published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

published_or_final_version / Zoology / Doctoral / Doctor of Philosophy

published_or_final_version / Biological Sciences / Master / Master of Philosophy

G-protein–coupled receptors are the largest superfamily in the human genome, and involved in critical cellular signaling processes in living cells. Protein structural fluctuations are the key for GPCR function that is driven and modulated by a variety of factors that are not well understood. This dissertation focusses on understanding the activation of GPCRs using the visual receptor, rhodopsin as the prototype. Rhodopsin is an ideal candidate for this study, as it represents the largest class of GPCRs, and is known to demonstrate more noticeable structural changes upon activation compared to the other GPCRs. What structural fluctuations occur, the role of water, and how the retinal cofactor regulates the protein dynamics during rhodopsin activation are specific research problems addressed in this work. Hypothesizing an ensemble activation mechanism, experiments were conducted using a variety of techniques to probe structural and dynamical fluctuations of rhodopsin in native membranes, as well as in membrane mimetics such as detergent micelles. Time-resolved wide-angle X-ray scattering (TR-WAXS), small-angle neutron scattering (SANS), quasielastic neutron scattering (QENS), and electronic spectroscopy are among the prominent techniques used to gain insights into the photo-intermediates that are key to understanding the rhodopsin activation process. The small-angle neutron scattering (SANS) experiments revealed a volumetric expansion of the protein molecule upon photoactivation of rhodopsin. Electronic spectroscopy together with the differential hydration study revealed the crucial role of water in rhodopsin signaling process and signal amplification by water. The quasielastic neutron scattering study conducted on powdered rhodopsin probed the changes in the local dynamics that are regulated by the retinal cofactor of the rhodopsin molecule. The increased local steric crowding in the ligand-free opsin is consistent with collapsing of the apoprotein structure in the absence of the retinal chromophore leading to inactive opsin conformation. Finally, a time-resolved wide-angle X-ray scattering study was conducted using the X-ray free electron laser at the SLAC national laboratory to probe the early structural fluctuations in rhodopsin photoactivation. The preliminary pump-probe experiments conducted on rhodopsin in CHAPS detergent micelles revealed a light-triggered protein quake that occurs during the early activation stages of rhodopsin photoactivation. Thus the protein fluctuations underlying the GPCR function are revealed by neutrons, X-rays, and other photons in a combined implementation of both spectroscopic and scattering techniques as applied to the investigation of rhodopsin activation.

The cell envelope of Aggregatibacter actinomycetemcomitans, a Gram-negative pathogenic bacterium implicated in human oral and systemic disease, plays a critical role in maintenance of cellular homeostasis, resistance to external stress, and host'pathogen interactions. Our laboratory has identified a novel gene product, morphogenesis protein C (MorC), deletion of which leads to multiple pleotropic effects pertaining to membrane structure and function. The MorC sequence was determined to be conserved in Gammaproteobacteria. Based on this bioinformatic analysis, the functional conservation of this protein was investigated utilizing an A. actinomycetemcomitans morC mutant as a model system to express homologs from four phylogenetically diverse representatives of the Gammaproteobacteria: Haemophilus influenzae, Escherichia coli, Pseudomonas aeruginosa, and Moraxella catarrhalis. MorC from all organisms restored at least one of the A. actinomycetemcomitans mutant phenotypes, implying that the protein is functionally conserved across Gammaproteobacteria. Further, deletion mutagenesis indicated that the last 10 amino acids of the carboxyl terminus were necessary to maintain the integrity of the membrane. The observed pleiotropic effects suggested alterations in the membrane protein composition of the morC mutant. Stable isotope dimethyl labeling in conjunction with mass spectrometry was employed to quantitatively determine the differences in the abundance of membrane proteins of the isogenic mutant and wild-type strains. A total of 665 envelope associated proteins were identified and functionally annotated using bioinformatic tools. All proteins, except MorC, were detected in the mutant strain. However, 12 proteins were found in lesser (10) or greater (2) abundance in the membrane preparation of the mutant strain. These proteins were ascribed functions associated with protein quality control, oxidative stress response, and protein secretion systems. One protein found to be reduced was a component of the fimbrial secretion system of A. actinomycetemcomitans. The significance of this finding was unclear due to the afimbriated nature of the laboratory strain used in the study. Therefore, the defect in fimbriation was identified and complemented in trans. The transformed strain displayed all of the hallmarks of a naturally fimbriated strain including: a distinct star-like colony morphology; robust biofilm formation; and presence of fimbriae as detected by electron microscopy. The isogenic morC mutant strain transformed with an identical plasmid did not display any fimbriated phenotypes. The role of MorC in fimbriae production of a naturally fimbriated strain was investigated by inactivation of morC in a clinical isolate. The mutant strain displayed phenotypes typically associated with inactivation of morC. However, fimbriae were still observed on the surface, although in lesser amounts on some individual bacteria, and this strain formed a biofilm with volume similar to the parent. Interestingly, significant changes in microcolony architecture of the biofilm were observed by confocal microscopy. MorC plays a critical role in maintaining secretion of major virulence determinants of A. actinomycetemcomitans. Specific changes in the protein composition of the cell envelope indicate a direct or compensatory role of these proteins in maintaining membrane physiology. The functional conservation of MorC also implies an important role for this protein in other Gram-negative bacteria. This work suggests a role of MorC as an accessory or a scaffold protein involved in secretion.

Signal transduction is critical during the lifetime of a neuron as it navigates to reach its targets, forms functional synaptic connections and adjusts the molecular architecture of these connections in an activity-dependent manner. Understanding the molecular organisation of components required for neuronal signalling will provide novel biological insight and can contribute to the design of therapeutics for neurodevelopmental and neurodegenerative disorders. The focus of the thesis is on determining mechanistic molecular details of a number of distinct cell surface systems implicated in neuronal signalling. Crystallographic studies on the cell surface complex between Eph receptor A4 and ephrinA5 contributed to understanding how the modes of higher order arrangements of receptors involved in guidance affect signal transduction across the membrane. A set of structural and biophysical studies addressed the proteoglycan regulation of RPTPσ-TrkCtrans-synaptic interaction and contributed to deciphering the principles of the switch from axonal growth to synapse establishment and formation. A crystallographic and biochemical analysis of the neuronal C1q-like family, enabled mapping their interactions with potential synaptic partners, and guided functional studies aimed at elucidating their roles in the maintenance of synaptic integrity. Preliminary work on the neuronal Sigma-1 receptor chaperone laid the foundations for the structural determination of this receptor.

Foram estudadas as proteínas da membrana eritrocitária de quarenta e seis espécies diferentes pertencentes à treze ordens da classe Mammalia, através de eletroforese em gel de poliacrilamida. Observamos que: 1. não parece haver correlação entre as concentrações relativas de espectrinas e o volume corpuscular médio. 2. não parece haver correlação entre as concentrações relativas de banda 3 e o volume corpuscular médio. 3. parece haver uma correlação inversa entre as concentrações relativas das anquirinas e o volume corpuscular médio. Assim, a maior parte dos animais pertencentes à ordem dos Artiodacty/a estudados (girafa, cervo nobre, veado campeiro, veado catingueiro, carneiro, cabra), que apresentam volume corpuscular baixo, exibe aumento significativo da concentração das anquirinas. 4. observou-se, o aparecimento de uma banda difusa na região 4.5 no Didelphis marsupialis (gambá), Arctocephalus tropicalis e Arctocephalus australis (lobo marinho subantártico e lobo marinho das ilhas do sul) e Panthera leo (leão) quando os estromas são submetidos à ação do NEM na concentração de 200 µM, e o não encontro desta banda nas demais espécies estudadas. Este fato poderia advir da hipótese de haver, nas três espécies, sequências de aminoácidos comuns que possibilitariam a ação química direta do NEM. 5. observou-se que, nos mamíferos estudados, embora houvesse variações qualitativas e quantitativas das proteínas da membrana nas espécies estudadas, práticamente todas elas apresentaram as proteínas descritas no Homo sapiens, mostrando serem todas elas importantes na manutenção do citoesqueleto e da membrana como um todo. 6. Com exceção da cutia, que exibe provavelmente quatro formas de espectrinas, todas as demais espécies exibiram duas formas, embora com variações qualitativas e quantitativas. 7. A banda 3 foi a proteína que mais exibiu variação, seja no peso molecular, com variação desde 95 kDa (Capra hircus, cabra) até 169 kDa (Alouatta sp, bugio), seja no aspecto difuso ou bem definido. 8. Houve na região 4.1/4.2 uma variabilidade grande, seja qualitativa, seja quantitativa com aparentes ausências dê\' uma ou de outra. Alguns roedores, a cobaia, cutia, hamster exibiram somente uma banda protéica na região 4.1/4.2. 9. Entre os primatas, todas as proteínas apresentaram o mesmo padrão entre os Hominoidea (macacos antropóides, Gorilla gorilla e Pongo pygmaeus e homem, Homo sapiens) e os do Velho Mundo (Cercopithecoidea, Papio cynocephalus e Erythrocebus pata). Mas, os primatas do Novo Mundo (Ceboidea, Cebus apella, Alouatta sp e AteIes paniscus chamek) apresentaram variação do peso molecular da banda 3, apresentando no entanto o mesmo aspecto difuso dos outros primatas descritos anteriormente. 10. Com exceção do Didelphis marsupialis (gambá) e Giraffa came/opardalis (girafa), que apresentaram evidência da ação de serina-proteases sobre uma anquirina, as demais espécies não exibiram proteases intra-eritrocitárias ativas nas condições estudadas. / Forty six different mammal species from thirteen regarding their erythrocyte membrane proteins, following data were observed: 1. No correlation was observed between mean corpuscular volume and spectrins concentration. 2. No correlation was observed between mean corpuscular volume and band 3 concentration. 3. It seems that there is inverse correlation between mean corpuscular volume and ankyrins concentration. Most of the studied animmals belonging to Artiodactyla, which exhibit low mean corpuscular volume, present higher ankyrin concentration. 4. A diffuse band in 4.5 region was observed among the Didelphis marsupialis, Arctocephalus tropicalis, Arctocephalus australis and Panthera leo when the ghosts were treated with 200 µM NEM, what was not found in the others species. This fact could be ascribed to hypothetical sequence of the Iysine, hystidine and cysteine which may be prone to NEM direct chemical action. 5. Although some qualitative and quantitative changes were observed among ali the studied species, ali of them seem to occur in the humans, disclosing that they are \"house-keeping\" proteins which participate as important pieces in the cytoskeleton structure and the membrane as well. 6. Ali the species presented two spectrins, with exception of Dasyprocta sp., which exhibited fours bands in the spectrins region. 7. The band 3 was the protein which showed the greatest variation, since 95 kDa in the Capra hircus up to 169 kDa in the Alouatta sp., as well as in the aspect, diffuse or well defined. 8. A great variability was observed in the 4.1/4.2 region, with apparent absence of one or another. Some Rodentia exhibited only one protein in the 4.1/4.2 region. 9. Among the Primates, the Hominoidea and the Cercopithecoidea presented an uniform pattern, but the New World monkeys (Cebus apella, Alouatta sp. and Ateies paniscus chamek) exhibited a striking molecular weight band 3 variation, although keeping the common diffuse pattern. 11. Ali the studied species did not present the evidence of protease action upon the membrane proteins, except the Didelphis marsupialis and Giraffa camelopardalis, which disclosed a proteolytic action upon the ankyrin.

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.

Ellen Lai Ping Chan. / Thesis (Ph.D)--Chinese University of Hong Kong, 1990. / Bibliography: leaves 238-272. / Chapter CHAPTER I --- LITERATURE REVIEW / Chapter 1. --- History --- p.1 / Chapter 2. --- Species Distribution of BAT --- p.3 / Chapter 3. --- Distribution of BAT --- p.4 / Chapter 4. --- Structure of BAT --- p.4 / Chapter 4.1. --- Macroscopic Appearance --- p.4 / Chapter 4.1.1. --- Innervation --- p.4 / Chapter 4.1.2. --- Blood supply --- p.5 / Chapter 4.2. --- Microscopic Structure of BAT --- p.6 / Chapter 4.3. --- Difference Between Brown Fat and White Fat --- p.9 / Chapter 5. --- Composition of BAT --- p.11 / Chapter 6. --- The Mechanisms of Brown Adipose Tissue Thermogenesis --- p.12 / Chapter 6.1. --- Factors Influencing Proton Transport by UCP --- p.16 / Chapter 6.2. --- Postulated Sequence of Events during Thermogenesis --- p.18 / Chapter 7. --- Measurements of thermogenic Capacity of BAT --- p.21 / Chapter 8. --- Age-related Differences in BAT --- p.28 / Chapter 9. --- Non-shivering Thermogenesis and BAT --- p.32 / Chapter 9.1. --- Changes In BAT During Cold Acclimation --- p.35 / Chapter 9.1.1. --- GDP Binding --- p.35 / Chapter 9.1.2. --- Concentration of UCP --- p.37 / Chapter 9.1.3. --- Metabolic changes in BAT during Cold Acclimation --- p.39 / Chapter 10. --- Diet-induced Thermogenesis and BAT --- p.41 / Chapter 10.1. --- Mechanism of DIT --- p.42 / Chapter 10.2. --- Controversies in DIT --- p.44 / Chapter 10.3. --- Nutritional Factors Inducing DIT --- p.46 / Chapter 10.4. --- DIT in Man --- p.47 / Chapter 10.5. --- Neuroendocrine Control of BAT in DIT --- p.48 / Chapter 10.6. --- Effects of Fasting in BAT --- p.51 / Chapter 11. --- Obesity and BAT --- p.53 / Chapter 11.1. --- NST and DIT in Obese Animals --- p.58 / Chapter 11.2. --- Regulation of BAT in Obese Animals --- p.59 / Chapter 11.2.1. --- Sympathetic Nervous System in Obese Animals --- p.59 / Chapter 11.2.2. --- Corticosteriods in Obese Animals --- p.61 / Chapter 11.2.3. --- Adrenergic Receptors in Obese Animals --- p.63 / Chapter 11.2.4. --- Insulin in Obese Animals --- p.64 / Chapter 12. --- Pregnancy and Lactation and BAT --- p.67 / Chapter 12.1. --- Energy Balance During Pregnancy and Lactation --- p.67 / Chapter 12.2. --- Some Metabolic Changes During X Pregnancy and Lactation --- p.68 / Chapter 12.3. --- Role of BAT in Pregnancy and Lactation --- p.70 / Chapter 12.4. --- Mechanism of Regulation of Thermogenesis during Pregnancy and Lactation --- p.71 / Chapter 13. --- Factors Controlling the Thermogenesis --- p.75 / Chapter 13.1. --- Sympathetic Nervous Control --- p.75 / Chapter 13.1.1. --- Studies of Administration of Noradrenaline --- p.75 / Chapter 13.1.2. --- Control of the Fuel Supply to BAT by Sympathetic Nervous System --- p.77 / Chapter 13.1.3. --- Sympathetic denervation --- p.78 / Chapter 13.2. --- Hormonal Control --- p.79 / Chapter 13.2.1. --- Thyroid Hormone --- p.79 / Chapter 13.2.2. --- Insulin --- p.81 / Chapter 13.2.3. --- Pituitary Hormones --- p.83 / Chapter 13.2.4. --- Glucocorticoids --- p.83 / Chapter 13.2.5. --- Corticotropin-Releasing Factor --- p.85 / Chapter 14. --- Aims of the Study --- p.87 / Chapter CHAPTER II --- ISOLATION AND PURIFICATION OF UCP AND DEVELOPMENT OF AN ENZYME LINKED IMMUNOSORBENT ASSAY FOR UCP / Chapter 1. --- INTRODUCTION --- p.88 / Chapter 2. --- MATERIALS AND METHODS --- p.89 / Chapter 2.1. --- Animals --- p.89 / Chapter 2.2. --- Collection of BAT --- p.89 / Chapter 2.3. --- Isolation of Mitochondria --- p.90 / Chapter 2.4. --- Electron Microscopy (EM) of Isolated BAT Mitochondria --- p.92 / Chapter 2.5. --- Measurement of Protein and Cytochrome C Oxidase Activity --- p.94 / Chapter 2.5.1. --- Measurement of Protein Concentration --- p.94 / Chapter 2.5.2. --- Measurement of Cytochrome C Oxidase Activity --- p.99 / Chapter 2.6. --- GDP Binding Assay of BAT Mitochondria --- p.101 / Chapter 2.6.1. --- GDP Binding Assay of Mitochondria by Centrifugation Method --- p.103 / Chapter 2.6.2. --- GDP: Binding Activity by Equilibrium Dialysis --- p.106 / Chapter 2.6.3. --- GDP Binding by Microfiltration Method --- p.108 / Chapter 2.7. --- Experiments Designed for Validation of GDP Binding Assay --- p.109 / Chapter 2.7.1. --- GDP Binding Activity in BAT Mitochondria after Noradrenaline Treatment --- p.109 / Chapter 2.7.2. --- GDP Binding Activity in BAT Mitochondria after Cold Acclimation and Noradrenaline Treatment --- p.110 / Chapter 2.7.3. --- Effect of Food Restriction on Cold Acclimated Rats --- p.110 / Chapter 2.7.4. --- GDP Binding Activity of BAT Mitochondria of Rats of Different Ages --- p.111 / Chapter 2.8. --- Isolation and Purification of UCP --- p.111 / Chapter 2.9. --- Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.115 / Chapter 2.10. --- Methods for Raising Anti-Rat-UCP Antibody and the Characterization of Antiserum --- p.120 / Chapter 2.10.1. --- Raising Rabbit Anti-Rat-UCP Antibody --- p.120 / Chapter 2.10.2. --- Western Blot Analysis For Cross Reactivity Study --- p.120 / Chapter 2.10.3. --- Immuno-Autoradiographic Method for Detection of Specificity of Rabbit Anti-Rat UCP Antiserum --- p.121 / Chapter 2.11. --- Enzyme Linked Immunosorbent Assay For UCP --- p.124 / Chapter 2.12. --- Experiment Designed to Validate the ELISA --- p.129 / Chapter 2.13. --- Statistical Analysis --- p.129 / Chapter 3. --- RESULTS --- p.130 / Chapter 3.1. --- Electron Microscopy of Isolated BAT Mitochondria --- p.130 / Chapter 3.2. --- GDP Binding Assay of BAT Mitochondria --- p.130 / Chapter 3.3. --- Experiments Designed for Validation of GDP Binding Assay --- p.133 / Chapter 3.3.1. --- GDP Binding Activity of BAT Mitochondria after Noradrenaline Injection --- p.133 / Chapter 3.3.2. --- GDP Binding Activity of BAT Mitochondria after Cold Acclimation and Noradrenaline Treatment --- p.136 / Chapter 3.3.3. --- Effects of Food Restriction on Cold Acclimated Rats --- p.136 / Chapter 3.3.4. --- GDP Binding Activity of BAT Mitochondria from Rats of Different Ages --- p.140 / Chapter 3.4. --- Isolation and Purification of UCP --- p.140 / Chapter 3.4.1. --- Results of SDS-PAGE --- p.143 / Chapter 3.4.2. --- Results of GDP Binding Activity --- p.149 / Chapter 3.5. --- Rabbit Anti-rat-UCP Antibody and the Characterization of Antiserum --- p.151 / Chapter 3.5.1. --- Immuno-autoradiography for Specificity of Rabbit Anti-rat-UCP Antiserum --- p.153 / Chapter 3.5.1.1. --- Cross-reactivity of the Rabbit Anti-rat-UCP Antiserum to Mitochondrial Proteins of BAT and from other Tissues --- p.153 / Chapter 3.5.1.2. --- Cross-reactivity of the Rabbit Anti-rat-UCP Antiserum to BAT Mitochondrial Protein from Different Rodent Species --- p.156 / Chapter 3.5.1.3. --- Dose Response of Rabbit Anti-rat-UCP Antiserum to UCP --- p.159 / Chapter 3.6. --- ELISA of UCP --- p.161 / Chapter 3.6.1. --- Determination of Maximum Amount of UCP Binding on Microtitre Plate --- p.161 / Chapter 3.6.2. --- Antibody Dilution Curve --- p.161 / Chapter 3.6.3. --- Incubation Time for Enzyme-Substrate Reaction --- p.163 / Chapter 3.6.4. --- Competitive ELISA --- p.163 / Chapter 3.6.5. --- Precision of ELISA --- p.167 / Chapter 3.7. --- Experiment Designed for Validation of ELISA by Measuring UCP in Cold Acclimated Rats --- p.170 / Chapter 4. --- DISCUSSION --- p.172 / Chapter 4.1. --- GDP Binding Assay of BAT Mitochondria --- p.172 / Chapter 4.2. --- Isolation and Purification of UCP --- p.176 / Chapter 4.3. --- Development and Evaluation of ELISA --- p.178 / Chapter CHAPTER III --- CHANGES IN BAT DURING PREGNANCY AND LACTATION AND ROLE OF PROLACTIN / Chapter 1. --- INTRODUCTION --- p.184 / Chapter 2. --- MATERIALS AND METHODS --- p.187 / Chapter 2.1. --- Animal --- p.187 / Chapter 2.2. --- Experimental Designs --- p.187 / Chapter 2.2.1. --- "Effects of Pregnancy, Lactation and Post Weaning on BAT" --- p.187 / Chapter 2.2.2. --- Effect of Metoclopramide on BAT --- p.188 / Chapter 2.2.3. --- Effect of Metoclopramide and Bromocriptine on BAT --- p.188 / Chapter 2.2.4. --- Effect of PRL Injection on BAT --- p.189 / Chapter 2.2.5. --- Continuous infusion of PRL --- p.189 / Chapter 2.6.6. --- Measurements of BAT Parameters --- p.191 / Chapter 2.2.7. --- RIA of serrum PRL --- p.191 / Chapter 2.2.8. --- PRL Receptors in BAT --- p.197 / Chapter 2.4. --- Statistical Analysis --- p.201 / Chapter 3. --- RESULTS --- p.202 / Chapter 3.1. --- Effects of Pregnancy and Lactation --- p.202 / Chapter 3.1.1. --- Food Consumption and Body Weight --- p.202 / Chapter 3.1.2. --- BAT --- p.205 / Chapter 3.1.3. --- Serum PRL level --- p.209 / Chapter 3.2. --- Effects of PRL njection --- p.213 / Chapter 3.3. --- Effects of Continuous Infusion of PRL on BAT --- p.213 / Chapter 3.4. --- Effects of Metoclopramide on BAT --- p.216 / Chapter 3.5. --- Effects of Bromocriptine and Metoclopramide on BAT --- p.216 / Chapter 3.6. --- PRL Receptor in BAT --- p.219 / Chapter 4. --- DISCUSSION --- p.223 / GENERAL CONCLUSION --- p.236