Dissertations / Theses: '3101 Biochemistry and cell biology'

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Hodzic, Denis. "Effects of EF-24 and Cisplatin on Cancer, Renal, and Auditory Cells." TopSCHOLAR®, 2019. https://digitalcommons.wku.edu/theses/3110.

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Cisplatin is a chemotherapy drug effective against several forms of cancer, but can also cause serious side-effects, including nephrotoxicity and ototoxicity. Curcumin, a natural plant compound, can increase cisplatin’s anti-cancer activity and counteract cisplatin’s deleterious effect on the auditory and renal systems. Unfortunately, curcumin exhibits poor bioavailability, which has promoted interest in the development of synthetic curcumin analogs (curcuminoids) that are soluble, target cancer, and do not cause side effects. This study investigated whether the curcuminoid (3E,5E)-3,5-bis[(2-fluorophenyl) methylene]-4-piperidinone (EF-24) increases the anti-cancer effects of cisplatin against a human ovarian cancer cell line (A2780) and its cisplatin-resistant counterpart (A2780cis), while preventing cisplatin-mediated side effects in a human kidney cell line (HEK-293T) and a mouse auditory hybridoma cell line (HEI-OC1). The effect of cisplatin and EF-24 on cellular viability was measured using the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay. The expression and activity of signal transduction proteins in several apoptotic pathways was measured using caspase luminescence assays. Reactive oxygen species (ROS) production was also measured using flow cytometry. Our data suggest that cisplatin and EF-24 are effective against ovarian cancer cell lines, but both compounds may also have adverse effects on auditory and renal cells. This project provides relevant information that may improve our understanding of how these compounds function in different tissues, facilitating improved cancer treatment and circumvention of side effects commonly associated with cisplatin treatment.

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Camacho, Diogo Mayo. "In silico cell biology and biochemistry: a systems biology approach." Diss., Virginia Tech, 2007. http://hdl.handle.net/10919/27960.

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In the post-"omic" era the analysis of high-throughput data is regarded as one of the major challenges faced by researchers. One focus of this data analysis is uncovering biological network topologies and dynamics. It is believed that this kind of research will allow the development of new mathematical models of biological systems as well as aid in the improvement of already existing ones. The work that is presented in this dissertation addresses the problem of the analysis of highly complex data sets with the aim of developing a methodology that will enable the reconstruction of a biological network from time series data through an iterative process. The first part of this dissertation relates to the analysis of existing methodologies that aim at inferring network structures from experimental data. This spans the use of statistical tools such as correlations analysis (presented in Chapter 2) to more complex mathematical frameworks (presented in Chapter 3). A novel methodology that focuses on the inference of biological networks from time series data by least squares fitting will then be introduced. Using a set of carefully designed inference rules one can gain important information about the system which can aid in the inference process. The application of the method to a data set from the response of the yeast Saccharomyces cerevisiae to cumene hydroperoxide is explored in Chapter 5. The results show that this method can be used to generate a coarse-level mathematical model of the biological system at hand. Possible developments of this method are discussed in Chapter 6.
Ph. D.

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Agüera-González, Sonia. "Cell biology on NKG2D ligands and NK cell recognition." Thesis, University of Cambridge, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.609348.

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Delorme, Marilyne. "Downregulation of ATRX disrupts cell proliferation and cell cycle progression." Thesis, University of Ottawa (Canada), 2008. http://hdl.handle.net/10393/27627.

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ATRX is a chromatin remodelling protein of the SNF2 family of chromatin remodelling proteins. Mutations in the ATRX gene have been shown to cause the ATR-X syndrome, an X-linked mental retardation disorder. ATRX is part of a chromatin-remodelling complex with Daxx that localizes to PML nuclear bodies or pericentromeric heterochromatin and is thought to regulate gene expression. In mice, Atrx inactivation results in embryonic lethality whereas conditional forebrain specific Atrx ablation showed impaired development and disorganization of the cortex. Furthermore, ATRX phosphorylation was shown to be cell cycle dependant, suggesting an important role for ATRX in cell cycle regulation. In this study we investigated the effects of ATRX downregulation in cell culture models, using siRNA transient transfection, a clone expressing an shRNA targeted to ATRX, and Atrxnull MEFs. ATRX downregulated cells showed reduced growth rates and cell cycle defects at the G1 and S phases of the cell cycle. Moreover, ATRX ablation was associated with an altered Rb phosphorylation status and decreased expression of the cyclin A and E2F-1 proteins. Taken together our results suggest that ATRX may play a significant role in cell cycle progression that is pertinent for proper development.

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Ye, Qing. "LIPASE-KINASE ASSOCIATIONS INVOLVING PLD2, JAK3 AND FES THAT UNDERLIE CANCER CELL PROLIFERATION AND INVASION." Wright State University / OhioLINK, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=wright1421939242.

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Chu, Wei. "Mouse Mast Cell Proteases: Induction, Molecular Cloning, and Characterization." Digital Commons @ East Tennessee State University, 1991. https://dc.etsu.edu/etd/2656.

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Tryptase, a mast cell-specific serine protease with trypsin-like specificity, has been identified in a mouse mast cell line (ABFTL-6) based on it's enzymatic activity, inhibition properties, and cross-reactivity to a human mast cell tryptase antibody. The effects of fibroblast-conditioned medium and sodium butyrate on ABFTL-6 mast cell differentiation and tryptase expression have been examined. ABFTL-6 mouse mast cells undergo phenotypic changes upon culturing in media supplemented with fibroblast-conditioned media at 50% or 1 mM sodium butyrate. The induced cells increased in size, had larger and more metachromatic cytoplasmic granules, and increased their total cellular protein about four-fold. Tryptase activity increased 13- and 6-fold upon fibroblast-conditioned media and butyrate induction, respectively. However, tryptase antigen levels increased dramatically from 2.3 $\mu$g/10$\sp6$ uninduced cells to 125 (54-fold) and 75 (33-fold) $\mu$g/10$\sp6$ cells induced with fibroblast-conditioned media or butyrate, respectively. A cDNA library was constructed in $\lambda$gt10 from ABFTL-6 cell poly(A)$\sp+$ RNA, and screened with dog mast cell tryptase and rat mast cell chymase cDNAs. Clones encoding two distinct tryptases (mouse tryptases I and II), a chymase (mouse chymase I) and a novel carboxyl terminal chymase (mouse chymase II) were isolated and sequenced. Mouse tryptases I and II have 75% and 70% sequence identity at the nucleotide and amino acid levels, respectively. The deduced amino acid sequence for the mature active enzyme for each mouse tryptase contains 245 residues and all the characteristics of a serine protease. Asp is found in the substrate binding pockets, consistent with a trypsin-like specificity for Arg-X and Lys-X bonds. It is predicted that tryptases are synthesized with prepropeptides, requiring signal peptidase processing and removal of a three amino acid propeptide for activation. Mouse chymase I consists of a 226 amino acid catalytic portion and a 21 amino acid preprosequence. An Asn occurs in the substrate binding pocket, a feature that has not been observed in any other serine protease.

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Bainor, Anthony J. "Elucidating the Role of SIN3B as a Regulator of Cell Cycle Exit." Thesis, New York University, 2017. http://pqdtopen.proquest.com/#viewpdf?dispub=10604607.

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Progression through the mammalian cell cycle is a tightly regulated process that allows cells to replicate their genomes and divide properly. In growth factor-deprived conditions or in response to stress, the cell will exit the cell cycle either reversibly through quiescence, or permanently via senescence. Studies have shown that the SIN3 family of proteins plays a crucial role in these cell cycle exit processes. SIN3 proteins are highly conserved, and exist in mammals as two family members: SIN3A and SIN3B, which function as flexible scaffolding proteins to assemble co-repressor complexes. Our laboratory has recently implicated SIN3B as a critical mediator of each of these cell cycle exit processes. However, its mechanism of action and the consequences of its disruption pertaining to cancer progression have not been comprehensively elucidated. Here we demonstrate that SIN3B is required for the induction of senescence in a mouse model of prostate cancer, and thus prevents the progression to aggressive and invasive carcinoma. In addition, through interaction analysis, we uncovered a novel and robust association between SIN3B and the DREAM complex. The DREAM complex, comprised of p107/p130, E2F4/5, DP1 and the MuvB core complex, is responsible for the repression of hundreds of cell cycle-related transcripts during quiescence. We determined that the deletion of SIN3B resulted in the derepression of DREAM target genes during quiescence, but was not sufficient to allow quiescent cells to resume proliferation. However, the ectopic expression of APC/C^CDH1 inhibitor EMI1 was sufficient for SIN3B deleted cells, but not wild-type cells, to reenter the cell cycle. These studies demonstrate a critical role for SIN3B in the senescence and quiescence programs, and provide important mechanistic insight into the molecular pathways that exquisitely regulate cell cycle exit.

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Reimer, Michael. "Characterization of IQGAP1 Protein in Areas of Cell Retraction." Thesis, Southern Illinois University at Edwardsville, 2015. http://pqdtopen.proquest.com/#viewpdf?dispub=1582876.

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IQGAP1 interacts with numerous binding partners through a calponin homology domain (CHD), a WW motif, IQ repeats, a Ras GAP related domain (GRD), and a conserved C-terminal (CT) domain. Among various biological and cellular functions, IQGAP1 plays a role in cell-matrix interactions and actin cytoskeleton dynamics in membrane ruffling and lamellipodium protrusion. Phosphorylation in the CT domain regulates intramolecular interaction and IQGAP1 cellular activity. In a recent study, we discovered that IQGAP1 surprisingly localizes to actively retracting edges, instead of protruding areas, in B16F10 mouse melanoma cells and some other cells types. In these current studies we examined localization of IQGAP1 mutants to retracting versus protruding areas in phorbol ester-stimulated B16F10 cells. Cells were co-transfected with GFP-IQGAP1 full length (GFP-IQGAP1-FL), as an internal control, and one of five Myc-tagged IQGAP1 constructs (FL, CA, ΔCHD, ΔGRD or ΔCT). The cotransfected cells were plated onto laminin for 30 minutes, stained with anti-Myc and anti-WAVE2 antibodies, and normalized fluorescence measurements were made in retracting and protruding areas. Retracting cell areas were defined as GFP-IQGAP1-FL positive and WAVE2 negative, while protruding cell areas were defined as GFP-IQGAP1-FL negative and WAVE2 positive. In retracting areas there were large decreases in both ΔGRD and ΔCT localization, a slight decrease in ΔCHD localization, and normal localization of the CA mutant. In areas of cell protrusion there were large increases in both ΔGRD and ΔCT localization, and normal localization of ΔCHD and CA mutants. These results indicate that two domains, GRD and CT, are essential for normal localization of IQGAP1 to retracting cell areas. Furthermore, our results suggest a model in which IQGAP1 in the areas of cell retraction is in the open, phosphorylated, conformation. Additionally we investigated the knockdown of IQGAP1 in B16F10 cells by means of actin images. Cells were exposed to the lentil virus which contained short hairpin Ribonucleic acid (shRNA) that would silence IQGAP1. Two controls were used in the experiment, untransfected B16F10 cells and B16F10 cells which were exposed to the lentil virus without any shRNA. We found that the knockdown cells were in general much more compact and that they did not polarize. Surface stiffness was investigated in the effect it would have on B16F10 cells. Polyacrylamide gels were made and cross-linked using sulfo-SANPAH. Laminin was added to the cross-linked gels and B16F10 cells were placed on top of the laminin coated hydrogels. Investigation of the cells by means of actin images revealed that surface stiffness had an effect on cell morphology. The 1 kPa surfaces did not allow for spreading of the cells, while the surfaces greater than 100 kPa exhibited normal cell behavior.

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Hussey, P. J. "Studies on the molecular and cell biology of plant tubulin." Thesis, University of Kent, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.376791.

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Upcher, William R. "Biochemical studies of the cohesin complex." Thesis, University of Oxford, 2012. http://ora.ox.ac.uk/objects/uuid:e9a77a9c-40a5-466d-a894-c7fefeddef52.

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The accurate inheritance of genetic material depends upon the establishment and maintenance of sister chromatid cohesion. Replicated chromosomes are topologically encircled by the large, tripartite protein complex cohesin, allowing bi-orientation in mitosis. To entrap and reversibly dissociate from DNA, the annular complex structure must be disrupted at either the hinge domain between Smc1 and Smc3, or the interfaces created by the kleisin subunit Scc1 bridging the two ABC-like ATPase domains. The aim of this work was to characterise the cohesin complex loading and releasing mechanisms by examining the biochemical requirements for these processes. Although the identity of a chromosomal cofactor could not be assigned, the loading reaction was found to necessitate engagement of ATPase domains in an ATP-dependent manner. Notions of allosteric modulation of ATP binding and NBD engagement by acetylation were discredited. Likewise, a direct and stable physical association of hinge domains with NBDs was shown to be an unlikely conformational intermediate in a reaction thought to promote hinge opening for loading of cohesin onto DNA. The Smc3-Scc1 kleisin interface might be exploited during the opposing process of release of cohesin from DNA. Therefore, a novel protein- protein cross-linking system was adapted for use in S. cerevisiae, with a view to (1) confirming the well-founded role of hinge dissociation in topological entrapment, and (2) validating the Smc3- Scc1 interface as the recently conjectured exit gate. Despite promising preliminary kinetics in vitro, the SpyTag-SpyCatcher system was considerably less efficient in vivo. It was thus deemed unsuitable in its current format for investigating the process of interface dissociation in live cells. Finally, the large, cohesin complex-associated HEAT repeat protein Pds5 has been either speculated or shown to participate in all of the aforementioned processes, potentially modulating the hinge and Smc-kleisin interfaces. Acting as a regulatory node in cohesin function, it was pursued as an informative target for structural studies. Although no diffractive material could be obtained, Pds5 was confirmed to bind a short, N-terminal sequence of Scc1 and was in turn bound at its N- terminus by Wapl. Together, these findings contribute to defining the structures and states of the cohesin complex.

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Ng, Shyh Chang. "Regulation of Stem Cell Metabolism by the Lin28/let-7 Axis." Thesis, Harvard University, 2013. http://dissertations.umi.com/gsas.harvard:11217.

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My PhD thesis is focused on two fundamental aspects of stem cell metabolism: (1) the role of Lin28 in programming stem cell metabolism, and (2) how metabolism in turn fuels and governs pluripotency. Our studies led us to discover that the stem cell factor Lin28a promotes gigantism by enhancing glucose metabolism in mice, coinciding with discoveries that LIN28B polymorphisms influence height variation in human GWAS. Subsequently, we discovered that the Lin28/let-7 pathway controls glucose metabolism by orchestrating the upregulation of multiple insulin-PI3K-mTOR components, particularly in skeletal muscle progenitors. Since let-7 accumulates with aging, our discoveries suggest that let-7 could represent a new drug target for treating insulin resistance and type 2 diabetes during aging. During these studies, we also observed that Lin28a enhances tissue regeneration in adulthood. Regeneration capacity has long been known to decline with aging, but why juvenile organisms show enhanced tissue repair had remained unexplained. We found that Lin28a reactivation improved the regrowth of skin, hair, cartilage, bone and mesenchyme after injuries. Let-7 repression was necessary but insufficient to explain these phenotypes. In parallel, Lin28a bound to and enhanced the translation of mRNAs for several oxidative enzymes, thereby increasing OxPhos. Lin28a-mediated tissue repair was negated by OxPhos inhibition, whereas a pharmacologically-induced increase in OxPhos promoted wound repair. Thus, Lin28a enhanced tissue regeneration in adults by reprogramming cellular bioenergetics. My interest in the central principles of stem cell metabolism also led us to map the metabolic pathways associated with pluripotency during iPS reprogramming and Lin28/let-7 perturbation. Surprisingly, we found that Thr-Gly-S-adenosylmethionine (SAM) metabolism consistently showed the best correlation with pluripotency. 13Carbon isotope metabolomics further revealed that Thr was catabolized to generate Gly and acetyl-CoA, and ultimately SAM - essential for all methylation reactions. Thr is required for SAM and histone H3K4 methylation in mouse ESCs, thus regulating the open euchromatin and pluripotency of ESCs. Our study shed light on a novel amino acid pathway in stem cells, and demonstrated that metabolic conditions can direct cell fate. In summary, my work has helped us to understand how we can reprogram and manipulate metabolic networks to regulate stem cell homeostasis.

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Sipe, Conor W. "Cloning and Functional Characterization of Hypoxia-Inducible Factor 1alpha Upstream Regions in Xenopus laevis." W&M ScholarWorks, 2003. https://scholarworks.wm.edu/etd/1539626407.

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Chambers, Jeremy W. "Studies in the biochemistry and cell biology of Trypanosoma brucei hexokinases." Connect to this title online, 2007. http://etd.lib.clemson.edu/documents/1202500780/.

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Trotman, Jackson B. "New Insights into the Biochemistry and Cell Biology of RNA Recapping." The Ohio State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=osu1523896565730483.

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Buchanan, Fritz G. "Endogenous Alkylglycerol Functions As a Mediator of Protein Kinase C Activity and Cell Proliferation." Digital Commons @ East Tennessee State University, 1997. https://dc.etsu.edu/etd/2885.

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To explore the possibility that 1-O-alkyl-sn-glycerol (alkylglycerol) may serve a regulatory role in the control of cell proliferation or PKC activity, we examined the ability of alkylglycerol to influence PKC activity and subcellular distribution as well as the ability of alkylglycerol to effect cell proliferation. MDCK cells grown to confluence show a loss of PKC activity associated with the membrane, as reported in fibroblasts. Preconfluent cultures of MDCK cells have a high level of PKC activity associated with the membrane. However, treatment of preconfluent cultures with alkylglycerol causes a reduction of PKC activity. A similar inhibition was observed with alkylglycerol when cells were treated with TPA, an activator of PKC. To confirm that alkylglycerol was exerting an effect directly on PKC, alkylglycerol was shown to inhibit PKC activity in vitro in a dose dependent manner. Since PKC exists as a family of closely related isozymes, we have determined the effects of growth arrest and alkylglycerol treatment on PKC $\rm\alpha,\ \epsilon,\ and\ \zeta$ (expressed in MDCK cells). The active forms of PKC $\alpha$ and $\epsilon$ are lost early in the growth of MDCK cells during the endogenous accumulation of alkylglycerol and synthetic alkylglycerol inhibits the membrane form of PKC $\alpha$ and $\epsilon.$ However, alkylglycerol inhibits the TPA induced translocation of PKC $\alpha$ but not $\epsilon$ suggesting a differential inhibition among these isoforms. Neither TPA or alkylglycerol had any effects on the distribution of PKC $\zeta.$ To examine the effect of alkylglycerol on cell proliferation, Swiss 3T3 cells were used. GLC analysis shows that 3T3 cells accumulate alkylglycerol in a similar manner as MDCK cells. Since this accumulation occurs just prior to cell growth arrest, the effects of alkylglycerol on preconfluent cells was observed. Preconfluent cultures of 3T3 cells were treated with alkylglycerol on day 1 of growth. After 8 days of culture, the treated group showed a slower growth rate and saturation density. Furthermore, after these cells were reseeded in the absence of alkylglycerol, the original growth rate and saturation density returned. Thus alkylglycerol induces a decrease in cell proliferation without causing any detrimental effects. Similarly, alkylglycerol was found to inhibit the induction of mitogenesis by TPA (a PKC dependent pathway) and these effects were shown not to be stereospecific. To further investigate the effect of alkylglycerol on cell proliferation, the content of the monoglycerides in ras-transformed cells was analyzed. These cells have lost contact dependent growth arrest indicating a disruption of cell growth regulation. We observed a massive increase in the content of alkylglycerol during the culture of ras transformed cells. This increase is 3 fold higher than MDCK or 3T3 cells. This raises the possibility that alkylglycerol may be the end result of an increased number of cell-cell contacts. We have observed an increase in the accumulation of alkylglycerol in normal and ras-transformed cells. This accumulation is accompanied by a decrease in PKC activity and alkylglycerol was shown to be a potent in vitro inhibitor of PKC. Similarly, alkylglycerol was shown to inhibit PKC $\alpha$ under stimulation by TPA. Alkylgylcerol is a inhibitor of the TPA induced induction of mitogenesis and slows the growth rate of proliferating cultures of 3T3 cells. These results indicate that the endogenous ether-linked glycerolipid, alkylglycerol, is a regulator of cell proliferation through its inhibitory effects on protein kinase C. (Abstract shortened by UMI.)

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Arojo, Omotooke Ajoke. "The Role of Sin 1-mTORC2 in the Regulation of T Cell Homeostasis." Thesis, Yale University, 2018. http://pqdtopen.proquest.com/#viewpdf?dispub=10783434.

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In healthy individuals, T cells generally exist in a state of equilibrium or homeostasis. This homeostasis is achieved by a tight and dynamic regulation of signaling cascades that control T cell numbers, localization, metabolism, and differentiation at any given time. Dysregulation of T cell homeostasis underlies several immunopathologic conditions including autoimmune diseases and cancer, hence the need to better understand molecular mechanisms that control T cell homeostasis. The mechanistic target of rapamycin (mTOR) is a critical hub within several important signaling cascades that are operational throughout the life of a T cell from thymic development to antigen encounter and beyond. While significant progress has been made in uncovering the roles of mTOR in various aspects of T cell immunity, the molecular mechanisms that dynamically regulate the threshold of mTOR signaling at various stages of the T cell life cycle and how this contributes to T cell homeostasis are poorly understood. mTOR functions as the enzymatic nucleus of two multiprotein complexes designated mTOR complex 1 (mTORC1) and mTORC2. We investigated the role of mTORC2 in naïve T cell homeostasis and the dynamic biochemical regulation of mTORC2 signaling by Sin1 and its isoforms. This led us to uncover a previously unappreciated but fundamental aspect of T cell biology; active mTORC2 signaling in naïve T cells suppresses bone marrow homing and in this manner allows T cells recirculate more efficiently through secondary lymphoid organs for better immune-surveillance. Furthermore, our exploration of the dynamic biochemical regulation of mTORC2 in naïve versus activated T cells revealed a potential mechanism through which dynamic mTORC2 signaling is achieved. We found that the overall strength of mTOR signaling is controlled at the level of transcriptional expression of mTOR and its adaptors by TCR stimulation. More broadly, we also investigated the biochemical regulation of mTORC2 by Sin1 isoforms, taking advantage of their domains to understand how various upstream signals might control mTORC2 activity. Through these investigations, we established that Sin1 isoforms differentially regulate mTORC2 response to Pi3K and non-Pi3K signals as well as mTORC2 target substrate specificity.

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Sakanovic, Alenko. "EphrinB2 reverse signaling in endothelial cell migration and actin remodeling." Thesis, University of Ottawa (Canada), 2005. http://hdl.handle.net/10393/27030.

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Angiogenesis, the formation of new blood vessels from preexisting vasculature, is exaggerated in cancer. A ligand that may be involved in angiogenesis is ephrinB2, which upon binding to its cognate receptor on an adjacent cell, becomes phosphorylated on tyrosine residues and transduces an as yet uncharacterized signal inside the cell. To examine reverse signaling pathways mediated by ephrinB2, ephrinB2 mutants were generated in which five tyrosine residues in the cytoplasmic tail were mutated to phenylalanine by site directed mutagenesis to effectively block the putative signal transduction pathways. Upon overexpression of wildtype and mutant ephrinB2 in endothelial cells, cellular phenotype changed drastically, with impeded proliferation and severe impact on actin remodeling. Observed cytoskeletal changes suggested that the Rho-like GTPases were involved in the actin remodeling mediated by the cytoplasmic tail of ephrinB2. Thus, activation of Rac and Rho upon ephrinB2 stimulation with soluble EphB6, a cognate receptor of ephrinB2, was examined. Adenoviral vectors encoding wildype and mutant ephrinB2 were also generated and used to infect endothelial cells. Wounding assays were used to monitor cell migration of ephrinB2 overexpressing cells to characterize the role of ephrinB2 in this process. Our data suggest that ephrinB2 may play an important role in the migration of endothelial cells during angiogenesis and future work will identify the signaling components of this pathway.

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Ibarra-Sánchez, Maria. "Biochemical and cellular function of t-cell protein tyrosine phosphatase." Thesis, McGill University, 2003. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=82895.

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Tyrosine phosphorylation is one of the most important post-translational modifications used by cells to respond to external stimuli. Two different sets of enzymes control the tyrosine phosphorylation levels of proteins: the protein tyrosine kinases and the protein tyrosine phosphatases. The identification of the cellular function of these enzymes is a key step in the understanding of many biological processes, such as proliferation, differentiation and apoptosis. We used a mouse model deficient in the protein tyrosine phosphatase TC-PTP to identify its biological and biochemical functions. Analysis of the phenotype of murine embryonic fibroblasts from TC-PTP-/- embryos revealed that TC-PTP-/- cells have an impaired proliferation due to a slow progression during the early G1-phase of the cell cycle. In these cells, there is a delayed expression of cyclin D1 through a defective IKK/NF-kappaB signaling pathway. These data confirmed that TC-PTP plays a positive role in cell proliferation.
In the second part of this thesis, I studied the putative role of TC-PTP in response to DNA damage. I demonstrated that mice and cells lacking TC-PTP have a high sensitivity to genotoxic stress, which seems to be associated with an increased stability and elevated phosphorylation of serine-15 of the tumor suppressor p53. We found that the p53 inhibitor, Mdm2 is highly tyrosine phosphorylated in TC-PTP-/- cells. High tyrosine phosphorylation levels on Mdm2 are associated with more stability of p53. Although, it was not possible to show that Mdm2 is a direct substrate for TC-PTP, this seems to be the most likely mechanism used by TC-PTP to control p53. Interestingly, I also showed that double deficient mice for both TC-PTP and p53 live longer and display a normal spleen compared with the TC-PTP single knockout. However, another phenotype like the anemia is not rescued in the double knockout mice. Murine embryonic fibroblasts from TC-PTP-/- p53-/- embryos also have a better survival after genotoxic stress when compared with the TC-PTP-/- cells. Thus, we propose that TC-PTP plays a role in response to DNA damage by affecting the tyrosine phosphorylation levels of Mdm2, and its associated increased stability of the tumor suppressor p53
In the last part of my thesis, I tested the genetic interaction between the protein kinase c-Abl and the protein tyrosine phosphatase TC-PTP. Taking in account the knockout phenotypes of both enzymes, we generated double deficient mice for both TC-PTP and cAbl. TC-PTP-/- c-Abl-/- mice are viable, but there was no difference in the time of survival of these animals when compared with the single knockouts. However, data obtained with primary fibroblasts indicated that indeed there is a genetic interaction between TC-PTP and c-Abl. Double deficient primary fibroblasts proliferate better than TC-PTP-/- cells, and also present a better survival in response to genotoxic stress. The induction of p53 is also reduced in comparison with TC-PTP-/- cells. These results indicate that a genetic interaction between both enzymes exists and that this phenomenon is likely to be resulting from the existence of at least one common substrate.

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Miller, Christina Roshek 1969. "Photosensitive liposome-cell interaction in vitro." Diss., The University of Arizona, 1998. http://hdl.handle.net/10150/288913.

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Bennett and O'Brien [Biochemistry 1995 34, 3102] showed that the ultraviolet light exposure of two-component large unilamellar liposomes (LUV) composed of a 3:1 molar mixture of dioleoylphosphatidylethanolamine (DOPE) and 1,2-bis[10-(2'-hexadienoyloxy)-decanoyl]-sn-glycero-3-phosphatidylcholine (bis-SorbPC) facilitated liposome fusion. The rate and extent of fusion was dependent on the extent of photopolymerization, the temperature, and the pH. Here, the effect of the molar lipid ratio of DOPE/bis-SorbPC liposomes on the temperature for the onset of fusion, was characterized by changing the relative amounts of unreactive polymorphic lipid, and reactive lamellar lipid. The cellular uptake of liposomes is mediated by nonspecific adsorption of liposomes onto the cell surface and subsequent endocytosis. This research compared the effect of liposome surface charge on liposomal binding and endocytosis by a human cancer cell line, HeLa, and a murine macrophage cell line, J774. LUV were composed of dioleolylphosphatidylcholine with and without either a cationic lipid, dioleoyldimethylammonium propanediol, or an anionic lipid, dioleolylphosphatidylserine. HeLa cells endocytosed cationic liposomes to a greater extent than either neutral or anionic liposomes and with PEG- LUV, a neutral PEG-lipid over the anionic PEG-PE2000. In contrast, the extent of liposome endocytosis by J774 cells was quite similar for both cationic and anionic liposomes, both greater than neutral liposomes. Incorporation of a neutral PEG lipid may minimize interactions with cells of the RES, yet strongly interact with proliferative cells. Clapp et al., [Macromolecules 1997 30, 32] demonstrated that certain amphiphilic cyanine dyes are capable of sensitizing lipid polymerization to visible light. The individual effects of pH, light intensity, temperature, and the requirement for oxygen suggested that the polymerization process is initiated by electron transfer from the dye excited state to oxygen, to yield superoxide anion, which in aqueous media combines to form hydrogen peroxide. Here, irradiation of cell-associated visible light sensitive liposomes sensitized with either the cationic dye, N, N' -dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine, DiIC(18)3, or a sulfonated derivative, DiI-DS, caused cell membrane damage and cytoplasmic delivery of liposomal contents could not be confirmed.

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Westrate, Laura Michelle. "Quantitation of Mitochondrial Dynamics Reveals Critical Roles for Mitochondrial Morphology in Cell Cycle Progression and Apoptosis." Thesis, Van Andel Research Institute, 2017. http://pqdtopen.proquest.com/#viewpdf?dispub=10274820.

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The mitochondrion is a complex, double membrane organelle that serves several important cellular functions including ATP synthesis, Ca ²⁺ buffering, and ROS homeostasis. Although classic mitochondrial diagrams depict the mitochondrion as a simple oval or “bean” shaped organelle, the mitochondria can form extensive tubular networks or numerous small spheres in response to various cellular environments through two opposing processes, mitochondrial fission and fusion. Deregulation of mitochondrial dynamics has been implicated in a wide range of diseases, including Parkinson’s disease, heart disease and cancer. While significant emphasis for the last 15 years has been placed on the identification of the protein machinery responsible regulating mitochondrial morphology, it remains less clear how mitochondrial morphology affects various cellular functions and cellular fate outcomes. This thesis summarizes our findings on how mitochondrial morphology regulates cellular fate in the context of mitotic cell division and apoptosis. Using live cell microscopy and image analysis software we characterized mitochondrial dynamics with single cell resolution. We found that loss of key components of the mitochondrial fission machinery promotes a defect in cell cycle progression, characterized by an inability for cells to exit G2/M. Prolonged periods of mitochondrial fusion induced potent cell death, suggesting a novel mechanism to target the replicative potential of cancer cells. We also found that mitochondrial fission and fusion can alter the kinetics of cell death following apoptotic stimuli by inducing mitochondrial fusion prior to the commitment step in apoptosis, mitochondrial membrane permeabilization. This thesis summarizes our work in trying to elucidate how the structure of the mitochondria influences both mitochondrial and cellular fate.

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Rodrigues, Sonia Patricia. "The function of the Crk adapter proteins in cell migration and invasion /." Thesis, McGill University, 2004. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=80866.

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Epithelial morphogenesis is an important signaling program in normal embryonic development involving cell proliferation, migration, invasion and cell matrix turnover and also during tumourigenesis. The Crk adaptor family of proteins has been implicated as promoters and mediators of such biological responses downstream of various extracellular signals. Our laboratory has defined the involvement of Crk adaptor proteins in the signaling of Met-dependent anchorage-independent growth, cell dispersal, cell invasion and epithelial morphogenesis. In an MDCK epithelial cell model CrkII overexpression was sufficient to promote cell spreading. This initial biological response was dependent on Rac activity. In Chapter 3 I demonstrate that MDCK cells overexpressing CrkII unstimulated, or stimulated with HGF, exhibit higher levels of activated Rac1. In an attempt to validate the function of Crk during cell migration and invasion I have developed targeted siRNAs to CrkI/II and CrkL and assessed their impact in the disruption of migration and invasion of cancer cell lines. In Chapter 4 I demonstrate that siRNAs promote a specific decrease in the level of Crk adaptor proteins. Notably this decrease impairs motility and invasion of several human tumor cell lines. I provide evidence that a role for Crk during cell migration or invasion is to promote the formation of cell matrix adhesion contacts necessary for cell spreading and migration. These studies have established that Crk adaptor proteins are required and may be key promoters of cell migration events during tumorigenesis.

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22

He, Jiang. "Fluorescence Imaging of Virus-Host Cell Interaction and Super-Resolution Imaging of Neuronal Cytoskeleton." Thesis, Harvard University, 2015. http://nrs.harvard.edu/urn-3:HUL.InstRepos:26718714.

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To study biological molecules, pathways and processes, fluorescence microscope has become an indispensable tool in modern biology. The major advantages of using fluorescence microscope include its ability to achieve molecule-specific visualization for targets of interest, and the compatibility of live cell imaging. With the advent of super-resolution imaging techniques, fluorescent microscope, for the first time, allows researchers to study biological processes on the nanometer scale. In this dissertation, I present the application of fluorescence imaging to characterize the role of host factors in influenza virus infection, and super-resolution imaging to study the developmental mechanism and prevalence of a periodic membrane skeleton in neurons. In Chapter 2 and 3, my colleagues and I studied the role of CD81 and COPI complex, two host factors identified in large-scale screens, for influenza infection. We found that CD81 regulates two distinct steps during influenza infection cycle: virus uncoating during entry and virus budding during egress. Depleting CD81 led to a significant defect in viral uncoating and viral gene replication during entry, while during virus egress, CD81 depletion resulted in virions that failed to detach from the plasma membrane and a marked decrease in progeny virus production. For COPI complexes, we found that COPI plays a direct role in viral membrane protein expression and assembly post-viral entry. In Chapter 4 and 5, we used super-resolution imaging to study the developmental mechanism and prevalence of a newly discovered periodic membrane skeleton in axons, formed by actin, spectrin and associated molecules. We found that the periodic membrane skeleton is highly prevalent in different neuronal types. It forms early during neuronal development, and originates from regions closer to the cell body and propagates toward the distal ends of axons. The lattice structure further matures by recruiting additional molecular components and appears to be highly stable once formed. The local concentration of βII spectrin is a key determinant for the formation of this periodic membrane skeleton. In addition, we identified ankyrin B as a critical molecular component for the polarized distribution of βII spectrin in neurites.
Biology, Molecular and Cellular

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23

Saleh, Maya. "Protein-protein interactions and cell signaling in the regulation of HOX.PBX functions." Thesis, McGill University, 2001. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=37620.

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HOX proteins are homeodomain-containing transcription factors essential for embryonic patterning. Despite amino acid differences, all HOX homeodomains recognize highly similar sites on DNA. One mechanism by which HOX proteins achieve specificity is through interaction with cofactors of the PBX and MEIS/PREP1 families. Higher order complexes between HOX, PBX and MEIS/PREP1 proteins form in vivo and are essential for target recognition and transcriptional regulation. Another level of control of HOX function is the nuclear availability of its cofactors. This thesis addresses the regulation of the nuclear availability of the PBX protein by MEIS/PREP1 family members. We identified two nuclear localization signals (NLS) in the PBX homeodomain and showed that the NLS are masked in the absence of MEIS/PREP1. Upon a conformational change in PBX induced by MEIS/PREP1 binding, the NLS are exposed and a receptor-mediated active transport of PBX into the nucleus is allowed. This thesis also investigates the mechanisms of transcriptional regulation by the HOX·PBX complexes. We show that HOX·PBX complexes repress transcription and are switched to transcriptional activators in response to cell signaling. We demonstrate that PBX mediates the repression function by recruiting histone deacetylases (HDACs) to HOX target promoters. Inhibition of HDAC activity or stimulation of protein kinase A (PKA) signaling converts the HOX·PBX complex into a net activator of transcription. The activation function is mediated by the HOX protein through its recruitment of CREB-binding protein (CBP), a coactivator with histone acetyl-transferase (HAT) activity. We propose a model whereby HOX·PBX transcriptional activity is determined by cell signaling, and is mediated by the local modification of chromatin structure in the promoter of downstream targets.

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24

Niedowicz, Dana M. "The mechanisms of hyperglycemia-induced oxidative stress in human erythrocytes." [Bloomington, Ind.] : Indiana University, 2006. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:3204307.

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Thesis (Ph. D.)--Indiana University, Dept. of Biology, 2006.
Source: Dissertation Abstracts International, Volume: 67-01, Section: B, page: 0043. Adviser: David L. Daleke. "Title from dissertation home page (viewed Feb. 21, 2007)."

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25

Abrol, Meena. "The role of Sir2alpha in myogenesis." Thesis, University of Ottawa (Canada), 2003. http://hdl.handle.net/10393/26434.

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Protein acetylation is becoming recognized as an important modification in the regulation of many cellular pathways. The silent information regulator 2 (Sir2) proteins are a family of NAD-dependent protein deacetylases. Sir2 protein function in yeast has extensively been characterized as an essential mediator of gene silencing that occurs at the mating type loci, the telomeres and the rDNA regions. There are Sir2 homologues from yeast to mammals, but little is known about the role of Sir2 in the mammalian system. There is evidence that myogenesis is regulated by the acetylation state of the histones in the promoter region of muscle-specific genes as well as the myogenic transcription factors. MyoD is a myogenic transcription factor that plays a key role in myogenesis. Its activity is required to initiate and maintain transcription of muscle-specific genes. In addition to this, acetylation of MyoD is required for its activity. We set out to determine if MyoD is a target for deacetylation by Sir2alpha, and thus determine if Sir2alpha has a role in myogenesis. We determined that MyoD transactivation activity is not inhibited by Sir2alpha deacetylation. Over-expression of Sir2alpha-wt or a catalytically inactive mutant Sir2alpha-H355Y in C2C12 cells also showed that Sir2alpha deacetylase activity does not effect myogenesis. In addition to this, nicotinamide, a Sir2 inhibitor, had no effect on myogenesis while TSA significantly inhibited myogenesis in the C2C12 cells, suggesting that Class I and II HDACs have a more important role than Sir2 in regulating MyoD activity.

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26

Gangaraju, Sandhya. "Role of mitofusin2 in the regulation of mitochondrial dynamics." Thesis, University of Ottawa (Canada), 2003. http://hdl.handle.net/10393/26483.

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Mitochondria in all cell types undergo frequent fission and fusion events, and these dynamics determine the overall morphology of the organelle in cells. Two important GTPases have been recently identified that regulate mitochondrial membrane activity, a dynamin related protein (DRP1) required for fission, and the novel fusion GTPase, Mitofusin2. Mitofusin2 is an outer mitochondrial membrane protein and, like other GTPases involved in membrane fusion events, the N-terminal GTPase domain is exposed to the cytosol, such that it could interact with and recruit potential cytosolic proteins. The work documented in this thesis aims towards understanding the specific role of this unique GTPase in regulating mitochondrial fusion events, and to identify potential interacting proteins that work together with Mfn2 to carry out this complex biochemical event. Two independent approaches were taken to identify interacting proteins, both a yeast two-hybrid screen and affinity chromatography using recombinant bacterial expressed Mfn2 protein as bait. (Abstract shortened by UMI.)

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27

Taylor, Rebecca Ann C. "VSV infection of resting and activated T lymphocytes." Thesis, University of Ottawa (Canada), 2004. http://hdl.handle.net/10393/26782.

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Resting T lymphocytes are uniquely resistant to VSV even at high multiplicities of infection but they can be rendered fully permissive for VSV replication following in-vitro activation with monoclonal anti-CD3 and PMA with ionomycin. The block to VSV replication is at the level of viral RNA production and is independent of transcription following infection. T lymphocytes must be activated before infection for at least 24 hours to be rendered susceptible to VSV and transcription is an absolute requirement during this process. Fusion of resting and activated T cells results in a resistant cell indicating that the resistant state is dominant. Resting T cells do not produce interferon (IFN) alpha or beta in response to VSV infection whereas activated T cells produce both but have a down modulated response to the antiviral activity of type I IFNs. Gene expression array analysis demonstrates that the interferon response factors, IRF-4 and IRF-8, are up regulated during activation and a number of interferon stimulating genes (ISG), including ISG20, MxA and GBP-1 are down regulated during activation. IRF-4 and IRF-8 have both been described to bind to and inhibit transactivation from the interferon stimulated response elements (ISRE) in the promoters of a number of ISGs and this could explain the down modulated antiviral response to type I IFNs in activated T cells. The IFN-independent constitutive expression of these ISGs in resting T cells is a possible mechanism for their unique resistance to VSV replication.

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28

Dunkley, Rosamund. "Ubiquitous over-expression of human X-linked inhibitor apoptosis (XIAP) in a transgenic mouse and implications for tumorigenesis." Thesis, University of Ottawa (Canada), 2005. http://hdl.handle.net/10393/26895.

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The suppression of programmed cell death is an essential alteration in the transformation from normal to neoplastic growth. The X-linked inhibitor of apoptosis (XIAP) is a potent suppressor of apoptosis. It is a member of the Inhibitor of Apoptosis (IAP) family and functions by inhibiting caspases 3, 7 and 9 critical proteases in the process of programmed cell death. XIAP expression levels are frequently elevated in cancer cell lines and tumors, yet the link between XIAP expression and tumorigenesis has not been demonstrated experimentally. The objective of this project is to determine whether XIAP over-expression predisposes mice to cancer. A XIAP transgenic mouse model has been created with expression of the transgene driven by the ubiquitin C promoter. The UbiC-6-myc XIAP transgenics demonstrate ubiquitous over-expression of the human homolog of XIAP. Transgene over-expression was detected by western blot in all tissues tested including brain, retina, thymus, lung, heart, liver, kidney, pancreas, and spleen. The mice develop normally and show no unusual phenotype. Homozygous mice have been bred that show a further two-fold over-expression relative to their heterozygous littermates. Suppression of apoptosis has been documented in in vitro studies of mouse embryo fibroblasts and hepatocyte cultures. Transgenic XIAP provides protection against injury-induced apoptosis in vivo in a high dose streptozotocin induced pancreatic beta cell damage model in adult mice. The mice have been bred with an inducible c-myc oncogene transgenic strain that expresses c-myc in the beta cells of the islets of Langerhans when activated by tamoxifen (obtained from Dr. Gerard Evan). The myc/XIAP double transgenics responded in the same manner as the single c-myc transgenics and the beta cells underwent apoptosis. These results suggest a model where XIAP suppresses injury or stress induced apoptosis but is unable to block genetically pre-determined or oncogene-activated apoptosis.

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29

Czechura, Pawel. "Saturated neoglycopolymers for tissue engineering." Thesis, University of Ottawa (Canada), 2006. http://hdl.handle.net/10393/27121.

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Norbornene monomers bearing carbohydrate groups of relevance for tissue engineering were synthesized via the norbornene acid chlorides, transformed into their ROMP polymers, and reduced to yield saturated neoglycopolymers. These materials bear either O-glycoside groups designed to cross-link collagen via reaction of the ring-open sugar with free NH2 groups of lysine, or C-glycoside groups. The latter are intended for use as the central block in triblock copolymers terminated with blocks capable of crosslinking: they serve only as potentially biocompatible "spacers" to help span the interlamellar distance in collagen. A third ROMP monomer bearing a succinimide group as an alternative crosslinking agent was also prepared and polymerized. The O-glycoside monomer, bis(1,2;3,4-di-O-isopropylidene-D-galactopyranos-6-O-yl) 5-norbornene-2,3-dicarboxylate 9, and the succinimide monomer, 5-norbornene-2-carboxylic acid N-hydroxysuccinimide ester 11, were prepared by the Diels-Alder synthesis of 5-norbornene-2,3-dicarbonyl chloride 7 and 5-norbornene-2-carboxylic acid chloride 8, and subsequent nucleophilic substitution of the acid chlorides. (Abstract shortened by UMI.)

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30

Dykstra, Natalie. "The investigation of microbial-intestinal epithelial cell interactions in the gut and their effects on inhibitor of apoptotic proteins (IAPs)." Thesis, University of Ottawa (Canada), 2008. http://hdl.handle.net/10393/27978.

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Probiotic bacteria such as Lactobacillus plantarum strain 299v (Lp299v) have been shown to upregulate facets of innate immunity. This study assessed whether Lp299v prevents apoptosis in intestinal epithelial cells (IECs) when faced with cytokine challenge. Both in vitro and in vivo systems were exposed for a pre-determined time to Lp299v or strain Lpadh---a non-adherent derivative of Lp299v. HT-29 cells were then challenged with cytokine mixture (TNF-alpha, IFN-gamma, and IL-1a) to imitate conditions of inflammation. To assess for apoptosis, we evaluated both TUNEL analysis and caspase activity assays. Results of the assays indicate a marked decrease in apoptosis in samples treated with Lp299v. The adherent negative Lpadh-strain showed similar but less pronounced prevention of cytokine-induced apoptosis. IAPs were assessed both in vitro and in vivo and HIAP1 and HIAP2 were determined to be involved in this process. This is consistent with NF-kB activation by Lp299v. Probiotics may confer exogenous effects on IECs to elaborate protective mechanisms and alter cell homeostasis through alteration of caspase-dependent apoptosis.

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31

Ewing, Robyn. "Analysis of the subcellular trafficking of the glucocorticoid receptor and properties of the ligand binding domain." Thesis, University of Ottawa (Canada), 2008. http://hdl.handle.net/10393/27979.

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The glucocorticoid receptor (GR) is a ligand dependent transcription factor and member of the nuclear receptor superfamily. Nuclear import and export of transcription factors is accomplished through nuclear localization signals (NLS) and nuclear export signals (NES), respectively. We have determined that L687 and L690 of rat GR are necessary for the characteristically slow nuclear export of GR and may be included in the signal sequence responsible for directing post-agonist withdrawn GR from the nucleus to the cytoplasm. We also suggest that L687 and L689 of rat GR are required for efficient NL2-mediated nuclear translocation. Substitutions L687A and L689A mildly affect steroid binding and steroid off-rate, yet significantly increase the concentration of steroid required for inducing nuclear import of naive GR. When introduced into GRNL1-, these substitutions compromise the receptor's ability to transfer to the nucleus, suggesting they partially abrogate NL2 activity. We have also observed that NL1-dependent transfer does not begin until 10 -7M steroid, demonstrating that NL2 is the primary physiological mediator of GR nuclear import.

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32

Guzzo, Rosa M. "Sarcolemmal membrane associated proteins: Structure-function analyses and localization studies." Thesis, University of Ottawa (Canada), 2003. http://hdl.handle.net/10393/29048.

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Previous work from our lab identified a novel cDNA encoding a family of tail-anchored coiled-coil integral membrane proteins termed SLMAPs (sarcolemmal membrane associated proteins) (Wigle et al., 1997). Subsequent studies determined that SLMAPs are encoded by a single gene, and alternative splicing yields three SLMAP isoforms, including two muscle-specific variants (SLMAP1, SLMAP2) and a ubiquitously expressed isoform (SLMAP3). Here, I report a series of studies designed to examine putative novel and isoform specific functions of SLMAPs in striated muscle and fibroblast cells. The tissue distribution and subcellular localizations of SLMAPs were examined in developing and adult mouse tissues in order to correlate SLMAP expression with specific physiological or developmental process(es). Immunohistochemical staining using polyclonal anti-SLMAP antibodies revealed that SLMAPs are highly expressed in developing somites and cardiac tissue. Confocal microscopy determined that SLMAPs localized within the discrete membrane structures (sarcoplasmic reticulum and T-tubules) of developing and mature skeletal and cardiac muscle, respectively. These localization studies suggest a correlative role for SLMAPs in excitation-contraction (EC) coupling mechanisms. In vivo expression of SLMAPs in pre-fusion myoblasts indicated a possible involvement in skeletal myogenesis. An additional SLMAP protein was expressed under conditions that promote differentiation in cultured myoblasts. Deregulation of SLMAPs by ectopic expression in myoblasts resulted in a potent inhibition of fusion without affecting the expression of muscle-specific genes. Protein-protein interaction assays demonstrated that the leucine zipper motifs in SLMAPs mediate SLMAP homodimer formation. Proteomic analysis further revealed that a muscle-specific SLMAP variant binds a component of the contractile apparatus (cardiac myosin heavy chain). The expression of a cardiac-specific SLMAP isoform that resides in distinct membranes, self assembles and interacts with the contractile apparatus further suggest a unique role for this molecule in excitation-contraction coupling mechanisms. Alternative splicing mechanisms generate SLMAP variants with divergent carboxyl-terminal hydrophobic segments, which target SLMAPs to different membrane compartments. Immunocytochemistry studies revealed that the expression of the first transmembrane domain directs a 6Myc-SLMAP fusion protein to the endoplasmic reticulum in COS7 cells. (Abstract shortened by UMI.)

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33

Bou, Khalil Maroun. "Biophysical and biochemical properties of the mammalian male germ cell specific sulfogalactosylglycerolipid (SGG): Contribution to the structure and zona pellucida affinity of pig sperm raft membranes." Thesis, University of Ottawa (Canada), 2004. http://hdl.handle.net/10393/29079.

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Raft membranes are implicated in cell adhesion. Here, we demonstrated that rafts isolated from hypermotile capacitated pig sperm as low-density Triton X-100 insoluble membranes had the ability to bind specifically to homologous zonae pellucidae (ZP). This binding was dependent on pig ZP3alpha sulfoglycoprotein, a major player in intact sperm binding. The male germ cell specific sulfogalactosylglycerolipid (SGG) was a sperm raft component and participated in sperm raft-ZP binding, since rafts pretreated with anti-SGG IgG/Fab had decreased ability to bind to the ZP dose dependently. SGG may also partake in raft formation. Fourier transform infrared (FTIR) spectroscopic studies of mixed SGG-cholesterol liposomes revealed that the sulfoglycolipid interacted with cholesterol (a significant raft component) via intermolecular hydrogen bonding. Moreover, mixtures of SGG and cholesterol were insoluble in 1--2% Triton X-100. Since cholesterol is significant for raft formation and since sperm capacitation is associated with cholesterol efflux, we determined whether raft levels in capacitated sperm were the same as those in control sperm. Interestingly, our results revealed an increase in raft levels in the capacitated sperm, as well as an enhanced ZP affinity of these membrane domains, compared to those of control sperm. These results corroborated the implication of rafts in cell adhesion and strongly suggested that the enhanced ZP-binding ability of capacitated sperm may be attributed to increased levels of sperm rafts, with a greater affinity for the ZP.

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34

Stewart, Nicolas Andre Stirling. "In vacuo chemical modifications of proteins and peptides." Thesis, University of Ottawa (Canada), 2004. http://hdl.handle.net/10393/29170.

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A novel approach for the chemical modification of proteins and peptides was developed, namely the chemical modification under vacuum (in vacuo) of lyophilized proteins and peptides. This method was used with iodomethane to produce peptides with a permanent positive charge by converting their amino groups to trimethylammonium derivatives for analysis by matrix assisted laser desorption ionization mass spectrometry (MALDI MS). Peptides with such a permanent charge have a higher ionization yield under MALDI MS resulting in a dramatic increase in detection. The signal intensity of the trimethylammonium derivative in MALDI MS is much greater compared to the unmodified peptide. The trimethylammonium derivative of alpha-amino groups of peptides derived from test lyophilized peptides and a lyophilized protein, and an epsilon-amino group of a peptide derived from the solitary lysine residue of the oxidized B-chain of insulin were shown to have a large increase in signal intensity compared to the unmodified peptides. The in vacuo methylation procedure is readily amenable to the use of a combination of isotopes, e.g. 12CH3I and 13CH3I or CH3I and CD3I, yielding a doublet signal to discriminate between peptide and non-peptide signals in the mass spectrum providing an even greater increase in sensitivity. This strategy was employed to isolate an N-terminal peptide derived from an enzymatic digest of human hemoglobin and to facilitate the analysis of spectra. Proteins and peptides lyophilized from slightly alkaline solutions yielded the greatest amount of derivatization. However, a peptide lyophilized under acidic conditions could also be methylated to give an increased MALDI signal. In light of these results a mechanism is proposed where the vacuum facilitates the removal of gaseous counter-ions to drive the reaction to the trimethylammonium derivative. In order to selectively isolate the C-terminal peptides from enzymatic digests of proteins, lyophilized proteins were reacted with iodomethane under conditions where reaction occurred mainly with carboxylate groups. A proof of concept has been demonstrated for a method of general applicability for the determination of the C-terminal sequences of proteins. Separation and isolation of the C-terminal peptides generated from enzymatic digests for further sequencing either by chemical means (N-terminal Edman degradation chemistry) or by tandem mass spectrometry (MS/MS) was accomplished by the use of two dimensional (2D) high voltage paper electrophoresis (HVPE). (Abstract shortened by UMI.)

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35

Korley, Robert E. C. "Characterization of a major protein of the mouse perinuclear theca." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0035/MQ64383.pdf.

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36

Harrouk, Wafa. "Mitogen-activated protein kinase during oocyte growth in the mouse." Thesis, McGill University, 1994. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=55499.

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In this study, the role of MAP kinase in the acquisition of meiotic competence in growing oocytes was investigated. The results presented in this thesis show that two species of MAP kinase, p42 and p44, are present in their unphosphorylated forms in oocytes as early as 5 days of age. At this age, oocytes are small and have not acquired the capacity to resume meiosis. They are referred to as meiotically incompetent. MAP kinase continues to be present throughout the growth phase and up to the acquisition of meiotic competence.
In growing mouse oocytes, a group of partially competent oocytes are abundant. Such oocytes arrest at metaphase I where they assemble a morphologically normal spindle. Immunoblotting results of partially competent oocytes show that MAP kinase is present and becomes phosphorylated following culture as is indicated by the retarded mobility on the SDS gels.
Okadaic acid, an inhibitor of protein phospharases 1 and 2A, induces incompetent oocytes to enter metaphase. These oocytes contain the slow migrating phosphorylated forms of p42 and p44, indicating that okadaic acid causes the phosphorylation of MAP kinase. A time course study shows that the okadaic acid-induced phosphorylation of MAP kinase occurs coincidentally with entry into metaphase in incompetent oocytes. In fully competent oocytes, this phosphorylation occurs after entry into metaphase. In addition, these oocytes do not assemble a spindle, indicating that phosphorylation of MAP kinase, although it may be necessary, is not a sufficient event to induce spindle formation.

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37

Schroeder, Hans R. "S-acylation and intracellular targeting of lipid-modified proteins and model lipopeptides." Thesis, McGill University, 2000. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=36812.

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Three related studies were performed to better characterize the intracellular process of protein S-acylation in mammalian cells. The first study focused on the use of cysteinyl-containing fluorescent lipopeptides which mimic the N-terminal of various S-acylable intracellular regulatory proteins. These lipopeptides diffuse rapidly between membranes and are efficiently S-acylated by a variety of mammalian cells. S-acylation appears to be enzymatic by various criteria and is highly selective for cysteinyl as opposed to serinyl residues. Fluorescence microscopy revealed that the plasma membrane is the predominant site for intracellular S-acylation but that a second potential site localized to the Golgi apparatus may also exist.
The second study examined the intracellular S-acylation of lipopeptides which mimic the carboxy-terminus of N-ras. S-acylation of lipopeptides was again specific for cysteinyl residues as opposed to serinyl residues. The exact structure of the attached prenyl group, for example the farnesyl versus geranyl group, does not affect the ability of the lipopeptide to undergo S-acylation, however, an attached moiety with sufficient hydrophobicity to promote high affinity but rapidly reversible interactions with membranes is required for efficient S-acylation. Fluorescence microscopy suggests that S-acylation of these peptides and likely N-ras protein itself occurs at the plasma membrane of mammalian cells.
The third study examined the hypothesis that proteins of different sequences may be S-acylated by distinct S-acyltransferases. Lipopeptides bearing sequences mimicking the N-termini of src-like nonreceptor protein tyrosine kinases or heterotrimeric G-protein alpha-subunits, incorporate similar ratios of [3H]palmitate/[3H]stearate following incubation of lipopeptide with cells and equal activities of [3H]palmitate and [3H]stearate. In contrast, lipopeptides which mimic the C-terminus of N-ras exhibit ratios which are significantly different from the ratios for those lipopeptides described above. These results suggest that these two groups of structurally-different lipopeptides are S-acylated by distinct S-acyltransferases within the plasma membrane. Further, lipopeptides bearing nonphysiological sequences which are preferentially S-acylated in the Golgi, incorporate radiolabeled fatty acids in ratios which are significantly different from those determined for most lipopeptides S-acylated at the plasma membrane, suggesting that distinct S-acyltransferases may exist in the plasma membrane and Golgi apparatus of mammalian cells.

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38

Lee, Menq-Jer. "Biochemical characterization of statin, a protein marker specific fofor nonproliferating cells, and identification of its associated proteins in cultured human diploid fibroblasts." Thesis, McGill University, 1993. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=41296.

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Statin, a 57 kilodalton (kDa) protein expressed specifically in nuclei of non-proliferative cells (Wang, 1985a, c), is a phosphoprotein, phosphorylated at serine residues. By comparing immunoprecipitation and immunoblotting analyses, I find that p57$ sp{ rm statin}$ in vivo associates with other cellular proteins with molecular masses of 45 kDa (p45) and 110 kDa (p110).
The p45 statin-associated protein is a serine/threonine kinase. The specificity of the association of statin and p45 kinase is strongly supported by the fact that the kinase activity of p45 correlates both qualitatively and quantitatively with the amount of statin present in immunoprecipitates from cultured fibroblasts having different states of growth or from different subcellular fractions. In addition, the p45 kinase can phosphorylate statin in vitro, suggesting that statin may be one of the physiological substrates for the p45 kinase in vivo. The biochemical evidence indicates that p45 is not a member of the casein kinase II, cAMP-dependent kinase, calmodulin-dependent kinase, or protein kinase C families. The relationship of this kinase to other families is yet to be investigated.
The p110 statin-associated protein reacts with antibodies specific for the retinoblastoma susceptibility gene product, Rb. Also present in anti-Rb immunoprecipitates is 57 kDa protein which is recognized by anti-statin antibody (S44). Both p57 and p110 are co-precipitated from ($ sp{35}$S) -labelled quiescent cell extracts with either anti-Rb or anti-statin antibodies. However, in ($ sp{35}$S) -labelled extracts of growing cells, neither p110 nor p57 is present in S44-immunoprecipitates; and anti-Rb immunoprecipitates contain several protein species, the phosphorylated forms of Rb (112-116 kDa), but not p57. Comparisons of V8 digests indicate that p110 in S44-immunoprecipitates is Rb, and p57 in anti-Rb immunoprecipitates is statin. Confocal microscopical analysis shows that statin and Rb are colocalized within fibroblasts, predominantly in the nuclear envelope and matrix regions. These results strongly suggest that statin also associates with unphosphorylated species of Rb in the nuclei of nonproliferative cells.
Finally, the kinetics of statin expression in two states of cellular growth arrest, i.e. in young quiescent and in in vitro aged fibroblasts, was examined. I found that 95% of serum-starved cells express statin, independent of their in vitro age. After the addition of serum, the majority of senescent cells (CPDL $>$ 50) persistently express statin, whereas young cells (CPDL $<$ 20) lose statin after feeding. I also directly demonstrated that statin expression and DNA synthesis cannot occur simultaneously in the same cell. Furthermore, one additional nuclear protein possessing a common antigenic epitope to p57$ sp{ rm statin}$, with a molecular mass of 80 kDa, is detected only in senescent cells, but not in their young quiescent counterparts. Analysis of peptide maps indicates that this 80 kDa protein is different from p57$ sp{ rm statin}$. This finding implies that this unidentified 80 kDa protein may be a protein marker specific for senescent cells.

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39

Brignole, Claudine. "The adenovirus E4orf4 protein induces G2 / M arrest and cell death by inhibiting PP2A phosphatase activity regulated by the B Alpha subunit /." Thesis, McGill University, 2004. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=81604.

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The human adenovirus E4orf4 protein is toxic in human tumor cells and yeast. In both cases, interaction of E4orf4 with the Balpha/Cdc55 regulatory subunit of protein phosphatase 2A (PP2A) appears to be critical for cell killing; however, the effect of E4orf4 binding on PP2A is not known. Balpha is one of several B subunits that form holoenzymes with A and C subunits and regulate PP2A substrate specificity. In the present studies the effects of E4orf4 on PP2A activity was examined, as well as the role of PP2A in E4orf4-mediated cell death. Previously it was shown that binding of E4orf4 had little effect on the dephosphorylation of a universal peptide substrate by purified PP2A, however this study demonstrates that E4orf4 reduced PP2A activity to varying degrees against two substrates, phosphorylase a and histone H1, the latter known to represent a class of substrate against which Balpha-containing PP2A complexes are highly active. Expression of E4orf4 was also found to result in the accumulation of hyperphosphorylated forms of two PP2A substrates, 4EBP-1 and p70S6K (Balpha specific) in vivo, indicating a significant inhibition of PP2A activity against these substrates. The importance of such inhibition in cell killing by E4orf4 was supported by the fact that low levels of okadaic acid (OA) or expression of the heat stable I1 PP2A inhibitor, both of which inhibit PP2A activity globally, enhanced E4orf4-induced cell death. Furthermore, E4orf4 killing was reduced by overexpression of a Y307F mutant form of the PP2A catalytic Calpha subunit, which is insenstitive to downregulation by tyrosine phosphorylation. E4orf4 was found to induce a significant G2/M arrest, an effect that was enhanced both by treatment with OA, which alone at high levels has similar effects, and by expression of simian virus 40 small T antigen (SV40-ST), which binds to PP2A holoenzymes and preferentially replaces the Balpha subunit. These results suggest that E4orf4 induce

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40

Roudaia, Liya. "Characterization of RasGAP-SH3-binding protein's functions." Thesis, McGill University, 2004. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=82418.

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RasGAP-SH3 binding protein (G3BP) is a player in the ras signal transduction pathway and is involved in mRNA turnover. G3BP is a multifunctional protein affecting cell growth, cell stress responses, and cell transformation. The goal of this study was to define ex vivo function(s) of G3BP in several systems. We developed two RNA interference methods to knockdown G3BP in murine cell lines. We observed that the partial plasmid-mediated RNAi knockdown of MmuG3BP1 does not affect muscle cell differentiation. To improve efficiency, we successfully developed lentiviral-mediated RNAi against MmuG3BP1 resulting in significant knockdown in NIH-3T3 cells. We also developed vectors for pull-down experiments designed to identify G3BP1's binding proteins. Furthermore, we generated an efficient polyclonal anti-GST-G3BP1 antibody. Thus, we developed and tested tools to define the ex vivo function(s) of G3BP1.

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Cardillo, Marricco Nadia. "Characterization of casein kinase I-c2, as a serinethreonine protein kinase associated with the adaptor protein, Nck." Thesis, McGill University, 1998. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=21603.

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CKI-gamma2 is a serine/threonine protein kinase of unknown biological function. This study is the first to detect CKI-gamma2, as mRNA and protein, widely expressed in mouse tissues and mammalian cells. In addition, CKI-gamma2 as a protein of 75kDa was immunologically detected coprecipitating with Nck, an adaptor protein involved in receptor tyrosine kinase (RTK) signaling. CKI-gamma2 coprecipitated with Nck presents similar enzymatic properties as the recombinant and endogenous CKI-gamma2, demonstrating that the immunoreactive p75 CKI-gamma2 associated with Nck is indeed CKI-gamma2. These results suggest that Nck-mediated signaling downstream of activated RTKs could involve CKI-gamma2 as an important component. The finding that Nck is a substrate of CKI-gamma2, suggests that the function of Nck may be regulated by CKI-gamma2. Furthermore, observations that insulin negatively regulates p75 CKI-gamma2 activity provide evidence for a role of the Nck-CKI-gamma2 complex in insulin signaling. Understanding the insulin regulation of CKI-gamma2 may point to the biological function of this enzyme.

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Park, Minwoo. "2',3'-Cyclic nucleotide 3'-phosphodiesterase : investigation of interaction with Fyn tyrosine kinase during the development of nervous system, and mitochondrial import of CNP2 isoform." Thesis, McGill University, 2004. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=81423.

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2',3'-Cyclic nucleotide 3'-phosphodiesterase is a protein highly expressed in oligodendrocytes in the central nervous system, and it is believed to be an important regulator of myelination. In this report, two aspects of CNP are investigated, each introduced in their own chapters.
First, a possible interaction of CNP with Fyn tyrosine kinase during myelination is investigated. Fyn is an important factor known to be active during the process of myelination, and CNP contains a number of possible tyrosine phosphorylation sites. Furthermore, their presence in an isolated domain of cell membrane called lipid rafts, further strengthened the possibility of interaction. However, no evidence was found which could support the possibility, neither in new born mouse brain nor in in-vitro experiment using transfected cells expressing Fyn and CNP.
Secondly, the role of CNP2 isoform in mitochondria is investigated. CNP2 is found to be associated with mitochondria, and its 20 amino acid segment at the N-terminus possesses characteristics of a mitochondrial import signal. Furthermore, two known sites of serine phosphorylation in the N terminus of CNP2 show influence on mitochondrial localization of the protein. However, results collectively suggests that CNP2 is not imported into mitochondria, as pulse chase did not show the typical processing of what was suspected to be the N-terminal import signal sequence. Furthermore, CNP was degraded when partially purified mitochondria was subjected to protease action, showing that CNP is not enclosed by mitochondrial membranes. Two serine residues at positions 9 and 22 in the N-terminus of CNP2 are likely phosphorylated by both PKA and PKC, and is responsible for the decrease in mitochondrial localization of CNP2.

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Minuk, Jeffrey. "Studies on the intracellular sorting of the two myelin-associated glycoprotein isoforms." Thesis, McGill University, 1995. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=23414.

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Myelin-associated glycoprotein (MAG) is amongst the earliest expressed myelin specific proteins. Its precise function remains unknown, though evidence supports a role for cell-cell adhesion. MAG may also act as a spacer molecule and could be involved in signal transduction. MAG is expressed as two isoforms, designated as L-MAG and S-MAG. Both share identical extracellular and transmembrane domains but differ in their cytoplasmic domains. L-MAG is expressed earlier during myelination than S-MAG. These features, as well as others, suggest that the isoforms have different functions. To confirm this hypothesis, both isoforms were expressed transiently and stably in Madin-Darby canine kidney (MDCK) epithelial cells and the localization of the isoforms was studied. In both transiently and stably transfected cells, L-MAG sorted primarily to the basolateral membrane. In single transfected cells, S-MAG sorted primarily to the apical membrane. When groups of adjacent cells became transiently transfected, S-MAG accumulated at areas of cell-cell contact within the basolateral membrane. In stably transfected cells S-MAG sorted to the basolateral membrane. The data suggest that L-MAG contains an invariable basolateral sorting signal, but that the sorting of S-MAG is dependent upon extrinsic factors, such as the co-expression by adjacent (contacting) cells. As MDCK cells sort the MAG isoforms differently, these data support the hypothesis that the MAG isoforms do perform different functions.

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Côté, Richard Gaston. "Characterization of the retinoblastoma binding protein 2 (RBP2)." Thesis, McGill University, 2000. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=33388.

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The retinoblastoma (RB) family of proteins plays a pivotal role in cell cycle regulation. Its members, p105/Rb, p107 and p130, interact with the E2F and DP family of transcription factors to regulate transcription of essential cell cycle and DNA synthesis genes. Several reports have mapped the regulation of E217 by RB family members to the "pocket" domain of these proteins. We demonstrate here that RBP2, a pocket-binding protein that encodes multiple DNA-binding and protein-protein interaction domains, is a transcriptional repressor. Overexpression of RBP2 inhibits E2F-dependent transcription and inhibits cellular growth. The growth suppression activity of RBP2 could not be associated with a single domain within the protein but the transcriptional repression activity can be mapped to a minimal 17 kDa fragment located at the extreme N-terminus of RBP2 that can repress transcription when expressed alone but requires the C-terminus in the context of the full-length protein.

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Tremblay, Gilles B. "Methylenetetrahydrofolate dehydrogenases in eucaryotic cells." Thesis, McGill University, 1995. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=40012.

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Methylenetetrahydrofolate dehydrogenases are expressed in mammalian cells and are required for the interconversian of one-carbon substituted folates. The trifunctional NADP-dependent dehydrogenase-cyclohydrolase-synthetase is localized in the cytoplasm whereas the bifunctional NAD-dependent dehydrogenase-cyclohydrolase is expressed in mitochondria suggesting they play different metabolic roles. Insect tissues also express both proteins but when immortalized into cell lines, the trifunctional enzyme is not detectable and the bifunctional enzyme is localized predominantly in the cytoplasm. The change in localization coupled with the observation that insect cell lines are not purine auxotrophs, indicate the NAD-dependent dehydrogenase now fulfills the role of interconverting one-carbon units when the NADP-dependent dehydrogenase is not present. The cDNA encoding the insect NADP-dependent trifunctional enzyme was cloned and Northern analysis revealed the gene is transcriptionally down-regulated n insect cell lines. The cytoplasmic localization of the NAD-dependent dehydrogenase in these cells appears to be due to the transcription of shortened mRNAs which translate into a protein lacking a mitochondrial targeting signal. Targeted disruption of the gene in embryonic stem cells showed the enzyme is not required for initiation of protein synthesis in mitochondria as was originally hypothesized, and raises new questions as to its metabolic role.

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Ajdukovic, Boris. "Dystrophin expression during skeletal myogenesis." Thesis, McGill University, 1992. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=56988.

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The factors that regulate dystrophin accumulation, and its association with other proteins in culture, are largely unknown. In this study I have examined the effects of chemical agents on dystrophin accumulation in culture, as well as determined whether dystrophin in culture has associations with glycoproteins in a similar fashion to that found in normal muscle tissue. My results show that the onset of detectable dystrophin accumulation occurs shortly after myoblast fusion has taken place, and increases rapidly thereafter as a percentage of total cell protein. The effects of depolarizing concentrations of potassium ion, resulting in the inhibition of myotube contraction, are negligible, consistent with the fact that dystrophin is a cytoskeletal protein. I have found that 5-bromo-2$ sp prime$-deoxyuridine, known to affect terminal differentiation in myoblasts, markedly inhibits dystrophin accumulation in culture. Finally, I have found that dystrophin in cultured cells associates with Wheat Germ Agglutinin-binding proteins, as reported for adult skeletal muscle tissue, implying that the expression of these glycoproteins either precedes or occurs coordinately with dystrophin expression in culture.

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Taheri, Maryam. "Characterization of the structural determinants of two functions of human carcinoembryonic antigen (CEA) : intercellular adhesion and differentiation inhibition." Thesis, McGill University, 2000. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=36841.

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Human carcinoembryonic antigen (CEA) is a heavily glycosylated cell surface protein that belongs to the immunoglobulin superfamily (IgSF). It is overexpressed in a wide variety of human cancers. CEA functions in vitro, at least, as an intercellular adhesion molecule. It has also been shown that ectopic expression of CEA can block myogenic differentiation of rat L6 and mouse C2C12 myoblasts. Both the intercellular adhesion function of CEA as well as the inhibition of myogenic differentiation are dependent on homophilic binding of its extracellular domains. Previous studies have demonstrated that homophilic binding of CEA is mediated by double reciprocal bonds between the N and A3B3 domains of anti-parallel CEA molecules on apposed cell surfaces. Treatment of nonfusing L6 (CEA) with four fusion polypeptides representing the different domains of CEA showed that only N and A3B3 peptides were able to release the myogenic differentiation block. This information provoked studies designed to identify the precise subdomain (s) within the N domain that could be used to develop inhibitory agents to both disrupt the intercellular adhesion function and to release the myogenic differentiation block. Moreover, we show here that in addition to anti-parallel CEA-CEA interactions, parallel CEA-CEA interactions on the same cell surface are involved in the myogenic differentiation block. In this study, it is demonstrated by site directed mutagenesis, that at least three different subdomains, G30YSWYK, N42RQII, and Q 80NDTG, in the N-terminal domain are required for homophilic binding of CEA. Two cyclized peptides representing subdomains, N42RQII and Q80NDTG, were effective in blocking intercellular adhesion mediated by CEA. We also identified the binding epitope of an anti-CEA monoclonal antibody (A20), known to block homophilic binding of CEA, and showed that it bridges N42RQII and G30YSWYK. Furthermore, it was shown that G30YSWYK, N42RQII, and Q80NDTG are all involved in the diffe

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Oliva, Rossella Norma. "Characterization of the isoform gamma 2 ([gamma] 2) of the serinethreonine protein kinase Casein Kinase I (CKI-[gamma] 2)." Thesis, McGill University, 2003. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=81369.

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Casein Kinase I (CKI) is one of the first serine/threonine protein kinase activities to be isolated and characterized. In vertebrates, CKI represent a multigene family with seven isoforms, alpha, beta, delta, ε, gamma1-3 for which, very little is known with respect to their biological functions. This study reports that stable overexpression of CKI-gamma2 into fibroblasts impairs cell morphology and the organization of the actin cytoskeleton in a kinase-dependent mechanism and cell proliferation through a kinase-independent mechanism. In addition, by immunofluorescence we determined the subcellular distribution of CKI-gamma2 to perinuclear vesicles. Furthermore, we established that a putative prenylation motif present in the C-terminal extension of CKI-gamma2 determines its proper subcellular localization.

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Terrone, Donato Gerardo. "Ras protein targeting in mammalian cells." Thesis, McGill University, 2005. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=98506.

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The Ras proteins (H-Ras, N-Ras and K-Ras4B) are monomeric GTP-binding proteins that play key roles in cell regulation. It has been proposed that correct plasma membrane localization of the Ras proteins requires 'two signals', the processed farnesylation sequence and an adjacent palmitoylation site(s) or polybasic sequence. First, to investigate potential proteinaceous binding partners for the K-Ras4B targeting sequence at the plasma membrane, an in vivo crosslinking analysis was carried out. Consistent with current suggestions that K-Ras4B interacts via electrostatic interactions with plasma membrane lipids, no indication of a K-Ras4B plasma membrane binding partner was found. In the second portion of this thesis, I describe the development of a new approach, using fluorescence microscopy and the technique of rapamycin-induced protein heterodimerization, to demonstrate that prenylated proteins lacking a 'second signal' are not specifically sequestered in the endoplasmic reticulum and Golgi compartments as suggested previously but can distribute freely and rapidly between different cellular membranes.

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Li, Jing 1957. "Studies on iron-dependent regulation of transferrin receptor gene expression in chicken HD3 cells." Thesis, McGill University, 1996. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=27368.

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Expression of the transferrin receptor (TfR) gene is developmentally regulated in mammalian cells (1), an iron-dependent post-transcription regulatory mechanism has been demonstrated in non-erythroid cell (2,3). In order to understand the molecular mechanism underlying the iron-dependent regulation of the TfR gene expression in the differentiating erythroid cell, a virus-transformed chicken erythroblast cell line, HD3 was employed in this study. We have investigated the possible effects of iron on the TfR gene expression that might involve regulatory mechanisms at the transcriptional and/or the post-transcriptional level during the HD3 cell differentiation.
We found that treatment of the differentiating HD3 cell with Pyridoxal Isonicotinoyl Hydrazone (PIH) causes an increase in TfR mRNA accumulation as a result of increased TfR gene transcription. No evidence was obtained for stabilization of TfR mRNA. These data indicate that there may be another distinct iron-dependent regulatory mechanism for the TfR gene expression during erythroid cell differentiation.

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Dissertations / Theses on the topic '3101 Biochemistry and cell biology'

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