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Title: 2B4 IS A CHECKPOINT MOLECULE FOR iNKT CELL ANTI-TUMOR RESPONSE
Creator: Bahal, Devika Naresh
Date: 2022
Collection: Electronic Theses & Dissertations
Description: Invariant natural killer T (iNKT) cells are robust cytotoxic effectors and immune modulators, which makes them ideal candidates for cancer immunotherapy. However, the use of iNKTs for cellular therapy against cancer has been limited due to their transient response in pre-clinical trials. Although TCR-CD1d interactions are generally required for iNKT cell cytotoxicity, the receptors and signaling mechanisms that co-operate with the TCR to promote maximal anti-tumor responses are poorly...
Show moreInvariant natural killer T (iNKT) cells are robust cytotoxic effectors and immune modulators, which makes them ideal candidates for cancer immunotherapy. However, the use of iNKTs for cellular therapy against cancer has been limited due to their transient response in pre-clinical trials. Although TCR-CD1d interactions are generally required for iNKT cell cytotoxicity, the receptors and signaling mechanisms that co-operate with the TCR to promote maximal anti-tumor responses are poorly understood. Therefore, elucidating the mechanisms that regulate anti-tumor responses is critical for the development of effective iNKT-based therapies. Our efforts have shown that 2B4, a SLAM receptor, when expressed on iNKTs reduces their cytotoxic response against lymphoma cells. Surprisingly, 2B4 is not expressed on resting iNKTs but gets rapidly upregulated via stimulation through the TCR. 2B4 has two isoforms, which are splice variants of each other, of which the inhibitory long form is predominantly expressed in activated iNKTs. Our data show that 2B4 is a checkpoint molecule and has an inhibitory role in iNKT cell cytotoxicity. Indeed, when we overexpressed 2B4 in an iNKT cell hybridoma, the killing capacity of the iNKT cell line was abrogated. Moreover, 2B4 can be converted to a potent activating receptor by swapping its intracellular domains with proline motifs, which drastically augments tumor cell lysis. Taken together, this study highlights the important role of 2B4 in iNKT cell cytolysis and broadens the knowledge of immunoregulatory receptors in iNKT cells for future applications in cancer therapy.
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Title: Biochemical analysis of the chloroplast division proteins FtsZ1 and FtsZ2
Creator: Olson, Bradley Jesse Stanford Carnahan
Date: 2008
Collection: Electronic Theses & Dissertations

Title: Biochemical analysis of transformation-sensitive alterations in the substratum associated material of chicken embryo fibroblasts
Creator: Blenis, John
Date: 1983
Collection: Electronic Theses & Dissertations

Title: Biomolecular engineering of siRNA therapeutics
Creator: Gredell, Joseph A.
Date: 2009
Collection: Electronic Theses & Dissertations

Title: Cytology and seed set studies in the pansy, Viola tricolor Hortensis L
Creator: Emino, Everett Raymond, 1942-
Date: 1967
Collection: Electronic Theses & Dissertations

Title: Detecting intracellular metabolites and the resulting cell function by merging microfluidic and microtitre plate technologies
Creator: Tolan, Nicole Villiere
Date: 2009
Collection: Electronic Theses & Dissertations

Title: Dietary manipulation of natural killer cell biology through refeeding of previously calorically restricted mice
Creator: Clinthorne, Jonathan F.
Date: 2013
Collection: Electronic Theses & Dissertations
Description: The dynamic role of natural killer (NK) cells in immunology has been demonstrated in numerous fashions, proving they are much more than "natural killers". However, NK cells are perhaps still the least well understood lymphocyte, due to their relatively low abundance and the limited transgenic models available for study. An innate immune cell that plays a critical role in providing early immunity against viral infections and cancers, NK cells are becoming increasingly recognized for shaping...
Show moreThe dynamic role of natural killer (NK) cells in immunology has been demonstrated in numerous fashions, proving they are much more than "natural killers". However, NK cells are perhaps still the least well understood lymphocyte, due to their relatively low abundance and the limited transgenic models available for study. An innate immune cell that plays a critical role in providing early immunity against viral infections and cancers, NK cells are becoming increasingly recognized for shaping and directing immune responses. Accounting for approximately 5-25% of peripheral blood mononuclear cells in humans and 5-10% of lymphocytes in murine circulation, NK cells have very similar functional attributes in both species, making mice an ideal model system for the study of NK cell biology. Studies in mice have revealed the critical importance of NK cells in providing tumor surveillance as well as early protection from viral infections. Preclinical studies have underscored the utility of using NK cells as an immunotherapeutic technique to combat various cancers, while NK cell function is often associated with positive or negative outcomes in various disease states. Furthermore, various lifestyle factors have been found to positively or negatively influence NK cell function. Among these lifestyle factors, diet has gained notoriety as being capable of influencing the homeostasis and function of NK cells. However, the mechanisms by which diet influences NK cells are not fully understood, highlighting the need for a better understanding of the molecular and cellular mechanisms by which diet influences NK cell development. Thus, our laboratory has extensively studied the effects of the restriction of energy intake, or caloric restriction (CR) on NK cell function and biology at the organismal, cellular, and molecular level. Here we describe a series of experiments investigating the role of energy intake on immunity to influenza virus, with a focus on NK cells. We describe a series of studies that identify the specific changes to NK cells induced by CR, both beneficial and potentially damaging. In these experiments we show that CR results in fewer NK cells with a mature phenotype, and that expression of transcription factors critical for NK cell maturation are reduced by CR. We also demonstrate that thymic derived NK cells are present in normal numbers in CR mice and have enhanced function. In a series of experiments demonstrating the intricate relationship between immunity and metabolism we show how refeeding of CR mice restores NK cell homeostasis and function, both before, and during influenza infection. Using in vitro techniques combined with ex vivo analysis of metabolic signaling pathways, we provide potential mechanisms by which CR impairs NK cell maturation. These studies serve to highlight the critical role of optimal nutrition in maintaining NK cell homeostasis and function. To our knowledge, this dissertation is the first data presented that clearly details the effects of CR on NK cell development and homeostasis, as well as the molecular and biochemical pathways mediating this effect.
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Title: EXPRESSION AND ROLES OF BLASTOCYST LINEAGE-DETERMING GENES DURING SOMATIC CELL REPROGRAMMING
Creator: Moauro, Alexandra
Date: 2022
Collection: Electronic Theses & Dissertations
Description: In order to properly use stem cells, it is important that we first understand how these cells are establish and maintained. One of the most widely used stem cells are induced pluripotent stem cells (iPSCs) which provide great therapeutic promise and a novel source of ethical stem cells for research models. iPSCs are created by overexpression Oct4, Sox2, Klf4 and c-Myc (OSKM) in a somatic cell. As studies have sought to improve reprogramming efficiency and develop the most embryonically...
Show moreIn order to properly use stem cells, it is important that we first understand how these cells are establish and maintained. One of the most widely used stem cells are induced pluripotent stem cells (iPSCs) which provide great therapeutic promise and a novel source of ethical stem cells for research models. iPSCs are created by overexpression Oct4, Sox2, Klf4 and c-Myc (OSKM) in a somatic cell. As studies have sought to improve reprogramming efficiency and develop the most embryonically identical stem cells, our lab has uncovered that OSKM is not a specific cocktail for pluripotency formation. Instead OSKM induces additional cell fates including the formation of a multipotent stem cell termed induced extraembryonic endoderm stem (iXEN) cells. This raises the question as to how two distinct stem cell types arise in parallel. Interestingly, in embryo development we observe the same pluripotent and multipotent extraembryonic endoderm lineages form in parallel. Using our knowledge of normal embryo development, I set out to identify what blastocyst lineage markers can help us identify early iPSC and iXEN colonies as they start to form and mature. Of these markers, we observed that endogenous OCT4 is expressed in both iXEN and iPSC colonies. Based on the expression pattern of the key embryonic transcription factor, OCT4, we further focused on how this transcription factor may have a dual role in establishing iPSC and iXEN fates. Lastly, we altered the reprogramming cocktail using additional embryonic transcription factors to determine how these factors affect the propensity for pluripotency or extraembryonic endoderm fate.
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Title: Engineering of the small cytosolic retinoid binding proteins into pH-sensing probes and novel fluorescent protein tags
Creator: Berbasova, Tetyana
Date: 2014
Collection: Electronic Theses & Dissertations
Description: Reprogramming the biological systems to perform a process other than that dictated by nature illustrates the power of the modern synthetic biology. Protein engineering represents one of the research areas in this field. Crystal structure guided mutagenesis studies allow for a rational protein redesign. Utilizing this de novo approach, Cellular Retinoic Acid Binding Protein II (CRABPII) has been reengineered into a Schiff base forming protein. The reaction is performed deep inside the protein...
Show moreReprogramming the biological systems to perform a process other than that dictated by nature illustrates the power of the modern synthetic biology. Protein engineering represents one of the research areas in this field. Crystal structure guided mutagenesis studies allow for a rational protein redesign. Utilizing this de novo approach, Cellular Retinoic Acid Binding Protein II (CRABPII) has been reengineered into a Schiff base forming protein. The reaction is performed deep inside the protein cavity and fascilitates a complete ligand closure from the aqueous media.An initial aim of this project was to design a functioning rhodopsin mimic from CRABPII for the wavelength regulation studies. The strategy combined the principles we learned in the earlier work on CRABPII and human Cellular Retinol Binding Protein II (hCRBPII). The rational modifications of the CRABPII binding cavity provides the effective charge delocalization along the protein-embedded all- trans-retinal-PSB that leads to the regulation of absorption. Reengineered CRABPII is capable of spanning the visible spectrum in the range of 474 - 640 nm. Additionally, CRABPII mutants exhibit an extraordinary range of the iminiumpKa values, ranging from 1.8 to 8.1. The latter phenomena allowed for thechromophoric pH-probe design utilizing the protein-bound chromophoric aldehyde, an isoform of Vitamin A. Moreover, the fusion of the two selected pigments provides a ratiometric protein based pH-sensor.The aldehydes other than retinal might yield alternative pH-sensing ligands with the engineered CRABPII and hCRBPII mutants. With fluorescence being the most widely used detection method in biology, fluorescent and fluorogenic retinal analogs are the primary targets in the ligand redesign. Synthetic julolidine retinal analog meets most of the criteria for an effective fluorogenic probe with pH- sensing properties upon PSB formation. In the protein-bound form this chromophore accounts for the imine-iminium equilibrium with changes in fluorescence intensity. Only iminium yields a light emitting pigment. Moreover, the wavelength shift in response to the pH changes provided the platform for a single protein ratiometric pH-probe design. This pH-dependent shift of absorption is a result of the more effective charge delocalization along the chromophore after protonation of the anionic carboxylate residue in the iminium region.Merocyanine retinal analog shows bright fluorescence and penetrates cells easily. These are valuable criteria for live-cell imaging applications. Optimization of the reaction half-life between hCRBPII mutants and merocyanine retinal analog provided an hCRBPII-tag with instantaneous red fluorescence. Designed method comprises a unique fluorescence recovery after the photobleaching when the cells are treated again with merocyanine.
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Title: From skin to neurons : examining variations in reprogramming efficiency
Creator: Keaton, Sarah A.
Date: 2013
Collection: Electronic Theses & Dissertations
Description: Cellular reprogramming is a newly emerging field with promising clinical applications. The ability to generate non-dividing crucial cell types from rapidly proliferating cell types, the potential to heal diseased people who do not have many treatment options, being able to bypass immune rejection, and avoid invasive surgery has captured the media's attention. However, there have been disparities in the efficiency of reprogramming and these needs to be addressed before cellular reprogramming...
Show moreCellular reprogramming is a newly emerging field with promising clinical applications. The ability to generate non-dividing crucial cell types from rapidly proliferating cell types, the potential to heal diseased people who do not have many treatment options, being able to bypass immune rejection, and avoid invasive surgery has captured the media's attention. However, there have been disparities in the efficiency of reprogramming and these needs to be addressed before cellular reprogramming can be applicable in a clinical setting. To better understand the variations of cellular reprogramming, human and mouse fibroblasts were converted into induced neural cells in an attempt to unveil the impact of disease state, tissue origin and genetics. The experimental results indicate reprogramming efficiency was reproducible within a primary fibroblast line however there was a dramatic difference between lines even from an isogenic source. Testing a larger number of fibroblast lines, even lines with the identical genetic backgrounds and tissue origins, is likely the most direct means of improving reprogramming efficiency and enabling this procedure to be available for therapeutic use.
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Title: Impact of ploidy on morphological variation in Arizona phlox, Phlox amabilis (Polemoniaceae)
Creator: Chansler, Matthew Thomas
Date: 2015
Collection: Electronic Theses & Dissertations
Description: Polyploidy is an important factor in the evolution and ecology of flowering plants. A better understanding of the kind and degree of morphological differentiation among ploidy levels within a species can help explain further how polyploidy affects biodiversity. How widespread is the impact of ploidy across the phenotype of a species? Which aspects of morphology vary, and do they vary consistently? How does ploidy relate to overall morphological diversity? Do ploidy levels have detectable...
Show morePolyploidy is an important factor in the evolution and ecology of flowering plants. A better understanding of the kind and degree of morphological differentiation among ploidy levels within a species can help explain further how polyploidy affects biodiversity. How widespread is the impact of ploidy across the phenotype of a species? Which aspects of morphology vary, and do they vary consistently? How does ploidy relate to overall morphological diversity? Do ploidy levels have detectable phenotypic profiles? Finally, are there morphological differences between populations, potentially due to environment or evolutionary changes since formation apparent in natural populations? I assessed morphological variation within Arizona phlox, Phlox amabilis. This species of conservation concern is endemic to Arizona, and prior work has detected diploid, tetraploid, and hexaploid populations. I sampled 11 populations of P. amabilis, covering a large portion of the species’ range. A wide array of morphological features, including characters that described cell size, overall habit, leaf dimensions, and floral dimensions, were measured for up to 25 plants at each population. Significant differences were detected in 15 out of 27 characters using mixed GLM. A large amount of overall morphological variation is explained by the differences between ploidy levels, and each ploidy level can be described by a specific multivariate phenotype with 95% accuracy. Finally, although overall structuring was influenced by ploidy, differences among populations still contributed a high degree of variation in the morphospace of Phlox amabilis. This morphological assessment will be integrated with ecological and genetic data to build a more complete understanding of the interplay between these factors and ploidy in Phlox amabilis.
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Title: In vivo analysis of Arabidopsis FtsZ isoforms
Creator: Schmitz, Aaron James
Date: 2011
Collection: Electronic Theses & Dissertations
Description: Chloroplasts are organelles derived from the ancient endosymbiosis between a cyanobacterium and a primitive eukaryote. These organelles are essential for algae and plants for their many functions, including photosynthesis, biosynthesis of a wide array of essential molecules, and reduction of sulfur and nitrogen. Chloroplasts cannot form de novo and their numbers are maintained through the process of binary fission. This division process requires several genes originally encoding bacterial...
Show moreChloroplasts are organelles derived from the ancient endosymbiosis between a cyanobacterium and a primitive eukaryote. These organelles are essential for algae and plants for their many functions, including photosynthesis, biosynthesis of a wide array of essential molecules, and reduction of sulfur and nitrogen. Chloroplasts cannot form de novo and their numbers are maintained through the process of binary fission. This division process requires several genes originally encoding bacterial cell division factors. FtsZ is one of these genes. FtsZ is present in most bacteria and encodes a cytoskeletal protein that is structurally similar to tubulin. FtsZ polymerizes into a ring structure (Z-ring) at mid-cell prior to cell division in bacteria and also forms a Z-ring within the stroma of chloroplasts and other plastids types in plants. However, in the green lineage FtsZ has split into two phylogenetically-distinct families called FtsZ1 and FtsZ2. Both families have been shown to colocalize to the Z-ring and interact with themselves and each other. Chloroplast division, like cell division in bacteria, is sensitive to small decreases or increases in FtsZ protein levels, which result in division defects and fewer, enlarged chloroplasts. Therefore, distinguishing the relationship between the encoded FtsZ protein isoforms based upon ftsZ null or overexpression mutants is not feasible. The focus of this work was to resolve the functional relationship and distinguishing features of FtsZ isoforms in Arabidopsis - our chloroplast division model. Stable transformation of Arabidopsis ftsZ mutants followed by careful examination of complemented chloroplast division defects and FtsZ protein levels was the predominant approach for these studies.FtsZ2-1 complemented chloroplast division defects of plants lacking FtsZ2-2, and vice versa, near the previously quantified protein levels expected for complete FtsZ2 substitution. Therefore, I conclude that the two AtFtsZ2 isoforms are functionally redundant. Subsequently, I determined that FtsZ1 cannot substitute for FtsZ2 protein, and vice versa, since chloroplast division defects remained. In a related study, though both FtsZ1 and FtsZ2 are required for maintenance of chloroplast numbers, the generation of fully viable Arabidopsis plants lacking FtsZ, yet maintaining one chloroplast per cell, indicated that an FtsZ-independent mode of chloroplast partitioning exists in higher plants.FtsZ1 and FtsZ2 proteins diverge significantly at their C-termini where only the FtsZ2 family has a conserved motif found in bacteria. This motif is critical for the interaction with Z-ring promoting factors in bacteria and with ARC6 in plants. By swapping the C-termini and substituting the resulting chimeric FtsZ proteins in vivo, I demonstrate that neither C-terminus fully defines the unique functions of FtsZ1 or FtsZ2. Though I also show that the C-termini are required for the full function of each FtsZ family, these results indicate that other regions contribute significantly to FtsZ function. Related experiments also indicate that ARC3, a negative regulator of Z-ring formation, interacts with FtsZ2 in addition to FtsZ1. Together these results have clarified FtsZ functional relationships and laid significant groundwork for future analyses of FtsZ and their regulators.
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Title: Jasmonate regulation of defense responses in tomato (Lycopersicon esculentum)
Creator: Liu, Guanghui
Date: 2004
Collection: Electronic Theses & Dissertations

Title: Nanoengineered tissue scaffolds for regenerative medicine in neural cell systems
Creator: Tiryaki, Volkan Mujdat
Date: 2013
Collection: Electronic Theses & Dissertations
Description: Central nervous system (CNS) injuries present one of the most challenging problems. Regeneration in the mammal CNS is often limited because the injured axons cannot regenerate beyond the lesion. Implantation of a scaffolding material is one of the possible approaches to this problem. Recent implantations by our collaborative research group using electrospun polyamide nanofibrillar scaffolds have shown promising results in vitro and in vivo. The physical properties of the tissue scaffolds have...
Show moreCentral nervous system (CNS) injuries present one of the most challenging problems. Regeneration in the mammal CNS is often limited because the injured axons cannot regenerate beyond the lesion. Implantation of a scaffolding material is one of the possible approaches to this problem. Recent implantations by our collaborative research group using electrospun polyamide nanofibrillar scaffolds have shown promising results in vitro and in vivo. The physical properties of the tissue scaffolds have been neglected for many years, and it has only recently been recognized that significant aspects include nanophysical properties such as nanopatterning, surface roughness, local elasticity, surface polarity, surface charge, and growth factor presentation as well as the better-known biochemical cues.The properties of: surface polarity, surface roughness, local elasticity and local work of adhesion were investigated in this thesis. The physical and nanophysical properties of the cell culture environments were evaluated using contact angle and atomic force microscopy (AFM) measurements. A new capability, scanning probe recognition microscopy (SPRM), was also used to characterize the surface roughness of nanofibrillar scaffolds. The corresponding morphological and protein expression responses of rat model cerebral cortical astrocytes to the polyamide nanofibrillar scaffolds versus comparative culture surfaces were investigated by AFM and immunocytochemistry. Astrocyte morphological responses were imaged using AFM and phalloidin staining for F-actin. Activation of the corresponding Rho GTPase regulators was investigated using immunolabeling with Cdc42, Rac1, and RhoA. The results supported the hypothesis that the extracellular environment can trigger preferential activation of members of the Rho GTPase family, with demonstrable morphological consequences for cerebral cortical astrocytes. Astrocytes have a special role in the formation of the glial scar in response to traumatic injury. The glial scar biomechanically and biochemically blocks axon regeneration, resulting in paralysis. Astrocytes involved in glial scar formation become reactive, with development of specific morphologies and inhibitory protein expressions. Dibutyryl cyclic adenosine monophosphate (dBcAMP) was used to induce astrocyte reactivity. The directive importance of nanophysical properties for the morphological and protein expression responses of dBcAMP-stimulated cerebral cortical astrocytes was investigated by immunocytochemistry, Western blotting, and AFM. Nanofibrillar scaffold properties were shown to reduce immunoreactivity responses, while PLL Aclar properties were shown to induce responses reminiscent of glial scar formation. Comparison of the responses for dBcAMP-treated reactive-like and untreated astrocytes indicated that the most influential directive nanophysical cues may differ in wound-healing versus untreated situations.Finally, a new cell shape index (CSI) analysis system was developed using volumetric AFM height images of cells cultured on different substrates. The new CSI revealed quantitative cell spreading information not included in the conventional CSI. The system includes a floating feature selection algorithm for cell segmentation that uses a total of 28 different textural features derived from two models: the gray level co-occurance matrix and local statistics texture features. The quantitative morphometry of untreated and dBcAMP-treated cerebral cortical astrocytes was investigated using the new and conventional CSI, and the results showed that quantitative astrocyte spreading and stellation behavior was induced by variations in nanophysical properties.
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Title: On the isolation of basal bodies from Tetrahymena pyriformis
Creator: Linney, Elwood A.
Date: 1967
Collection: Electronic Theses & Dissertations

Title: Peroxisome associated proteolytic processes in Arabidopsis
Creator: Kaur, Navneet
Date: 2013
Collection: Electronic Theses & Dissertations
Description: Peroxisomes are small single-membrane-bounded organelles that play key roles in development and metabolism in most eukaryotic organisms. Peroxisome functions encompass ß-oxidation of fatty acids, detoxification of hydrogen peroxide and the metabolism of a range of biochemical compounds including glyoxylate, glycolate, urate, polyamines, benzoate, phylloquinone, bile acids and plasmalogen to name a few, as well as synthesis of plant hormones such as jasmonic acid (JA) and indole-3-butyric acid...
Show morePeroxisomes are small single-membrane-bounded organelles that play key roles in development and metabolism in most eukaryotic organisms. Peroxisome functions encompass ß-oxidation of fatty acids, detoxification of hydrogen peroxide and the metabolism of a range of biochemical compounds including glyoxylate, glycolate, urate, polyamines, benzoate, phylloquinone, bile acids and plasmalogen to name a few, as well as synthesis of plant hormones such as jasmonic acid (JA) and indole-3-butyric acid (IBA). Peroxisomes exhibit great functional diversity largely because of a plastic proteome that varies greatly depending on the environmental condition, tissue type or developmental stage of the specific organism. Proteins in peroxisomes are imported by the actions of conserved machinery that includes several proteins known as peroxins that are important for peroxisome biogenesis. Proteins destined for the peroxisome matrix contain peroxisome targeting signal (PTS) enabling their recognition by cytosolic receptor proteins that transport them to the peroxisome. Although we have an increased understanding of how peroxisomal protein import is accomplished, we know little about how proteins in peroxisomes are degraded. In this research, I provide evidence that RING domains of three peroxisomal membrane proteins AtPEX2, AtPEX10 and AtPEX12 have E3 ligase activity. I further show that AtPEX2 specifically interacts with two homologous ubiquitin receptor proteins, DSK2a and DS2Kb that have been implicated as adapters linking ubiquitination and 26S proteasome-based degradation events. DSK2 amiRNA lines lacked obvious plant growth phenotypes and were not compromised in peroxisome functions, suggesting that functional redundancies exist among ubiquitin receptor proteins. My results indicate that Arabidopsis RING peroxins and DSK2s can together form a peroxisome membrane associated degradation system. I also explored the role of a predicted ovarian tumor-like cysteine protease (OCP1) in Arabidopsis. OCP1 was found to be a novel plant specific peroxisomal protein with a canonical C-terminal PTS1 and a novel N-terminal PTS2. Analysis of mutant lines revealed that OCP1 influences IBA metabolism in the peroxisome. Further, ocp1 mutants show retarded degradation of two transiently expressed seedling peroxisome enzymes, isocitrate lyase (ICL) and malate synthase (MS) suggesting that OCP1 has a role in the timely removal of ICL from seedling peroxisomes. In summary, these studies add significantly to our knowledge of proteolysis in plant peroxisomes and open up several avenues for future investigations that may have ramifications in agriculture and biomedical applications.
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Title: Rat colon epithelial cells : isolation, cultivation, and benzo(a)pyrene metabolism
Creator: Skrypec, Daniel J.
Date: 1981
Collection: Electronic Theses & Dissertations

Title: Reprogramming to the nervous system : a computational and candidate gene approach
Creator: Alicea, Bradly John
Date: 2013
Collection: Electronic Theses & Dissertations
Description: The creation of stem-like cells, neuronal cells, and skeletal muscle fibers from a generic somatic precursor phenotype has many potential applications. These uses range from cell therapy to disease modeling. The enabling methodology for these applications is known as direct cellular reprogramming. While the biological underpinnings of cellular reprogramming go back to the work of Gurdon and other developmental biologists, the direct approach is a rather recent development. Therefore, our...
Show moreThe creation of stem-like cells, neuronal cells, and skeletal muscle fibers from a generic somatic precursor phenotype has many potential applications. These uses range from cell therapy to disease modeling. The enabling methodology for these applications is known as direct cellular reprogramming. While the biological underpinnings of cellular reprogramming go back to the work of Gurdon and other developmental biologists, the direct approach is a rather recent development. Therefore, our understanding of the reprogramming process is largely based on isolated findings and interesting results. A true synthesis, particularly from a systems perspective, is lacking. In this dissertation, I will attempt to build toward an intellectual synthesis of direct reprogramming by critically examining four types of phenotypic conversion that result in production of nervous system components: induced pluripotency (iPS), induced neuronal (iN), induced skeletal muscle (iSM), and induced cardiomyocyte (iCM). Since potential applications range from tools for basic science to disease modeling and bionic technologies, the need for a common context is essential.This intellectual synthesis will be defined through several research endeavors. The first investigation introduces a set of experiments in which multiple fibroblast cell lines are converted to two terminal phenotypes: iN and iSM. The efficiency and infectability of cells subjected to each reprogramming regimen are then compared both statistically and quantitatively. This set of experiments also resulted in the development of novel analytical methods for measuring reprogramming efficiency and infectability. The second investigation features a critical review and statistical analysis of iPS reprogramming, specifically when compared to indirect reprogramming (SCNT-ES) and related stem-like cells. The third investigation is a review and theoretical synthesis which stakes out new directions in our understanding of the direct reprogramming process, including recent computational modeling endeavors and results from the iPS, iN and induced cardiomyocyte (iCM) experiments. To further unify the outcomes of these studies, additional results related to Chapter 2 and directions for future research will be presented. The additional results will allow for further interpretation and insight into the role of diversity in direct reprogramming. These future directions include both experimental approaches (a technique called mechanism disruption) and computational approaches (preliminary results for an agent-based population-level approximation of direct reprogramming). The insights provided here will hopefully provide a framework for theoretical development and a guide for traditional biologists and systems biologists alike.
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Title: SIGNALING MECHANISMS OF PULMONARY ARTERIAL HYPERTENSION
Creator: Ji, Yajing
Date: 2022
Collection: Electronic Theses & Dissertations
Description: Pulmonary arterial hypertension (PAH) is a severe and life-threatening disease that is characterized by elevated pulmonary blood pressure. A challenge in treating PAH is that while the current generation of therapeutics alleviate symptoms, they fail to target the underlying causes of the disease. Initially it was thought that PAH is caused by increased pulmonary vasoconstriction; it is now understood that PAH mainly results from remodeling of the pulmonary vasculature. Further...
Show morePulmonary arterial hypertension (PAH) is a severe and life-threatening disease that is characterized by elevated pulmonary blood pressure. A challenge in treating PAH is that while the current generation of therapeutics alleviate symptoms, they fail to target the underlying causes of the disease. Initially it was thought that PAH is caused by increased pulmonary vasoconstriction; it is now understood that PAH mainly results from remodeling of the pulmonary vasculature. Further characterization of the underlying mechanisms of PAH will identify newpharmacological targets to treat PAH. In this dissertation I seek to address this challenge from three distinct perspectives. In Chapter 2, I investigated the signaling network downstream of TGFβ and highlighted the MRTF/SRF pathway as potential therapeutical targets for PAH given its pivotal role regulating expression of contractile proteins in PASMCs. In Chapter 3, I aim to test whether TGFβ and the silencing of BMPR2, a member of the TGFβ family of receptors, contribute to the activation of lung fibroblasts in vitro. My results presented do not replicate the role of BMPR2 silencing found in other studies. This could be caused by the relatively short duration of BMPR2 silencing in our system. Finally, in Chapter 4, I perform a combined meta-analysis of several publicly available transcriptomic datasets of lung tissues from PAH patients. Using this approach, I identify PAH-associated signaling pathways, and chemical compounds which reverse a PAH-associated gene expression signature. My findings also suggest that while we bin PAH patients into various subtypes in the clinic, on a transcriptional level, PAH patients tend to group into distinct gene expression clusters without relying on their clinical subtype. These findings improve our understanding of PAH biology and also highlight several potential drug targets for PAH.
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Title: SULFATED AND SIALYLATED N-ACETYL-LACTOSAMINE AS BIOMARKER OF SUBPOPULATIONS OF PANCREATIC DUCTAL ADENOCARCINOMAS
Creator: Hsueh, Peter Yiping
Date: 2019
Collection: Electronic Theses & Dissertations
Description: The sialyl Lewis A (sLeA) glycan forms the basis of the CA19-9 blood test and is the current biomarker for pancreatic ductal adenocarcinoma (PDAC). However, it is not elevated in approximately 25% of PDAC patients and it also has difficulties in diagnosing early-stage PDAC. My overarching goal was focusing on improving precision of overall PDAC diagnostics. I hypothesized that other glycans within the Lewis blood group family besides sLeA are aberrantly increased in the subpopulation of PDAC...
Show moreThe sialyl Lewis A (sLeA) glycan forms the basis of the CA19-9 blood test and is the current biomarker for pancreatic ductal adenocarcinoma (PDAC). However, it is not elevated in approximately 25% of PDAC patients and it also has difficulties in diagnosing early-stage PDAC. My overarching goal was focusing on improving precision of overall PDAC diagnostics. I hypothesized that other glycans within the Lewis blood group family besides sLeA are aberrantly increased in the subpopulation of PDAC patients who do not secret sLeA into their blood. To test the hypothesis, two specific approaches were implemented in this study: 1) Profile an isomer of sLeA, named sialyl-Lewis X (sLeX), and glycans with fucosylated motifs in the plasma of sLeA-low PDAC patients using antibody and lectin microarray method; and 2) Test the sulfated and sialylated glycans derived from type 2 N-acetyl-lactosamine precursor in subpopulations of PDACs using a novel on-chip analysis method.In the first approach, I profiled the levels of multiple glycans and glycosylated mucins in plasma from two cohorts of 200 and 116 test subjects with PDACs and non-malignant disease patients. From these screens, I found significant increases in two categories of glycans: sialyl Lewis X variants, presented both in sulfated and non-sulfated forms, and the sialylated type 1 N-acetyl-lactosamine. These glycans are increased in distinct groups of PDAC patients and contribute to the improved accuracy of a biomarker panel.Thus, I concluded that detecting other glycans within the Lewis blood-group besides sLeA has the potential to improve diagnoses of PDAC patients.To further elucidate the structural nuances of sialyl Lewis X variants from initial screen, I developed a new assay called On-chip Glycan Modification and Probing and a complementary computational algorithm to accurately analyze novel sulfated and sialylated glycans from plasma of pancreatic cancer patients. In detailed structural information, I observed strong evidences of sulfated and sialylated type 2 N-acetyl-lactosamine glycans overexpressed in plasma of PDAC patients and pancreatic cancer cell lines, but not in the plasma of healthy people. In addition, the sulfated and sialylated type 2 N-acetyl-lactosamine glycans presented on a specific mucin, MUC5AC, was statistically associated (p < 0.001) with short time-to-progression of PDAC patients, but CA19-9 test was not. I concluded sulfated and sialylated type 2 N-acetyl-lactosamine glycans presented on MUC5AC were new serological biomarkers that could improve precision of current practices for diagnosis and prognosis of PDACs patients.
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