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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.
21

Characterization and phosphorylation of an MPM-2 reactive sperm protein complex involved in zygotic aster formation

Duncan, Richard Peter 01 January 1997 (has links)
The zygotic centrosome, contributed by the sperm, forms an array of microtubules that spread throughout the ooplasm. These microtubules are used by the female pronucleus to come into juxtaposition with the male pronucleus in order to form the zygotic nucleus. Zygotic centrosome regulation has been shown to involve an MPM-2 phosphoepitope that is dephosphorylated prior to aster formation. The MPM-2 reactive candidate appears as a triplet of proteins with MWapp of 85, 81, and 77 kDa on SDS-PAGE. The objectives of this work were to investigate the relationship among the triplet proteins, determine physical characteristics of the MPM-2 reactive protein, and investigate potential kinases that may act on the MPM-2 target epitope. Amino acid analysis and peptide mapping show that the triplet proteins are nearly identical with all of the peptide fragments of the smaller proteins contained in the largest of the triplet. Physical properties, immunoreactivity, and isolation characteristics demonstrate that the reactive protein is part of the outer dense fiber/segmented column complex. The protein is highly insoluble in aqueous solutions and requires a reducing agent and a chaotropic agent, such as urea, to remain in solution. Kinase studies reveal that the MPM-2 reactive protein is maintained in a phosphorylated state by a kinase that is Maturation Promoting Factor (MPF) or dependent on MPF. Further, the epitope is dephosphorylated coincident with a drop in MPF in activated oocyte extract while MAP kinase levels remain high. This information indicates that the MPM-2 reactive sperm protein is part of the segmented columns or dense fibers and likely exerts its influence on the zygotic centrosome indirectly, perhaps through blocking. The epitope on the protein appears to be regulated through the action of kinases that maintain the phosphorylation of the protein. Dephosphorylation is thought to occur by ubiquitous phosphatases that are favored upon deactivation of MPF and downstream kinases.
22

A post-import pathway for protein targeting to chloroplast inner membrane

Li, Ming 01 January 2008 (has links)
The chloroplast envelope plays critical roles in the synthesis and regulated transport of key metabolites, including intermediates in photosynthesis and lipid metabolism. Despite this importance, the biogenesis of the envelope membranes has not been investigated in detail. To identify the determinants of protein targeting to the inner envelope membrane (IM), I investigated the targeting of the nucleus-encoded integral IM protein, atTic40. I found that pre-atTic40 is imported into chloroplasts and processed to an intermediate size (int-atTic40) before insertion into the IM. Int-atTic40 is soluble and inserts into the IM from the internal stromal compartment. I also show that atTic40 and a second IM protein, atTic110, can target and insert into isolated IM vesicles in vitro. These in vitro studies are further supported by in vivo evidence showing pre-atTic40 directly engineered into the chloroplast genome and expressed within chloroplasts can efficiently target to the inner envelope membrane. Taken together, my experiments are consistent with a "post import" mechanism in which the IM proteins are first imported from the cytoplasm and subsequently inserted into the IM from the stroma.
23

Cytokinesis in plant cells and the role of the cytoskeleton

Valster, Aline Hendrika 01 January 2000 (has links)
Cytokinesis in plant cells involves the formation and insertion of a new cell wall that separates daughter nuclei after mitosis. The deposition of the new wall is regulated by a structure called the phragmoplast which contains microtubules, actin filaments and membranes. The experiments described in this thesis, emphasize the participation of the actin filaments in the cytokinetic process. Caffeine inhibition of cytokinesis shows that two distinct phases cell plate development can be distinguished; an early initiation phase and a late lateral expansion phase. Caffeine completely inhibits the latter stage. I have looked at the dynamics of actin filaments, as well the microtubules, during the inhibition of cytokinesis by caffeine. I found that formation of the cytoskeletal torus, associated with the later stages of cell plate formation, is completely inhibited in the presence of caffeine. The functional role of actin filaments was probed by microinjection of the actin monomer binding protein profilin. The results show a dose-dependent effect on cytokinesis, in which the effects range from delayed cell plate formation to complete inhibition. To asses the localization of profilin, I microinjected a fluorescent profilin probe into living cells and analyzed the localization throughout mitosis. Profilin is accumulated in the nucleus during interphase and prophase and is released in the accessible volume after nuclear envelope break down at the onset of metaphase. It remains in the accessible volume during the subsequent mitotic stages until it re-accumulates in the telophase nuclei. No specific localization of profilin in the phragmoplast was observed. Also, the role of actomyosin in plant cytokinesis was studied by using the myosin ATPase inhibitor, 2,3-butanedione monoxime (BDM). Cytokinetic cells, treated with BDM, displayed buckled and thin cell plates that were often tilted at a significant angle. In addition, late lateral cell plate expansion was inhibited and actin filaments in the phragmoplast lost their proper orientation. These results show that actomyosin plays a crucial role in cell plate alignment and late lateral expansion of the cell plate. Finally, a new cell plate formation model is presented that combines some of the results of this thesis with existing models and previous findings.
24

Characterization of the sperm factor responsible for initiating [calcium (2+)](i) oscillations during fertilization in mammalian eggs

Wu, Hua 01 January 2000 (has links)
During fertilization the sperm activates the mammalian egg by eliciting [Ca2+]i oscillations. However, how the sperm triggers [Ca2+]i oscillations in mammalian eggs remains unknown. To test the possibility that a factors) from the sperm is able to elicit [Ca 2+]i oscillations, sperm fractions (factors) were prepared from different species and injected into mammalian oocytes or eggs. The results show that injection of sperm factor from either porcine or human sperm triggered long-lasting [Ca2+]i oscillations in mouse oocytes and bovine eggs in a pattern similar to the physiological responses observed in each of these species, implying that the sperm factor is functionally conserved among mammalian species. In addition, sperm factor-induced [Ca2+] i oscillations appeared to be mediated by the IP3 receptor, while the ryanodine receptor may be involved in the modulation of these oscillations. Furthermore, [Ca2+]i oscillations induced by sperm factor are capable of initiating normal egg activation and parthenogenetic development. In order to isolate and characterize the unknown active molecule(s) in sperm factor, different tissues or cell extracts were screened for the presence of sperm factor like activity. It appears that the [Ca2+] i oscillation-inducing activity is sperm/testis specific. In addition, the results showed that the active component contains a protein moeity and that the same single active component of porcine sperm factor is present in both soluble and in less soluble sperm compartments. We also demonstrated that gpd/oscillin, a proposed active component of mammalian sperm factor, is not responsible for the [Ca2+]i oscillation-inducing activity as shown by the lack of effects on Ca2+ responses or absence in active fractions following immunodepletion or a combination of fractionation techniques. Similarly, our results also showed that neither PLCγ1, PLCγ2 nor tr-c-Kit is likely to be the active component of sperm factor. Although we were unable to identify a specific candidate molecule, these experiments have led to the identification of three polypeptides in the final active fraction after sequential chromatographic steps. Our results also show that the active component has an isoelectric point of 6.5–7.0 and a relative molecular weight ranging from 29–68 kDa. These polypeptides will be sequenced and used to raise monoclonal or polyclonal antibodies, which will be used to screen a testis cDNA library. This last step is expected to lead to the isolation of the gene encoding sperm factor.
25

Microtubule organization, movement and turnover in motile and non-motile cells

Yvon, Anne-Marie C 01 January 2000 (has links)
Microtubules are required for several cellular processes including vesicle transport, cytoplasmic organization, cell motility, maintenance of cellular polarity, and mitosis. The mechanisms by which microtubule arrays are established, maintained and remodeled are important for understanding these vital processes. I have used fluorescence analog chemistry and live cell imaging approaches in mammalian cells to address these questions. I have documented the de novo formation of microtubules in peripheral regions of epithelial-like cells, at sites distant from the centrosome, challenging the traditional doctrine that microtubules are nucleated solely at microtubule organizing centers (MTOCs). Historically, the behavior of minus ends has been difficult to study in vivo, due to the high density of microtubules in central cellular regions; however, the peripheral position of the non-centrosomal microtubules allowed me to quantify the dynamic behavior of both microtubule ends. The results demonstrate that individual minus ends are remarkably stable, suggesting that their stability is either intrinsic to their structure or is the result of a molecular cap. During cell motility, microtubules must populate the advancing lamella and be removed from retracting regions; however, the mechanisms that cells utilize to remodel microtubule arrays are complex and poorly defined. My experiments, using photoactivated fluorescent tubulin to mark the microtubule lattice, demonstrate that microtubules are transported in motile cells, and that transport is a two component process. The first component is the unidirectional transport of microtubules in the direction of the dominant actomyosin-generated contractile force; the second is the bidirectional movement of individual microtubules. In cells with numerous noncentrosomal microtubules, transport is likely to play a significant, and previously unrecognized, role in microtubule reorganization. The mechanism of the bidirectional component was analyzed; inhibition of myosin II abolished microtubule movement, while inhibition of cytoplasmic dynein increased it. In addition, the absence of myosin function resulted in slower turnover of the microtubule array, which is the first direct evidence for myosin-generated forces modulating the kinetic behavior of microtubules. The results support a role for cytoplasmic dynein in tethering microtubules and resisting actomyosin-generated forces, suggesting that the antagonistic forces of these motors contribute to the organization of the microtubule array.
26

The role of EDEM1 in the quality control and degradation of misfolded glycoproteins

Cormier, James H 01 January 2009 (has links)
Immature or incompletely assembled proteins that do not fold correctly are retained in the endoplasmic reticulum (ER) by quality control factors. Terminally misfolded glycoproteins are eventually sorted for dislocation out of the ER and ubiquitinated, leading to degradation by the 26S proteasome in a process termed ER-associated degradation (ERAD). Mannose-trimming of glycans has been proposed to act as a sorting mechanism for the degradation of misfolded glycoproteins. EDEM1 (ER degradation-enhancing α-mannosidase-like 1) is hypothesized to extract misfolded proteins out of the calnexin cycle and sort them for degradation by recognizing a mannose-trimmed glycan. This extraction is proposed to be assisted by a direct interaction between the transmembrane domain of calnexin and a putative transmembrane domain of EDEM1. To investigate the role of EDEM1 in the quality control and degradation of misfolded glycoproteins, initially, the fundamental properties of EDEM1 were characterized. We observed that endogenous EDEM1 matures to a soluble protein that is heterogeneously glycosylated. This predominantly soluble phenotype was in disagreement with the proposed mechanism for EDEM1 extraction of misfolded glycoproteins from the calnexin binding cycle. EDEM1 binding to misfolded proteins has been proposed to be mediated through the presence of a mannose-trimmed glycan on the misfolded substrate. After establishing an EDEM1 binding assay, we found that EDEM1 bound transiently associated with misfolded glycoproteins in a glycan-independent manner. EDEM1 was also found to bind SEL1L, an ERAD dislocation and ubiquitination complex adapter glycoprotein. Inhibition of mannose trimming with kifunensine or disruption of the EDEM1 mannosidase-like domain by mutation had no effect on EDEM1 substrate binding, but diminished its association with the SEL1L. Therefore, we propose a model where EDEM1 binds to misfolded glycoproteins in a glycan-independent manner and delivers the non-native cargo to the ERAD dislocation and ubiquitination complex using its mannosidase-like domain, which associates with SEL1L. This investigation presents an alternative hypothesis of the function of EDEM1 in the degradation of misfolded glycoproteins while also emphasizing the importance of glycans in the degradation of misfolded glycoproteins.
27

Functional analysis of Hic-5/ARA55 isoforms in C2C12 myogenesis

Gao, Zhengliang 01 January 2006 (has links)
Hic-5 is a focal adhesion protein of paxillin superfamily that was initially cloned from mouse osteoblasts as a TGF-β or H2O2 inducible cDNA. As well, Hic-5 was independently identified as an Androgen receptor activator (ARA55). Conflicting data have implicated Hic-5 in opposing processes. With two Hic-5 isoforms documented, I hypothesized that multiple Hic-5 isoforms may exist that have both overlapping and isoform-specific functions, which may explain those discrepancies. To test this hypothesis, I have utilized C2C12 myoblasts and analyzed the roles of Hic-5 isoforms in development and homeostasis. 1. I have confirmed the presence of the two previous reported Hic-5 isoforms (α and β) and uncovered 10 additional novel Hic-5 transcripts. Conceptually translated proteins from these transcripts significantly differ at the N-terminal region and likely have distinct binding properties and functions. Hic-5 isoforms have distinct tissue distribution and are developmentally regulated in the mouse mammary gland in vivo (Chapter two). 2. I found that: (a) myoblasts express multiple Hic-5 isoforms; (b) the two predominant isoforms, Hic-5α and Hic-5β, are differentially expressed during myogenesis; (c) any experimentally-induced change in Hic-5 expression results in a substantial increase in apoptosis during differentiation; (d) ectopic expression of Hic-5α is permissive to differentiation while expression of either Hic-5β or antisense Hic-5 reduces myoblast chemo-differentiation and blocks fusion; (e) Hic-5 localizes to focal adhesion in C2C12 myoblasts and perturbation of Hic-5 leads to defects in cell spreading; (f) perturbations of Hic-5 expression interfere with the normal expression dynamics of laminin; and (g) the rescue of myoblast survival and differentiation by laminin but not fibronectin suggests that Hic-5 isoforms differentially regulate myogenesis due to their different impacts on cell-ECM interaction, focal adhesion dynamics and integrin signaling (Chapter Three). In summary, the roles Hic-5 may assume in development and homeostasis are complex and the different Hic-5 isoforms may mediate distinct physiological and/or pathological responses in cells. Therefore, a more precise analysis of Hic-5 isoforms is required to more fully understand the roles of not only Hic-5, but also integrin signaling in normal and diseased cells ( Chapter four and five).
28

The role of ovarian hormones in p53-mediated resistance to mammary tumorigenesis

Dunphy, Karen A 01 January 2008 (has links)
A full term pregnancy reduces breast cancer by up to 50%. In rodents, pregnancy or treatment with estrogen and progesterone to mimic pregnancy reduces mammary tumor incidence. The molecular mechanisms for parity-induced resistance to mammary tumorigenesis appear to involve a sustained increase in p53 responsiveness to cellular stresses. The following experiments tested whether pregnancy levels of ovarian hormones alter molecular pathways that prime p53 to be more responsive to DNA damage and if these pathways confer resistance to mammary tumors in a mouse model of Li-Fraumeni syndrome. Mice were treated with estrogen and progesterone (E+P) for 14 days, neonatally or at maturity. At 10 weeks of age, radiation-induced nuclear accumulation of p53 and apoptosis were increased similarly in the mammary epithelium from E+P-treated and parous mice compared to placebo. This effect was sustained for at least 7 weeks after E+P treatment and did not depend on the continued presence of ovarian hormones. Hormone-stimulation also enhanced apoptotic responses to ionizing radiation in BALB/c- Trp53+/- mice, a model of Li-Fraumeni syndrome. The appearance of spontaneous mammary tumors was delayed by parity in BALB/c-Trp53+/- mice. However, this protective mechanism was not preserved within epithelial progenitor cells because apoptotic responses to ionizing radiation and tumor incidence in epithelial transplants from E+P-treated donors was not different from nulliparous epithelial outgrowths. Therefore, E+P and parity confer a sustained increase in p53-mediated apoptosis within the mammary epithelium and suppresses mammary tumorigenesis, but this was not retained in epithelial outgrowths. Parity reduces the expression of estrogen receptor alpha (ERα). Activation of ERβ with an ERβ-specific agonist represses the expression of ERα. Parity-related alteration in the expression ratio of the two estrogen receptors in the mammary gland could regulate p53 priming to enhance its responsiveness to genomic stress in the parous individual.
29

A human induced pluripotent stem cell in vitro system to model the inception of lung adenocarcinoma

Vedaie, Marall 01 December 2020 (has links)
Lung adenocarcinoma is responsible for significant global mortality with limited effective treatments. Although some studies suggest that these tumors arise from alveolar epithelial type 2 cells (AEC2s), there is scant information regarding the early events that might occur in human AEC2s at the inception of oncogenesis. This limitation, is partially due to a lack of human model systems that recapitulate the initiation of oncogenesis in AEC2s. Unfortunately, primary AEC2s from patients are difficult to access in vivo or stably maintain in cell cultures. Hence, we sought to develop an in vitro system to model the early stages of oncogenesis utilizing human induced AEC2s (iAEC2s) generated through the directed differentiation of induced pluripotent stem cells (iPSCs). To this end, we selected a normal human iPSC line we have previously engineered to carry fluorochrome reporters targeted to lung epithelial-specific loci, NKX2-1 GFP and SFTPC tdTomato that enable monitoring and purification of alveolar lung epithelial cells. To test the effects of adenocarcinoma oncogene induction in these cells, we targeted a third locus, AAVS1 using gene editing to engineer a doxycycline-inducible cassette encoding mutant KRAS G12D, the most commonly found oncogene in lung adenocarcinomas. Successful induction of KRAS G12D with doxycycline was demonstrated in both the targeted undifferentiated iPSCs as well as in the iAEC2s derived from these cells. We profiled the downstream effects of KRAS G12D induction in iAEC2s, comparing dox vs vehicle exposed cells by cell counting, FACS for NKX2-1 GFP/SFTPC tdTomato, RT-qPCR, deep proteomic and phosphoproteomic analyses, and scRNA-sequencing. Through this characterization, we found that induction of KRAS G12D robustly activates MAPK signaling resulting in a shift of iAEC2s away from their mature alveolar program towards a distal lung epithelial progenitor phenotype, indicated by the upregulation of lung progenitor and proliferation markers (e.g. SOX9, ETV4, LEF1, TM4SF1, MKI67, and TOP2A) while maintaining NKX2-1 expression, at the expense of mature alveolar markers (e.g. SFTPC, SFTPB, NAPSA, and LPCAT1). Successful modeling of lung adenocarcinoma with this model system has a variety of future applications, including testing unknown mechanisms for oncogenesis, discovery of novel biomarkers of disease, or development of new effective treatment methods through drug screening.
30

Small molecule screen to identify mitotic kinesin KIF18A inhibitors

Mauch, Austen 23 November 2021 (has links)
Mitotic cell division is critically reliant on consistent and faithful segregation of genetic information into newly forming daughter cells. Any perturbation in this process can have catastrophic intracellular effects and as such, many natural mechanisms occur during mitosis to prevent these anomalous events. Kinesins make up 14 families of motor proteins, using ATP-driven movement along microtubules to fulfill a range of functions, including organelle and vesicular transport. One such kinesin, plus-end directed KIF18A of the Kinesin 8 family, has been extensively investigated and found to be critically associated with restricting chromosome oscillation during chromosome alignment at the metaphase plate. Depletion of KIF18A subtly increases mitotic duration, chromosome oscillations, lagging chromosomes, and micronucleus formation in normal diploid cells and has minimal effects on cell viability. By contrast, loss of Kif18A induces severe mitotic defects that significantly impair viability in whole genome-doubled and highly aneuploid cells. As tetraploidization or whole genome doubling has been linked to large percentages of cancer types, this dependence on KIF18A has been proposed as a possible therapeutic target for cancer treatment. Two known KIF18A inhibitors exist; however, their function is either limited (BTB-1) or the efficacy remains to be publicly validated (Amgen). In this thesis, a screening protocol involving fluorescent-based live cell imaging was formulated to test several small molecule compounds that were suspected to have compatibilities with a binding site on KIF18A. Through the screening process, Compound 3 was identified and found to demonstrate a phenotype similar to KIF18A depletion—prolonged mitosis, decreased cell proliferation/viability, and induction of chromosome oscillation. Further examination of this compound as a potential therapeutic and elucidation of its specific mechanism of action appear to be warranted for future studies.

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