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Host colonization is mediated through the secretion of effector proteins in order to neutralize host immune responses. However, the mechanism of the effector delivery during biotrophic invasion is not well defined in M. oryzae. In this thesis, I define the role of the exocyst complex, an evolutionarily conserved octameric protein complex involved in vesicle docking to the plasma membrane (composed of Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70 and Exo84), during infection-related development in M. oryzae. Like other filamentous fungi, M. oryzae, exocyst components localize to the vegetative hyphal tip distinct from the Spitzenkörper. However, at the initial stage of infection-related development all the exocyst components localise as a ring at the cortex of the appressorium and re-assembles around the appressorium pore in an actin-dependent manner in mature appressoria. I report that the septin network is required for the transition of exocyst ring from periphery to the appressorium pore. Deletion of Exo70 and Sec5 showed significant reduction in protein secretion and plant infection. I show that Sec6 is required for the exocyst assembly around the appressorium pore and effector secretion from the appressorium. I report that, during biotrophic invasion, effectors are secreted through a distinct pathway. Apoplastic effectors, Bas4 and Slp1 are secreted via a Golgi-dependent pathway while secretion of cytoplasmic effectors, Pwl2 and Bas1 meditates through a Golgi-independent pathway in which exocyst components Exo70 and Sec5 are involved.

Knowledge of the structure and dynamics of cellular protein complexes is essential for understanding their functionally relevant interactions. In Gram-negative bacteria, the complex machinery associated with the type II secretion system (T2SS) polymerises inner membrane pseudopilin proteins into thin filaments, to export substrates such as toxins, hydrolases and cytochromes. Here, computational simulations were used to study proteins from the Klebsiella oxytoca T2SS, focusing on the substrate pullulanase PulA, the major pseudopilin PulG, and the putative chaperone PulM. Chapter 3 contains an in silico study of both post-translationally acylated PulA (lipoPulA) and non-acylated PulA (PulANA) in association with a lipid bilayer, representing an approximation of the biological state prior to secretion; this study examined PulA dynamics and the possible role of the acyl tail in protein-membrane interactions before secretion. Novel insights into the interactions of a key residue necessary for Type 2 secretion were gained via simulations performed on a PulANA D2S variant, extending prior in vitro results. In Chapter 4, PulA was simulated in conditions closer to the physiological environment, using counter-ions to investigate the possible effect of the high periplasmic calcium concentration on protein conformation and lipid interactions prior to secretion. In Chapter 5, variants of the major pseudopilin PulG containing one transmembrane helix were simulated, demonstrating N-terminal interactions made possible by wild-type methylation of residue Phe1. Simulations of several monomeric PulG variants provided insight into the roles of the essential residue Glu5 and Phe1 methylation, previously identified by experimental work to be important. Simulations of the PulG dimer demonstrated the dynamic nature of the membrane-embedded dimer interface, and showed how computational analysis can predict in vivo contacts. Finally, Chapter 6 extended the T2SS studies to coarse-grained methods, sampling possible conformations and predicting the PulG-PulM interface within the membrane, prior to PulG presentation to the remaining secretion apparatus.

by Wai-on Fu. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1993. / Includes bibliographical references (leaves 88-95). / Chapter Chapter I --- Introduction --- p.1 / Chapter I .1 --- Structure and functions of the epididymis --- p.1 / Chapter I.2 --- Cellular mechanisms for electrolyte secretion --- p.3 / Chapter I.3 --- Second-messenger modulation of chloride secretion in epididymis --- p.5 / Chapter I.3.1 --- Cyclic AMP pathway --- p.5 / Chapter I.3.2 --- Ca2+ as second messenger --- p.6 / Chapter I.4 --- Pathophysiology of electrolyte transport in the epididymis --- p.6 / Chapter I.5 --- Objectives of the study --- p.9 / Chapter Chapter II. --- Materials and Methods --- p.10 / Chapter II. 1 --- Materials --- p.10 / Chapter II. 1.1 --- Culture media and enzyme --- p.10 / Chapter II. 1.2. --- Drugs --- p.10 / Chapter II. 1.3 --- Chemicals --- p.11 / Chapter II. 1.4. --- Animals and human tissue --- p.11 / Chapter II.2 --- Preparation of solutions --- p.12 / Chapter II. 3. --- Preparation of cultured cells --- p.12 / Chapter II.3 .1 --- Culture of rat epididymal epithelial cells --- p.12 / Chapter II.3.2 --- Cultured of human epididymal epithelial cells --- p.16 / Chapter II.4. --- Patch-Clamp technique --- p.21 / Chapter II.4.1 --- Electrode --- p.23 / Chapter II.4.2 --- Pulling of electrode --- p.23 / Chapter II.4.3 --- Coating of electrode --- p.23 / Chapter II.4.4 --- Polishing of the electrode --- p.24 / Chapter II.4.5 --- Filling of the electrodes --- p.26 / Chapter II.4.6 --- Mounting of Electrode to the Headstage Pipette Holder --- p.26 / Chapter II.4.7 --- Electrical Isolation --- p.28 / Chapter II.4.8 --- Vibration Isolation --- p.28 / Chapter II.4.9 --- "Formation of ""Giga-seal"" and Whole-cell configuration" --- p.28 / Chapter II.4.10 --- Correction for liquid junction potential --- p.30 / Chapter II.4.11 --- "Data Acquisition and Analyses," --- p.32 / Chapter II.4.12 --- Statistics --- p.34 / Chapter Chapter III. --- Results --- p.35 / Anion Secretion in human epididymal cells --- p.35 / Chapter III. 1 --- Whole-cell current in human epididymal cells --- p.35 / Chapter III. 2 --- Effect of adrenergic receptor blockers on whole-cell current --- p.38 / Chapter III.3 --- Effect of inhibitors of the cyclic AMP pathway --- p.42 / Chapter III.4 --- Effect of altering intracellular calcium concentration --- p.42 / Anion secretion in rat epididymal cells --- p.46 / Chapter III. 5 --- Effect of cAMP on whole cell C1- current in rat epididymis --- p.46 / Chapter III.6 --- Effect of ionomycin on whole cell C1- current --- p.53 / Chapter III.7 --- Differences between cAMP- and ionophore- dependent C1- current --- p.57 / Chapter III. 8 --- The swelling-induced whole-cell currents --- p.63 / Chapter III.9 --- The swelling-induced current was mainly C1- selective --- p.65 / Chapter III. 10 --- Anion selectivity of the swelling-induced C1- current --- p.68 / Chapter III. 11 --- Inhibition of the swelling-induced C1- conductance by anion channel blockers --- p.68 / Chapter III. 12 --- The role of Ca2+ and cAMP in swelling-induced C1- conductance --- p.74 / Chapter Chapter IV. --- Discussion --- p.79 / Signal transduction mechanism of adrenaline stimulated C1- current in human epididymal cell --- p.79 / Anion secretion in rat epididymal cell The role of Ca2+ and cAMP --- p.83 / Chapter Chapter V. --- Reference --- p.88

Chan Ling Nga. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 143-153). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgements --- p.vi / Table of Contents --- p.vii / List of Figures --- p.xi / List of Tables --- p.xiv / Abbreviations --- p.xv / Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- The Human Endometrium --- p.1 / Chapter 1.1.1 --- The Structure of the Endometrium --- p.1 / Chapter 1.1.2 --- Cyclic Changes in the Endometrium --- p.1 / Chapter 1.1.3 --- Physiological Roles of the Endometrium --- p.5 / Chapter 1.1.4 --- Roles of Luminal Epithelium in Implantation --- p.5 / Chapter 1.1.5 --- Exocrine Functions of the Endometrial Epithelium --- p.6 / Chapter 1.2 --- Review of Epithelial Ion Channels --- p.8 / Chapter 1.2.1 --- Epithelial Na+ Channels (ENaC) in Absorbing Epithelia --- p.9 / Chapter 1.2.2 --- Epithelial C1- Channels in Secretory Epithelia --- p.13 / Chapter 1.2.3 --- Na+ and C1- Channels in Endometrial Epithelia --- p.15 / Chapter 1.3 --- Review of the Intracellular Signal Transduction Pathways --- p.15 / Chapter 1.3.1 --- The cAMP-Mediated Signal Transduction Pathway --- p.17 / Chapter 1.3.2 --- The cAMP-Mediated Chloride Channels in Epithelial Cells --- p.17 / Chapter 1.3.3 --- Ca2+-Dependent Signal Transduction Pathway --- p.21 / Chapter 1.4 --- Physiological Roles of some Neurohormonal Agents in Uterine Functions: Selected Examples --- p.23 / Chapter 1.4.1 --- Roles of Adrenaline on the Endometrial Ion Transport --- p.23 / Chapter 1.4.2 --- Prostaglandin (PG) E2 and PGF2α --- p.24 / Chapter 1.4.3 --- Biological Effect of Extracellular Nucleotides --- p.26 / Chapter 1.5 --- Objective of this Study --- p.28 / Chapter 2 --- Materials and Methods --- p.31 / Chapter 2.1 --- Materials --- p.31 / Chapter 2.1.1 --- Culture Media and Enzymes --- p.31 / Chapter 2.1.2 --- Drugs --- p.31 / Chapter 2.1.3 --- Chemicals --- p.32 / Chapter 2.1.4 --- Experimental Tissues and Animals --- p.32 / Chapter 2.2 --- Preparations --- p.32 / Chapter 2.2.1 --- Previous Support for Cell Growth --- p.32 / Chapter 2.2.2 --- Growth Medium --- p.33 / Chapter 2.2.3 --- Culture of Mouse Endometrial Epithelial Cells --- p.35 / Chapter 2.2.4 --- Solutions for the Short-Circuit Current Measurements --- p.36 / Chapter 2.2.5 --- Solutions for the Patch-Clamp Experiments --- p.38 / Chapter 2.2.6 --- Running Buffers for RNA and DNA Gel Electrophoresis --- p.39 / Chapter 2.2.7 --- UTP-free UDP --- p.40 / Chapter 2.2.8 --- Electrodes for the Short-Circuit Current Measurement --- p.40 / Chapter 2.3 --- Protocols --- p.41 / Chapter 2.3.1 --- Characterization of Neurohormonal Agents-induced Ion Channels --- p.41 / Chapter 2.3.2 --- Possible Interaction between CFTR and ENaC --- p.41 / Chapter 2.3.3 --- Characterization of Pyrimidinoceptors-mediated Conductances --- p.42 / Chapter 2.4 --- Methods of Measurements --- p.42 / Chapter 2.4.1 --- The Patch-Clamp Technique --- p.42 / Chapter 2.4.1.1 --- The Patch-Clamp Setup --- p.43 / Chapter 2.4.1.2 --- Shielding and Grounding --- p.45 / Chapter 2.4.1.3 --- Pipette Fabrication --- p.45 / Chapter 2.4.1.4 --- Pipette Holder and Electrodes --- p.48 / Chapter 2.4.1.5 --- Experimental Procedures --- p.49 / Chapter 2.4.1.6 --- Signal Recording and Data Acquisition --- p.54 / Chapter 2.4.1.7 --- Data Analysis --- p.54 / Chapter 2.4.2 --- The Short-Circuit Current Technique --- p.55 / Chapter 2.4.2.1 --- The Short-Circuit Current Setup --- p.56 / Chapter 2.4.2.2 --- Experimental Procedures --- p.56 / Chapter 2.4.2.3 --- Data Analysis --- p.61 / Chapter 2.4.3 --- Reverse Transciption - Polymerase Chain Reaction (RT-PCR) --- p.61 / Chapter 2.4.3.1 --- RNA Isolation --- p.61 / Chapter 2.4.3.2 --- RNA Gel Electrophoresis --- p.62 / Chapter 2.4.3.3 --- Reverse Transcription (RT) --- p.63 / Chapter 2.4.3.4 --- Polymerase Chain Reaction (PCR) --- p.64 / Chapter 2.4.3.5 --- DNA Gel Electrophoresis --- p.66 / Chapter 2.4.4 --- Statistical Analysis --- p.66 / Chapter 3 --- Results --- p.67 / Chapter 3.1 --- Activation of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) in Response to Hormonal Stimuli --- p.67 / Chapter 3.2 --- Inhibition of Na+ Absorption by CFTR --- p.89 / Chapter 3.3 --- Pyrimidinoceptors-activated Ca2+-dependent C1- Conductance --- p.111 / Chapter 4 --- General Discussions --- p.132 / Appendix --- p.140 / Chapter A --- RNA Isolation --- p.140 / Chapter B --- Reverse Transcription --- p.141 / Chapter C --- Polymerase Chain Reaction --- p.142 / References --- p.143

In eukaryotic cells, proteins continuously flux through the organelles of the secretory pathway in an essential cellular process called protein secretion. This dynamic process originates at the endoplasmic reticulum (ER), where translating ribosomes push linear peptides into the ER membrane and lumen. ER chaperones assist in folding nascent peptides into three-dimensional conformations and proteins are concentrated into membrane-encapsulated vesicles bound for the Golgi apparatus. ER to Golgi transport is mediated by a set of cytosolic coat proteins called COPII. The COPII coat polymerizes into a lattice on the ER membrane that is able to bend the membrane around secretory cargo and bud off a spherical vesicle. Protein secretion is subject to rapid changes as a cell responds to its environment and requirements for viability alter. In addition to accommodating short-term demands, such as translational up-regulation, evolved complexity of secretory proteins over time, has also required that secretory components adapt. In both cases changes in secretory demands require that the COPII proteins have an inherent flexibility to navigate these changes without disrupting secretory flux. In this work I have examined a family of quintessential secretory cargo, p24 proteins, that challenge protein secretion. This family of proteins forms a hetero-tetrameric complex that cycles between the ER and the Golgi and mediates transport of glycosylphosphatidylinositol-anchored proteins (GPI-APs). Here I present evidence that suggests, when present in vesicles, both p24 proteins and their GPI-AP cargo present a challenge to vesicle formation. I posit that three attributes of these proteins present a local barrier to membrane bending: Lumenal asymmetric distribution across the membrane, high cellular abundance and affinity for ceramide rich membranes. I have also elucidated mechanisms that the coat has evolved to accommodate troublesome cargo such as p24 proteins, which enhance structural scaffolding and increase average vesicle size. Finally I present preliminary findings indicating that p24s also contribute to ER homeostasis by preventing aberrant incorporation of proteins into vesicles. Comprehensively, these findings have shed light on the role of p24 proteins in vesicles. Traditionally thought to be canonical ER cargo receptors, these proteins also appear capable of contributing to the composition of the vesicles in which they reside, and impacting trafficking efficiency in two ways: First by directly mediating transport of GPI-APs and second by uniformly packing vesicles to avoid wasteful secretion. My work has contributed to a growing notion in the field that secretory cargo are not inert passengers but active participants in vesicle mediated secretion.

Pancreatic beta-cells regulate blood glucose by secreting insulin in response to nutrients. The development of Type 2 Diabetes (T2D) is characterized by elevated insulin secretion in the fasted state and a failure to adequately respond to nutrient influx, particularly glucose. Current dogma states that insulin resistance is the initiating event in the development of T2D, with compensation by beta-cells necessary to maintain glucose homeostasis. An alternative model, which will be a central theme throughout this thesis, is that hypersecretion of insulin is the initiating and sustaining event in the development of T2D. The underlying cause of insulin hypersecretion is unclear. Determining this is important in order to test this alternative model as a viable target for prevention and treatment of T2D. Because of the association between obesity and hyperinsulinemia, we hypothesized that exposure of the β-cell to high levels of nutrients stimulates insulin hypersecretion. We found that chronic incubation of β-cells in high glucose and/or oleate, which mimics nutrient conditions in obesity, lowered the half-maximal response for glucose to stimulate insulin secretion. The degree of the left-shift correlated with lipid stores. We determined that heightened sensitivity of granule exocytosis to Ca2+ was driving this left-shift. Thus glucose, while not necessarily abnormal in obesity, may cause hypersecretion of insulin due to altered sensitivity of the β-cell to this secretagogue. Iron stores are increased in obesity and are predictive of T2D development. We found that iron acutely stimulated both basal and glucose-stimulated insulin secretion (GSIS) in a reactive oxygen species dependent manner. Interestingly, iron did not increase insulin secretion via Ca2+ influx. Thus, both iron and glucose/oleate induce insulin hypersecretion via an aspect of the triggering pathway that is not Ca2+, the putative triggering signal. Previous work in our laboratory documented that exogenous mono-oleoyl-glycerol, an endogenous lipid signaling molecule and food additive, increases basal insulin secretion. We found that inhibition of monoacylglycerol lipase, which increases cellular monacylglycerol species, reduced GSIS, possibly via a reduction in long-chain CoA. Collectively, our works supports the hypothesis that chronic exposure to high nutrient levels drives insulin hypersecretion in obesity. / 2018-06-15T00:00:00Z

There are six known secretion systems in Gram negative bacteria, referred to as Type 1 to Type 6 respectively, which are dedicated to moving substrate across the outer membrane. Secretion systems are broadly separated into those that move their substrate across the cell envelope in a single translocation event (one-step systems), and those that are dependent on the Sec or Tat machineries for export to the periplasm (two-step systems). Serratia marcescens is an important opportunistic human pathogen and has gathered a lot of interest due to its repertoire of secreted proteins. These include the haem-scavenging protein HasA, which is secreted by a Type 1 secretion system, and the cytotoxic haemolysin ShlA, which is secreted as part of a two-partner Type 5 secretion system. Serratia marcescens also encodes a Type 6 secretion system, which is known to translocate at least six effector molecules directly into other bacterial target cells. Serratia marcescens is a model organism in terms of its ability to degrade the quite intractable polymer chitin, for which it produces three chitinase enzymes ChiA, ChiB, ChiC and a chitin-binding protein Cbp21, which hydrolyse the ß-1,4 link in the chitin chain and promote binding of chitinase to the chitin substrate respectively. These chitinolytic enzymes are utilised by S. marcescens for both basic physiology and also in pathogenesis. In this work, genetic, biochemical and proteomic approaches identified, for the first time, genes that are essential for the secretion of all three chitinases as well as Cbp21. A genetic screen identified genes encoding a holin-like membrane protein (ChiW) and a putative L-alanyl-D-glutamate endopeptidase (ChiX). Subsequent quantitative proteomics experiments and biochemical analyses established that ChiW and ChiX were required for secretion of the entire chitinolytic machinery. Chitinase secretion was observed to be blocked at a late stage in the mutant strains as normally secreted enzymes were found to accumulate in the periplasm, thus implicating ChiW and ChiX in a novel outer membrane protein translocation process. It is proposed that the bacterial genome-encoded holin-like protein and endopeptidase identified represent a putative secretion system utilised by Gram-negative bacteria. In addition to this, genes encoding the chitinolytic machinery and the putative secretion apparatus were shown to be bimodally regulated and co-ordinately expressed.

abstract: Synechocystis sp. PCC 6803 is a readily transformable cyanobacteria used to study cyanobacterial genetics, as well as production of biofuels, polyesters, and other industrial chemicals. Free fatty acids are precursors to biofuels which are used by Synechocystis cells as a means of energy storage. By genetically modifying the cyanobacteria to expel these chemicals, costs associated with retrieving the products will be reduced; concurrently, the bacteria will be able to produce the products at a higher concentration. This is achieved by adding genes encoding components of the Escherichia coli AcrAB-TolC efflux system, part of the resistance-nodulation-division (RND) transporter family, to Synechocystis sp. PCC 6803. AcrAB-TolC is a relatively promiscuous multidrug efflux pump that is noted for expelling a wide range of substrates including dyes, organic solvents, antibiotics, and free fatty acids. Adding components of the AcrAB-TolC multidrug efflux pump to a previously created high free fatty acid producing strain, SD277, allowed cells to move more free fatty acids to the extracellular environment than did the parent strain. Some of these modifications also improved tolerance to antibiotics and a dye, rhodamine 6G. To confirm the function of this exogenous efflux pump, the genes encoding components of the AcrAB-TolC efflux pump were also added to Synechocystis sp. PCC 6803 and shown to grow on a greater concentration of various antibiotics and rhodamine 6G. Various endogenous efflux systems have been elucidated, but their usefulness in expelling products currently generated in Synechocystis is limited. Most of the elucidated pumps in the cyanobacteria are part of the ATP-binding cassette superfamily. The knowledge of the resistance-nodulation-division (RND) family transporters is limited. Two genes in Synechocystis sp. PCC 6803, slr2131 and sll0180 encoding homologs to the genes that encode acrB and acrA, respectively, were removed and the modifications resulted in changes in resistance to various antibiotics and a dye and also had an impact on free fatty acid secretion. Both of these deletions were complemented independently with the homologous E. coli gene and the resulting cyanobacteria strains had some of the inherent resistance restored to chloramphenicol and free fatty acid secretion was modified when compared to the wild-type and a high free fatty acid producing strain. / Dissertation/Thesis / Doctoral Dissertation Microbiology 2018

A rota de biossíntese de colesterol tem sido estudada pelo seu envolvimento com a secreção de hormônios. A secreção de insulina depende do colesterol e de intermediários da sua biossíntese como o geranilgeranil pirofosfato. Para compreender a contribuição destes elementos na secreção de insulina foram utilizados inibidores de diferentes pontos da rota de biossíntese do colesterol. Estudou-se a contribuição do geranilgeranil pirofosfato e do colesterol na secreção de insulina estimulada pela glicose e analisados parâmetros físicos de membrana dos quais decorrem aspectos funcionais da célula beta. Sinvastatina inibiu a secreção de insulina sem afetar o conteúdo celular de colesterol, mas inibindo a geranilgeranilação de proteínas. A diminuição do conteúdo de colesterol aumenta a fluidez de membrana desorganizando lipid rafts. A suplementação com colesterol recupera a função secretória. Por tanto, a inibição da síntese de colesterol diminui a secreção de insulina através da redução da isoprenilação de proteínas e da alteração estrutural de membranas celulares. / Cholesterol biosynthesis pathway has been studied for its involvement with the hormone secretion process. Insulin secretion depends on cholesterol and its biosynthesis intermediates such as geranylgeranyl pyrophosphate. For understand their contribution on the glucose stimulated insulin secretion, cholesterol biosynthesis pathway key steps inhibitors were used. The geranylgeranyl pyrophosphate and cholesterol contribution to the insulin secretion was studied so as physical parameters of the membrane, from where derive key functions of it. Simvastatin inhibited insulin secretion without affecting total cellular cholesterol, but inhibiting protein geranylgeranylation. The cholesterol reduction increased the membrane fluidity with consequent disorganization of membrane lipid rafts, in which secretion machinery is organized. Cholesterol supplementation recovers cellular secretory function. We conclude that cholesterol biosynthesis inhibition reduces insulin secretion through protein isoprenylation impairment and structural disarrangement of the cellular membranes.