothelial cell proliferation, vessel diameter, and vascular permeability. Endothelial cells express Notch1, whereas pericytes express both Notch1 and Notch3. Here we show that Notch signaling is essential for pericyte function. Through genetic manipulation and pharmacological tools we show that Notch regulates pericyte recruitment and pericyte/endothelial cell interactions. Notch1^+/-;Notch3^-/- mutant mice display decreased pericyte coverage and altered pericyte association with the retinal vascular plexus. Notch deficiency is associated with vascular anomalies where Notch1^+/-;Notch3-/- mice display retinal arteriovenous malformations (AVM) characterized by dilated vessels, vascular tangles and arteriovenous shunts that are similar to human brain AVMs. Disruption of pericyte/endothelial cell association is accompanied by an increase in vascular density, venule enlargement, and increased vascular permeability observed prior to AVM formation. In the ovary, we show that Jagged is essential for pericyte association with the endothelium where inhibition of Jagged-specific Notch activation results in luteal vessel dilation and hemorrhaging following ovarian hyperstimulation. By in vitro analysis of cultured pericytes we show that Notch1 and Notch3 induce plated derived growth factor receptor-β (PDGFR-β) expression to regulate cell migration. These findings expand the role for Notch in angiogenesis by demonstrating that Notch signaling in pericytes is essential for vascular development and function.
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Building a Genetic System in Yeast to Search for High Affinity Proteins in Sequence SpaceMerguerian, Matthew Douglas January 2013 (has links)
Binding proteins (both natural and man-made) have the ability to bind tightly and specifically with small molecules and other biopolymers. Binding proteins can function as human therapeutics, diagnostics, and as tools for scientific research. Given the wide range of potential applications, there is great interest in both academia and industry to develop methods for discovering novel binders. An important step in discovering new binders is called affinity maturation, when an initial hit that shows some ability to bind the target is further mutated in additional steps to improve binding affinity, specificity, solubility, pharmacokinetic profile. Ideally, the methods for affinity maturation would allow for cookbook protocols, be successful for arbitrary targets of interest, and be minimally resource intensive. Although traditional methods for affinity maturation have had some stunning successes over the past, the state of the field is still far from this ideal. In Chapter 1, I discuss the current state of the protein engineering field. In Chapter 2, I discuss the use of phenotypic selection for yeast-display protein binders, and test these systems on simple loop libraries. In Chapter 3, I construct a genetic system in yeast that can mutate a protein loop via homologous recombination, and test its recombination function. In Chapter 4, I mate libraries that target two different loops, and run FACS on the combinatorial libraries. In Chapter 5, I discuss future directions for the project.
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Trimethoprim-Based Chemical Tags for High Resolution Live Cell ImagingJing, Chaoran January 2013 (has links)
Over the past decade chemical tags have been developed to complement the use of fluorescent proteins in live cell imaging. Chemical tags retain the specificity of protein labeling achieved with fluorescent proteins through genetic encoding, but provide smaller, more robust tags and modular use of organic fluorophores with high photon-output and tailored functionalities. The trimethoprim-based chemical tag (TMP-tag) was initially developed based on the high affinity interaction between E.coli dihydrofolatereductase and the antibiotic trimethoprim and subsequently rendered covalent and fluorogenic via proximity-induced protein labeling reactions. To date, the TMP-tag is one of the few chemical tags that enable intracellular protein labeling and high-resolution live cell imaging. In this dissertation I first focused on the development of the fluorogenic TMP-tag that enabled high resolution live cell imaging with redeced background noise. Then some efforts to further improve the fluorogenic TMP-tag were described. Furthermore, I worked with collaborators to apply the TMP-tag technology to study the dynamics of the focal adhesion complex at the single-molecule level. Finally, I also tried to develop a short peptide tag for protein labeling with minimal perturbation of the function and dynamics of the target molecule. Together, these studies exemplified the maturation of the TMP-tag technology from the proof-of-principle stage to real-world biological applications.
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