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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.

Structural characterization of primary cilia using accelerated piezoelectrically driven STED nanoscopy

Nathwani, Bhavik Bharat January 2012 (has links)
Primary cilia are non-motile, hair-like projections occurring on most mammalian cell types. They play essential roles in transduction of chemical and mechanical signals across the cell membrane. For example, primary cilia are able to transduce sonic hedgehog signals, necessary in embryonic development and adult stem cell functions. Recent work on primary cilia has demonstrated correlations between primary cilia morphology and its ability to sense/transduce signals. Several such studies have underscored the need for detailed study of morphology of primary cilia and structure-function mapping of its morphology with its ability to transduce signals. However, the size scale of the primary cilium makes it very challenging to extract biologically relevant morphometric features using conventional imaging techniques. The molecular architecture of the primary cilium is beyond the resolvability of conventional diffraction limited optical imaging techniques. Data from non-optical tools such as electron microscopy have been limited by the need for dehydration during sample prep. Advent of superresolution optical imaging approaches has only recently made it possible to probe primary cilia morphologically to study its structure in physiologically interesting environments. Signaling pathways regulated by primary cilia are critical to embryo development and organogenesis. Therefore, it would be interesting to study primary cilia both in somatic (adult) cells while simultaneously comparing and contrasting it with their occurrence on stem cells. Human induced pluripotent stem cell (hiPSC) reprogramming possesses enormous potential in stem cell research and disease modeling. Chemical and mechanical signaling has been implicated in maintenance of pluripotency of hiPSCs and their differentiation pathways toward various lineages, where primary cilia have been shown to play a critical role in mechano-chemical signaling across a wide spectrum of cell types. The functions of primary cilia in hiPSCs and their characteristic changes during the reprogramming process remain largely vague. Therefore, in order to study primary cilia morphology on both somatic cells as well as hiPSCs, we developed a superresolution nanoscopy system using the stimulated emission depletion (STED) technique with novel accelerated piezoelectric control (apSTED). This improved STED system achieved a reduction in photobleaching rates from ~80% to ~10% while maintaining superresolution, ~50 nm at the focal plane for biological samples. Subsequently, we focused on conducting comparative morphometric studies of primary cilia found on somatic cells and hiPSCs. Our work was the first to systematically demonstrate the existence of primary cilia on hiPSCs. Using quantitative PCR assays, we demonstrated high levels of expression of primary cilia signaling partners, such as Patched1, Smoothened, and members of Gli family. Comparative morphometric analysis revealed that the mean length of reprogrammed cells was shorter than those of parental human fibroblasts. Morphometric analyses revealed that reprogramming resulted in an increase in curvature of primary cilia from ~0.015 µm to 0.064 µm, indicating an underlying ~4-fold decrease in their rigidity, and a decrease in length of primary cilia from ~2.38 µm to ~1.45 µm. Furthermore, reprogramming resulted in fewer primary cilia displaying either kinked or punctated geometries. Custom-built software scripts were developed to extract and analyze superresolution apSTED imaging data collected on fibroblast primary cilia. Using apSTED, we were able to measure local variations in primary cilia curvature. A review of confocal data revealed that such variations in curvature were either completely missed or were significantly underestimated. We also utilized our technique to study macromolecular complexes within transition zone; a structure found at the base of primary cilia that plays a significant role in ciliogenesis and in maintaining structural integrity of primary cilia. Our data provides the first visualization of two important transition zone members, Tctn-2 and Cep290. We were able to demonstrate structural detail heretofore impenetrable to conventional imaging techniques. Furthermore, quantification of spatial distribution of these molecules, ~160 nm for Tctn-2 and ~180 nm for Cep290, provides evidence to indicate the relative positioning of these molecules within the transition zone. These studies highlight the advantages of using apSTED to study primary cilia and provide tools that could enable the deciphering of the architecture of the transition zone in primary cilia.

Molecular Mechanisms of Mitotic Spindle Assembly and Accurate Chromosome Segregation

Guo, Yige January 2013 (has links)
During the cell cycle, duplicated DNA in S phase is segregated, in the form of chromatids, into two daughter cells in mitosis. The accuracy of chromosome segregation is essential as two daughter cells have the same genetic contents as the mother cell. Two major mechanisms are utilized by the cell to ensure accurate chromosome segregation. First, interactions between the dynamic microtubules and kinetochores, the proteinaceous structures built on centromeres of mitotic chromosomes that act as the attachment site for microtubules, serve as major forces to position each pair of chromosomes to the metaphase plate. Secondly, a surveillance system, known as the mitotic checkpoint, put the anaphase onset on hold until each pair of sister chromosomes are aligned at the metaphase plate and appropriately attached with microtubule plus ends by kinetochores. In the first part (Chapter 2) of this thesis, I illustrate the role of the auto-phosphorylation of BubR1, a mitotic checkpoint protein, in kinetochore-microtubule attachment and the mitotic checkpoint. Using a phospho-specific antibody against the auto-phosphorylation site identified by mass spectrometry, I demonstrate that kinetochore-associated BubR1 phosphorylates itself in human cells in vivo and that this phosphorylation is dependent on its binding partner, the kinetochore-associated kinesin motor CENP-E. Studies using cells expressing a non-phosphorylatable BubR1 mutant revealed that the CENP-E-dependent BubR1 phosphorylation at unattached kinetochores is important for a full-strength mitotic checkpoint to prevent single chromosome loss. Furthermore, replacing endogenous BubR1 with the non-phosphorylatable BubR1 mutant or depletion of CENP-E, the BubR1 kinase activator, results in metaphase chromosome misalignment and increased incidents of syntelic attachments. Using indirect immunofluorescence, I have discovered a decreased level of Aurora B-mediated Ndc80 phosphorylation at the kinetochore of cells expressing the non-phosphorylatable BubR1 mutant, which might contribute to the alignment defect. Moreover, expressing a phosphomimetic BubR1 mutant substantially reduces the incidence of polar chromosomes in CENP-E-depleted cells, further supporting a signaling cascade function of CENP-E and BubR1 on the kinetochore. Thus, the state of CENP-E-dependent BubR1 auto-phosphorylation in response to spindle microtubule capture by CENP-E is important for kinetochore functions in achieving accurate chromosome segregation. In the second part (Chapter 3), my colleague and I demonstrate a novel mechanism of mitotic spindle assembly in Xenopus egg extracts and mammalian cells. I show that the MRN (Mre11, Rad50, and Nbs1) complex is required for metaphase chromosome alignment. Consistent with the result of my colleague using Xenopus egg extracts, disruption of MRN function by depleting Mre11 using an inducible shRNA system, or Mre11 inhibitor mirin, triggers a metaphase delay and disrupts the RCC1-dependent Ran-GTP gradient. Addition of mirin to mammalian cells reduces RCC1 association with mitotic chromosomes and changes the confirmation of RCC1. Thus, the MRN-CtIP pathway contributes to Ran-dependent mitotic spindle assembly by modulating RCC1 chromosome association. In summary, my novel findings have revealed a pair of molecular mechanisms not known previously, which are important to the mitosis field.

C-terminal lysines modulate Connexin32 turnover and its ability to suppress growth of Neuro-2a cell cultures

Alaei, Sarah Rose January 2013 (has links)
The extent of gap junction (GJ)-mediated coupling can be modulated through GJ channel gating. However, the amount of connexin protein available for incorporation into GJ, efficiency of channel assembly, trafficking to the cell surface, and disassembly also contribute to the regulation of cell-cell communication. In addition to their function in GJ, connexins also regulate a variety of physiological processes by forming hemichannels that are involved in paracrine signaling (Sanchez, Orellana et al. 2009) and through interactions with other proteins in the cytoplasm(Francis, Xu et al. 2011) and at the plasma membrane(Fowler, Akins et al. 2013). These other connexin functions are also likely to be influenced by the channel assembly dynamics, trafficking, and fast turnover of connexin proteins. The aim of this work was to determine if post-translational modifications, such as lysine acetylation, regulate connexin function through the fine-tuning of protein turnover or some other aspect of GJ dynamics. We chose to focus specifically on Cx32 for several reasons. Cx32 is an important regulator of neuronal myelination and loss of Cx32 GJ function is a common cause of the demyelinating neuropathy, Charcot-Marie-Tooth disease. Most studies addressing post-translational modifications of connexins focus on Cx43, which shares little sequence homology with Cx32 in the domains that are most-often subject to post-translational modification. We surmised that our results could be compared to what is known about Cx43 in order to determine if shared post-translational modifications regulate evolutionarily divergent connexins in similar ways. Here we show that Cx32 is an acetylated protein and that acetylated Cx32 is found in the cytoplasm and in the plasma membrane, where it is incorporated into GJ. For many proteins, acetylation has been implicated in pathways that modulate protein turnover(Caron, Boyault et al. 2005), thus we tested whether acetylation could regulate Cx32 protein level, resulting in the modulation of Cx32 functions. Our results demonstrate that acetylation is a positive regulator of Cx32 protein level, which increases the amount of Cx32 at the cell surface. Inhibition of the cytoplasmic deacetylase, HDAC6, results in hyperacetylation and accumulation of Cx32, which is dependent upon cytoplasmic C-terminal lysines. Mutational analysis revealed that these C-terminal lysines influence the ubiquitination and turnover rate of Cx32 protein. Comparison of the subcellular localization of WT Cx32 to that of mutants that either abolish acetylation sites while maintaining the original amino acid charge (K → R) or mimic constitutive acetylation (K → Q) suggests that acetylation does not simply alter lysine occupancy, thus preventing Cx32 ubiquitination and subsequent turnover. Instead, it seems likely that acetylation modulates protein-protein interactions that influence the amount of Cx32 in the plasma membrane and the role of Cx32 as a regulator of growth of cell cultures. K → Q Cx32 accumulates at the cell-surface more than WT Cx32, while K → R behavior resembles that of WT. Further, K → Q Cx32 suppresses the expansion of N2a cell cultures, whereas WT and K → R Cx32 do not. Interestingly, none of the mutations resulted in detectable alterations of cell-cell communication, suggesting that the suppression of cell culture growth observed when cells expressed the K → Q mutant may be independent of cell-cell communication. These results suggest that Cx32 acetylation is a positive regulator of Cx32 mediated suppression of proliferation or enhancement of pro-apoptotic signaling and provide rationale for future studies to determine which protein-protein interactions are modulated through Cx32 acetylation.

Adult Neural Stem Cells and Their Perivascular Niche

Crouch, Elizabeth January 2013 (has links)
Stem cells reside in specialized niches that support their selfrenewal and differentiation. A balance between intrinsic and extrinsic signals mediates stem cell quiescence, activation and proliferation. In the mammalian subventricular zone (SVZ), the stem cells are a subset of GFAP+ astrocytes. A quiescent pool of GFAP+ stem cell astrocytes generates activated (actively dividing) GFAP+EGFR+ stem cell astrocytes. These in turn generate EGFR+ transit amplifying cells, which give rise to neuroblasts that migrate to the olfactory bulb. In the SVZ niche, dividing cells localize next to blood vessels. SVZ stem cells and transit amplifying cells also directly contact blood vessels at sites that lack glial end feet and pericyte coverage, a feature unique to SVZ vasculature. Diffusible signals from transformed endothelial cell lines have been shown to increase survival, proliferation and neurogenic differentiation of SVZ neural stem cells and their progeny in vitro. However, the effect of primary endothelial cells is unknown. Furthermore, previous studies have not elucidated whether vascular signals from neurogenic and non-neurogenic regions are different and/or act on specific stages of the neural stem cell lineage. Moreover, the role of pericytes in the SVZ stem cell niche has not been defined. Here we describe a FACS methodology to isolate pure, primary endothelial cells and pericytes from neurogenic and non-neurogenic brain regions and perform studies in vitro to examine their effect on distinct stages of the SVZ neural stem cell lineage. Primary endothelial cells from both cortex and SVZ support proliferation and neuronal differentiation of activated stem cell astrocytes and transit amplifying cells in the absence of any exogenous growth factors. Notably, their signals are more potent than those secreted from the immortalized bend.3 endothelial cell line. Proliferation of activated stem cell astrocytes and transit amplifying cells with conditioned medium from primary cortical cells was shown to depend on EGFR in vitro. Here we define for the first time the effect of pericytes on SVZ neural stem cells. Pericytes promote the proliferation of activated stem cell astrocytes and transit amplifying cells, but to a lesser extent than endothelial cells. Strikingly, activated stem cell astrocytes and transit amplifying cells generate proportionally more neurons in response to pericyte conditioned medium than other conditions, and SVZ pericyte signals are particularly potent on activated stem cell astrocytes. Little is known about the heterogeneity of pericytes in the brain. After culturing FACS-purified pericytes, we observed multiple in vitro phenotypes of pericytes from both cortex and SVZ. Over time, both cortical and SVZ pericyte cultures became dominated by a rapidly proliferating cell with a progenitor morphology, which could be serially passaged. In preliminary studies, this passaged pericyte exhibited features of mesenchymal stem cells. To probe pericyte heterogeneity in the brain, we used mesenchymal stem cell markers. Novel pericyte subpopulations could be prospectively purified from both the cortex and SVZ using CD13, CD146, and CD105. Interestingly, CD13+CD105-CD146- pericytes were the most proliferative population from both the SVZ and cortex, but only those from SVZ could be passaged. Staining with these markers in vivo demonstrated specific morphologies and staining patterns on different sized vessels in the SVZ. Fractones, an ECM structure unique to the SVZ, arose from pericytes. As an endothelial marker, CD146 displayed different patterns of staining on different sized vessels, and stained naked vessels that lacked a basement membrane. While the SVZ vascular bed is largely quiescent, we also detected rare CD146+ tip cells. Collectively, these studies demonstrate the use of a powerful methodology to directly purify endothelial cells and pericytes from the brain in a neurogenic region, the SVZ, and a non-neurogenic region, the cortex. We uncover previously undescribed vascular cell diversity in the brain, and a novel role for brain pericytes on neural stem cells and their progeny. In addition to elucidating novel roles of vascular cells in the SVZ niche, this protocol offers a flexible and effective platform to obtain pure and contextually precise cells for future experiments in other brain regions or stem cell niches. Defining the different components of the niche is central to understanding the regulation of stem cells under homeostatic conditions and conversely how these signals are lost or perturbed during aging and tumorigenesis.

Evaluation of Chloride Intracellular Channels 4 and 1 Functions in Developmental and Pathological Angiogenesis

Tung, Jennifer Jean January 2012 (has links)
Members of the chloride intracellular channel (CLIC) protein family have been implicated as regulators of tubulogenesis, a critical step in the formation of new blood vessels during angiogenesis. We sought to determine CLIC4 and CLIC1 function in angiogenesis. We hypothesized that CLIC4 and CLIC1 act in endothelial lumen formation and promote both developmental and pathological angiogenesis. Using in vitro studies, we found that CLIC4 promotes endothelial proliferation, network formation, capillary-like sprouting, and lumen formation. In vivo, Clic4 knockout mice display a mild defect in retinal vascular development and an apparent decrease in retinal macrophage content. By implanting murine tumor cells in Clic4 knockout mice, we discovered that Clic4 affects the establishment of lung metastases. Endothelial and smooth muscle cell content of tumors are comparable to wild type, but overall vessel architecture is altered. In studying CLIC1, we found that CLIC1 knockdown results in reduced endothelial proliferation, directed migration, network formation, and capillary-like sprouting in vitro. In vivo analysis revealed no apparent angiogenic phenotype in the developing retinas of Clic1 knockout mice. We developed Clic4-/-;Clic1-/- double mutant embryos, which were unable to develop beyond 9.5 dpc. Whole mount staining of Clic4-/-;Clic1-/- 9.5 dpc embryos for vasculature revealed an angiogenic defect, most notable along the intersomitic vessels and in the brain. Endothelial content is reduced in Clic4;Clic1 double knockout embryos, and double nullizygous embryos were growth retarded. Preliminary histological analysis of Clic4-/-;Clic1-/- 9.5 dpc embryos suggests altered aortic development, reduced proliferation, and increased apoptosis. I conclude that CLIC4 and CLIC1 function in endothelial proliferation and morphogenesis and that Clic4 and Clic1 are required for embryonic development. Together, our findings indicate that CLIC4 and CLIC1 are important in developmental angiogenesis and should be considered in elucidating the molecular mechanisms of tubulogenesis.

A Mother’s Sacrifice: The contribution of asymmetric cell division to lifespan regulation in Saccharomyces cerevisiae.

Higuchi-Sanabria, Ryo January 2015 (has links)
Aging determinants are asymmetrically distributed during cell division in S. cerevisiae, which leads to production of an immaculate, age-free daughter cell. During this process, damaged components are sequestered and retained in the mother cell, while higher functioning organelles and rejuvenating factors are transported to and/or enriched in the bud. Here, we will describe the key quality control mechanisms in budding yeast that contribute to asymmetric cell division of aging determinants, with a specific focus on mitochondria. We find that the actin cytoskeleton, which drives transport of many cellular components in yeast, plays a crucial role in segregating fit from less fit mitochondria between mother and daughter cells. Since actin cables are dynamic structures that undergo retrograde flow, treadmilling from the bud towards the mother cell, they acts as filters to prevent damaged, dysfunctional mitochondria from being inherited by the daughter cell. This asymmetry has a direct impact on regulation of daughter cell fitness. A direct counterpart to mitochondrial motility events is anchorage of the organelle, which occurs in the mother tip, mother cortex, and bud tip in budding yeast. We find that mitochondrial fusion, together with tethering protein, serves to promote anchorage and accumulation of mitochondria at the bud tip. This anchorage must be properly maintained, as ectopic increase in mitochondrial anchorage can disrupt quality control mechanisms aimed at promoting asymmetric cell division.

Engineering a heterologous transforming growth factor-alpha promoter that is glucose-responsive in 293T cells /

Reale, Virginia Danielle Hikel. January 2002 (has links)
Thesis (M.A.)--Central Connecticut State University, 2002. / Thesis advisor: Thomas King. " ... in partial fulfillment of the requirements for the degree of Master of Arts in Biological Sciences." Includes bibliographical references (leaf 35). Also available via the World Wide Web.

Studies on the permeability of the internal cytoplasm of animal and plant cells ...

Kite, George Lester. January 1915 (has links)
Thesis (Ph. D.)--University of Chicago, 1913. / "Reprinted from the American journal of physiology, vol. 37, no. 2, May, 1915." Includes bibliographical references (p. 299).

Studies on the permeability of the internal cytoplasm of animal and plant cells ...

Kite, George Lester. January 1915 (has links)
Thesis (Ph. D.)--University of Chicago, 1913. / "Reprinted from the American journal of physiology, vol. 37, no. 2, May, 1915." Includes bibliographical references (p. 299).

Studies on the permeability of the internal cytoplasm of animal and plant cells ... /

Kite, George Lester. January 1915 (has links)
Thesis (PH. D.)--University of Chicago, 1913. / "Reprinted from the American journal of physiology, vol. 37, no. 2, May, 1915." Bibliography: p. 299. Also issued online.

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