published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

Fan, Dahua. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 117-155). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese.

PURPOSE: To determine, under flow conditions, whether reduced connections between Schlemm’s canal (SC) inner wall (IW) and juxtacanalicular tissue (JCT) cells play a role in giant vacuole (GV) formation; and whether decreased amount of cell margin overlap between adjacent IW cells promotes paracellular pore formation using serial block-face scanning electron microscopy (SBF-SEM). METHODS: Normal human eyes were immersion-fixed (0 mmHg, N=2) or perfusion-fixed (15 mmHg, N=1). Frontal and radial sections of SC were processed for SBF-SEM. IW and JCT cells, GVs, and pores were 3D-reconstructed. In each IW cell, total number of connections with underlying JCT cells/matrix was determined. Total cell margin length (TCML) and zero-overlap length (ZL) of each IW cell were measured to calculate percent zero-overlap length (PZL=ZL/TCML). All data were compared between the eyes fixed at 0 and 15 mmHg. RESULTS: Total number of IW/JCT connections in individual IWs significantly decreased in the eye fixed at 15 mmHg (33±5, N=5 cells) compared to those fixed at 0 mmHg (189±12, N=4 cells, p<0.001). The summed GV volume in individual cells significantly increased in the eye fixed at 15mmHg (218.03±19.65 μm3) compared to those fixed at 0 mmHg (82.33±27.22 μm3, p=0.0043). PZL increased 26.68% (p=0.001) in the eye fixed at 15mmHg vs. those fixed at 0mmHg, and all paracellular pores were found only in regions where the overlap length was 0 μm. CONCLUSIONS: Cellular connections between IW/JCT and IW/IW cells play a role in GV and pore formation in normal human eyes under flow conditions. Our results provide a baseline for future comparison with primary open angle glaucoma eyes.

Accumulation of misfolded proteins in the endoplasmic reticulum (ER) causes numerous human pathologies. Biochemical evidence suggests that soluble misfolded proteins are retrotranslocated out of the ER, via the endoplasmic reticulum associated degradation (ERAD) pathway, for proteosome-mediated cytoplasmic degradation. Excess, misfolded- or insoluble proteins, are suggested to cause induction of “overflow” degradation pathways. For soluble proteins, overflow to vacuole-mediated destruction is suggested to occur via two Golgi-to-vacuole (Gvt) routes, the alkaline phosphatase (ALP), direct route, or, a carboxypeptidase Y- (CPY-), prevacuolar compartmentvacuole, indirect route, though only the CPY route is thought to degrade soluble proteins. Insoluble aggregate-containing structures are suggested to be degraded by engulfment by membranes of unknown origin and trafficking to the vacuole for destruction, via an autophagic pathway. To confirm biochemical evidence, wild-type (BY4742), autophagosome- (W303/ATG14), CPY- and autophagy pathway- (W303/VPS30), and proteosome (WCG/2) mutants of S. cerevisiae yeasts were transformed with a high expression pYES plasmid and mutant (Z) human alpha-1- proteinase inhibitor (A1PiZ), giving rise to the derivatives cells BY4742/Z, W303/ATG14/Z, W303/VPS30/Z and WCG/2/Z, respectively. Electron microscopy using gold labeling for A1PiZ, markers for the ER, the ERAD ER channel protein, Sec61, or the chaperone, binding protein (BiP), ALP for the ALP pathway, and CPY for the CPY pathway, was used. Overexpression of A1PiZ seems to result in targeting to the vacuole via a prevacuolar, CPY-like compartment (PVC, 200-500 nm), though CPY and A1PiZ appears not to colocalise, unconvincingly confirming collaborative biochemical data. Large amounts of A1PiZ localise in the cytosol, possibly indicating a largely proteasome-mediated degradation. ER-resident A1PiZ targeting to the vacuole seems also to occur by the budding of the ER and peripheral plasma membrane or ER membrane only. This occurs in all cells, but especially in ATG14 gene (ΔATG14) mutants, possibly indicating autophagosome-mediated degradation independence, in the latter mutants. The ATG14 mutation gave rise to crescent-shaped, initiating membranelike (IM-like) structures of approximately Cvt vesicle-diameter, possibly indicating that ΔATG14 blocks autophagosome- (500-1000 nm) and Cvt vesicle (100-200 nm) enclosure, after core IM formation. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2008.

Thesis (MSc (Plant Biotechnology))--University of Stellenbosch, 2005. / Vacuolar Pyrophosphatase (V-PPase) has never been studied in sugarcane before and to date nothing is known about V-PPase in sugarcane, except for the sequences of a few expressed sequence tags (ESTs). The aim of this project was to characterize V-PPase expression in several hybrid sugarcane varieties that differ significantly in sucrose content, with the main objective of the study to assess whether V-PPase is correlated in any way to the sucrose storage phenotype. Therefore, the goals of this project were to (i) develop molecular tools for the detection and quantification of V-PPase on a DNA, RNA, protein and enzyme level and (ii) to use these tools to characterize the expression of V-PPase within the culm of the three hybrid varieties. The cDNA sequence of the catalytic subunit of the sugarcane V-PPase gene was cloned, expressed in a bacterial system and the V-PPase peptide was purified. This peptide was used for the immunization of mice and the production of polyclonal anti-VPPase antiserum. Anti-VPPase antiserum reacted specifically with a single polypeptide among vacuolar membrane proteins. Moreover, anti-VPPase antiserum recognized V-PPase from various monocotyledons and dicotyledons. The anti-VPPase antiserum was used for the establishment of an ELISA system to determine V-PPase protein content in vacuolar membrane preparations. This system proved to have several advantages over the protein blotting technique and shared a strong linear relation with V-PPase specific activity, showing that these two tests are compatible and reliable. The optimisation of sugarcane V-PPase zero-order kinetics was fundamental in order to measure V-PPase specific activity accurately. It had a relative broad pH optimum, retaining more than 90% of its maximum activity between pH 6.50 and 7.25. V-PPase required both Mg2+ and K+, in addition to PPi, for maximum activity in vitro. The reported kinetic variables are within range of previous data determined for other species, including mung bean, red beet and sugar beet. V-PPase protein level and specific activity within the sugarcane culm followed a similar trend , withoiofofoenaobserved for sucrose accumulation rates observed in sugarcane. Moreover, V-PPase protein contents and specific activity share the same general trend as total sucrose content in a specific tissue compared among the three varieties. No significant differences were observed in V-ATPase activity among the three varieties. Our findings suggest that V-PPase may play a role in sucrose accumulation in sugarcane.

Orientador: Tomomasa Yano / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-21T18:53:11Z (GMT). No. of bitstreams: 1 Nascimento_CatiaMariaGeraldadosSantos_M.pdf: 1717590 bytes, checksum: 6c341cf3cd058cf34bc633755af508d8 (MD5) Previous issue date: 2012 / Resumo: Neste estudo foram analisadas oito isolados de Plesiomonas shigelloides, obtidos da bacterioteca do Laboratório de Fatores de Virulência em Bactérias, Instituto de Biologia -UNICAMP - Campinas/SP: sete isolados são provenientes de água doce do Rio Cambé na região de Londrina/PR e, um isolado de ATCC 14029. Os sobrenadantes de culturas de P. shigelloides foram capazes de induzir efeitos anticarcinogênicos in vitro (em células He-La e HEp-2), apesar de apresentarem efeitos citotóxicos em linhagens celulares não cancerígenas (em células CHO e Vero). A atividade hemolítica dos cultivos de P. shigelloides foi detectada em placas com ágar sangue (meio sólido) e em microplaca (meio líquido) contendo hemácias de carneiro. A atividade citotóxica e hemolítica de Plesiomonas shigelloides apresenta característica de termolabilidade. Os isolados de P. shigelloides aderiram em células HEp-2 e em superfícies inertes como o plástico (microplaca de poliestireno) e vidro (lamínula) e, ainda apresentaram a capacidade de formar biofilme in vitro, sendo possível a colonização e formação de biofilmes em células epiteliais e em dispositivos médicos implantáveis como cateteres, próteses e/ou sondas. A produção de exoenzimas hidrolíticas como lípase e proteases (caseinase e elastase) foi detectada nos isolados de P. shigelloides. No teste de sensibilidade antimicrobiana pelo método de difusão de discos, os isolados de P. shigelloides se mostraram sensíveis à maioria dos antibióticos utilizados. Entretanto, outros testes de sensibilidade antimicrobiana se tornam necessários de modo a assegurar uma terapia antibiótica eficiente em casos de confirmação de diagnóstico clínico microbiológico. Neste estudo, os isolados de P. shigelloides expressaram fatores de virulência in vitro como citotoxicidade, adesão, exoenzimas e resistência antimicrobiana que podem potencialmente estar envolvidos na sua patogenicidade, de modo especial às gastroenterites ou complicações extra-intestinais (septicemia) em indivíduos imunocomprometidos (HIV-soropositivo, câncer ou ainda doenças hepatobiliares) / Abstract: We analyzed eight isolates Plesiomonas shigelloides, obtained from the bacterial collection Laboratory of Virulence Factors in Bacteria, Biology Institute - UNICAMP - Campinas/SP: seven isolates are from freshwater in Cambé River, region of Londrina/PR and a isolate ATCC 14029. The culture supernatants P. shigelloides were able to induce anticarcinogenic effects in vitro (He-La and HEp-2 cells), although having cytotoxic effects on non-cancer cell lines (CHO and Vero cells). The hemolytic activity of cultures P. shigelloides was detected on blood agar plates (solid medium) and microplates (liquid medium) containing sheep erythrocytes. The hemolytic and cytotoxic activity P. shigelloides features characteristics of thermo ability. Isolates P. shigelloides joined in HEp-2 cells and inert surfaces such as plastic (polystyrene microplate) and glass (coverslips) and also had the ability to form biofilm in vitro, it being possible colonization and biofilm formation in epithelial cells and devices implantable medical devices such as catheters, implants and/or probes. The production of hydrolytic exoenzymes such as lipase and protease (caseinase and elastase) was detected in isolates P. shigelloides. In antimicrobial susceptibility testing by the disc diffusion method, isolates P. shigelloides were sensitive to most antibiotics. However, other antimicrobial susceptibility testing has become necessary to ensure efficient antibiotic therapy in cases of clinical diagnostic microbiological confirmation. In this study, isolates P. shigelloides expressed virulence factors in vitro as cytotoxicity, adhesion, exoenzymes and antimicrobial resistance that may potentially be involved in this pathogenicity, specially to gastroenteritis or extraintestinal complications (septicemia) in immunocompromised individuals (HIV-seropositive, cancer or diseases hepatobiliary) / Mestrado / Clinica Medica / Mestra em Clínica Médica

Apolipoprotéines L est une famille nouvellement caractérisée en humain sans une fonction patho- physiologique définitive. Ces protéines sont classiquement considérées être impliquées dans le transport et métabolisme des lipides, principalement due à l'association de son premier membre de la famille sécrétée l’apolipoprotéine L1 aux particules des lipoprotéines de haute densité. Néanmoins, le reste des membres sont des protéines intracellulaires (absence de domaine de peptide signal). Apolipoprotéine L1 a été initialement identifiée comme l'élément clé du facteur trypanolytique dans le sérum humain. L'exploration de la séquence des différents apolipoprotéines L a révélé un domaine distinct «B cell lymphoma-2 homology domain 3» ayant des similitudes structurelles et fonctionnelles avec le domaine B cell lymphoma-2 homology domain 3 des protéines de la famille B cell lymphoma-2. Ainsi la découverte de ce domaine peut contribuer à la compréhension de la fonction et rôle des apoLs dans différents mécanismes et processus tels que la mort cellulaire programmée, la prolifération cellulaire, le métabolisme cellulaire .Notre étude visait à caractériser les fonctions de patho- physiologique du premier membre de la famille «apolipoprotéine L1 ». L’expression de l’apolipoprotéine L1 ARNm, à partir de 48 carcinomes papillaires de la thyroïde, a été évaluée par des études à haut débit et normalisée à un pool de tissus normal de la thyroïde. Une confirmation de PCR en temps réel valide ainsi la surexpression d’apoL1 dans 91,67 % des cas testés. Le niveau élevé de l’apolipoprotéine L1 ARNm est en corrélation avec une expression protéique élevée dans les échantillons histologiques (70%), et détermine que les cellules folliculaires de la thyroïde dans la zone de la tumeur sont les cellules principales responsables de l’expression spécifique de l’apolipoprotéine L1. Nous avons étudié l'expression apolipoprotéine L1 dans le modèle de cancer pour approfondir notre compréhension des relations reliant cette expression distincte dans le cancer papillaire de la thyroïde et son rôle et fonction concernant le métabolisme du cancer (de reprogrammation métabolique :effet Warburg).7En outre, la localisation de l’apolipoprotéine L1 dans la mitochondrie des cellules cancéreuses de la thyroïde ainsi que dans la mitochondrie de levure, a été le point de départ de la recherche dans ce nouveau modèle, il nous a permis de révéler et d'introduire de nouvelles hypothèses pour expliquer l'effet inhibiteur de l’apolipoprotéine L1 en fonction des conditions métabolique variantes et l’effet pléotropiques de l’apolipoprotéine L1 sur la levure (dommages des mitochondries et vacuoles). Dans ce manuscrit, nous avons décrit nos efforts à mettre en évidence la spécificité d'expression de l’apolipoprotéine L1 dans le cancer papillaire thyroïdien notamment au niveau de la transcription ainsi que la localisation mitochondriale et l'interférence probable avec les voies métaboliques. / Option Biologie moléculaire du Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Depuis plus de 30 ans, de nombreuses publications se sont attachées à expliquer la physiopathologie des anomalies morphologiques du spermatozoïde humain et ont mis en évidence des corrélations entre le pourcentage de formes normales et certaines anomalies fonctionnelles et des relations avec certains facteurs d'exposition. Le caractère physiologique de la plupart des " traits " morphologiques du spermatozoïde humain rend très difficile l'interprétation du spermocytogramme en dehors des syndromes d'anomalies monomorphes. A ces difficultés d'interprétation, viennent se rajouter le manque cruel de fiabilité analytique des techniques actuelles pour l'évaluation de la morphologie du spermatozoïde humain rendant très difficile l'utilisation de seuil décisionnel pour le choix d'une technique d'Assistance Médicale à la Procréation. Cette morphologie est classiquement observée sur lame après coloration (de Schorr le plus souvent). Il a récemment été développé une technique d'observation des spermatozoïdes mobiles associant un contraste interférentiel de Nomarski et un fort grossissement numérique et optique de x6000 au total permettant de mieux déceler certaines anomalies et notamment des vacuoles au niveau de la tête des spermatozoïdes. Cette technique est appelé MSOME (Motile Sperm Organelle MorphologyExamination). L'origine de ces vacuoles reste controversée, sont-elles d'origine nucléaire ou acrosomique ? Certains auteurs ont mis en évidence une relation entre la présence de larges vacuoles et des anomalies de condensation de la chromatine. De plus, beaucoup de questions subsistent quant au réel intérêt diagnostic du MSOME. Au cours d'un premier travail j'ai essayé d'amener un argument supplémentaire à l'origine des vacuoles. J'ai étudié le cas de deux patients atteints de globozoospermie totale (patients porteurs de spermatozoïdes sans acrosome confirmé par microscopie électronique). Au cours de ce travail, outre l'analyse morphométrique des vacuoles avec le MSOME, j'ai utilisé des techniques d'immunofluorescence (lectines PNA et anticorps anti-CD46) pour analyser le statut acrosomique et des techniques TUNEL (terminal deoxynucleotidyltransferase-mediateddUTPnick-end labelling assay) et SCSA (spermchromatin structure assay) pour le statut nucléaire du spermatozoïde. Les deux patients avaient un nombre et une surface vacuolaire (6,3% et 5%) très similaires à celles de 12 témoins fertiles (5%) permettant d'exclure une origine acrosomique de la plupart de ces vacuoles. Je me suis ensuite intéressé à l'impact de la congélation du sperme (très utilisé en pratique clinique) sur l'apparition de ces vacuoles. Après analyse morphométrique précise à l'aide d'un logiciel d'analyse d'image de plus de 2000 spermatozoïdes avant et après congélation (27 individus) je n'ai retrouvé aucune différence statistiquement significative en termes de nombre, surface et position de ces vacuoles entre avant et après congélation. De nombreux auteurs plaident en faveur d'une utilisation en pratique clinique de cette évaluation des vacuoles à fort grossissement sans qu'il n'y ait aucune donnée sur la présence de ces vacuoles dans une population de référence (témoins fertiles). J'ai donc réalisé une analyse morphométrique précise de ces vacuoles dans une population de 50 témoins fertiles. La très grande fréquence de ces vacuoles (95,8 %) dans cette population souligne le caractère physiologique de la plupart d'entre elles. / For over 30 years, many publications have sought to explain the pathophysiology of human sperm morphological abnormalities and showed correlations between the percentage of normal forms and functional defect and some relationships with some exposure factors. The physiological nature of most of the "features" of human sperm morphology makes the interpretation of human sperm morphology very difficult except for monomorphic defects. To these difficulties of interpretation, come to add the lack of analytical reliability of current techniques for assessing human sperm morphology making very difficult the use of threshold of normal forms for the choice of medical treatment of infertile couple in Assisted Reproductive Treatment. Recently a new aspect of sperm morphology, the head vacuoles, has shown to be of interest. The vacuoles can be easily observed using Nomarski interference contrast microscopy at high magnification: ×6000 to 10000 (optical magnification ×1000 associated with digital enhancements that achieves a final magnification up to 6000) even on motile spermatozoa (MSOME: Motile Sperm Organellar Morphology Examination). The origin of these vacuoles raises many questions. Several studies have found a link between chromatin condensation defects and the presence of sperm head vacuoles and clinical interest of MSOME is still somewhat debated. To add new arguments concerning the origin of the sperm-head vacuoles observed under high magnification with interference contrast microscopy, we carried out in two patients with total globozoospermia confirmed using transmission electron microscopy (TEM), a detailed sperm morphometric analysis with MSOME, an acrosomal status analysis (using fluorescent labelling with peanut agglutinin (PNA) lectins and anti-CD46 antibodies) and a nuclear status analysis (using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling assay TUNEL, sperm chromatin structure assay SCSA and aniline blue staining). Our two patients with globozoospermia had relative sperm vacuole areas of 6.3% and 5%, similar to those observed in a reference population of 12 fertile men (5.9%). This study provides further information on the non-acrosomal origin of the sperm-head vacuoles. Since the development of MSOME for observing the cephalic vacuoles at high magnification, no study as yet assessed the effect of cryopreservation on these vacuoles, although sperm freezing-thawing procedures are known to affect sperm quality. In 27 sperm samples from fertile men, morphological analysis at high magnification (·6000) using image analysis software was performed before freezing and after thawing, there was no evidence for any difference in any vacuolar criteria (relative vacuole area, total vacuole area, vacuole area in the anterior, median and basal parts of the head, percentage of spermatozoa with a vacuole area < 6.5% and percentage of spermatozoa with a vacuole area >13%). Freezing-thawing procedures have no effect on human sperm vacuoles. In order to establish reference values concerning sperm vacuoles and to know if the assessment of sperm vacuoles at high magnification can contribute to the explanation of idiopathic infertility, I investigated the number, position and area of sperm head vacuoles using an image analysis software in 50 fertile men and 50 infertile men were within couples who had unexplained infertility and were consulting in our centre. After analysis, the characteristics of sperm head vacuoles (number, area, position) are no different between fertile controls and patients with unexplained infertility.

Primary open-angle glaucoma (POAG) is one of the leading causes of blindness worldwide. The only modifiable risk factor for POAG is elevated intraocular pressure, resulting from increased aqueous humor production or decreased drainage. Resistance to drainage in the aqueous outflow pathway is believed to reside in the juxtacanalicular connective tissue (JCT) and to be modulated by the inner wall (IW) endothelium of Schlemm’s canal (SC); however, the mechanisms that increase resistance in POAG remain unclear. To cross the IW, aqueous humor passes through I-pores on giant vacuoles (GVs) or B-pores between adjacent endothelial cells. Additionally, outflow around the circumference of the eye is segmental, or non-uniform, and fluorescent tracers can be used to label areas of high-flow and non-flow. The morphological differences in the endothelial cells of SC and their GVs in high- vs. non-flow areas have not been fully elucidated. In this project, we investigated the role of GVs and pores in the IW endothelial cells of SC in regulating segmental outflow in human eyes. We used serial block-face scanning electron microscopy to generate thousands of serial images and visualize these structures in 3D at the ultrastructural level. First, we 3D-reconstructed 45 individual IW cells and their GVs and quantified the number of connections each cell makes with the underlying JCT matrix/cells. We found that cells in high-flow areas made significantly fewer connections to JCT matrix/cells compared to cells in non-flow areas. Secondly, we analyzed 3,302 GVs for I-pores and basal openings and found a significantly greater percentage of GVs with both basal openings and I-pores in high-flow area compared to non-flow area, suggesting this type of GVs form a channel through which aqueous humor passes from JCT to SC. We also found that GVs with I-pores were significantly larger than those without I-pores. Our results suggest that decreasing number of cellular connections and increasing number of GVs with pores may be potential strategies to increase the amount of high-flow area and aqueous outflow for glaucoma treatment. Together, these studies add to our understanding of the role of GVs and pores in regulating segmental flow around the eye.

Aging of the hematopoietic system promotes various immune and systemic disorders and is driven in-part by dysfunction of life-long self-renewing hematopoietic stem cells (HSC). Autophagy is required for the benefit associated with activation of conserved longevity signaling programs and is essential for HSC function in response to various stressors. With age, some HSCs basally increase autophagy flux and maintain inert metabolic activity. This autophagy-activated subset is responsible for the residual regenerative capacity of old stem cells, but the mechanisms promoting autophagy activation in HSC aging remain unknown. Here, we demonstrate that autophagy is a response to chronic inflammation in the aging HSC niche. Chronic inflammation impairs glucose metabolism in young and old HSCs (oHSC) by impeding AKT-FOXO intracellular signaling networks. We find that autophagy enables metabolic adaptation of oHSCs to non-glucose energy substrates for functional maintenance. Notably, water-only fasting transiently further activates autophagy in oHSCs, and upon refeeding normalizes glucose uptake and glycolytic flux as well as regenerative output. Our results demonstrate that inflammation-driven glucose hypometabolism impairs oHSC regenerative capacity, that autophagy activation metabolically adapts oHSCs to an inflamed niche, and that autophagy is a modulable node to restore glycolytic and regenerative capacity during stem cell aging.