A thimet oligopeptidase (EP24.15) foi originariamente descrita como uma enzima metabolizadora de neuropeptídeos que não possui um peptídeo sinal para entrada na via secretória clássica, mas é secretada pelas células através de um mecanismo não-convencional. Nesse trabalho, identificamos uma nova interação cálcio-dependente entre EP24.15 e calmodulina I (CaM), que é importante para a secreção estimulada, mas não constitutiva, da EP24.15. A superexpressão da CaM em células HEK293 aumenta a secreção estimulada da EP24.15, podendo ser inibida pelo inibidor da CaM. O inibidor específico da PKA reduz a secreção estimulada de EP24.15. Nossos dados sugerem que a interação entre EP24.15 e calmodulina é regulada e relevante para a secreção estimulada da EP24.15 em células HEK293. Surpreendentemente, experimentos com slices (fatias) de cérebros de ratos sugerem que, fisiologicamente, a EP24.15 é secretada predominantemente de forma constitutiva, embora o tratamento com A23187 e forskolin sejam capazes de aumentar modestamente a secreção dessa enzima nessas preparações. / Thimet oligopeptidase (EC3.4.24.15; EP24.15) was originally described as a neuropeptide-metabolising enzyme that lacks a typical signal-peptide sequence for entry into the secretory pathway and is secreted by cells via an unconventional and unknown mechanism. Here, we identify a novel calcium-dependent interaction between EP24.15 and calmodulin I (CaM) that is important for the stimulated, but not constitutive, secretion of EP24.15. Overexpression of CaM in HEK293 cells increase the stimulated secretion of EP24.15, which can be inhibited by the CaM inhibitor. The specific inhibition of PKA with reduced the A23187-stimulated secretion of EP24.15. Our data suggest that the interaction between EP24.15 and calmodulin is regulated within cells and is important for the stimulated secretion of EP24.15 from HEK293 cells. Surprising, the rats brain slices experiments showed that, physiological, EP24.15 has a constitutive secretion, although the A23187 and forskolin treatment are able to increase a little this enzyme secretion in these preparations.

by Psyche, Sui-Ki Fong. / Thesis submitted in: December 1997. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 129-135). / Abstract also in Chinese. / Abstract in Chinese --- p.1 / Abstract in English --- p.2 / Chapter Chapter I. --- Introduction --- p.4 / Chapter I.1. --- Structure and functions of the uterus --- p.4 / Chapter I.1.1. --- Uterus: Gross structure and functions --- p.4 / Chapter I.1.2. --- Uterine wall: functional layers --- p.6 / Chapter a. --- Myometrium --- p.6 / Chapter b. --- Endometrium --- p.6 / Chapter I.1.3. --- Uterine functions : regulatory systems --- p.10 / Chapter a. --- Nervous regulation --- p.10 / Chapter b. --- Hormonal regulation --- p.11 / Chapter I.1.4. --- Uterotrophic agents : selected examples --- p.12 / Chapter a. --- Adrenaline and noradrenaline --- p.12 / Chapter b. --- Prostaglandin E2 and F2α --- p.15 / Chapter I.2. --- Endometrial epithelium and uterine fluid composition --- p.19 / Chapter I.2.1. --- Uterine fluid composition --- p.19 / Chapter I.2.2. --- Regulation of uterine fluid volume and composition --- p.19 / Chapter I.2.3. --- Role of endometrial epithelium in the regulation of uterine fluid composition --- p.20 / Chapter I.3. --- Pioneering works on ion transport across the endometrium --- p.21 / Chapter I.4. --- Objectives of study --- p.23 / Chapter Chapter II --- Materials and Methods --- p.24 / Chapter II.1. --- Materials --- p.24 / Chapter II.1.1. --- Culture media and enzyme --- p.24 / Chapter II 1.2. --- Drugs --- p.24 / Chapter II 1.3. --- Chemicals --- p.25 / Chapter II 1.4. --- Animals --- p.25 / Chapter II.2. --- Methods --- p.25 / Chapter II.2.1. --- Preparation of permeable support for cell culture --- p.25 / Chapter II.2.2. --- Preparation of culture medium for cell culture --- p.26 / Chapter II.2.3. --- Cell isolation and culture --- p.28 / Chapter II.2.4. --- Preparation of electrodes --- p.29 / Chapter II.2.5. --- Preparation of solutions --- p.29 / Chapter II.2.6. --- The short-circuit current technique --- p.31 / Chapter a. --- Experimental setup --- p.32 / Chapter b. --- Transepithelial conductance and resistance measurements --- p.37 / Chapter c. --- Experimental procedure --- p.37 / Chapter II.2.7. --- Statistics --- p.38 / Chapter Chapter III. --- Results --- p.39 / Chapter III.1. --- Electrogenic ion transport across the cultured mouse endometrial epithelium --- p.39 / Chapter III.2. --- Stimulation of anion secretion by β-adrenoceptors --- p.54 / Chapter III.3. --- Regulation of anion secretion by prostaglandin E2 --- p.78 / Chapter III.4. --- Cellular mechanisms of adrenaline-stimulated anion secretion --- p.98 / Chapter Chapter IV. --- General Discussion --- p.123 / Chapter Chapter V. --- Reference --- p.129

Tsang Po Ting. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 135-152). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / Abstract 摘要 --- p.iv / List of Figures --- p.vi / List of Tables --- p.viii / List of Abbreviations --- p.ix / Table of Contents --- p.xii / Chapter Chapter 1: --- General Introduction --- p.1 / Chapter 1.1 --- The Role of Glucose in Biological System --- p.1 / Chapter 1.2 --- Glucose Transporter Families --- p.1 / Chapter 1.2.1 --- Na+-Dependent Glucose Transporters --- p.2 / Chapter 1.2.2 --- Facilitative Glucose Transporters --- p.3 / Chapter 1.3 --- Glucose Transporter Type1 --- p.7 / Chapter 1.3.1 --- Primary Structure of the Glutl Protein --- p.7 / Chapter 1.3.2 --- Secondary Structure --- p.8 / Chapter 1.3.3 --- Tertiary Structure --- p.8 / Chapter 1.3.4 --- Kinetics Properties --- p.11 / Chapter 1.3.5 --- Tissue Distribution --- p.12 / Chapter 1.3.6 --- Multifunctional Property --- p.13 / Chapter 1.3.7 --- Characterization of GLUT1 Gene --- p.13 / Chapter 1.3.8 --- Regulation of GLUT1 Expression --- p.14 / Chapter 1.4 --- Glucose Transporter Type 1 and the Brain --- p.16 / Chapter 1.5 --- Glucose Transporter Type 1 Deficiency Syndrome (GIutlDS) --- p.19 / Chapter 1.5.1 --- Backgronnd of GIutlDS --- p.19 / Chapter 1.5.2 --- Clinical Features of GIutlDS --- p.23 / Chapter 1.5.3 --- Genotype-Phenotype Correlations --- p.24 / Chapter 1.5.4 --- Diagnosis --- p.26 / Chapter 1.5.5 --- Manage nent --- p.27 / Chapter 1.5.5.1 --- Ketogenic Diet --- p.27 / Chapter 1.6 --- Hypothesis and Objectives --- p.29 / Chapter Chapter 2: --- Biochemical and Molecular Analysis of GLUT1 in a Suspected GlutlDS Case --- p.31 / Chapter 2.1 --- Materials --- p.32 / Chapter 2.1.1 --- Clinical History of Suspected GlutlDS Patient --- p.32 / Chapter 2.1.2 --- Blood Samples --- p.32 / Chapter 2.1.3 --- Reagents and Buffers for Reverse Transcription --- p.32 / Chapter 2.1.4 --- Reagents and Buffers for TA Cloning --- p.34 / Chapter 2.1.5 --- Reagents for Genomic DNA Extraction --- p.34 / Chapter 2.1.6 --- Reagents and Buffers for Polymerase Chain Reaction (PCR) --- p.34 / Chapter 2.1.7 --- Reagents and Buffers for Agarose Gel Electrophoresis --- p.35 / Chapter 2.1.8 --- Reagents for Zero-trans 3-OMG Influx in Erythrocytes --- p.37 / Chapter 2.1.9 --- Reagents for Zero-trans 3-OMG Efflux from Erythrocytes --- p.38 / Chapter 2.1.10 --- Reagents for Erythrocytes Membrane Extraction and Detection --- p.39 / Chapter 2.2 --- Methods --- p.44 / Chapter 2.2.1 --- GLUT1 Gene Analysis --- p.44 / Chapter 2.2.2 --- Zero-trans 3-OMG Influx into Erythrocytes --- p.51 / Chapter 2.2.3 --- Zero-trans 3-OMG Efflux from Erythrocytes --- p.52 / Chapter 2.2.4 --- Glutl Protein Expression --- p.54 / Chapter 2.2.5 --- Statistics --- p.57 / Chapter 2.3 --- Results --- p.58 / Chapter 2.3.1 --- Molecular Analysis of the GLUT1 Gene of a Suspected GlutlDS Patient --- p.58 / Chapter 2.3.2 --- Functional Analysis of the GlutlDS Patient's Glutl Protein --- p.61 / Chapter 2.3.3 --- Glutl Protein Expression in the GlutlDS Patient --- p.64 / Chapter 2.4 --- Discussion --- p.66 / Chapter Chapter 3: --- Pathogenicity Studies of GLUT1 Mutations --- p.71 / Chapter 3.1 --- Materials --- p.72 / Chapter 3.1.1 --- Construction of Glutl-Encoding Vectors --- p.72 / Chapter 3.1.2 --- Cell Lire --- p.73 / Chapter 3.1.3 --- "Cell Culture Media, Buffers and Other Reagents" --- p.73 / Chapter 3.1.4 --- Cell Culture Wares --- p.75 / Chapter 3.1.5 --- Reagents for Transfection --- p.75 / Chapter 3.1.6 --- Reagents for Protein Determination and Western Blot Analysis --- p.76 / Chapter 3.1.7 --- Consumables for Confocal Microscopy --- p.77 / Chapter 3.1.8 --- Reagents and Buffers for Flow Cytometry --- p.77 / Chapter 3.1.9 --- Reagents for 2-DOG Uptake in CHO-K1 Cells --- p.77 / Chapter 3.2 --- Methods --- p.79 / Chapter 3.2.1 --- Cell Culture Methodology --- p.79 / Chapter 3.2.2 --- Construction of GLUT1 Mutants --- p.80 / Chapter 3.2.3 --- Establishment of Wild Type and Mutant Glutl Expressing Cell Lines --- p.84 / Chapter 3.2.4 --- Protein Expression Study --- p.85 / Chapter 3.2.5 --- 2-DOG Influx Assay in CHO-K1 Cells --- p.87 / Chapter 3.2.6 --- Confocal Microscopy Studies on Glutl Cellular Localization --- p.89 / Chapter 3.2.7 --- Statistics --- p.90 / Chapter 3.3 --- Results --- p.91 / Chapter 3.3.1 --- Molecular Analysis of 1034-1035Insl2 Mutation --- p.91 / Chapter 3.3.2 --- Expression of the Wild Type and Mutant GFP-Glutl Fusion Proteins --- p.92 / Chapter 3.3.3 --- Functional Analysis of the 1034-1035Insl2 Mutant --- p.95 / Chapter 3.4 --- Discussion --- p.97 / Chapter Chapter 4: --- GLUT1 Promoter Study --- p.100 / Chapter 4.1 --- Materials --- p.101 / Chapter 4.1.1 --- Construction of GLUT1 Promoter Vectors --- p.101 / Chapter 4.1.2 --- Cell Lines --- p.102 / Chapter 4.1.3 --- Cell Culture Media and Other Reagents --- p.103 / Chapter 4.1.4 --- Dual Luciferase Reporter Assay System --- p.103 / Chapter 4.2 --- Methods --- p.105 / Chapter 4.2.1 --- Bioinformatics --- p.105 / Chapter 4.2.2 --- Cell Culture --- p.105 / Chapter 4.2.3 --- Construetion of GLUT1 Promoter Vectors --- p.105 / Chapter 4.2.4 --- 5'-Deletion Analysis of GLUT1 Promoter --- p.108 / Chapter 4.2.5 --- Determination of the Activities of GLUT1 Promoter Fragments --- p.110 / Chapter 4.2.6 --- Statistics --- p.113 / Chapter 4.3 --- Results --- p.114 / Chapter 4.3.1 --- Determination of the Promoter Activity of the 5'-deletion Fragments --- p.114 / Chapter 4.3.2 --- Prediction of Transcription Factors in the 5'-deletion Fragments --- p.119 / Chapter 4.4 --- Discussion --- p.121 / Chapter Chapter 5: --- General Conclusion and Future Perspectives --- p.133 / References --- p.135

The chondrocyte is the only cell type in articular cartilage, and its role is to maintain cartilage integrity by synthesizing and releasing macromolecules into the extracellular matrix (ECM) or breaking down its damaged constituents (Stockwell, 1991). The two major constituents of the ECM are type II collagen and aggrecans (aggregating proteoglycans). Proteoglycans have a high negative charge which attracts cations and increases the osmolarity, while also lowering the pH of the interstitial fluid. The fibrillar collagen matrix constrains ECM swelling that results from the Donnan osmotic pressure produced by proteoglycans (Wilkins et al., 2000). Activities of daily living produce fluctuating mechanical loads on the tissue which also alter the mechano-electro-chemical environment of chondrocytes embedded in the ECM. These conditions affect the physiology and function of chondrocytes directly (Wilkins et al., 2000; Guilak et al., 1995; Guilak et al., 1999). Relatively few studies of in situ chondrocyte mechanics have been reported in the biomechanics literature, in contrast to the more numerous experimental studies of the mechanobiological response of live cartilage explants to various culture and loading conditions. Analyses of chondrocyte mechanics can shed significant insights in the interpretation of experimental mechanobiological responses. Predictions from carefully formulated biomechanics models may also generate hypotheses about the mechanisms that transduce signals to chondrocytes via mechanical, electrical and chemical pathways. Therefore, computational tools that can model the response of cells, embedded within a charged hydrated ECM, to various loading conditions may serve a valuable role in mechanobiological studies. Computational modeling has become a necessary tool to study biomechanics with complex geometries and mechanisms (De et al., 2010). Usually, theoretical and computational models of cell physiology and biophysics are formulated in 1D, deriving solutions by solving ordinary differential equations, such as cell volume regulation (Tosteson and Hoffman, 1960), pH regulation (Boron and De Weer, 1976), and Ca2+ regulation (Schuster et al., 2002). Cell modeling software, such as The Virtual Cell (vcell.org Moraru et al. (2008)), analyze stationary cell shapes and isolated cells. To model the cell-ECM system while accounting for ECM deformation, the fibrillar nature of the ECM, interstitial fluid flow, solute transport, and electrical potential arising from Donnan or streaming effects, we adopt the multiphasic theory framework (Ateshian, 2007). This framework serves as the foundation of multiphasic analyses in the open source finite element software FEBio (Maas et al., 2012; Ateshian et al., 2013), which was developed specifically for biomechanics and biophysics, and offers a suitable environment to solve complex models of cell-ECM interactions in 3D. In the studies proposed here, we will extend the functionality of FEBio to further investigate the cell-ECM system. These extensions and studies are summarized in the following chapters: Chapter 1: This introductory chapter provides the general background and specific aims of this dissertation. Chapter 2: Cell-ECM interactions depend significantly on the ECM response to external loading conditions. For fibrillar soft tissues such as articular cartilage, it has been shown that modeling the ECM using a continuous fiber distribution produces much better agreement with experimental measurements of its response to loading. However, evaluating the stress and elasticity tensors for such distributions is computationally very expensive in a finite element analysis. In this aim we develop a new numerical integration scheme to calculate these tensors more efficiently than standard techniques, only accounting for fibers that are in tension. Chapter 3: Cell-ECM interactions also depend significantly on accurate modeling of selective transport across the cell membrane. However, the thickness of this membrane is typically three orders of magnitude smaller than the cell size, which poses significant numerical challenges when modeling the membrane using the finite element method, such as element locking. To date, no existing finite element software offers a multiphasic membrane element. In this aim, we formulate and implement a new membrane element in FEBio, which can accommodate fluid and solute transport within the biphasic and multiphasic framework, to model passive and selective transport across the cell membrane. Chapter 4: This aim extends Aim 2 to incorporate reactions across multiphasic membrane elements in FEBio, to model the conformational reactions of cell membrane transporters, such as carrier-mediated transporters and membrane pumps. This implementation is verified against standard models for the regulation of cell volume, pH, and Ca2+. Chapter 5: This final chapter provides a summary of the advances contributed in this dissertation, along with suggestions for future aims related to the topics covered here. With the completion of these aims, we have extended the modeling capabilities for cell physiology and mechanobiology to more complex multicellular systems embedded within their ECM, while subjected to a range of varying mechanical, electrical or chemical loading conditions.

Zhang Li. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 148-159). / Abstracts in English and Chinese. / Acknowledgements --- p.I / Abstract --- p.II / Abstract (in Chinese) --- p.IV / Publications --- p.V / List of Abbreviations --- p.VI / List of Tables --- p.VII / List of Figures --- p.VIII / Table of Contents --- p.XIII / Chapter Chapter One. --- Introduction --- p.1 / Chapter 1.1 --- Flavonoids --- p.1 / Chapter 1.2 --- Tea --- p.4 / Chapter 1.2.1 --- Composition of green tea catechins (GTC) --- p.4 / Chapter 1.2.2 --- Pharmacological activity --- p.6 / Chapter 1.2.2.1 --- Anticarcinogenic activity --- p.6 / Chapter 1.2.2.2 --- Antioxidative activity --- p.7 / Chapter 1.2.2.3 --- Radical scavenge --- p.7 / Chapter 1.2.2.4 --- Cardiovascular activity --- p.8 / Chapter 1.2.3 --- Pharmacokinetics of GTC --- p.8 / Chapter 1.2.3.1 --- Absorption --- p.10 / Chapter 1.2.3.2 --- Distribution --- p.11 / Chapter 1.2.3.3 --- Elimination --- p.11 / Chapter 1.2.3.4 --- Metabolism --- p.12 / Chapter 1.2.3.4.1 --- Metabolism in the small intestine --- p.12 / Chapter 1.2.3.4.2 --- Metabolism in the liver --- p.13 / Chapter 1.2.3.5 --- Summary of the pharmacokinetics of GTC --- p.13 / Chapter 1.3 --- Hawthorn --- p.14 / Chapter 1.3.1 --- Composition of hawthorn --- p.14 / Chapter 1.3.2 --- Pharmacological activity --- p.16 / Chapter 1.3.2.1 --- Inotonic activity --- p.16 / Chapter 1.3.2.2 --- Antiarrhythmic activity --- p.17 / Chapter 1.3.2.3 --- Hypolipidemic activity --- p.17 / Chapter 1.3.2.4 --- Antihypertensive activity --- p.18 / Chapter 1.3.2.5 --- Antioxidative activity --- p.18 / Chapter 1.3.3 --- Pharmacokinetics of HF --- p.18 / Chapter 1.3.3.1 --- Absorption --- p.19 / Chapter 1.3.3.2 --- Distribution and elimination --- p.21 / Chapter 1.3.3.3 --- Summary of pharmacokinetic of HF --- p.22 / Chapter 1.4 --- Mechanisms of intestinal absorption --- p.22 / Chapter 1.4.1 --- Passive transcellular transport --- p.23 / Chapter 1.4.2 --- Paracellular transport --- p.23 / Chapter 1.4.3 --- Carrier-mediated transport --- p.23 / Chapter 1.5 --- ABC transporters --- p.24 / Chapter 1.5.1 --- Cellular location and tissue distribution --- p.25 / Chapter 1.5.2 --- Substrates and inhibitors of ABC transporters --- p.26 / Chapter 1.6 --- Oral absorption models --- p.31 / Chapter 1.6.1 --- Ussing chamber --- p.31 / Chapter 1.6.2 --- In situ intestinal perfusion model --- p.33 / Chapter 1.6.3 --- Cell culture model --- p.34 / Chapter 1.7 --- Aims of the study --- p.36 / Chapter Chapter Two. --- Transport mechanism of green tea catechins --- p.37 / Chapter 2.1 --- Introduction --- p.37 / Chapter 2.2 --- Materials --- p.38 / Chapter 2.2.1 --- Chemicals --- p.38 / Chapter 2.2.2 --- Materials for cell culture --- p.38 / Chapter 2.2.3 --- Instruments --- p.39 / Chapter 2.3 --- Methods --- p.39 / Chapter 2.3.1 --- Analytical methods --- p.39 / Chapter 2.3.1.1 --- Analytical methods for validation of Caco-2 model --- p.39 / Chapter 2.3.1.1.1 --- Fluorescence analysis of lucifer yellow --- p.39 / Chapter 2.3.1.1.2 --- HPLC analysis of propranolol --- p.39 / Chapter 2.3.1.1.3 --- HPLC analysis of verapamil --- p.40 / Chapter 2.3.1.1.4 --- HPLC analysis of quinidine --- p.40 / Chapter 2.3.1.2 --- Analytical methods for samples contained GTC --- p.41 / Chapter 2.3.1.2.1 --- HPLC analysis for each GTC --- p.41 / Chapter 2.3.1.2.2 --- Preparation of calibration curves for each GTC --- p.42 / Chapter 2.3.1.2.3 --- HPLC/MS analysis of samples containing mixtures of four GTC --- p.42 / Chapter 2.3.1.2.4 --- Preparation of calibration curves for samples containing GTC mixture --- p.43 / Chapter 2.3.1.2.5 --- Validation of the HPLC methods --- p.43 / Chapter 2.3.1.3 --- Identification of metabolites with HPLC/MS --- p.44 / Chapter 2.3.2 --- Determination of stability profile of GTC in phosphate buffer --- p.44 / Chapter 2.3.3 --- Cell culture --- p.45 / Chapter 2.3.4 --- Validation of Caco-2 cell monolayer model --- p.46 / Chapter 2.3.4.1 --- Integrity of Caco-2 cell monolayer at pH 6.0 --- p.46 / Chapter 2.3.4.2 --- Permeability of paracellular and transcellular markers at pH 6.0 --- p.46 / Chapter 2.3.4.3 --- Validation of the existence of P-glycoprotein (P-gp) transporterin Caco-2 monolayer model --- p.46 / Chapter 2.3.4.4 --- Cytotoxicity test --- p.47 / Chapter 2.3.5 --- Transport study of GTC using Caco-2 cell monolayer model --- p.48 / Chapter 2.3.5.1 --- Bi-directional transport experiment --- p.48 / Chapter 2.3.5.2 --- Preparation of different dosing formulations of GTC --- p.48 / Chapter 2.3.5.2.1 --- Preparation of individual pure GTC solutions --- p.48 / Chapter 2.3.5.2.2 --- Preparation of cocktail 1 solution --- p.49 / Chapter 2.3.5.2.3 --- Preparation of green tea extract solution --- p.49 / Chapter 2.3.5.2.4 --- Preparation of cocktail 2 solution --- p.50 / Chapter 2.3.5.3 --- Sample treatment --- p.50 / Chapter 2.3.5.3.1 --- Samples for direct analysis --- p.50 / Chapter 2.3.5.3.2 --- Samples for enzymatic hydrolysis treatment --- p.51 / Chapter 2.3.5.4 --- Further investigation of the transport mechanism of GTC --- p.51 / Chapter 2.3.5.4.1 --- Inhibition transport of EC and EGC --- p.51 / Chapter 2.3.5.4.2 --- Transport mechanism of metabolites of EC and EGC --- p.52 / Chapter 2.3.5.4.3 --- Metabolic competition between EGC and the other GTC --- p.52 / Chapter 2.3.6 --- Calculation --- p.53 / Chapter 2.3.7 --- Data analysis --- p.54 / Chapter 2.4 --- Results --- p.55 / Chapter 2.4.1 --- Validation of the HPLC methods --- p.55 / Chapter 2.4.2 --- Stability of the GTC --- p.55 / Chapter 2.4.3 --- Extract of green tea leaves --- p.55 / Chapter 2.4.4 --- Validation of Caco-2 model --- p.59 / Chapter 2.4.4.1 --- Integrity of Caco-2 cell monolayer --- p.59 / Chapter 2.4.4.2 --- Permeability of paracellular and transcellular markers at pH 6.0 --- p.59 / Chapter 2.4.4.3 --- Validation of P-glycoprotein --- p.60 / Chapter 2.4.4.4 --- Cytotoxicity test --- p.61 / Chapter 2.4.5 --- Transport study of GTC --- p.63 / Chapter 2.4.5.1 --- Bi-directional transport of individual pure GTC --- p.63 / Chapter 2.4.5.2 --- Bi-directional transport of GTC in different dosing formulations --- p.66 / Chapter 2.4.5.2.1 --- Absorption transport profile of GTC in different dosing formulations --- p.66 / Chapter 2.4.5.2.2 --- Secretion transport profile of GTC in different dosing formulations --- p.66 / Chapter 2.4.5.3 --- Identification of metabolites of each GTC formed during the transport in Caco-2 cell model --- p.71 / Chapter 2.4.6 --- Further investigation of the transport mechanism of GTC --- p.82 / Chapter 2.4.6.1 --- Inhibition transport of EC and EGC --- p.82 / Chapter 2.4.6.2 --- Transport mechanism of metabolites of EC and EGC --- p.82 / Chapter 2.4.6.3 --- Metabolic competition between EGC and the other GTC --- p.85 / Chapter 2.4.6.4 --- Contribution of GTC on the metabolism of EGC --- p.89 / Chapter 2.5 --- Discussion --- p.92 / Chapter 2.5.1 --- Stability of the four GTC --- p.92 / Chapter 2.5.2 --- Validation of Caco-2 cell model --- p.92 / Chapter 2.5.3 --- Bi-directional transport of GTC --- p.93 / Chapter 2.5.4 --- Structure related efflux --- p.97 / Chapter 2.5.5 --- Metabolism of GTC --- p.98 / Chapter 2.5.6 --- Relationship between metabolism and efflux transport of GTC --- p.99 / Chapter 2.5.7 --- Bi-directional transport of GTC in different dosing formulations …… --- p.100 / Chapter 2.5.7.1 --- Absorption transport profile of different dosing formulations --- p.100 / Chapter 2.5.7.2 --- Secretion transport profile of different dosing formulations --- p.101 / Chapter 2.6 --- Conclusion --- p.105 / Chapter Chapter Three. --- Transport mechanism of hawthorn flavonoids --- p.106 / Chapter 3.1 --- Introduction --- p.106 / Chapter 3.2 --- Materials --- p.107 / Chapter 3.2.1 --- Chemicals --- p.107 / Chapter 3.2.2 --- Materials for cell culture --- p.108 / Chapter 3.2.3 --- Instruments --- p.108 / Chapter 3.3 --- Methods --- p.109 / Chapter 3.3.1 --- Analytical methods for HF --- p.109 / Chapter 3.3.1.1 --- Analytical methods of individual pure compound of HF --- p.109 / Chapter 3.3.1.1.1 --- HPLC analysis of HP and IQ --- p.109 / Chapter 3.3.1.1.2 --- HPLC analysis of EC --- p.109 / Chapter 3.3.1.2 --- Preparation of calibration curves for individual pure HF --- p.109 / Chapter 3.3.1.3 --- HPLC/MS analysis of three HF in mixture --- p.110 / Chapter 3.3.1.4 --- Preparation of the calibration curves of three HF in mixture --- p.111 / Chapter 3.3.1.5 --- Validation of HPLC methods --- p.111 / Chapter 3.3.2 --- Analytical methods for identification of metabolites with HPLC/MS --- p.111 / Chapter 3.3.3 --- Cell culture --- p.112 / Chapter 3.3.4 --- Cytotoxicity test --- p.113 / Chapter 3.3.5 --- Transport studies of HF using Caco-2 monolayer model --- p.113 / Chapter 3.3.5.1 --- Bi-directional transport experiment --- p.113 / Chapter 3.3.5.2 --- Preparation of loading solutions in different dosing formulations of HF for Caco-2 cell model --- p.114 / Chapter 3.3.5.2.1 --- Preparation of individual pure HF solutions --- p.114 / Chapter 3.3.5.2.2 --- Preparation of cocktail 1 solution --- p.114 / Chapter 3.3.5.2.3 --- Preparation of hawthorn extract solution --- p.114 / Chapter 3.3.5.2.4 --- Preparation of cocktail 2 solution --- p.114 / Chapter 3.3.5.3 --- Sample treatment --- p.115 / Chapter 3.3.5.4 --- Further study of the transport mechanism of HF --- p.115 / Chapter 3.3.5.4.1 --- "Inhibition transport of EC, IQ, and HP" --- p.115 / Chapter 3.3.5.4.2 --- "Transport mechanisms of the metabolites of EC, HP, IQ" --- p.116 / Chapter 3.3.6 --- Calculation --- p.116 / Chapter 3.3.7 --- Data analysis --- p.117 / Chapter 3.4 --- Results --- p.118 / Chapter 3.4.1 --- Validation of the HPLC methods --- p.118 / Chapter 3.4.2 --- Cytotoxicity test --- p.118 / Chapter 3.4.3 --- Transport study of HF --- p.122 / Chapter 3.4.3.1 --- Bi-directional transport of individual pure HF --- p.122 / Chapter 3.4.3.2 --- Bi-directional transport of the HF in different formulations --- p.123 / Chapter 3.4.3.2.1 --- Absorption transport of different formulations of HF --- p.123 / Chapter 3.4.3.2.2 --- Secretion transport of different dosing forms --- p.123 / Chapter 3.4.3.3 --- Identification of metabolites of each HF formed during their transport in Caco-2 model --- p.126 / Chapter 3.4.4 --- Further study on the transport mechanism --- p.136 / Chapter 3.4.4.1 --- "Inhibition transport of EC, HP, IQ" --- p.136 / Chapter 3.4.4.2 --- Transport mechanism of metabolites of HF --- p.136 / Chapter 3.4.4.3 --- Transport profiles of HF metabolites upon the loading of different dosing formulations of HF --- p.138 / Chapter 3.5 --- Discussion --- p.140 / Chapter 3.5.1 --- Bi-directional transport of each HF --- p.140 / Chapter 3.5.2 --- Bi-directional transport of HF in different formulations --- p.141 / Chapter 3.6 --- Conclusion --- p.142 / Chapter Chapter Four. --- Limitations of the current study --- p.143 / Chapter Chapter Five. --- Overall conclusions --- p.146 / References --- p.148 / Appendices --- p.160

Brefeldin A (BFA) has been a useful tool for studying organelle dynamics and protein trafficking in plant cells. Using several Golgi (MAN1 and GONST1) and TGN (SCAMP1 and SYP61) fluorescent protein markers as tools, I have showed that BFA-induced aggregates from Golgi apparatus and TGN are morphologically distinct in the same plant cells. In addition, the internalized endosomal marker FM4-64 colocalized with the TGN-derived aggregates but separated from the Golgi aggregates. In the presence of the endocytosis inhibitor tyrphostin A23, SCAMP1 and FM4-64 are largely excluded from the TGN SYP61-positive BFA-induced aggregates, indicating homotypic fusion of TGN rather than de novo endocytic trafficking is important for the formation of TGN/EE-derived BFA-induced aggregates. Since the TGN also serves as an EE receiving materials from plasma membrane continuously, these data therefore support the notion that the secretory Golgi organelle is distinct from the endocytic TGN/EE in response to BFA treatment in plant cells. / Little is known about the trafficking mechanism of plasma membrane (PM) proteins in the endomembrane system of plant cells that contain several membrane-bound organelles including the endoplasmic reticulum (ER), Golgi, trans-Golgi network (TGN) of early endosome (EE), prevacuolar compartment (PVC) or late endosome (LE). Here, I study the transport pathway and sorting signals of secretory carrier membrane protein 1 (SCAMP1) by following its transient expression in tobacco BY-2 protoplasts and show that SCAMP1 reaches the PM via an ER-Golgi-TGN-PM pathway. Loss-of-function and gain-of-function analysis of various GFP fusions with SCAMP1 mutations further demonstrates that: (1) the cytosolic N terminus of SCAMP1 contains an ER export signal; (2) the transmembrane domain 2 (TMD2) and TMD3 of SCAMP1 are essential for Golgi export; and (3) SCAMP1 TMD1 is essential for TGN-to-PM targeting. Therefore, both the cytosolic N-terminus and TMD sequences of SCAMP1 play integral roles in mediating its transport to the PM via an ER-Golgi-TGN pathway. / Cai, Yi. / Adviser: Liwen Jiang. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 93-102). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Phosphate is an essential but often growth-limiting nutrient for bacteria. At low concentrations of phosphate in the growth medium, bacteria induce high-affinity uptake systems for phosphate, and this is usually the ABC-type phosphate specific transport system Pst. In the fully sequenced genomes of pathogenic species of mycobacteria, several copies of the genes encoding for the Pst system (pstSCAB) have been identified and some of these genes have been shown to be virulence factors. The reasons for the presence of multiple copies of pst genes in pathogenic mycobacteria are not understood, and phosphate transport by these bacteria, as well as the gene regulation involved, is poorly characterised. The fast-growing M. smegmatis contains only a single copy of the pst operon, but we recently identified a gene locus containing three genes, phnDCE, which encode for a putative ABC-type phosphate/phosphonate transport system, and a gene, phnF, which encodes for a putative transcriptional regulator of the HutC subfamily of GntR like regulators. To identify a function for the PhnDCE transport system and to characterise high-affinity phosphate transport in M. smegmatis, we created allelic exchange mutants in phnD and pstS, as well as a phnD pstS double deletion mutant. All three mutants failed to grow in minimal medium containing 10 mM phosphate, while the wildtype was able to grow in the presence of micromolar phosphate concentrations. No differences were observed in complex growth medium. Steady-state levels of [��P]-phosphate uptake were approximately 25% lower in all mutant strains as compared to the wildtype. Kinetics of phosphate uptake in the wildtype strain when grown at low phosphate concentrations (50 [mu]M P[i]) were biphasic, suggesting the presence of two inducible transport systems with apparent K[m] values of 16 [mu]M P[i] and 64 [mu]M P[i], respectively. Analysis of the kinetics of phosphate transport in the mutant strains led us to the proposition that the Pst system has an apparent Km value of ca. 16 [mu]M P[i], and the Phn system has an apparent Km of ca. 60 [mu]M P[i]. A third inducible phosphate transport system, which was active in the double mutant strain, had an apparent K[m] of ca. 90 [mu]M P[i]. Uptake of phosphate in all strains was not inhibited by the presence of excess phosphonates or phosphite, suggesting that all three transport systems were specific for phosphate. The study of phosphate transport in the presence of various metabolic inhibitors revealed that uptake by the Phn and Pst systems is driven by ATP-hydrolysis, consistent with ABC-type transport, while the third, unidentified transport system may be driven by the proton motive force. We showed that phnDCE formed an operon, and that the promoter area of the operon lies within 200 bp of the start of phnD. To investigate the regulation of the phn and pst genes, β-galacosidase activities of strains carrying transcriptional lacZ-fusions of the pstSCAB, phnDCE and phnF promoter areas, and levels of mRNA of the phn and pst genes were studied. All genes were induced when phosphate concentrations fell below a threshold value of 30 [mu]M, which coincided with a shift in the growth characteristics of M. smegmatis. Expression of the pst operon appeared to be controlled directly by the PhoPR two-component regulatory system, while the phn operon may be under direct or indirect control by PhoPR. To identify a role for PhnF in the regulation of phn gene expression, we created a phnF deletion mutant. PhnF appeared to repress transcription of phnDCE and phnF under phosphate-replete conditions. We identified two putative binding sequences for PhnF in the intergenic region between phnD and phnF with the sequence TGGTATAGACCA, which is similar to the proposed recognition consensus for HutC-like transcriptional regulators. Using site-directed mutagenesis of these sequences, we demonstrated that they are required for the repression of phnDCE and phnF. To prove PhnF binding to these potential binding sites, we attempted to express the M. smegmatis PhnF protein in E. coli, but could not obtain soluble recombinant protein. Electrophoretic mobility shift assays of the phnDCE promoter fragment using cell-free crude extracts of M. smegmatis were not successful. We propose that Pst and Phn both constitute high-affinity phosphate specific transport systems of M. smegmatis, and that a third inducible phosphate transport system is present in this bacterium. PhnF is required for repression of phnDCE and phnF transcription under phosphate-replete conditions, while induction of the pst operon, and possibly the phn operon, under phosphate-limited conditions involves the PhoPR system.

Ph.D.

Thesis (Ph. D.)--University of Missouri-Columbia, 2002. / Typescript. Vita. Includes bibliographical references (leaves 177-189). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 2002. / Typescript. Vita. Includes bibliographical references (leaves 177-189). Also available on the Internet.