Dissertations / Theses on the topic '060602 Animal Physiology - Cell'
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1
Silverman, David A. "Red blood cell spacing in capillaries of rat heart." Thesis, University of Ottawa (Canada), 1996. http://hdl.handle.net/10393/9668.
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Theoretical studies have demonstrated a pronounced effect of red blood cell (RBC) spacing on tissue oxygen supply. In spite of this, realistic values for RBC spacing and related capillary hematocrit (Hct) are not known in the heart. One of the possible reasons is the lack of proper methodology. Thus, the goal of this research study was twofold: (i) to develop a method to rapidly freeze rat heart in situ, following which RBCs and capillary walls could be simultaneously visualized, and (ii) to apply this methodology in rat hearts to establish whether differences exist in capillary Hct and RBC spacing in two distinct locations within the capillary bed, in the subendo- and midmyocardium, during diastole or systole. The results of this study suggest that different geometrical conditions (e.g. RBC spacing and capillary Hct) exist at the two distinct locations of the capillary bed studied. Presumably, the oxygen supply conditions in distal portions of the capillary bed are improved by these geometrical adjustments, preventing hypoxic or anoxic conditions in the tissue, which would be detrimental to cardiac function. (Abstract shortened by UMI.)
2
Bindon, Shawn. "Interrelationship between chloride cell proliferation and gas transfer in the rainbow trout." Thesis, University of Ottawa (Canada), 1994. http://hdl.handle.net/10393/6797.
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This thesis examines the morphological and physiological changes that occur in the gills of the rainbow trout, Oncorhynchus mykiss, as a result of chloride cell proliferation. In the first component of this thesis (Chapter 2), the effects of chloride cell hyperplasia and hypertrophy on ion transport capability and the morphological diffusing capacity of the gill were evaluated. These experiments were performed to test the hypothesis that chloride cell proliferation benefits ionic regulation at the expense of efficient gas transfer. Concomitant with the surface morphology results, significant increases in Na$\sp+$ (J$\rm\sb{in}Na\sp+)$ and Cl$\sp-$ uptake (J$\rm\sb{in}Cl\sp-)$ were observed following treatment with the individual combined hormone regimens. Conversely, the gas morphological diffusion capacity, as indicated by the morphological parameters measured, of hormone treated fish was reduced, and was inversely correlated to chloride cell fractional surface area. Surface morphometric analysis showed that the hormone treatment increased the average chloride cell surface area by 2.7 x and chloride cell density by 2.2 x; the combined effect was a five-fold increase in chloride cell fractional area. While the P$\rm\sb aO\sb2$ values of hormone-treated and control fish were similar at P$\rm\sb wO\sb2>12.0$ kPa, the arterial O$\sb2$ tensions of treated fish were significantly lower than those of the control group for P$\rm\sb wO\sb2\leq12.0$ kPa. In comparison, to control fish at all environmental O$\sb2$ tensions, the hormone-treated fish exhibited elevated P$\rm\sb aCO\sb2$ values and a significant acidosis. The effects of chloride cell proliferation on blood gas parameters in hormone-treated fish were accompanied by significantly elevated ventilation amplitudes and lowered ventilation frequency. (Abstract shortened by UMI.)
3
Zhang, Hui 1971. "The role of Rho GTPases in complement-mediated glomerular epithelial cell injury /." Thesis, McGill University, 2005. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=98529.
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In glomerular epithelial cells (GEC), the actin cytoskeleton is a key determinant of cell morphology and functions, including permselectivity. Complement C5b-9 induces sublytic GEC injury associated with GEC morphological changes and proteinuria. This study addressed the role of Rho GTPases in complement-mediated GEC injury. We demonstrated that the amount of active RhoA increased; while the amount of active Rac1 and Cdc42 were decreased in C5b-9 mediated sublytic GEC injury both in vitro and in glomeruli from rats with PHN in vivo. Complement mediated inactivation of p190RhoGAP may contribute to complement-induced RhoA activation. Overexpression of constitutively active or dominant negative mutants of RhoA, Rac1 and Cdc42 distinctly altered GEC morphology and F-actin pattern. Complement caused changes in GEC actin cytoskeleton, at least in part mediated by a downstream kinase of RhoA--Rho kinase (ROCK). Activation of RhoA exacerbated complement-mediated cytotoxicity in GEC, while inhibition of ROCK attenuated it.
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Sohi, Jasloveleen. "Investigation of factors regulating parathyroid cell proliferation." Thesis, McGill University, 1994. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=55530.
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Parathyroid glands are responsible for maintaining normal extracellular calcium concentrations through their release of PTH. Calcium and 1,25-(OH)$ rm sb2D sb3$ have been demonstrated to be potent regulators of PTH and CgA synthesis and release. Primary cultures of quiescent bovine parathyroid cells proliferate in response to high concentrations of serum. Next, I examined the role of c-myc in the proliferation of the PT-r cell line, which was cloned from rat parathyroid cells. In order to study the role of c-myc in PT-r cell proliferation more precisely, I used antisense RNA technology to inhibit c-myc mRNA.In summary, my studies have shown that parathyroid cells respond to selected growth factors. This proliferative response involves increased expression of c-myc, c-fos, c-jun and PTHrP. 1,25-(OH)$ rm sb2D sb3$ inhibits the expression or c-myc, and cell proliferation is inhited. The differentiated parathyroid cell expresses high levels of CgA and PTH. However, during proliferation these high levels are not sustained.
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Sayegh, Camil E. "The role of the B cell receptor complex in avian B cell development dissected by retroviruses /." Thesis, McGill University, 2000. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=37621.
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During embryogenesis, B cell precursors that have undergone productive Ig V(D)J rearrangement are selected to expand in oligoclonal follicles of the bursa of Fabricius. Because Ig V(D)J recombination in chickens results in minimal diversity, diversity being generated instead by gene conversion in the follicles of the bursa of Fabricius, B cell precursors express a limited range of Ig specificities prior to colonization of the bursa. It has been proposed that recognition of endogenous ligands by this 'pre-diversified' B cell receptor is critical to the progression of normal B cell development. To test this hypothesis, we constructed a truncated IgM receptor (Tmu) lacking the V and Cmu1 domain, which does not associate with Ig L chain proteins nor does it require IgL chains for surface expression, and used a retroviral gene transfer system to introduce it in developing chick embryos. In these embryos, Tmu+ B cell precursors productively colonized bursal follicles as efficiently as B cell precursors expressing endogenous sIg. Furthermore, we detected low but significant levels of IgL VJ rearrangements in Tmu + bursal cells. The analysis of these VJ junctions revealed no selection for in-frame products as these cells are maintained by the Tmu receptor. Interestingly, we showed that the rearranged VL segments derived from Tmu+ bursal cells underwent gene conversion indistinguishably from rearranged VL segments derived from bursal cells expressing endogenous sIg. Taken together, we have ruled out a role for V(D)J encoded determinants in the normal development of B cells in avian embryos. Sequence analysis of 80 IgL VJ segments derived from Tmu+ bursal allowed the unique opportunity to assess the efficiency of gene conversion in vivo, in the absence of selection. Using this system, we demonstrated that >97% of gene conversion events maintain the sequence in-frame. Following hatching, the bursa undergoes morphological changes initiated by the migration of bursal cells ba
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Komorowski, Joanna Irena. "Influence of protein kinase C activators and inhibitors on rat granulosa cell steroidogenesis in vitro." Thesis, University of Ottawa (Canada), 1993. http://hdl.handle.net/10393/6745.
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The present studies were undertaken to determine the involvement of protein kinase C (PKC) in the regulation of rat granulosa cell steroidogenesis in vitro. The effects of PKC activators (1-oleoyl-2-acetylglycerol (OAG); 1,2-dioctanoylglycerol (DiC$\sb8$) and phorbol 12-myristate 13-acetate (TPA)) and inhibitors (DL-Sphingosine (ESP) and 1-(5-Isoquinolinylsulfonyl)-3-methylpiperazine free base (H$\sb7)\rbrack$ on basal and FSH-, (Bu)$\sb2$cAMP-, forskolin- and calcium ionophore A23187-stimulated pregnenolone (P$\sb5$), progesterone (P) and 20$\alpha$-hydroxy-pregn-4-en-3-one (20$\alpha$-OH-P) secretion by granulosa cells were studied. OAG, when continually present in the culture medium (MEM), significantly stimulated P$\sb5$, P and 20$\alpha$-OH-P secretion during 6 to 24 h culture periods. It also markedly increased the conversion of exogenous P$\sb5$ to P and 20$\alpha$-OH-P and exogenous P to 20$\alpha$-OH-P during 24 h cultures. Pretreatment of granulosa cells with TPA for 1 h or treatment for up to 6 h resulted in a significant increase in P$\sb5$, P and 20$\alpha$-OH-P secretion. Except for 20$\alpha$-OH-P production, which was stimulated by the phorbol ester during all culture periods studied, secretion of P$\sb5$ and P (in the presence or absence of exogenous hormones and the inhibitors of steroidogenic enzymes) were substantially inhibited by TPA during a 24 h incubation. However, when granulosa cells were incubated with both OAG (20 $\mu$g/ml) and TPA (40 ng/ml), progestin secretion was increased irrespective of the duration of incubation. PKC inhibitors dose-dependently suppressed the stimulatory effect of OAG (20 $\mu$g/ml) and TPA (40 ng/ml) with complete inhibition noted at 100 $\mu$M of H$\sb7$ and 10 $\mu$M of ESP. Diacylglycerols and TPA exerted divergent effects on FSH-, (Bu)$\sb2$cAMP- and forskolin-stimulated progestin secretion. FSH-stimulated accumulation of P$\sb5$ throughout the culture periods (1-24 h) was markedly increased by OAG (20 $\mu$g/ml) but inhibited by TPA (40 ng/ml). OAG (5-80 $\mu$g/ml) and DiC$\sb8$ (20 $\mu$g/ml) significantly enhanced FSH-induced progestin secretion during 6 h and 24 h culture periods and increased steroid synthesis in 24 h cultures in the presence of (Bu)$\sb2$cAMP or forskolin. In contrast, TPA significantly inhibited FSH- and (Bu)$\sb2$cAMP-stimulated progestin secretion during both 6 h and 24 h of incubation. Pretreatment of granulosa cells with TPA (40 ng/ml) for 20 h to down-regulate PKC, decreased progestin secretion during subsequent incubation with FSH (150 ng/ml) and prevented any stimulation by OAG (20 $\mu$g/ml). The effects of OAG (20 $\mu$g/ml) and TPA (40 ng/ml) on FSH-induced steroid secretion appeared to be additive when both PKC activators were present together and differed significantly from those when OAG and TPA were present with FSH separately. Diolein (a nonpermeable diacylglycerol), 4$\alpha$-phorbol 12,13-didecanoate and phorbol 13-monoacetate (two phorbol esters with no tumor promoting activity) did not influence basal or FSH-stimulated steroid secretion by granulosa cells. (Abstract shortened by UMI.)
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Dubé, Gilles. "Post-translational processing of atrial natriuretic factor. A study using a novel cell culture system." Thesis, University of Ottawa (Canada), 1992. http://hdl.handle.net/10393/7779.
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In the present work, a reproducible cell culture system using adult rat atrial cardiocytes was developed to study ANF processing. Freshly isolated atrial cardiocytes stored high molecular weight ANF (38.0 $\pm$ 4.5 pg/$\mu$g of DNA) and released almost exclusively (83.3% $\pm$ 6.7%) low molecular weight ANF, at an average rate of 12 pg/hour/$\mu$g of DNA. The cell content and the rate of release of ANF decreased over 15 days in culture to 3.9 $\pm$ 1.2 pg/$\mu$g of DNA and 0.32 pg/h/$\mu$g of DNA $\pm$ 0.08 respectively and 62.7% $\pm$ 6.3% of the released peptide was of a low molecular weight. Cultures of non-cardiocytes, superfused with exogenous proANF, did not process the peptide. There was no correlation between the changes in cell population and the reduction in processing. Therefore, atrial non-cardiocytes are not involved in ANF processing. The results presented in this work vary from other reports which found that ANF processing in cultures is absent. The discrepancies may be due to differences related to serum-free culture conditions versus serum supplemented cultures. This suggests that factors present in the serum may be responsible for maintaining ANF processing activity in culture. (Abstract shortened by UMI.)
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Pietersen, Alexander Nicolaas Johannes. "Adenosinergic modulation of hippocampal gamma oscillations : from single cell to whole animal." Thesis, University of Birmingham, 2010. http://etheses.bham.ac.uk//id/eprint/1041/.
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Gamma oscillations, synchronous network activity between 30 and 100 Hz, have been linked to higher cognitive functions. Adenosine receptor modulation has been shown to alter cognitive function in animals and humans. In this thesis the effects of adenosine receptor modulation on in vitro and in vivo hippocampal gamma oscillations were investigated as well as the underlying mechanisms. \(A_1\)-receptor activation selectively decreased gamma oscillations while blocking \(A_1\)-receptors and activating \(A_{2A}\)-receptors increased gamma oscillations. Increasing endogenous adenosine levels suppressed gamma oscillations while decreasing endogenous adenosine levels facilitated gamma oscillations in vitro. Sharp electrode current clamp and whole-cell voltage clamp experiments showed that \(A_1\)-receptor activation hyperpolarised resting membrane potential, reduced firing rate and EPSP amplitude and shifted the IPSC reversal potential to more negative potentials. Blocking \(A_1\)-receptors increased pyramidal cell excitability and increased excitatory synaptic transmission. The results in vivo were more ambiguous but \(A_1\)-receptor activation decreased power in all frequency bands indicating that adenosine receptors can modulate hippocampal gamma oscillations in vivo. \(A_1\)-receptor blockage had no consistent effect on in vivo hippocampal gamma oscillations. Adenosine receptors modulate gamma oscillations in rodent hippocampal slices but are difficult targets for developing treatments that have cognitive benefits because of their ambiguous effects in vivo.
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Dhawan, Anil. "The role of oocyte- and embryo-secreted factors in cumulus cell differentiation and their relationship to embryo quality and developmental competence." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0028/MQ52295.pdf.
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Kim, Changsung. "Assessing the function of Caenorhabditis elegans Ror receptor tyrosine kinase CAM-1 in cell migration, cell polarity, and axon protrusion." [Bloomington, Ind.] : Indiana University, 2006. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3215192.
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Thesis (Ph.D.)--Indiana University, Dept. of Biology, 2006.Source: Dissertation Abstracts International, Volume: 67-04, Section: B, page: 1801. Adviser: Wayne C. Forrester. "Title from dissertation home page (viewed June 20, 2007)."
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Bernier, Suzanne M. (Suzanne Marie). "Regulatory influences of microenvironmental factors on osseous cell proliferation and differentiation in vitro." Thesis, McGill University, 1992. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=39329.
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The cellular activity in bone and cartilage tissue is controlled by substances present in the osseous environment. Using representative cell lines, modulation of osteoblastic and chondrocytic phenotypic markers such as hormone-responsiveness, enzymatic activities, and matrix production by osteotropic factors was examined in vitro. In UMR 106 cells, a rat osteoblast-derived osteosarcoma cell line, a differential distribution of EGF and PTH receptors over proliferating and quiescent cells, respectively, was observed. EGF treatment resulted in expansion of a less differentiated population characterized by decreased PTH- and CT-stimulated adenylate cyclase and binding capacity, and increased CGRP-responsiveness. Using immunocytolysis, a mixed osseous cell population (OBCK6) was derived from fetal rat calvarial cells. Subsequent dilution cloning yielded two cell lines, the CFK1 line with osteoblastic characteristics and the CFK2 line with chondrocytic properties. Although only the OBCK6 cells deposited a mineralized matrix in vitro, organic matrix components were produced by the other two lines. EGF stimulated proliferation in both CFK1 and CFK2 cells. Retinoic acid decreased cell proliferation, PTH-responsiveness, and alkaline phosphatase activity, caused cell shape change and population reorganization, and increased EGF receptors in CFK1 cells. Induction of cartilage specific matrix components (type II collagen, proteoglycan core protein, link protein, and thrombospondin) was observed in CFK2 cells maintained in culture without passaging and could be regulated by EGF, PTH, dexamethasone, and retinoic acid. Thus, peptidergic and steroidal factors have been employed to modulate functional activity and cell cycle kinetics, and have resulted in the characterization of discrete stages of differentiation of osteoblastic and chondrocytic populations in vitro.
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Siam, Rania. "Mechanisms of C. crescentus regulation of chromosome replication by a cell cycle regulator protein." Thesis, McGill University, 2001. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=37838.
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Caulobacter crescentus divides asymmetrically to produce two different progeny, a swarmer progeny that is replication incompetent, and a stalked cell progeny that is replication competent. Upon cell division, a cell cycle regulator protein (CtrA) was identified only in the swarmer cells, and additional circumstantial evidence links this protein to being a repressor of chromosome replication. For example, this response regulator protein binds to five specific sites in the replication origin (Cori) designated [a-e]. We carefully studied the binding characteristics of both phosphorylated (CtrA~P) and unphosphorylated forms of the protein to the five binding sites [a-e] in the replication origin (Chapter 2) and upstream of the ctrA gene (Chapter 3). We showed that phosphorylation significantly enhances binding affinity in the replication origin (Chapter 2) but not upstream of ctrA (Chapter 3). In addition a "pseudo-active" protein form (CtrA D51E) that resembles the phosphorylated form in vivo did not improve the binding characteristics (Chapter 3). These results suggest that enhanced binding on phosphorylation is not the only signal achieved on phosphorylation. In fact, CtrA half-site mutation binding studies shows that phosphorylation stimulates protein/protein interaction and cooperative binding between sites [a] and [b] in the replication origin (Chapter 2). We show that CtrA binding site [b] is the major contributor in the cooperative CtrA binding between [a] and [b] (Chapter 4). We demonstrate that cooperative binding of CtrA~P to sites [a] and [b] repress transcription from a strong promoter (Ps), which in turn blocks plasmid replication. In addition, mutating site [b] to block CtrA binding to [a] and [b] has a deleterious effect on chromosome replication (Chapter 4).This cooperative CtrA binding at [a] and [b] is independent from the upstream binding sites [c-e] (Chapter 2). CtrA∼P binding in the origin is altered in the presence of the histone-like protein (IHF) that also binds and overlaps CtrA binding site [c] (Chapter 5). In-fact, IHF binds and overlaps binding site [c] (Chapter 5). We propose a replication model in the stalked cell were IHF binding hinders active CtrA binding in the replication origin and regulates cooperative transcription that coincides with replication initiation.
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Nguyen, Hannah Anh-Quan. "Regulation of gene expression and cell growth by transcriptional proteins of the interferon system." Thesis, McGill University, 1998. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=35028.
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The Interferon Regulatory Factors (IRFs) are a family of interferon-inducible proteins which play distinct roles in diverse processes such as pathogen response, cytokine signalling, cell growth regulation and hematopoietic development. The objective of this research was to investigate the mechanisms by which IRF-1 and IRF-2 regulate gene expression and cell growth. Structure-function analyses of the IRF-2 protein demonstrate that transcriptional repression by IRF-2 is contained within the first 125 N-terminal amino acids and correlates directly with IRF-2 DNA binding. Overexpression of functional IRF-2 deletion mutant proteins in NIH3T3 cells results in oncogenic transformation and tumorigenesis, suggesting that IRF-2 oncogenicity correlates directly with transcriptional repression. Similar structure-function analyses localize IRF-1 transactivation to the C-terminus. Like IRF-1, hybrid constructs which fuse the DNA binding domain of IRF-1 and IRF-2 to the transactivation domain of NF-kappaB RelA(p65) are transcriptional activators. Inducible expression of IRF-1 and IRF/RelA in NIH3T3 cells results in reduced cellular growth and induction of apoptosis. Furthermore, expression of the PKR, STAT1(p91), and WAF1 growth regulatory proteins are elevated following induction of IRF-1 or IRF/RelA, correlating transactivation function and tumor suppressor activity of IRF-1 or IRF/RelA. By RNA fingerprinting, the secretory leukocyte protease inhibitor (SLPI) was identified as the first gene whose expression is downregulated by IRF-1 or IRF-1/RelA. A region in the SLPI promoter was identified that bound IRF-1, suggesting a direct mechanism for IRF-1 regulation of SLPI expression.
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Therriault, Pierre. "Apoptosis in the adult goldfish pituitary." Thesis, University of Ottawa (Canada), 2003. http://hdl.handle.net/10393/26401.
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This thesis research characterized apoptosis in the adult goldfish pituitary. Apoptosis is a form of programmed cell death that plays key roles in many biological processes by selectively removing old or unwanted cells. Morphological and biochemical signs of apoptosis established its presence within the gland. The pituitary gonadotrophs secrete gonadotropin-II to regulate sex steroid production, ovulation and sperm production.
It was possible to combine TUNEL with immunocytochemistry and demonstrate apoptosis in gonadotropin-II positive cells in the pars distalis of the pituitary, allowing for the fist time the identification of apoptotic gonadotrophs in situ. Other studies suggest that pituitary cell populations and function fluctuate through the seasonal reproductive cycle. Apoptosis could play a role in regulating secretion by regulating the cell number. Treatments with the dopamine agonist apomorphine, and with a gonadotropin-releasing hormone agonist failed to modulate the apoptosis levels measured by DNA laddering or by TUNEL. Finally, this study demonstrated that the calcium ionophore A23187, significantly increases apoptosis in whole pituitary gland cultures. These studies established a new method to identify apoptotic hormone-secreting cells.
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Azarani, Arezou. "The effect of parathyroid hormone (PTH) and parathyroid hormone related peptide (PTHRP) on Na+H+ exchanger activity and analysis of signal transduction mechanisms." Thesis, McGill University, 1996. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=40109.
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Parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHRP) regulate Na$ sp+$/H$ sp+$ exchanger (NHE) activity in various types of cells such as osteoblastic cells and renal proximal tubule OK cells. Na$ sp+$/H$ sp+$ exchangers are plasma membrane transporters catalyzing the electroneutral exchange of 1 H$ sp+$ for 1 Na$ sp+.$ Several mammalian isoforms of NHE have been so far identified with each mediating a variety of specific functions. Parathyroid hormone, playing an essential role in the physiology of blood Ca$ sp{2+}$ and phosphate homeostasis, inhibits renal proximal tubular bicarbonate reabsorption by inhibiting the apically located Na$ sp+$/H$ sp+$ exchanger. However, which specific isoform of NHE mediated this effect and the specific signaling components involved were unknown. In our studies we determined that Na$ sp+$/H$ sp+$ exchanger NHE-3 isoform is expressed in the renal proximal tubule OK cells and that N-terminal PTH and PTHRP analogues upon binding to their receptor stimulate both the PKA and the PKC pathways, each of which can independently lead to inhibition of this exchanger activity. NHEs also play an important role in the regulation of intracellular pH which is subject to fluctuation occurring during the process of hormone stimulated bone formation and bone remodeling. Again the specific NHE isoform(s) mediating this effect and the signaling pathways involved were unidentified. It is determined by our studies that NHE type 1 is expressed in osteoblastic cell line, UMR-106 cells, and that PTH and PTHRP stimulate this exchanger via a cAMP-dependent pathway exclusively. It was believed that motivation of NHE-1 in the UMR-106 cells and inhibition of NHE-3 in the OK cells by N-terminal analogues of PTH and PTHRP involves binding of these analogues to a common G protein-coupled receptor called the "classical" PTH/PTHRP receptor. However, with the recent discovery of other PTH and/or PTHRP receptor, this hypothesis is no longer clear
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Drake, Julie. "Effect of Choline on Ca2+ -activated K+ channels in bovine chromaffin cells." Thesis, McGill University, 1990. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=60447.
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The actions of internal choline on single "maxi" Ca$ sp{2+}$-activated K$ sp{+}$ channels were studied in excised patches of chromaffin cell membrane. The channels had a unit conductance of approximately 200 pS in a physiological K$ sp{+}$ gradient.Choline (20-70 mM) applied to the intracellular membrane surface dose dependently reduced outward current flow through the channel. The reduction in single channel current amplitude increased with depolarization.
These data suggest that choline is a fast blocker that binds within the channel pore. The K$ sb{ rm d}$(0) for the reaction is 90 mM while $ delta$ is 0.27, suggesting that the choline binding site senses 27% of the transmembrane electric field.
The average open state probability appeared not to be affected by choline at any of the membrane potentials that were studied.
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Poulter, Michael Owen. "The role of potassium conductances in regulating the excitability of myelinated axons /." Thesis, McGill University, 1990. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=74560.
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Previous results indicate that the functional properties of myelinated axons are considerably more complex than previously believed. The purpose of this study was to further examine the functional properties of potassium conductances in dorsal and ventral root (DRA; VRA) frog myelinated axons by intracellular microelectrode recording. The voltage responses to hyperpolarizing current injection demonstrated: (1) a nonlinear voltage current (V/I) relationship in the region just below resting membrane potential; (2) a linear peak and steady state V/I relationship below $ approx$ $-$95 mV; (3) a large effective capacitance of the internodal membrane; and (4) an attenuation of the voltage response reflecting the activation of a voltage dependent anomalous rectifying conductance (G$ sb{ rm AR}$). G$ sb{ rm AR}$ is dependent on the presence of sodium and potassium ions in the external medium and is blocked by cesium but not barium ions. (5) An afterhyperpolarizing potential (AHP) after a train of action potentials was accounted for by the presence of a sodium dependent potassium conductance (G$ sb{ rm K(Na)}$). The functional role of the voltage dependent potassium conductances and G$ sb{ rm K(Na)}$ was examined. G$ sb{ rm AR}$ appears to regulate the length of a post tetanic AHP. Early accommodation (EAcc) is regulated by G$ sb{ rm Kf1}$ in DRA and VRA. Late accommodation (LAcc) and adaptation (Adp) is regulated by G$ sb{ rm Ks}$ in both DRA and VRA. G$ sb{ rm Kf2}$ may also contribute to EAcc and Adp. Action potential repolarization in DRA and VRA is governed by G$ sb{ rm Kf2}$ and G$ sb{ rm Kf1}$ respectively. G$ sb{ rm K(Na)}$ may also contribute to LAcc and Adp. An evaluation of our experimental results using a computer model based Hodgkin-Huxley type equations suggests that the gating of the sodium conductance plays only a minor role in accommodation and adaptation. These results indicate that potassium conductances play a pivotal role in regulating the excitabilit
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Choi, Jiwon 1977. "The role of fibulin-5 : in the ultrastructural and biomechanical properties of skin." Thesis, McGill University, 2005. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=81611.
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Properly assembled elastic fibers play an important role in providing skin with the properties of elasticity and resilience to allow for considerable mobility, but the mechanisms involved in elastic fiber assembly remains unknown. Fibulin-5 is an extracellular 66kDa glycoprotein synthesized and secreted by fibroblasts in skin, and has the ability to bind to tropoelastin. This study addresses (1) the role of fibulin-5 in the ultrastructure of elastic fibers in skin, and (2) its function in the cutaneous mechanical properties by using wild type and fibulin-5 null mice. In the first part, skin of fibulin-5 null mice as well as wild type mice was investigated in order to gain insight into the function of fibulin-5 in elastic fiber assembly. Using light and electron microscopy, the dramatic defects of dermal elastic fibers in the absence of fibulin-5 were analyzed. Interestingly, in the immunoelectron microscopy for LOXL-1, an enzyme responsible for the elastin cross-links, fibulin-5 null mouse skin exhibited similar immunoreactivity for LOXL-1 to wild type skin. Moreover, in the wild type skin, fibulin-5 localized to the microfibril-elastin interface at the edges and within the elastic fiber. In the second part of this study, the function of fibulin-5 in skin biomechanics was studied in order to determine its role in skin strength and elasticity. By using tensile tests, fibulin-5 null mouse skin was found to be significantly stiffer and weaker than wild type skin. Taken together, the defective elastic fibers in the absence of fibulin-5 suggest that fibulin-5 is involved in a secondary step of cutaneous elastic fiber assembly and in the mechanical properties of adult mouse skin.
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Jia, Yanlin. "Nitric oxide and airway smooth muscle responsiveness." Thesis, McGill University, 1995. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=29052.
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Airway hyperresponsiveness to bronchoconstrictors has been found in asthma and related to the severity of the disease. The factors which result in hyperresponsiveness are not completely understood. A possible mechanism is an imbalance between endogenous bronchoconstrictors and dilators. NO is known to relax tracheal smooth muscle by activating soluble guanylate cyclase and increasing the level of intracellular cyclic guanosine monophosphate (GMP). The first hypothesis tested was that the NO-cyclic GMP-relaxant pathway is involved in the regulation of airway responsiveness. Inhibition of endogenous nitric oxide by N$ sp{ omega}$-nitro-L-arginine (L-NNA) significantly increased airway responsiveness to inhaled methacholine in normoresponsive Lewis rats but less so in hyperresponsiveness Fisher rats. In addition, carbachol increased cyclic GMP levels in tracheal tissues from both strains; this cyclic GMP accumulation in tracheal tissues was also less in Fisher than in Lewis rats and abolished by L-NNA in both strains, indicating that it was mediated by a NO-dependent mechanism. These results suggest that endogenous NO plays a role in regulation of airway responsiveness and contributes to the strain-related difference in airway responsiveness in rats. To investigate the NO-cyclic GMP-relaxant pathway in rat airway, the effect of sodium nitroprusside (SNP, a NO donor) on airway responsiveness to a cholinergic agonist was measured in hyperresponsive Fisher rats and compared with the less responsive Lewis strain. Fisher rats were resistant to SNP as evidenced by less relaxation of carbachol contracted tracheal rings by SNP and less cyclic GMP accumulation induced by SNP in cultured airway smooth muscle cells in Fisher rats compared with Lewis rats, indicating an impaired response to SNP in Fisher airways.NO is known to be synthesized from L-arginine in a reaction catalyzed by NO synthase (NOS). Liver cytochrome P450 also catalyzes the oxidative cleavage of C=N bonds of compounds containing a -C(NH$ sb2$)NOH function, producing NO in vitro. We hypothesized that the biosynthesis of NO in airway smooth muscle cells could result from P450 enzymes acting on appropriate substrates. NO can be synthesized in a number of lung cell types. However, to date, no constitutive form of NOS activity has been found in airway smooth muscle cells. We next examined the possibility that airway smooth muscle itself might be able to synthesize NO. Formamidoxime, a compound containing the -C(NH$ sb2$)NOH function, was found to produce NO in cultured airway smooth muscle cells. As well, formamidoxime relaxed pre-contracted trachealis and cyclic GMP accumulation in airway smooth muscle cells in culture. These effects were inhibited by P450 inhibitors but not by NOS inhibitors. Thus, an L-arginine-independent pathway for production of NO was demonstrable in airway smooth muscle cells. This NO production was catalyzed by P450 but not by NOS.
In conclusion, my studies have demonstrated an important role for endogenous NO production in determining the airway responsiveness of normal rats to inhaled cholinergic agonists. This mechanism contributes to strain-related differences in airway responsiveness in the rat.
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Dorato, Andrea. "Characterization of the structure and subcellular distribution of the rat hepatic prolactin receptor." Thesis, McGill University, 1993. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=39814.
Full textAbstract:
The structure of rat hepatic prolactin (PRL) receptors was examined in various subcellular fractions by immunoblotting. In all subcellular fractions, a single 42 kDa species was identified. Tri- and/or tetrantennary complex carbohydrates compose $ sp sim$7 kDa of the weight of the receptor. Enzymatic deglycosylation of the mature receptor does not appreciably reduce ligand binding or antibody recognition of the receptor. However, core glycosylation of the receptor is necessary for the acquisition of binding capacity and antibody recognition.The intracellular distribution of PRL receptors was determined by Percoll gradient centrifugation and application of the diaminobenzidine (DAB)-shift methodology. There was a sex-dependent distribution of PRL receptors, with females having a 3-fold higher concentration of receptors in early endosomes than males. This discrepancy disappeared when male rats were treated with estrogen. No such sex-dependent distribution of intracellular receptors were discovered for insulin receptors.
Colchicine treatment of estrogen-induced male rats prevented newly synthesized receptors from reaching the cell surface, and allowed the accumulation of at least some of these receptors into an endosomal compartment. These studies suggest that under conditions of colchicine blockade, PRL receptors may access an endosomal compartment by an entirely intracellular route. To date, such a route has only been described for mannose-6-phosphate receptors.
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Dumont, Nancy. "Characterization of transforming growth factor-b receptors in the human endometrium." Thesis, McGill University, 1995. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=23268.
Full textAbstract:
Transforming growth factor-$ beta$ (TGF-$ beta$) is a multifunctional polypeptide growth factor which is believed to play an important role in the growth and differentiation of uterine cells. Although the expression of TGF-$ beta$ in the uterus has been previously described, the receptors for TGF-$ beta$ in this tissue have not been characterized. In the present study, the cell surface receptors for TGF-$ beta$ were characterized on cultures of stromal cells prepared from human endometrial biopsies, and on a human endometrial epithelial cell line (RL95-2) using affinity labeling techniques. On stromal cells, five TGF-$ beta$ binding proteins were identified. Analysis of the sensitivity of these proteins to dithiothreitol and phosphatidylinositol-specific phospholipase C, together with results from immunoprecipitations with anti-TGF-$ beta$ receptor antibodies, confirmed that three of these binding proteins correspond to the cloned type I, II, and III TGF-$ beta$ receptors. The other two binding proteins exhibited characteristics of isoform-specific glycosyl-phosphatidylinositol-anchored TGF-$ beta$ binding proteins. On RL95-2 cells, three TGF-$ beta$ binding proteins, corresponding to the type I, II and III TGF-$ beta$ receptors, were identified. The number of receptors on endometrial cells and their relative affinity for TGF-$ beta$ was estimated by Scatchard analysis. These receptors are responsive to physiological concentrations of TGF-$ beta$ as demonstrated by the effect of TGF-$ beta$ on DNA synthesis in these cells. Accordingly, they have the potential to respond to TGF-$ beta$ expressed in the endometrium in an autocrine and/or paracrine manner.
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Paquet, Michel. "Calcium permeation of the a4b2 subtype of the neuronal nicotinic acetylcholine receptor expressed in Xenopus oocytes." Thesis, McGill University, 1998. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=21618.
Full textAbstract:
Intracellular calcium has been widely demonstrated as an important part of intracellular signalling pathways. Entry of calcium in cells has been shown to trigger a variety of processes such as contraction in smooth and skeletal muscle, release of neurotransmitter in neurons and activation of modifying proteins in most cells, which in turn will activate or deactivate other processes. For these reasons, calcium permeation through any ion-channel type, whether ligand or voltage-gated, expressed on the surface of a cell, might potentially influence the intracellular activities or status of that cell. Calcium permeation in muscle nicotinic acetylcholine receptors has long been shown unequivocally. Calcium permeation through neuronal nAChR's has also been shown, but these experiments were done on preparations of neurons. Typically, neurons might express two or three subunits of the same type. The lack of knowledge on the assembly of subunits into receptors and the possibility that subunits might be permutable in a receptor make the exact subunit composition of the receptors unknown. To test the hypothesis that calcium is a permeant ion of the alpha 4beta2 subtype neuronal nicotinic acetylcholine receptor (nAChR), I performed single-channel patch-clamp experiments on micro-injected Xenopus oocytes expressing alpha4beta2 neuronal nAChR. When recording from single cell receptors in increasing concentration of extracellular Ca2+ I found that the amplitude of the single-channel current decreased with each increment in concentration. The behaviour of the resulting current-voltage relationships is described as a direct influence of calcium permeation on single-channel current amplitude. Analysis with a theoretical model describing permeation at the single-channel level supported this conclusion.
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Jackson, Andrew R. 1972. "Estrogenic regulation of N-cadherin in the rat testis : an examination using agonists and antagonists of estradiol." Thesis, McGill University, 1996. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=27349.
Full textAbstract:
Intercellular junctions within the mammalian testes play an important role during spermatogenesis. The cadherins are a superfamily of integral membrane proteins that mediate calcium-dependent cell adhesion and are involved in the formation of intercellular junctions. The cadherin subtype, known as N-cadherin, mediates the interaction between the germ cells and Sertoli cells of the testis. Previous studies have shown that estrogens are able to modulate N-cadherin mRNA levels in the 7-day-old mouse testis.I have examined the estrogen effects on N-cadherin protein levels in the testes of 21-day-old rats. The rats were injected with either 17$ beta$-estradiol, the anti-estrogens Tamoxifen and ICI 182,780, or estradiol in combination with ICI 182,780. Immunoblotting analysis of testicular proteins shows that N-cadherin levels in the 21-day-old rat are stimulated by estrogen. Treatment with anti-estrogens decrease N-cadherin levels. Administration of estrogen was able to overcome the inhibitory effects of anti-estrogen treatment. The results obtained from these studies indicate that the effect of estrogen on testicular N-cadherin levels is mediated through the estrogen receptor. Estrogen receptor levels within the testis are not altered by exogenous estrogen, but can be affected by treatment with anti-estrogen.
These experiments support the hypothesis that estrogens play an important role in spermatogenesis. In addition, the results provide insight into how disruption of testicular estrogen responsiveness can reduce fertility and why chronic anti-estrogen treatment results in disorganization of the seminiferous epithelium.
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Cruz, Javier. "The internalization pathway of insulin in exocrine pancreatic cells /." Thesis, McGill University, 1990. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=74619.
Full textAbstract:
The pathway of receptor-mediated endocytosis in exocrine pancreatic cells of the rat was studied. The pathway was explored using insulin radiolabeled with iodine-125 detectable by the technique of radioautography.Insulin was seen at the cell membrane soon after administration of the radiolabeled hormone. After 10 and 20 minutes silver grains were found over compartments deep within the cells, at distances greater than 5 half-distances. Grains were also seen to be associated with endosomal vesicles characterized by heavily stained, often fuzzy, membrane. At this time and later times of 45 minutes, grains were observed over zymogen granules. A group of rats was treated with chloroquine to interfere with intracellular vesicle acidification. The results revealed an accumulation of insulin in endosomes and in presecretory and secretory granules.
Degradation of insulin was found to be significantly inhibited by chloroquine in the liver but not in the pancreas. Degradation of insulin in normal rats was found to be slow in the pancreas but fast in the liver.
The results suggest that insulin is internalized to endosomes within which it may be degraded. Internalized insulin was also seen in the trans Golgi network and zymogen granules, especially after chloroquine treatment. This suggests an access route from endocytic to exocytic compartments which may be related to the pathway of sorting of lysosomal proteins. It may also be significant in the transcytosis of insulin to the pancreatic duct.
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Lu, Jun 1969. "Currents induced by somatostatin-14 in monkey kidney cells (COS-7) stably expressing human somatostatin receptor subtype 4." Thesis, McGill University, 1996. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=24023.
Full textAbstract:
Recent studies have shown that a variety of K$ sp+$ channels can be enhanced by somatostatin (SST; inward and delayed rectifier, muscarinic regulated K$ sp+$ channel, Ca$ sp{2+}$-activated K$ sp+$ channel, ...). Since a somatostatin receptor (SSTR) is heterogeneous, it is not clear which SSTR subtype (five are identified) is involved in regulating each K$ sp+$ channel, nor whether the regulation is and if so to what extent receptor subtype specific. In this study we characterized electrophysiologically the effect of somatostatin-14 (SST-14) in monkey kidney cells (COS-7) stably expressing one of the five recently cloned SSTR subtypes, human somatostatin receptor subtype 4, using whole cell voltage clamp technique at room temperature (20-23$ sp circ$C).The outward current produced by a sequence of depolarizing steps from a holding potential of $-$80 mV was enhanced by $>$1 nM SST-14. Both the endogenous and SST-14 enhanced outward current had a high threshold ($ sim -$40 mV). The reversal potentials of their tail currents changed with a change of (K$ sp+ rbrack sb{ rm o}$ as expected for a current carried predominantly by K$ sp+$ ions. It was blocked by TEA (10-20 mM) and Ba$ sp{2+}$ (1 mM), but not by charybdotoxin (100 nM) and carbachol (50 $ mu$M) in the extracellular solution suggesting that it is largely due to a delayed rectifier K$ sp+$ channel.
A smaller inward current at potentials negative to $ sim -$60 mV was also observed and was enhanced by SST-14 in a similar concentration range. It was blocked by Ba$ sp{2+}$ (1 mM) and Cs$ sp+$ (1 mM) suggesting that SST-14 also enhances an inward rectifier K$ sp+$ current. Both outward and inward currents enhanced by SST-14 were pertussis toxin and cAMP sensitive.
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Smith, Margaret Snoke. "The role of axis formation in the evolution of direct development in the sea urchin Heliocidaris erythrogramma." [Bloomington, Ind.] : Indiana University, 2008. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3330785.
Full textAbstract:
Thesis (Ph.D.)--Indiana University, Dept. of Biology, 2008.Title from PDF t.p. (viewed on Jul 22, 2009). Source: Dissertation Abstracts International, Volume: 69-10, Section: B, page: 5873. Adviser: Rudolf A. Raff.
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Rudy, Diane E. "Myofibrillogenesis and the avian precardiac explant system." Diss., The University of Arizona, 2002. http://hdl.handle.net/10150/280248.
Full textAbstract:
Cardiac muscle contraction is critically dependent upon the extensive level of organization of cytoskeletal proteins found in the repeating sarcomeric units of individual myofibrils. Within these units, thick and thin filament systems are assembled and aligned to the precision of single molecules. For years, scientists have been challenged to uncover the mechanisms by which this is accomplished. To date, however, these mechanisms remain relatively unclear due in large part to the lack of suitable in vitro models that faithfully recapitulate the events of myofibril assembly observed in vivo. Several years ago, an avian embryo explant system was developed to investigate other aspects of heart development. Within this system, premyocardial cells differentiate in culture and commence beating in a temporal pattern that corresponds with cardiomyocyte differentiation in vivo. We hypothesized that premyocardial explants could also serve as a particularly advantageous system for investigating myofibrillogenesis. To test this, in Chapter 2, we characterized the temporal/spatial relationships between sarcomeric components during assembly using immunofluorescence microscopy. Our results indicated that events of myofibril assembly in explants mirrored those observed in vivo. Furthermore, these cells are accessible to experimental manipulation (Chapter 5). In Chapter 3, we utilized the precardiac explant system to investigate events of actin (thin) filament assembly during development. Immunofluorescence and ultrastructural analyses revealed that thin filament and sarcomere lengths increase gradually as cardiomyocytes mature. FRAP analyses also demonstrated that the thin filament pointed-end capping activity of E-Tmod is more dynamic during early assembly stages, a property that could dramatically affect the rate of actin monomer exchange/addition during myofibrillogenesis. Research continues in an attempt to identify potential mechanisms regulating E-Tmod dynamics. Finally, in Chapter 4, we investigated the function of a unique elastic region of I-band titin called titin-N2B. In this study, GFP-tagged constructs of titin-N2B were overexpressed in cardiomyocytes in an attempt to disrupt the potential interaction of endogenous N2B with an intracellular ligand. Our results suggested that the NH2-terminal domains of N2B are directly or indirectly critical for stabilizing thin filament structure; thus, N2B emerges as a unique region of titin that is critical for the maintenance of cardiac myofibrils.
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Ladd, Andrea Nicole. "Growth factor-mediated regulation of cardiac myogenesis during early avian embryogenesis." Diss., The University of Arizona, 1999. http://hdl.handle.net/10150/288995.
Full textAbstract:
Previous studies have identified two signaling interactions regulating cardiac myogenesis in avians, a hypoblast-derived signal acting on epiblast and an endoderm-derived signal acting on mesoderm. In this study, experiments were designed to investigate the potential role of TGFβ superfamily members in regulating these early steps of heart muscle cell development. While activin or TGFβ can potently induce cardiac myogenesis in pregastrula epiblast, they show no capacity to convert noncardiogenic mesoderm toward a myocardial phenotype. Conversely, BMP-2/BMP-4 can induce cardiac myocyte formation in mesoderm in a variety of contexts, but show no capacity to induce cardiac myogenesis in epiblast cells. Activin/TGFβ and BMP-2/BMP-4 therefore have distinct and reciprocal inducing capacities that mimic the tissues in which they are expressed, the pregastrula hypoblast and the anterior lateral endoderm, respectively. Experiments with follistatin and noggin provide additional evidence that BMP signaling lies downstream of activin signaling in the cardiac myogenesis pathway. BMP-2 or BMP-4 inhibit cardiac myogenesis prior to stage 3, demonstrating a dual role for BMPs in mesoderm induction. These and other published studies suggest a signaling cascade in which a hypoblast-derived activin/TGFβ signal is required prior to and during early stages of gastrulation, regulated both spatially and temporally by an interplay between BMPs and their antagonists. Later cardiogenic signals arising from endoderm, and perhaps transiently from ectoderm, act on emerging mesoderm within cardiogenic regions. These signals, mediated in part by BMPs, activate or enhance expression of cardiogenic genes such as GATA and cNkx family members, leading to cardiac myocyte differentiation. Members of the FGF and the EGF-related CFC families may also participate in this pathway. FGF-2/FGF-4 can induce posterior region epiblast to form heart muscle cells, and the CFC family member Cripto can convert posterior lateral mesoderm to a myocardial phenotype. The role of these factors in the cardiac myogenesis pathway is unclear.
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Sun, Guoxian. "A murine cell model for karyotype instability in chronic myelogenous leukemia." Thesis, McGill University, 1994. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=26154.
Full textAbstract:
Chronic myelogenous leukemia (CML) is a neoplastic disorder of pluripotent hematopoietic stem cells which follows a biphasic clinical course consisting initially of a relatively benign chronic phase followed by progression to a fatal acute leukemia (CML blast crisis). A clonal cytogenetic abnormality (i.e. the Philadelphia or Ph chromosome), resulting from the reciprocal translocation t(9;22)(q34;q11), is found in over 95% of patients. A hybrid gene is created at the breakpoint of Ph, derived from the ABL proto-oncogene (9q34) and from the BCR gene (22q11), which results in the expression of a 210 KDa fusion protein tyrosine kinase called P210BCR/ABL (P210). Expression of P210 alone is generally believed to be sufficient to induce chronic phase CML but, the deregulation of additional genes appears to be required for progression to CML blast crisis, as inferred by the presence of secondary cytogenetic abnormalities in over 80% of patients. To investigate the potential significance of P210 expression in the induction of genetic instability associated with CML progression, I studied cytogenetic changes in a murine cell line (32D) which expressed P210BCR/ABL from a retroviral vector. Using Giemsa-trypsin banding technique, I found a common marker chromosome t(4;12) in 13 subclones, a second new marker t(2;17) in 3/13 subclones and, additional clonal marker chromosomes in all of the subclones examined. Six subclones consisted of two or three karyotypically distinct cell populations. This study demonstrates that BCR/ABL can directly induce both numerical and structural chromosomal abnormalities in hematopoietic cells. This murine cell model may provide a useful tool to further study the causal relationship of cytogenetic instability and cooperative molecular events involved in the initiation and progression of CML.
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Fouche, Celeste. "Differential effects of TNfα on satellite cell differentiation." Thesis, Stellenbosch : Stellenbosch University, 2007. http://hdl.handle.net/10019.1/19596.
Full textAbstract:
Thesis (MSc)--University of Stellenbosch, 2007.ENGLISH ABSTRACT: Tumour necrosis factor alpha (TNFα) is a pleiotropic cytokine and has a wide variety of dose dependent cellular effects ranging from cell growth and differentiation, to inducing apoptosis. It has long been implicated in muscle and non-muscle inflammatory disorders, such as muscle wasting in chronic disease states, and rheumatoid arthritis. However, a physiological role for TNFα in muscle regeneration has been proposed as elevated levels of the cytokine are present when muscle regeneration processes are initiated: TNFα is secreted by infiltrating inflammatory cells, and by injured muscle fibres. Adult skeletal muscle contains a population of resident stem cell-like cells called satellite cells, which become activated, proliferate and differentiate following muscle injury to bring about repair of damaged muscle. Much research on the effects of TNFα on satellite cell differentiation has been conducted in recent years. It is however difficult to get a complete characterisation of the cytokine’s action as all models used slightly differ. We aimed therefore at providing comprehensive assessment of the effects of increasing doses of chronically supplemented TNFα on differentiating C2C12 cells. Cells were allowed to differentiate with or without TNFα supplementation for 7 days. Differentiation was induced at day 0. The effect on differentiation was assessed at days 1, 3, 5, and 7 by western blot analysis, and supplementary immunohistochemical analysis at days 1, 4, and 7 of markers of differentiation - muscle regulatory factors: MyoD and myogenin, markers of the cell cycle p21, PCNA, and the integral signalling molecule, p38MAPK. TNFα supplementation at day 1 tended to positively regulate early markers of differentiation. With continued supplementation however, markers of differentiation decreased dose dependently in treated cultures as the initial effect appeared to be reversed: A trend towards a dose dependent decrease in MyoD, myogenin and p21 protein existed in treated cultures at days 3, 5, and 7. These findings were significant at day 5 (p21, p<0.05), and day 7 (myogenin, p<0.05). A significant dose dependent decrease in p38 phosphorylation was evident at day 3 (p<0.05), while phospho-p38 was dose dependently increased at day 7 (p<0.05). Taken together, these data show that TNFα supplementation for 24 hours following the induction of differentiation in vitro, tends to increase levels of early markers of differentiation, and with continued TNFα supplementation decrease markers of differentiation in a dose dependent fashion. This study provides a comprehensive characterisation of the dose and time dependent effects of TNFα on satellite cell differentiaton in vitro. The model system used in the current study, allows us to make conclusions on more chronic disease states.
AFRIKAANSE OPSOMMING: Tumor nekrose faktor alfa (TNFα) is ‘n pleiotropiese sitokien wat ‘n wye verskeidenheid, dosis afhanklike, sellulêre effekte te weeg bring. Hierdie sellulêre effekte sluit sel groei en differensiasie tot sel dood in. TNFα is by beide spier en niespier inflammatoriese stoornisse soos spier tering in kroniese siektetoestande, en rumatiese artritis betrek. ‘n Fisiologiese rol vir TNFα is egter voorgestel aangesien verhoogde vlakke van die sitokien tydens inisiasie van spier herstel meganismes teenwoordig is: TNFα word deur infiltrerende inflammatoriese selle, asook deur beseerde spier vesels afgeskei. Volwasse skeletspier bevat ‘n populasie stamselagtige selle, sogenoemde satelliet selle. Laasgenoemde word geaktiveer, prolifereer en differensieër volgende spierbesering, om sodoende herstel van beskadigde spier te weeg te bring. Baie navorsing op die effekte van TNFα op satelliet sel differensiasie is onlangs uitgevoer. Dit is egter aansienlik moeilik om volgens hierdie navorsing‘n algehele beeld van TNFα se aksies te vorm aangesien alle modelle wat gebruik word verskil. Ons doel was daarom om ‘n omvangryke assessering van toenemende konsentrasies kronies gesupplementeerde TNFα op differensieërende C2C12 selle op ‘n enkele model uit te voer. Selle was vir 7 dae met of sonder TNFα supplementasie gedifferentieër. Differensiasie was by Dag 0 geïnduseer. TNFα se effek op differensiasie is op dae 1, 3, 5, en 7 deur middel van western blot analise geassesseer. Aanvullende immunohistochemiese bepalings op dae 1, 4, en 7 is verder deurgevoer. Merkers vir differensiasie het die spier regulatoriese faktore MyoD en miogenien, sel siklus merkers p21 en PCNA, asook die integrale sein transduksie molekule p38MAPK ingesluit. TNFα supplementasie by dag 1 het geneig om vroeë merkers van differensiasie positief te reguleer. Met voortdurende supplementasie is die vroeë positiewe effekte (op ‘n dosis afhanklike manier) egter omgekeer: ‘n neiging teenoor (‘n dosis afhanklike) vermindering in MyoD, miogenien en p21 proteïen het in behandelde kulture op dae 3, 5, en 7 bestaan. Hierdie bevindinge was beduidend by dag 5 (p21, p<0.05), en dag 7 (miogenien, p<0.05). A beduidende dosis afhanklike afname in p38 fosforilasie was duidelik by dag 3 (p<0.05), terwyl fosfo-p38 by dag 7 verhoog het met verhoogde konsentrasie TNFα (p<0.05). Bogenoemde saamgevat, dui aan dat TNFα supplementasie 24h volgende die induksie van differensiasie in vitro, verhoogde vlakke van vroeë differnsiasie merkers te weeg bring. Met voortdurende TNFα supplementasie, word differensiasie merkers egter met toenemende dosis verminder. Hierdie studie voorsien ‘n omvattende karakterisering van die dosis- en tyd afhanklike effekte van TNFα op satelliet sel differesiasie in vitro. Die model sisteem in hierdie studie gebruik, maak afleidings oor meer kroniese siektetoestande moontlik.
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MacDougall, Stephen L. (Stephen Lindsay). "Effector:target interactions in the human natural killer cell system : characterization of the target structures." Thesis, McGill University, 1990. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=74570.
Full textAbstract:
Natural killers (NK) are a subpopulation of lymphocytes that are defined by their pattern of cytotoxicity against other normal and neoplastic cells of predominantly hemopoietic origin. Although they have been programmed to recognize a limited spectrum of targets, their activity can be augmented by certain immunoregulatory substances. The molecules that mediate the recognition events have not yet been identified.I describe here the derivation and characteristics of a variant clone (Clone I) of the human leukemic cell line K562. These cells, selected for decreased binding to peripheral blood lymphocytes, were less sensitive than the parent to lysis by NK in the resting, but not in the augmented state. Although their major plasma membrane proteins appeared identical to those of K562, they contained an additional minor group of fucosylated glycolipids. A later subclone of Clone I, selected for resistance to Concanavalin A, reverted to an NK sensitive pattern and exhibited the parental profile of glycolipids.
The results illustrate in an in vitro model how a leukemic cell can modulate its membrane to escape surveillance by NK cells, and suggest that the glycolipids might be involved (directly or indirectly) in the mechanism.
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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.
Full textAbstract:
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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Austin, Emily. "Homeostatic regulation of induced [beta]-cell mass expansion in mice." Thesis, McGill University, 2006. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=101701.
Full textAbstract:
Current therapies do not prevent the devastating complications associated with type 1 and 2 diabetes. Novel therapies seek to restore a functional beta-cell mass through stimulating endogenous beta-cell mass expansion. Whilst there is considerable evidence that the beta-cell mass is under homeostatic regulation in the normal pancreas, it is unclear if such regulation exists in the context of induced beta-cell mass expansion. The aim of this study was to demonstrate that beta-cell mass expansion resulting from the induction of islet cell neogenesis is subject to long-term homeostatic control in the normoglycemic mouse adult pancreas.A pentadecapeptide fragment of Islet Neogenesis Associated Protein (INGAP 104-118) was administered daily to adult C57BL/6J mice for 12 weeks. Four animals from the INGAP104-118 treatment group and control group were sacrificed each week. The pancreas was removed from each mouse and stained for insulin. beta-cell mass was calculated as the organ weight multiplied by the percent of insulin+ area of total tissue area. Contrary to our expectations, there was no change in the total beta-cell mass in INGAP104-118-treated animals compared to control. Reanalysis of the stained tissue sections was preformed, and insulin+ structures were classified as being: (1) a duct islet, (2) a cluster of insulin+ cells, or (3) a mature islet. The density (#/mm2) of duct islets, clusters, and total structures in INGAP 104-118-treated animals was significantly increased; conversely, the density of mature islets was significantly decreased. The increase in cluster density suggests that INGAP104-118 induced neogenesis in the pancreas of treated animals. Poisson regression revealed 9th order polynomial time trends in the structure densities. Though these time trends differed between the classes of structures, they were identical in INGAP104-118 and control animals for each class of structure, suggesting an external stimulus was acting equally on both groups.
While this study did not determine if there is homeostatic regulation of induced beta-cell mass expansion, it did reveal important aspects for the design of a future study to address this issue. The definitions for structure classification must be well-established and rates of beta-cell replication should be determined.
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Middlebrook, Aaron J. "Nicotine and TNF alpha, modulators of T cell signaling-effects on T cell development in fetal thymus organ culture." Diss., The University of Arizona, 2004. http://hdl.handle.net/10150/280628.
Full textAbstract:
T cell development is regulated by signals generated in the interactions between developing thymocytes and the thymic stroma. Using fetal thymus organ culture (FTOC) as a model of T cell development, we investigated the ability of two potent signal modulators to influence this process. These studies show that both nicotine and tumor necrosis factor-alpha have the ability to influence T cell receptor (TCR) signaling and the maturational capacity of treated cultures. FTOC treated with low concentrations of nicotine produced more immature T cells and fewer mature T cells. These expanded populations of cells also expressed CD69, CD95 (FAS) and elevated levels of recombinase activating genes (RAG). This phenotype reflects the fact that these cells have received a positive selection signal, are for apoptosis and are likely attempting secondary TCR rearrangements. Nicotine effects were partially blocked by the nicotinic antagonist, d-tubocurarine. Furthermore, d-tubocurarine alone blocked the development of T cells entirely, suggesting the presence of an endogenous ligand that may engage nicotinic acetylcholine receptors and regulate normal thymopoeisis. These observations underscore the linkage between the nervous and the immune systems, not only in terms of shared resources, but also in terms of direct interactions between these two systems. In another study we used FTOC and an associated in vitro Type 1 diabetes mellitus model to reconcile the role of TNF-alpha in thymopoiesis with its role in diabetes. Our data indicate that thymocytes from NOD FTOC express lower levels of TNF receptors and produce more TNF-alpha compared to non-diabetic C57BL/6 (B6) FTOC. Neutralization of endogenous TNF-alpha in NOD FTOC with a soluble TNF receptor (sTNF R1) rescued insulin production in our in vitro diabetes model. NOD FTOC treated with TNF-alpha produced greater numbers of mature T cells and a higher percentage of cells expressing CD95L (Fas ligand). Treatment with sTNF R1 had the opposite effect. TNF-alpha's known ability to attenuate TCR signaling coupled with these observations suggest that its overproduction in these animals may be driving T cells to maturity, altering the process of negative selection and ultimately enhancing the survival of potentially diabetogenic T cells resulting in disease susceptibility in these animals.
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Lu, Yarong 1971. "Pancreatic-specific insulin-like growth factor I gene deficiency on islet cell growth and protection." Thesis, McGill University, 2006. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=111827.
Full textAbstract:
The role of insulin-like growth factor I (IGF-I) in pancreatic islet cell growth and development has been debated in recent years. The dogma that IGF-I stimulates pancreatic islet growth has been challenged by combinational targeting of IGF or IGF-IR genes, as well as beta-cell-specific IGF-IR gene deficiency. In order to assess the physiological role of locally produced IGF-I, we have developed pancreatic-specific IGF-I gene deficiency (PID) by crossing Pdx1-Cre and IGF-I/loxP mice. PID mice were normal except for decreased blood glucose level and a 2.3-fold enlarged islet cell mass. When challenged with low doses of streptozotocin, control mice developed hyperglycemia after 6 days that was maintained at high levels for at least 2 months. In contrast, PID mice only exhibited marginal hyperglycemia after 12 days, maintained throughout the experiment. Furthermore, streptozotocin-induced beta-cell apoptosis (TUNEL assay) was significantly prevented in PID mice. PID mice also exhibited a delayed onset of type 2 diabetes induced by a high-fat diet, accompanied by super enlarged pancreatic islets and preserved sensitivity to insulin. As the phenotype is unlikely a direct consequence of IGF-I deficiency, we used oligonucleotide DNA microarray to explore possible activation of pro-islet genes in PID mice, which revealed upregulation of multiple new members of the Reg family genes (Reg2, 3alpha and 3beta) in the pancreas. The results were subsequently confirmed by Northern blot and/or realtime PCR, which exhibited 2 to 8 fold increases in the level of their mRNAs. Moreover, these Reg family genes were also activated following streptozotocin-induced beta-cell damage and diabetes. Our results reveal a possible mechanism of islet growth and protection in PID mice, thus serving a potential strategy in combating diabetes.
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Knudson, Jennifer Caroline. "Cardiotrophin-1 as an ex vivo activator of a stem-cell like population in the murine heart." Thesis, University of Ottawa (Canada), 2005. http://hdl.handle.net/10393/26946.
Full textAbstract:
The identification of a multi-potent stem cell-like population (SP) within the adult murine heart infers new methods of cardiac repair, i.e. stem cells compensate for damage by generating new cardiomyocytes. Several studies have emphasized the dominance of the tissue microenvironment on the differentiation & functional properties of stem cells. Of particular interest was Cardiotrophin-1 (CT-1), a member of the Interleukin-6 (IL-6) family of cytokines. To assess whether CT-1 functioned as an activator of cardiac SP cells in the murine heart, an adenovirus containing the full length CT-1 driven by a ubiquitous promoter was generated. The adenovirus was administered via intra-cardiac injections and the effects of CT-1 were primarily assessed using fluorescence-activated cell sorting (FACS) analysis. Analysis showed a temporary increase in the cardiac SP of CT-1 injected hearts. A closer look at the SP cells in other tissues (liver, skeletal muscle and bone marrow) demonstrated that this phenomenon was cardiac specific. Interestingly the cardiac SP expressed the Leukemia Inhibitory Factor (LIF) receptor, which is required for CT-1 signaling through the 130 pathway. Protein analysis also showed that STAT3, a downstream member of the 130 pathway becomes activated in the heart during CT-1 injections. Preliminary results from co-culture experiments suggested that this increase was also accompanied by SP cell differentiation. These results proposed that CT-1 not only increased the cardiac SP size but may have also activated a cardiac differentiation program. The existence of a potential biologic stimulant (CT-1) for cardiac stem cells is an exciting prospect and offers support to the notion that cardiac repair may become a viable therapeutic option in the not too distant future.
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Szymanska, Irena. "Exploration of lentiviral vectors and TAT-fusion proteins for the delivery of XIAP protein to neonatal retinal progenitor cells." Thesis, University of Ottawa (Canada), 2008. http://hdl.handle.net/10393/27790.
Full textAbstract:
Post-transplant apoptosis is a major obstacle to successful cell replacement therapy for retinitis pigmentosa. Over-expression of the X-linked inhibitor of apoptosis (XIAP) protein could increase transplant survival leaving a greater numbers of cells available to replenish photoreceptors lost during retinal degeneration. Sonic Hedge hog expanded C57BL/6 mouse neonatal retinal progenitor cells (Hh-RPCs) were infected with two lentiviral constructs, encoding XIAP/GFP or GFP only, as well as with the TAT-fusion protein, TAT-eGFP. The optimal delivery conditions and expression patterns were assessed. It was found that lentiviral infection, in conjunction with fluorescence activated cell sorting (FACS) allowed for the creation of a nearly pure line of Hh-RPCs which over-expressed XIAP protein for at least one month. Although the TAT-fusion protein efficiently transduced Hh-RPCs, its nuclear localization made it unsuitable for XIAP protein delivery. These results demonstrated two methods of transducing primary retinal progenitor cells and represent an important first step towards efficient cell replacement therapy in the retina.
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Wang, Yifang. "Role and regulation of X-linked inhibitor of apoptosis protein expression during development of the rat ovarian follicle in vitro." Thesis, University of Ottawa (Canada), 2003. http://hdl.handle.net/10393/29006.
Full textAbstract:
Follicle stimulating hormone (FSH) is an important survival factor in the ovarian follicular development. Inhibitor of apoptosis proteins (IAPs) is a family of intracellular anti-apoptosic proteins. X-linked IAP (XIAP) has been shown to be involved in multiple biological activities (e.g. inhibition of caspase activities, promotion of ubiquitin-proteasome-mediated protein degradation, regulation of cell signaling pathways). The present thesis research project examines: (1) the role and gonadotropic regulation of XIAP expression in rat granulosa cells during ovarian follicular development and atresia; (2) the possible involvement of intra-ovarian factors such as transforming growth factor alpha (TGFalpha) in the FSH-induced XIAP expression and follicular development; and (3) the signal pathways involved in the gonadotropic up-regulation of XIAP during follicular development in vitro.
A follicle culture system coupled to an adenoviral gene manipulation procedure has been established. FSH significantly increased follicular growth as evident by increases in follicular size, cell number and DNA contents in vitro. While cultured pre-antral or early-antral follicles showed a low XIAP content and evidence of apoptosis in the absence of FSH, gonadotropin addition increased XIAP content and suppressed apoptosis. At low FSH concentration, adenoviral XIAP sense cDNA expression increased follicular cell XIAP and DNA contents, reduced apoptosis, and enhanced follicular growth, while XIAP antisense elicited opposite responses. FSH-induced XIAP up-regulation appeared mediated, in part, by the secretion and action of follicular TGFalpha. In cultured rat follicles, FSH-stimulated estradiol production, TGFalpha secretion, XIAP expression and follicular growth were suppressed by intra-follicular injection of a neutralizing anti-TGFalpha antibody or addition of the estradiol antagonist ICI 182780 to the culture media. These results support my hypothesis that the FSH induces follicular growth by stimulating granulosa cell proliferation via theca TGFalpha secretion and action in response to increased granulosa cell estradiol synthesis.
Since the promoter region of XIAP gene has nuclear kappa B (NFkappaB) binding site, it is possible that the transcription of XIAP is mediated via NFkappaB activation. FSH increased rat granulosa cell XIAP mRNA abundance and protein content. While the gonadotropin induced granulosa cell NFkappaB translocation from cytoplasm to nucleus and increased NFkappaB-DNA binding activity, pretreatment with an NFkappaB translocation inhibitor suppressed FSH-stimulated XIAP expression. (Abstract shortened by UMI.)
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Sheng, Yinglun. "G protein signaling and G protein coupled receptor (GPCR) pathway in Xenopus oocyte maturation." Thesis, University of Ottawa (Canada), 2005. http://hdl.handle.net/10393/29262.
Full textAbstract:
Xenopus laevis oocytes are physiologically arrested at the first meiotic prophase. Progesterone reinitiates meiosis (maturation) through inhibition of an oocyte adenylyl cyclase (AC) and reduction of intracellular cAMP. However, the mechanism by which progesterone regulates AC activity and cAMP level still remains unclear.
In this thesis, I summarize work I conducted that collectively helps elucidate how high levels of cAMP might be achieved in G2 arrested oocytes. In Chapter 2, I describe our finding that inhibiting endogenous G-protein betagamma subunits, through the use of two structurally distinct Gbetagamma scavengers, causes hormone-independent oocyte maturation. In contrast, overexpression of Xenopus Gbeta1, alone or together with bovine Ggamma2, inhibits progesterone-induced oocyte maturation. These results for the first time implicate that an endogenous G protein coupled receptor system releases a Gbetagamma complex as the dominant meiosis inhibitor.
Chapter 3 describes my research aiming to reveal the identity of the oocyte AC responsible for generating meiosis-inhibiting cAMP. I provide further evidence here that the ability of Gbetagamma to inhibit meiosis is attributed to the activation of an endogenous AC, rather than other possible Gbetagamma effectors. Through molecular cloning and biochemical characterization, I discovered that the likely AC candidate is Xenopus AC7, an isoform that is activated by Gbetagamma, but only in the presence of GTP-bound Gsalpha. The identification of xAC7 suggests that the maintenance of high levels of cAMP may require the cooperation of Gsalpha and Gbetagamma.
Finally, in Chapter 4, I describe our efforts in identifying the GPCR(s) responsible for activating the cAMP signaling in prophase-arrested oocytes. A screening of known antagonists of GPCR(s) led to the identification of ritanserin, a potent antagonist of serotonin receptors, as a potent maturation inducer in Xenopus oocytes. Pharmacological and molecular studies, however, have ruled out the involvement of a known serotonin receptor in meiosis arrest. Instead, the most likely candidate is a "constitutively activated" GPCR that bears structural similarities to Xenopus serotonin receptor 7.
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Zhao, Qing 1966. "Prosaposin : a glycoprotein with multiple functions and dual destinations." Thesis, McGill University, 2000. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=36857.
Full textAbstract:
Prosaposin is a multifunctional glycoprotein with different molecular masses and dual destinations. A 65 kDa form of prosaposin is targeted to lysosomes and converted by partial proteolysis, into four smaller non-enzymatic saposin A-D required for the hydrolysis of glycosphingolipids. However, the 65 kDa protein may be further glycosylated to a 70 kDa secretory form that is found in various biological fluids and suspected to have a trophic activity. Mutations of the prosaposin gene are linked to several lysosomal disorders. This thesis examines various aspects of the synthesis, targeting and function of prosaposin, and for practical purposes, the results and discussion were divided in three main sections. The first part deals with the cloning of the mouse prosaposin gene, the analysis of its transcribed mRNA and translation products. The second section examines the mechanism of targeting of the 65 kDa protein to lysosomes using mutagenic analyses. The third part deals with the effect of the inactivation of the prosaposin gene on the development of the male reproductive system. Sequence analysis revealed that the mouse prosaposin gene is over 20 kb in length and composed of 15 exons and 14 introns. Two forms of alternatively spliced mRNA (including or excluding exon 8) were found by RT-PCR in a tissue specific manner. Structure analysis and secondary structure predictions among mouse, rat and human prosaposins illustrated a common framework of amino acids forming amphiphatic helices enclosing an internal hydrophobic core implicated on their interaction with lipids. Mutagenic deletions of functional domains of prosaposin demonstrated that its C-terminus was required for the lysosomal targeting of this protein. Further evidence from chimeric constructs of albumin attached to various functional domains of prosaposin, suggested that the C-terminus plus at least one saposin domain are necessary for the targeting of albumin to lysosomes. Investigation of the effect of p
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Patel, Bharatkumar N. "Ceruloplasmin : novel form and function in the central nervous system." Thesis, McGill University, 2000. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=37807.
Full textAbstract:
Ceruloplasmin oxidizes the toxic ferrous (Fe(II)) form of iron to the ferric (Fe(III)) form, and promotes iron loading onto transferrin. It is produced by the liver and secreted into the plasma. My work has shown that a novel membrane-anchored form of ceruloplasmin is the major form of this enzyme in the rat brain.The membrane-anchored form of ceruloplasmin was identified using a monoclonal antibody (mAb 1A1) that specifically labels the surface of astrocytes. Using mAb 1A1 immunoaffinity chromatography, the antigen was purified from detergent extracts of rat C6 glioma cells. Amino acid microsequencing, and additional experiments, revealed that this antigen was highly similar to ceruloplasmin. Further experiments revealed that this form of ceruloplasmin was directly anchored to the surface of astrocytes and C6 glioma cells by a glycosylphosphatidylinositol (GPI) anchor.
Screening of a C6 glioma cDNA library identified a novel alternatively-spliced transcript that codes for this GPI-anchored form of ceruloplasmin. HEK293T cells transfected with the novel full-length cDNA expressed ceruloplasmin on the cell surface. Treating these transfected cells with phosphatidylinositol-specific phospholipase C (PI-PLC), an enzyme that specifically cleaves GPI anchors, eliminated the cell surface localization of ceruloplasmin, confirming that the novel cDNA codes for the GPI-anchored form of the protein. RNase protections on rat brain and liver RNA revealed that GPI-anchored ceruloplasmin is the major form in the brain, whereas the liver expresses predominantly the secreted form.
To better understand the role of ceruloplasmin in vivo, a ceruloplasmin gene knockout mouse was generated. The knockout mice accumulate large amounts of iron in the liver and have reduced serum iron levels. Older knockout mice (15--17 months) have increased iron accumulation in different parts of the CNS, including the cerebellum, spinal cord, and retina. Increased lipid peroxidation is also observed in some regions of the CNS. These biochemical changes were accompanied by deficits in motor skills. Furthermore, dissociated cell cultures of the cerebellum from knockout mice were more susceptible to free radical injury when treated with hydrogen peroxide. These data suggest that ceruloplasmin plays an important role in iron metabolism in the CNS and provides protection against free radical injury.
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Zhu, Ping jun. "Adenosine release and neuronal depression during energy deprivation : electrophysiological studies." Thesis, McGill University, 1996. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=40305.
Full textAbstract:
This thesis focuses on the effects of adenosine release on neuronal activity in the CA1 region of rat hippocampal slices, especially during energy deprivation, and related events. The overall aim was to find out how brain function is suppressed when metabolism is greatly reduced by the lack of oxygen or glucose, or by cyanide applications, and therefore to provide some insights in the cellular mechanisms underlying these depressant effects. Experiments were carried out on hippocampal slices kept submerged and constantly superfused with oxygenated saline at 33-34$ sp circ$C. Energy deprivation was produced by either removal of oxygen or glucose, or applying cyanide. Extra-/intracellular and whole-cell patch recordings (both in current- and voltage-clamp mode) were used to assess synaptic function and postsynaptic properties.A most interesting phenomenon is the reversible suppression of neuronal function that occurs in the very early phase of energy deprivation. Adenosine receptor antagonists reversibly reduce this suppression. In contrast, neither glibenclamide, a blocker of ATP-sensitive K$ sp+$ channels, nor a nitric oxide synthase inhibitor prevents the suppression of neuronal activity induced by energy deprivation. The depressant effect acts selectively on excitatory synapses, since in the presence of excitatory receptor antagonists, anoxia causes only a small reduction of monosynaptic inhibitory responses. Also adenosine antagonists, but not the K$ rm sb{ATP}$ channel blocker, reversibly attenuate anoxia- and cyanide-induced post-synaptic hyperpolarizations.
Furthermore, under normoxic conditions, ongoing adenosine release exerts an inhibitory tone on excitatory synapses but not on inhibitory synapses. The effects of ongoing adenosine release are mainly on synapses, since after blockade of transmitter actions, ongoing adenosine release has no detectable effect on membrane conductance.
The evidence presented in this thesis shows that increased adenosine release induced by energy deprivation is a major cause of the reversible loss of synaptic transmission and fall in membrane resistance, and therefore indicates a mechanism by which adenosine release contributes to the reversal depression of neuronal activity seen during energy deprivation.
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Di, Falco Marcos Rafael. "Development of growth factor-cytokine fusion proteins with increased hematopoietic activity : physiological and cellular effects." Thesis, McGill University, 2002. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=82853.
Full textAbstract:
Hematopoietic precursor cells express cell surface receptors for a variety of cytokines and growth factors. These hematopoietic secreted humoral factors can influence the proliferation, differentiation, survival and mobilisation of blood stem cells. Moreover, simultaneous exposure to different combinations of hematopoietic hormones can synergistically affect one or more of these cellular responses. Our laboratory has been involved in studying the role played by insulin-like growth factors (IGFs) in hematopoiesis. IGFs are known to stimulate the expansion of precursor cells from various hematopoietic lineages when administered in vivo. However, difficulties in producing recombinant IGFs with conventional bacterial or yeast expression systems make the use of IGFs as therapeutic hematopoietic agents a cost prohibitive concept. A baculovirus based expression system was developed for the production of large amounts of properly folded and biologically active secreted IGF analogues (BOMIGFs). This system was later adapted for the synthesis of a fusion protein consisting of BOMIGF and intedeukin-3 (BOMIGF-IL-3). The in vitro and in vivo activities of this chimeric molecule were subsequently studied.The BOMIGF-IL-3 chimera promoted greater thymidine incorporation activity into TF-1 and bovine fetal erythroid cells than observed with the combined administration of BOMIGF and IL-3. This chimera also stimulated the formation of BFU-Es, CFU-GMs and, in particular, the highly proliferative macroscopic colonies in peripheral blood cell hematopoietic colony formation assays. These effects were reproduced in colony formation assays from bone marrow- and spleen-derived colony-forming cells from mice treated with BOMIGF-IL-3. This chimera also helped in the recovery of weight loss, anemia and neutropenia in an AZT-mediated myelossuppression mouse model. The chimera was shown to be significantly better than the corresponding equimolar mixture of the single factors at promoting the survival of TF-1 cells. This effect is associated with a sustained activation of PI-3 kinase, STAT5 phosphorylation and BclxL expression. The chimera was also better than the co-addition of BOMIGF and IL-3 at stimulating the migration of TF-1 cells across Transwell plates. The chimera-dependent potentiation of migration appears to be mediated by at the very least, an enhancement of PI-3 kinase activity.
This recombinant BOMIGF-IL-3 fusion molecule could prove useful for the therapeutic treatment of conditions with decreased production of blood cells such as in AZT- or chemotherapy-induced anemia. Other useful applications may include the mobilisation of stem cells and their ex vivo expansion for the purpose of stem cell transplantation.
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Cohen, Ricky Israel. "Rat brain oligodendrocytes express muscarinic and adrenergic receptors." Thesis, McGill University, 1996. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=42006.
Full textAbstract:
The aim of the studies underlying this thesis was to characterize the muscarinic and adrenergic receptors expressed in rat brain oligodendrocytes' (OLs); determine if ligand binding alters second messenger levels classically associated with these families of receptors, such as inositol phosphates (InsP), intracellular calcium ( (Ca$ rm sp{2+} rbrack sb{i}),$ and cyclic AMP (cAMP); and the role of neurotransmitters, acetylcholine (Ach) or norepinephrine (NE) on oligodendrocyte growth.CCH (carbachol), a stable Ach analog, caused a concentration and time dependent increase in the accumulation of InsPs and the mobilization of (Ca$ rm sp{2+} rbrack sb{i},$ which was inhibited by atropine, a specific muscarinic antagonist, and was negatively regulated by acute activation of protein kinase C by the phorbol ester TPA. CCH also negatively regulated the $ beta$-adrenergic-stimulated increase in cAMP levels. Using subtype m1 and m2 specific muscarinic receptor oligonucleotide primers RT-PCR confirmed the presence of, at least, these two muscarinic receptor subtypes. CCH caused a time and concentration-dependent increase in c-fos proto-oncogene mRNA levels as determined by Northern blot analysis. The CCH-stimulated c-fos increase was mediated through a non-phorbol ester sensitive PKC isozyme, and was dependent upon intra and extracellular calcium. Moreover, CCH stimulated DNA synthesis in OLPs, as measured by both ($ sp3$H) -thymidine and BrdU incorporation.
Lastly, the NE-stimulated signal transduction pathway was characterized in developing OLPs. Using selective agonists and antagonists, we determined that NE increased the formation of InsPs through $ alpha sb1$ adrenoceptors. We further subclassified the $ alpha sb1$ receptor to the $ rm alpha sb{1A}$ subtype using more selective reagents; WB4101, a selective antagonist for $ rm alpha sb{1A}$ receptors blocked the response to NE, while chloroethylclonidine, an $ rm alpha sb{1B}$ antagonist had no effect. Furthermore, Pertussis toxin, a bacterial toxin that ADP-ribosylates and inactivates certain G-proteins, EGTA, a calcium chelator, or CdCl$ sb2,$ an inorganic calcium channel blocker, all significantly blocked the NE-stimulated InsP formation. Together these results suggest that OLPs express $ alpha sb1$-adrenoceptors characteristic of the $ rm alpha sb{1A}$ subtype.
In toto, these studies demonstrate that developing OLs express functional muscariaic and adrenergic receptors, and suggest that Ach may function as a trophic factor. These results help to define a mechanism whereby neurons, and OLs may use neurotransmitters to communicate both during development and in the mature central nervous system.
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Khatchadourian, Karine. "Abnormalities in the testis, reduced sperm counts and decreased motility parameters in huntingtin- interacting protein 1 (HIP1) deficient mice." Thesis, McGill University, 2005. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=84045.
Full textAbstract:
Huntingtin interacting protein-1 (HIP1) is an endocytic protein that associates with clathrin on coated vesicles, but it also binds directly to actin and microtubules. Close histological examination of the testis of HIP1-/- mice from 7-30 wks of age revealed that HIP1-deficiency was particularly detrimental to spermatids. The testis showed a significant decrease in the diameter of seminiferous tubules, a reduction in the number of late spermatids and the sloughing of germ cells, which were evident as round cells in the epididymal lumen. Major abnormalities in spermatids included structural deformations of heads, bent flagella, the presence of proacrosomic vesicles with the complete or partial absence of an acrosome or its detachment from the nucleus, and retention of the cytoplasm enveloping the spermatid head. Abnormalities with respect to the association of elongating spermatids with ectoplasmic specializations of Sertoli cells were noted as well. Sperm counts and sperm motility parameters were significantly decreased in HIP1-/- mice compared to their wild-type littermates and these differences accounted for reduced fertility levels noted in HIP1-/- mice.Taken together, differences in sperm counts, morphology and their motility parameters suggest a functional role for HIP1 in relation to actin and microtubules during sperm development in the testis.
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Walker, Angela. "Electrochemical study of vesicular release in bovine chromaffin cells." Thesis, McGill University, 1995. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=23431.
Full textAbstract:
The time course of the spontaneous current spikes produced by the release of the catecholamines from individual vesicles was examined in bovine chromaffin cells by using the carbon filament technique in the amperometric mode.Frequency histograms of the rise and decay times of the current spikes showed a paucity of very short duration events. Scatterograms of the rise and decay times consistently showed a positive relation, and the best fitted lines intercepted the ordinate (the axis of the decay time) at: 16.06 $ pm$ 6.45 msec (n = 11).
The effect of temperature changes upon the time course of release of content of individual vesicles in chromaffin cells was also examined. The amplitudes of the current spikes did not change significantly, whereas the rise times and the decay times diminished from (23.2 $ pm$ 11.6 to 11.9 $ pm$ 2.7 msec, and from 76.6 $ pm$ 25.4 to 47.3 $ pm$ 9.3 msec respectively) as the temperature was raised from 15$ sp circ$C to 35$ sp circ$C (n = 5). Nevertheless, the Q$ sb{10}$ values of the rise and decay times were surprisingly low.
The experimental findings suggest that in bovine chromaffin cells the duration of the release of content of single vesicles is much longer than in synapses. The results also suggest that this mechanism does not involve processes that are strongly temperature sensitive.
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Ma, Weiya. "Substance P sensory fiber innervation of CNS target tissues in two experimental models." Thesis, McGill University, 1995. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=40185.
Full textAbstract:
The aim of this thesis study was to investigate the substance P (SP)-immunoreactive (IR) sensory fiber innervation of CNS target tissues in two experimental models. In the first model, we examined the SP-IR boutons apposed to three functional types of dorsal horn neurons and their morphological interaction with calcitonin gene-related peptide (CGRP) and enkephalin (ENK) in the cat spinal cord, using a combination of intracellular electrophysiological recording and horseradish peroxidase injection with ultrastructural immunocytochemistry. In addition to SP-IR only boutons, we detected boutons co-localizing SP plus CGRP and SP plus ENK immunoreactivities presynaptic to nociceptive neurons. Quantitatively, significantly higher numbers of SP-IR, SP+CGRP-IR and SP+ENK-IR boutons were apposed to nociceptive neurons. Non-nociceptive neurons were rarely innervated by boutons which were SP-IR, SP+CGRP-IR and SP+ENK-IR. In contrast, ENK-IR only boutons innervated non-nociceptive neurons considerably. Boutons co-localizing SP and CGRP were considered as originating from primary sensory afferents. Most nociceptive neurons contained ENK immunoreactivity, but non-nociceptive neurons were never ENK-IR. The interaction of SP and $ gamma$-aminobutyric acid (GABA) in the superficial dorsal horn of the cat and rat spiral cord was also investigated. The co-localization of SP and GABA in axonal terminals was detected for the first time in the superficial layers of the dorsal horn of the cat, but not rat, spinal cord.In the second model, we used immunocytochemistry to study the SP-IR fiber innervation of the white matter of transgenic mice expressing NGF in myelinating oligodendrocytes driven by a MBP promoter. SP-IR fibers were observed in the white matter of the CNS of both transgenic and control mice from postnatal day 0 to day 2. From day 5 on, however, these SP-IR fibers increased markedly to become ectopic fibers in transgenic mice, but decreased dramatically, and finally disappeared, in control mice. The ectopic SP-IR fibers of transgenic mice persisted throughout adulthood. Capsaicin treatment abolished all ectopic SP-IR fibers, indicating their primary sensory origin.
In conclusion, SP-IR fibers specifically innervated nociceptive neurons and co-localized with CGRP, ENK and GABA in the cat dorsal horn. The finding provides anatomical substrates for roles of SP in nociception and for functional interactions of SP with ENK and GABA. Ectopic SP-IR fibers innervated the white matter of the CNS of transgenic mice where NGF was abnormally-produced.
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An, Jing 1962. "Microenvironmental influences on the growth of normal and leukemic myeloid cells in the rat bone marrow." Thesis, McGill University, 1996. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=41964.
Full textAbstract:
The hemopoietic microenvironment of the bone marrow is an essential regulator of in vivo hemopoiesis. In addition to supporting the growth of normal blood cells, it also influences the growth of leukemia. This thesis describes the use of a rat model to examine three aspects of the function of the hemopoietic microenvironment. First, using a myeloid leukemia cell line (BNML), we showed that the pattern of growth of these cells differed in the bone marrow and spleen, that their presence was associated with a relocalization of normal hemopoietic stem cells from marrow to spleen, and that factors (yet to be defined) released from spleen cells altered the pattern, possibly to create a more permissive environment. Second, we showed that the "ST3" marker of marrow fibroblasts was associated with the Thy-1 molecule, and either directly or indirectly contributed to the in vitro adhesion reaction between marrow fibroblastoid cells and normal and leukemic myeloid precursors. Third, we showed that ectopic bony ossicles induced by subcutaneous implantation of recombinant human bone morphogenetic protein-2 contained marrow expressing the full range of hemopoiesis, including stem cells with a potential for long-term repopulation (demonstrated using a rat Y-chromosome specific DNA probe that we developed), and contained fibroblastoid cells differentiated to express the ST3 antigen in a manner similar to those from femoral bone marrow. These results provide further evidence for, although not final proof of, the hypothesis that the ST3 antigen participates in the function of the rat hemopoietic microenvironment, and points the way to future experiments on the interactions between stromal elements and normal and leukemic myeloid precursors.
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Ghamari, Langroudi Masoud. "Analysis of caesium sensitive membrane conductances in neurones of supraoptic nucleus." Thesis, McGill University, 2001. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=37893.
Full textAbstract:
The release of vasopressin and oxytocin from the nerve terminals of magnocellular neurosecretory cells (MNCs) is optimised by increases in firing frequency and the adoption of a phasic pattern of firing in the soma. The temporal summation of post-spike depolarising afterpotentials (DAPs) of consecutive action potentials has been proposed to contribute to the generation of phasic firing. There has not been, however, any experimental evidence to support this hypothesis. In this study, a direct blocker of the DAP is introduced as being Cs +. Using this blocker, it is shown that the DAP plays an important role in the generation of phasic firing. Furthermore, these experiments reveal that external Cs+ causes depolarisation of MNCs when the membrane potential is held between action potential threshold and near -80 mV. External Cs+, however, is also known as a classical blocker of the hyperpolarisation activated inward current (IH). If I H is present in rat MNCs, the blockade of the IH by external Cs+ should lead to hyperpolarisation rather than the observed depolarisation. Using a recently introduced blocker of IH, ZD 7288, I show that IH is indeed expressed in rat MNCs, and that it also plays an important role in excitability and phasic firing of these cells. Finally, the ionic basis for the depolarising effects of external Cs + in rat MNCs is investigated. It is concluded in that extracellular Cs+ blocks both IH and a leakage K+ current that contributes significantly to the resting potential.
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Kuhn, Hallie. "Regulation of Yolk Catabolism in Early Embryogenesis." Thesis, Harvard University, 2015. http://nrs.harvard.edu/urn-3:HUL.InstRepos:23845424.
Full textAbstract:
Yolk provides an important source of nutrients during the early development of oviparous (non-platental) organisms. In addition to phosphate and lipids, it is com- posed mainly of vitellogenin proteins packed into membrane-bound compartments called yolk platelets. Catabolism of yolk is initiated by acidification of the yolk platelet, leading to the activation of cathepsin-like proteases that degrade yolk contents, but it is unknown how this process is triggered. Using maternal shRNA technology in Drosophila melanogaster embryos we find that yolk catabolism depends on components of the Tor pathway, a well-characterized regulator of cellular metabolism. Knockdown of Tor also leads to severe nuclear fragmentation, abnormal gastrulation, and an increased ratio of AMP/ATP. This phenotype is more severe than inhibition of Tor in later development, or in cell culture models, suggesting that Tor may have additional functions during early development.
Additionally, we identify a downstream target of Tor, Atg1, as necessary for yolk catabolism. Atg1 is responsible for initiation of autophagy, a process that de- grades both protein and organelles within the cell. While Atg1 is required for a burst of spatially-regulated autophagy during late cellularization, autophagy is not required for yolk catabolism. We find that knockdown of Atg1, but not downstream autophagy proteins, can rescue shRNA-Tor embryos, suggesting that Atg1’s role in yolk cataboilism may be through regulation of Tor. Last, we find that Rheb, a GTPase responsible for activation of Tor on the lysosome membrane, is present on Xenopus laevis yolk platelets. Therefore, regulation of yolk catabolism by the Tor pathway may function in a similar manner to Tor’s activity on the lysosome. Together, this work connects the conserved Tor and Atg1 metabolic sensing pathways to yolk catabolism, and may provide insight into the metabolic regulation of lysosomes more generally.Systems Biology