L'épithélium intestinal se renouvelle tous les 4 à 6 jours chez les mammifères grâce aux cellules souches localisées au fond des cryptes. Le renouvellement dépend des signaux émis par le microenvironnement et requiert une phase de prolifération des cellules souches, de différenciation et d'apoptose/desquamation des cellules épithéliales. La signalisation Wnt, joue un rôle majeur dans l'homéostasie intestinale par l'action de deux gradients inversés le long de l'axe crypte/lumière ; la signalisation Wnt canonique, active au fond des cryptes, contrôle la prolifération alors que la signalisation non-canonique, active vers le haut des cryptes contrôle la différenciation. Il a été montré que ces deux voies contrôlent l'activité de la GTPase atypique RhoU/Wrch1. RhoU fait partie des GTPases qui s'activent spontanément, son activité est donc directement proportionnelle à son niveau d'expression dans la cellule. Enfin, cette GTPase atypique est sous exprimée dans de nombreuses tumeurs gastriques et colorectales.Compte tenu de ces données, nos objectifs étaient donc de caractériser les changements morphologiques induits par l'invalidation conditionnelle de RhoU dans l'épithélium intestinal murin et d'en déterminer les mécanismes d'action. Nos résultats montrent que la déplétion de RhoU n'est pas létale, cependant elle a induit une augmentation de 20% de la densité cellulaire et une désorganisation de la structure de l'épithélium dans le haut des cryptes du colon. Cette augmentation concerne aussi bien les lignages sécrétoires et absorptifs, cependant, l'absence de RhoU a induit une sur-représentation du lignage sécrétoire. Dans la lignée de tumeur colorectale DLD-1, nous avons montré que l'absence de RhoU mime le phénotype d'augmentation de la densité cellulaire observé chez la souris. L'invalidation de RhoU ne modifie pas la distribution des phases du cycle cellulaire ni de celle de la mitose, cependant, elle réduit le nombre des cellules en apoptose dans le colon des souris et dans les cellules DLD-1. L'invalidation de RhoU a réduit la signalisation Hippo et a altéré la contractilité cellulaire via une augmentation de la phosphorylation de la protéine MLC2. Des travaux récents ont montré que la diminution du niveau MLC2 phosphorylée est nécessaire pour l'activation des caspases par un stimulus apoptotique. Ceci suggère que cette perturbation de la contractilité peut être à l'origine de cette diminution de l'apoptose qui est la cause majeure responsable de ce phénotype. En conclusion, RhoU est un régulateur de l'homéostasie intestinale chez la souris via son rôle modérateur de la mort cellulaire. / In Mammals, the intestinal epithelium is renewed every 4-6 days through the stem cells located at the bottom of crypts. The renewal depends on signals from the micro-environment and requires a proliferation phase of stem cells, then a differentiation and apoptosis/desquamation phases of epithelial cells. Wnt signaling plays a major role in intestinal homeostasis by the action of two reversed gradients along the axis crypt/ lumen: canonical Wnt signaling, active in the bottom of crypts, control proliferation while non canonical signaling, active in the top of the crypts control cell differentiation. It was shown that these two pathways are regulator of the atypical GTPase RhoU/Wrch1. The RhoU protein activates spontaneously, its activity is directly proportional to its expression level in the cell and is expressed as in gastric and colorectal tumors. In view of these informations, our objectives were therefore to characterizethe morphological changes induced by conditional invalidation of RhoU in the intestinal epithelium of mice and to determine the mechanisms of action. Our results show that RhoU depletion is not lethal. However, it induces an increase of cell density (+20%) and a disruption of the epithelium structure in the top of the colonic crypts. This increase affects both absorptive and secretory lineages. However, the absence of RhoU induced over-representation of secretory lineage. In colorectal tumor cell line DLD-1, we have shown that the absence of RhoU mimics the phenotype of cell density increase observed in mice. RhoU invalidationdid not change the distribution of cell cycle phases and mitosis, however, it reduces the number of apoptotic cells in the colon of mice and in the DLD-1 cells. RhoU invalidation reduced Hippo signaling and altered cell contractility via the increase of the protein MLC2 phosphorylation. Recent work has shown that the reduction of MLC2-P level is necessary for the caspase protein activation by an apoptotic stimulus. Suggesting that the perturbation of contractility may be the cause of this apoptosis decrease which is the main cause responsible of this phenotype. Finally, RhoU is a regulator of the intestinal homeostasis in micevia its moderating role of cell death.

Abstract The carbonic anhydrases (CAs) catalyse the hydration of CO2 to bicarbonate at physiological pH. This chemical interconversion is crucial since HCO3- is the substrate for several biosynthetic reactions. Carbonic anhydrases are involved in many physiological processes connected with respiration and transport of CO2/bicarbonate between metabolising tissues and the lungs, pH homeostasis and electrolyte secretion in a variety of tissues/organs. The present work was undertaken to study the distribution and expression of CA isoenzymes in the normal and neoplastic gastrointestinal tissues. The expression of CA I, II, IX and XII in the human intestine and colorectal tumours was investigated by immunohistochemistry and western blotting. In the present study, immunohistochemical methods were also used to examine the location of CA IX and XII in the human pancreas and pancreatic tumours. The expression of CA XIV in the murine liver and intestine was studied using immunostaining and northern blotting. The present results suggest that transmembrane CA XII is absent from the small intestine, but is expressed in all segments of the normal large intestine. The positive signal for CA XII was confined to the basolateral plasma membranes of the epithelial cells of the surface epithelial cuff. In tumours, the signal for CA XII became stronger in the deep part of the lesion. The intensity of the immunostaining for CA I and II was clearly found to decrease in benign lesions and became very weak in malignant colorectal tumours. The reciprocal pattern of expression observed for membrane-associated (CA IX and XII) and cytoplasmic (CA I and II) isoenzymes in intestinal samples suggests that CA IX and XII may be functionally involved in tumour progression to malignancy and/or in invasion. CA I and II, which are thought to play important physiological roles in the normal colorectal mucosa, may not be required for growth of colorectal cancers and their expression consistently diminishes with progression to malignancy. In the human pancreas CA IX and XII appeared to be sporadically expressed in the basolateral plasma membrane of the normal acinar and ductal epithelium. The increased expression of CA IX in hyperplastic ductal epithelium may contribute to the pancreatic tumourigenesis. CA XIV was expressed in the hepatocyte plasma membrane and its localization on both apical and basolateral membrane domains suggests an important role for this isoenzyme in the regulation of ion and pH homeostasis in the liver.

La biopharmacie s'intéresse au devenir du médicament au contact d'un épithélium. Cela conditionne la biodisponibilité du principe actif, rendant les études biopharmaceutiques indispensables au développement des médicaments. L'obtention de données biopharmaceutiques in vitro passe par l'utilisation de cellules de diffusion. La variabilité des résultats expérimentaux obtenus nous a conduits à développer un nouveau dispositif nommé VitroPharma, destiné à optimiser l'étude de la perméabilité épithéliale. Son originalité réside dans la possibilité d'employer les milieux récepteurs semi-solides en substitution des milieux liquides habituels.Ce travail propose une exploration des capacités de VitroPharma en vue de sa validation. Le dispositif est testé en modalités de dose (i) finie et (ii) infinie, sur (i) peau porcine et (ii) membrane artificielle de silicone, respectivement. Des milieux récepteurs (i) liquide, (ii) semi-solide (hydrogel d'agarose) et (iii) thermogélifiant (hydrogel de poloxamer) sont évalués. La caféine et la testostérone sont utilisées comme composés modèles. Les résultats sont comparés à une méthode de référence.De plus, deux problématiques expérimentales peu étudiées dans la littérature sont évaluées : (i) la formation de bulles à l'interface milieu récepteur-membrane et (ii) l'altération de l'hydratation des membranes biologiques en cours d'essai. Il ressort que l'utilisation de VitroPharma combinée à un milieu récepteur thermogélifiant permet (i) de limiter la formation de bulles et (ii) de contrôler le contenu en eau des explants cutanés.Ainsi le dispositif VitroPharma apparait comme adapté à la tenue d'essais de perméabilité épithéliale, apportant notamment, (i) une manipulation facilitée, (ii) une optimisation des conditions d'essai et (iii) l'obtention de résultats cinétiques de pénétration. Des exemples de développements cliniques mettant à profit les performances de VitroPharma sont présentés en conclusion / Biopharmacy studies the outcomes of contact between a medicine and its administration site epithelium, thus determining active compound bioavailability. Hence, biopharmaceutical studies are paramount to drug development processes. Biopharmaceutical data are obtained in vitro using experimental devices (i.e., diffusion cells) but show high variability. To overcome this limitation we development a new experimental device, called VitroPharma, meant to optimize the study of epithelial permeability. Distinctiveness of this innovating diffusion cell is due to substitution of classical liquid receptor medium with semi-solid medium.In this work, validation studies of VitroPharma are detailed including (i) finite and (ii) infinite dosing protocols using (i) biological membrane (i.e., pig ear skin) and (ii) artificial silicone membrane, respectively. Three different types of receptor medium are employed: (i) liquid medium, (ii) semi-solid medium (i.e., 2% agarose hydrogel) and (iii) thermogelifying medium (i.e., 20% poloxamer 407 hydrogel). Caffeine and testosterone are used as model compounds. Permeability results are displayed and compared to that obtained using reference Franz static diffusion cell.Furthermore, two experimental pitfalls often mentioned but scarcely studied in literature are evaluated, that is (i) bubble formation at the membrane-receptor medium interface and (ii) biological membrane hydration modification over assay time. VitroPharma combined to thermogelifying receptor medium was found efficient (i) in reducing bubble formation and (ii) enabling control of biological membrane water content.Therefore, VitroPharma appears adapted to the in vitro study of epithelial permeability, enabling (i) easy handling, (ii) optimized experimental parameters and (iii) dual penetration and permeation determination. To conclude this work, examples of clinical outcomes that could advantageously use VitroPharma are presented

La protéine CCSP (« Club Cell Secretory Protein »), produite par les cellules Club au niveau de l’épithélium respiratoire, se retrouve déficiente chez les patients atteints de Bronchopneumopathie Chronique Obstructive (BPCO). Le but de ce travail était de comprendre la régulation et les différents rôles de la protéine CCSP afin d’en évaluer son potentiel intérêt thérapeutique. Nous avons dans un premier temps observé les effets du polymorphisme connu de CCSP au niveau de sa région promotrice, la mutation G38A, sur la transcription même du gène. Nous avons constaté in vivo dans une étude clinique prospective sur 1 an comprenant 66 patients souffrant de BPCO, et confirmé in vitro dans un modèle de cellules BEAS-2B transfectées, que la fumée de cigarette était un répresseur de la transcription de CCSP et que ce phénomène était amplifié par la présence de la mutation G38A. De plus, in vitro, certains facteurs de transcription tels que p53 et Nkx2.1, ainsi que les lipopolysaccharides, affectaient l'efficacité du promoteur de CCSP.Ensuite, nous avons caractérisé les cellules qui sécrètent cette protéine dans un modèle ex vivo de culture en interface air-liquide de cellules primaires épithéliales bronchiques. Nous avons observé par microscopie électronique à balayage des cellules en dôme, forme caractéristique des cellules Club, et par microscopie électronique à transmission des cellules contenant des granules de sécrétion contenant la protéine CCSP. Nous avons constaté par immunofluorescence que les cellules marquées CCSP+ étaient également MUC5AC+ (marqueur de cellules à mucus), P63+ (marqueur de cellules basales) ou encore KI-67+ (marqueur de prolifération). Nous suggérons donc que les cellules Club sont des cellules progénitrices, permettant ainsi la régénération de l’épithélium bronchique. Par ailleurs, nous avons évalué l’implication de la protéine CCSP dans le recrutement des neutrophiles, cellules inflammatoires prépondérantes dans la BPCO. Une étude pharmacologique a d’abord permis d’évaluer les effets de CCSP sur des neutrophiles de sujets témoins. Le déplacement des neutrophiles, stimulé par l’IL8 ou le fMLP (tous deux puissants agents chemoattractants), était inhibé par CCSP. Puis, par une étude in vitro, nous avons déterminé la modulation du sécrétome de l’épithélium bronchique par CCSP. Lorsque les sécrétions d’épithélia reconstitués ex vivo à partir de biopsies de fumeur et de BPCO étaient mis en présence de neutrophiles, un chimiotactisme exagéré des neutrophiles étaient constaté. Lorsque les épithélia étaient traités avec la protéine CCSP, à l’état de base ou stimulés par de la fumée de cigarette, ce chimiotactisme exagéré était alors diminué.Enfin, dans une dernière partie, nous nous sommes intéressés à la régulation de la protéine, dans un modèle de culture cellulaire NCI-H292, lignée de cellules bronchiques cultivées en monocouche. Nous avons supplémenté ces cellules en CCSP exogène afin d’analyser les variations du profil protéomique des secrétions engendrées (méthode LC-MS/MS). De façon générale, il s’avérait que la supplémentation en CCSP permettrait une restauration de la « machinerie » du protéasome avec une augmentation des protéines de la famille des tubulines.Ce travail de thèse a démontré que la protéine CCSP était un acteur potentiel de la physiopathologie de la BPCO. L’étude de sa régulation a montré que la synthèse de CCSP était effectivement diminuée dans la BPCO. Ainsi, une supplémentation en CCSP pourrait être une piste thérapeutique. / A defective in Club Cell Secretory Protein (CCSP) produced by nonciliated Club cells was observed in COPD (Chronic Obstructive Pulmonary Disease) airways. Our aim was to understand CCSP biological mechanisms of action and its dysregulation in COPD and whether it might be a therapeutic axis in COPD.First, the influence of the CCSP G38A polymorphism on CCSP transcription levels and its regulatory mechanisms were analyzed. Our in vivo study conducted in a 1 year prospective cohort consisting of 66 COPD patients confirmed that circulating CCSP levels were associated with smoking. Moreover, the CCSP G38A polymorphism and the smoking status significantly repressed CCSP serum levels. Our in vitro study conducted in BEAS-2B transfected cells supported those findings as CSE repressed the CCSP transcription of the A carrying transfected cells more intensely than the wild type cells. Noteworthy, LPS, Nkx2.1 and p53 transcription factors also modulated the CCSP promoter efficiency in vitro. Furthermore, CCSP producing cells were characterized in an air-liquid interface (ALI) culture model of bronchial epithelial cells. Transmission electron microscopy, scanning electron microscopy and confocal microscopy confirmed the pseudostratified organization of the reconstituted epithelium. Evidences of full differentiation were identified and labeled with MUC5AC (goblet cells), tubulinIV (ciliated cells), P63 (basal cells) and CCSP (club cells). Moreover, the ex vivo reconstituted COPD epithelium released higher levels of IL8 and MUC5AC. Ki-67 and collocating antibodies with CCSP argued for an accessory stem cell and a transitory differentiating roles for CCSP+ cells.Then, we aimed to investigate whether exaggerated airway neutrophilia was driven by the CCSP-defective COPD airway epithelium. CCSP action on healthy neutrophil chemotaxis was evaluated in a pharmacological study demonstrating that CCSP directly inhibited neutrophil chemotaxis induced by fMLP and IL8. Then, in an in vitro study, ALI-reconstituted COPD airway epithelium in a clean environment promoted an exaggerated neutrophilic chemotaxis compared to smokers and controls at steady state. Treating the airway epithelium with exogenous CCSP prevented baseline and CSE-induced neutrophil chemotaxis.Finally, CCSP regulation was studied in NCI-H292 cells, a human pulmonary cell line. The cells were supplemented with CCSP. Proteomic profile (LC-MS/MS method) of the bronchial epithelium in response to CCSP treatment demonstrated that the proteasome machinery and the tubulin family members were upregulated.This work supported the potential implication of CCSP in the pathophysiology of COPD. CCSP was confirmatively defective in COPD patients, therefore, restoring physiological concentrations of CCSP by exogenous supplementation may be a therapeutic perspective.

La structure polygonale des épithéliums mono-stratifiés exerce une certaine fascination sur les biologistes depuis les observations originales par Robert Hooke en 1665. Cependant, il est difficile d‘expliquer comment la stabilité de la morphogenèse est atteinte, i.e. comment les structures polygonales maintiennent la régularité au sein d'un individu, entre les individus et au sein des phylums. Dans ces travaux, nous introduisons une nouvelle mesure quantitative de la stabilité de la morphogenèse entre individus appelée l'homéostasie topologique. Nous démontrons que les épithéliums non-prolifératifs, formés par un processus d'accrétion, sont plus stables que les épithéliums prolifératifs. Dans le contexte de prolifération, l'homéostasie topologique dépend du rapport apoptose/mitose comme en témoigne le modèle Drosophila où l'homéostasie épithéliale diminue drastiquement quand l'apoptose est inhibée dans les disques imaginaux. Ainsi, l'apoptose agit comme un régulateur positif dans la canalisation de la stabilité de la morphogenèse. En outre, des simulations numériques reproduisant la morphogenèse épithéliale, basées sur la physique des milieux divisés, décrivent comment les mécanismes d'accrétion dans les épithéliums non prolifératifs et l'apoptose dans les épithéliums prolifératifs sont des moyens efficaces pour parvenir à la stabilité morphogénétique. / The polygonal structure of mono-stratified epithelia exerts a unique fascination among biologists since the original observations of Robert Hooke in 1665. However, it is always unclear how the stability of morphogenesis is achieved, i.e., how these polygonal structures maintain regularity among individual, between individuals and among all phyla, and among individuals for each tissue within each species. Here, we introduce a new and quantitative measure of the level of morphologic stability between individuals, referred to as topological homeostasis. We demonstrated that non-proliferative epithelia, formed by an accretion process, are significantly more regularly stabilized than proliferative ones. In proliferative context, topological homeostasis directly depends on the apoptosis/mitosis ratio, as evidenced in the Drosophila imaginal disc model, where topological homeostasis drastically drops down when apoptosis is inhibited. Apoptosis therefore acts as an unexpected positive regulator in the canalization of morphogenetic stability. In addition, numerical simulations of epithelial morphogenesis, based on the physics of devided media, described how accretion mechanisms in non-proliferative epithelia, and, apoptosis in proliferative ones, are efficient means to achieve morphogenetic stability.

The ovarian surface epithelium, a very small portion of the total mass of the ovary, is generally thought to be the site of origin of over 85% of ovarian cancers. Such cancers are classified with the "Common Epithelial Tumours" of the ovary. In most industrialized countries, malignancies of the ovary rank fourth in cancer deaths in women; over 70% of these neoplasms have spread beyond the ovary when first diagnosed. Experimental approaches to the study of carcinogenesis in this tissue have been limited by the lack of pure populations of ovarian surface epithelial cells. Studies done on rodents in vivo suggest that both chemicals and C-type RNA viruses can induce ovarian cancers similar to those which are said to arise from the surface epithelium. However, the cell of origin cannot be proven in such studies. The purpose of this project was to develop a model for ovarian cancers of surface epithelial origin based on-carcinogenesis in vitro. To this end a method was devised to culture the rat ovarian surface epithelium in pure form. These cultured cells, whose identity has been confirmed by morphological, histochemical and ultrastructural means, are polygonal with clear cytoplasm, have well-defined borders, and grow in confluent monolayers. Their morphology is quite distinct from those of other ovarian cells in vitro. Cultured rat ovarian surface epithelial cells are histochemically positive for 17β-hydroxysteroid dehydrogenase, and negative for Δ5-3β-hydroxysteroid dehydrogenase, the same as in frozen sections of whole rat ovary. Ultrastructurally, cultured surface epithelial cells have basal laminae, microvilli, apical intercellular junctions, large nuclei, abundant rough endoplasmic reticulum, Golgi complexes, perinuclear bundles of microfilaments, and numerous vesicles. Although the ovarian suface epithelium is suspected of being an estrogen target tissue, there is no previous report of estrogen receptors in these cells. In this study cultured rat ovarian surface epithelial cells have been shown by autoradiographic means to exhibit estrogen receptor-like activity. Translocation of tritiated estradiol from cytoplasm to nucleus, and estrogen-specific binding have been demonstrated. Estradiol was shown to be mitogenic for cultured ovarian surface epithelial cells. From these results, the surface epithelial .cells of the ovary should be considered an estrogen target tissue. Kirsten murine sarcoma virus was used to produce three transformed cell lines from pure, first passage cultures of these cells. These three lines retained 170-hydroxysteroid dehydrogenase activity and showed slight Δ5-3β-hydroxysteroid dehydrogenase activity. Tumours resulting when these cells were injected into immunosuppressed female rats were highly malignant and resembled histologically human endometrioid stromal sarcomas of the ovary. This neoplasm is classed with the "Common Epithelial Tumours" of the ovary, but is generally not considered a derivative of the surface epithelium. In light of this study, perhaps this tumour should: be considered, to be of surface epithelial origin. A continuous cell line arising from pure cultures of rat ovarian surface epithelial cells produced structures in vitro resembling those found in ovarian serous papillary cystadenomas of borderline malignancy. This tumour is classed as a common epithelial ovarian tumour. Hence, in this study the rat ovarian surface epithelium has been cultured in pure form, has been characterized for a number of properties by several investigative techniques, and has been shown to be susceptible to transformation by an oncogenic virus; This work supports the theory that the "Common Epithelial Tumours" of the ovary are, in fact, derived from the surface epithelium. The availability of cultured ovarian surface epithelial cells should allow investigation into factors which make this tissue so susceptible to malignant transformation. From such cultures could come markers suitable for use in tests to detect ovarian cancers at an early stage. The culture of pure rat ovarian surface epithelium, as described herein, could readily be used to study chemical, physical and viral carcinogenesis in this tissue to produce experimental models of cancers arising in the ovarian surface epithelium. / Science, Faculty of / Zoology, Department of / Graduate

Orientadores: Jacqueline Mendonça Lópes de Faria, José Butori Lopes de Faria / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-24T10:41:17Z (GMT). No. of bitstreams: 1 Rosales_MarianaAparecidaBrunini_D.pdf: 28953374 bytes, checksum: e9a2824bad639c7cbd3628c24c4308ad (MD5) Previous issue date: 2014 / Resumo: A patogênese da retinopatia diabética (RD) está associada ao estresse nitrosativo. Alterações na barreira hemato-retiniana (BHR) externa, formada pelas células do epitélio pigmentar da retina (EPR), estão associadas às fases precoces da RD e podem acarretar no desequilíbrio da manutenção dos fotorreceptores e consequentemente promoverem mudanças nas células neuronais da retina. O estresse nitrosativo como conseqüência do aumento da produção de óxido nítrico (NO¿) produzido pela super expressão da óxido nítrico sintetase induzida (iNOS) esteve presente em todas as camadas da retina, inclusive no EPR em condições de RD experimental in vivo precoce ou na linhagem celular humana do EPR (ARPE-19) expostas à alta concentração de glicose. O tratamento com agentes químicos como a S-nitrosoglutationa (GSNO), ou naturais (cacau enriquecido com polifenol) atuaram em diferentes vias de inibição da iNOS, prevenindo o estresse nitrosativo. Para o estudo in vivo com o colírio de GSNO (artigo I) foram utilizados animais espontaneamente hipertensos (SHR) com 4 semanas de idade. O diabetes (DM) foi induzido por STZ. Após a confirmação do DM (48 horas), os animais foram divididos em 6 grupos: controles (CTs) veículo; GSNO 900nm e GSNO 10?m ou DMs veículo; GSNO 900nm e GSNO 10?m. O efeito do tratamento com colírio de GSNO foi dependente da presença ou ausência da condição do DM. Nos animais CT, o GSNO atuou como um agente nitrosativo e nos animais DM preveniu o aumento da expressão da iNOS, preservando a retina funcional. Os estudos in vitro, demonstraram que o efeito do GSNO foi deletério ou protetor dependente da concentração de glicose. Nas células ARPE-19 expostas a condições normais de glicose, o tratamento promoveu um aumento na produção de NO¿ sem aumentar a expressão de iNOS e nas células sob alta glicose induziu uma modificação pós-translacional de proteína, a S-glutationilação da iNOS prevenindo o estresse nitrosativo. No estudo do cacau (artigo II), foi avaliado in vitro (ARPE-19 exposta a alta concentração de glicose) o seu efeito protetor dependente da concentração de polifenóis. Para isso foram testadas duas formulações de cacau que diferiram somente na concentração de polifenol: 0,5% para o cacau com baixo teor de polifenol e 60,5% para o cacau com alto teor de polifenol. A epicatequina (EC), encontrada na concentração de 12% no cacau com alto teor de polifenol foi tão eficaz quanto o próprio e esteve envolvida no controle da expressão da iNOS através da estimulação do receptor ?-opióide (DOR) diminuindo os níveis de TNF-?. A modulação da iNOS, preveniu a S-nitrosilação da caveolina-1 (CAV-1) e diminuição da expressão das junções intercelulares claudina-1 e ocludina através da prevenção da interação CAV-1?junções. Em ambos os estudos, o alvo terapêutico foi a iNOS em duas diferentes modalidades: modificação pós-translacional de proteína e modulação do TNF-? via DOR no EPR em modelos experimentais de RD. Os tratamentos apresentados neste trabalho demonstraram a iNOS como alvo terapêutico e mostraram-se eficaz em conter danos funcionais e morfológicos promovidas pela situação de mimetismo do DM no EPR demonstrando o importante papel da iNOS no desenvolvimento da RD / Abstract: The pathogenesis of diabetic retinopathy (DR) is associated with nitrosative stress. Changes in outer blood-retinal barrier (BRB), formed by retinal pigment epithelium cells (RPE) are associated in the early stages of DR and can cause imbalance in the maintenance of photoreceptors and thereby cause changes on retinal neuronal cells. The nitrosative stress as a result of increased production of nitric oxide (NO) produced by overexpression of nitric oxide synthase (iNOS) was present in all layers of the retina and mainly in RPE cells in early in vivo experimental DR or in human RPE cell line (ARPE-19) exposed to high glucose condition. Therapy with chemical agents such as S-Nitrosoglutathione (GSNO) or natural compounds (enriched cocoa polyphenol) acted in different pathways of iNOS inhibition, preventing nitrosative stress. For the in vivo study with GSNO eye drops (article I), it were used spontaneously hypertensive rats (SHR) rats with 4 week old. Diabetes (DM) was induced by streptozotocin (STZ). After DM confirmation (48 hours), the animals were divided into 6 groups: controls (CTs) vehicle; GSNO 900nm and GSNO 10?m or DMs vehicle; GSNO 900nm e GSNO 10?m. The effects of treatments were dependent on glucose concentration. In CT animals, GSNO acted as a nitrosative agent and in DM rats prevented iNOS overexpression, preserving the retina function. In vitro study showed that GSNO protective or deleterious effects were dependent on the glucose concentration. In ARPE-19 cells exposed to normal glucose, the treatment promoted an increase of NO¿ production without increase iNOS expression and in cells under high glucose (HG) condition induced post-translational protein modification, S-glutationylation of iNOS, preventing nitrosative stress. In the study with cocoa (article II), it was evaluated its protective effect dependent on concentration of polyphenols in ARPE-19 cells under HG condition. For this study, the composition of cocoa was the same in both preparations with the only difference in the amounts of polyphenol, 0.5% for low polyphenol cocoa (LPC) and 60.5% for high polyphenol cocoa (HPC). Epicatechin (EC), found in 12% of HPC was similarly protective compare to HPC and it was involved in controlling iNOS expression by stimulation of the delta opioid receptor decreasing TNF- ? levels. The modulation of iNOS prevented S-nitrosylation of caveolin-1 (CAV-1) and decreased expression of claudin-1 and occluding tight junctions by preventing CAV-1/junctions interactions. The treatments presented here showed iNOS as a therapeutic target containing functional and morphological changes promoted by DM milieu in RPE showing the important role of iNOS in the development of DR / Doutorado / Clinica Medica / Doutora em Clínica Médica

Dans un contexte d’augmentation du nombre d’amétropes dans la population mondiale, et en conséquence, de l’accroissement du recours aux techniques de corrections chirurgicales, la compréhension et l’amélioration de celles-ci sont un enjeu crucial. Nous avons cherché à améliorer la prédictibilité de certains résultats postopératoires dans le cas d’un LASIK (Laser-Assisted In-Situ Keratomileusis), d’une PKR (Photorefractive Keratectomy) ou d'une chirurgie de la cataracte, et ainsi formuler des recommandations pratiques qui contribueraient au développement de stratégies de traitement davantage personnalisés. Pour cela, nous avons utilisé prospectivement des méthodologies de « contrôle de qualité » des chirurgies sur de larges échantillons de patients. Dans un premier temps, nous avons étudié la dynamique pupillaire dans le cadre de chirurgies au LASIK et notamment le rôle du centre pupillaire, point de référence important dans les stratégies de centrage. Nous avons également évalué la dynamique du diamètre pupillaire et les modifications du segment antérieur sur des yeux subissant une chirurgie de la cataracte. La seconde partie du travail s’est focalisée sur le rôle de l’épithélium dans la topographique cornéenne. Nous avons comparé les topographies spéculaires de l'épithélium et de la couche de Bowman sur des cornées saines et des cornées kératoconiques, présentant une myopie faible à modérée corrigée par PKR. Enfin, dans la dernière partie de notre recherche, nous nous sommes intéressés aux changements de paramètres anatomiques de l'oeil, des performances visuelles et de la qualité de vision subjective survenant dans un échantillon d’yeux myopes après un LASIK réalisé avec le laser WaveLight® Refractive Suite (Alcon® Laboratories Inc., USA). / While the number of ametropic eyes in the world’s population and consequently the use of surgical correction techniques is increasing, understanding and improving these techniques is a crucial issue. We sought to improve the predictability of certain postoperative results in the case of LASIK (Laser-Assisted In-Situ Keratomileusis), PRK (Photorefractive Keratectomy) and cataract surgery, and thus to formulate practical recommendations that would contribute to the development of more personalized treatment strategies. To achieve this objective, we have prospectively used "quality control" methodologies to assess surgeries performed on large samples of patients. First, we studied the pupillary dynamics in LASIK surgery and in particular the role of the pupillary centre, an important point of reference in the centration strategies. We also assessed the dynamics of pupillary diameter and anterior segment changes on eyes undergoing cataract surgery. The second part of the work focused on the role of the epithelium in the corneal topography. We compared specular topographies of the epithelium and Bowman's layer in healthy and keratoconus corneas with mild to moderate myopia corrected by PRK. Finally, in the last part of our research, we were interested in the changes in anatomical parameters of the eye, visual performance and subjective quality of vision occurring in a sample of myopic eyes after LASIK performed with the WaveLight® Refractive Suite (Alcon® Laboratories Inc., USA).

Vitamin A is an essential nutrient; it is not synthesized by mammals and therefore must be obtained through the diet. During times of fasting or dietary vitamin A insufficiency, retinol, the alcohol form of the vitamin is released from the liver, its main storage tissue, for circulation in complex with retinol-binding protein 4 (RBP) to provide an adequate supply to peripheral tissues. Stimulated by Retinoic Acid 6 (STRA6), the transmembrane RBP receptor, mediates retinol uptake across blood-tissue barriers such as the retinal pigment epithelium of the eye, the placenta and the choroid plexus of the brain. Our understanding as to how this protein functions has been greatly enhanced by the high-resolution 3D structure of zebrafish STRA6 in complex with calmodulin (CaM) solved by single-particle cryogenic-electron microscopy. However, the nature of the interaction of STRA6 with retinol remains unclear. Here, I present the high-resolution structures of zebrafish and sheep STRA6 reconstituted in nanodisc lipid bilayers in the presence and absence of retinol. The nanodisc reconstitution system has allowed us to study this protein in a close to physiological environment and examine its interaction with the cell membrane and relationship with its ligand, retinol. We also present the structure of sheep STRA6 in complex with human RBP. The structure of the STRA6-RBP complex confirms predictions in the literature as to the nature of the protein-protein interaction needed for retinol transport. Calcium-bound CaM is bound to STRA6 in the RBP-STRA6 structure, consistent with a regulatory role of this calcium binding protein in STRA6-RBP interaction. The analysis of the three states of STRA6 – pre, post and during interaction with retinol – provide unique insights into the mechanism of STRA6-mediated cellular retinol uptake.

Similarities between the acanthomatous epulis and ameloblastomas resulted in debate regarding the nature and origin of the acanthomatous epulis found in dogs. In an attempt to elucidate the origin and character of the acanthomatous epulides, this study aimed to find suitable cell markers to identify odontogenic epithelium versus oral epithelium in developing dog teeth in order to use in future research on the pathogenesis and pathology of odontogenic neoplasms in dogs. As specific markers for odontogenic epithelium have not been described in dog tissue, proposed markers of odontogenic epithelium of human and rat tissues were tested on developing dog teeth. Keratin 14, keratin 19, amelogenin, p75 neurotrophin receptor and calretinin have been proposed as markers for inner enamel epithelium and/or ameloblasts in human and rat tissue and was therefore included in this study. Keratin 14 and keratin 19 can not be regarded as specific markers of odontogenic epithelium as various other types of epithelium also stained positive with these markers. Amelogenin could be a promising marker to distinguish between odontogenic tumours and non-odontogenic tumours as it was only detected in odontogenic tissues in this study. However, amelogenin has also been observed in other tissues in dogs and rats, and therefore further studies on this protein will be needed to elucidate the expression profile of amelogenin in odontogenic versus non-odontogenic tissues in dogs. p75 Neurotrophin receptor expression was restricted to certain regions of the inner enamel epithelium and no staining was observed in other epithelial cells. It therefore seems to be a promising marker to differentiate between odontogenic and non-odontogenic epithelium, but the widespread staining observed in the mesenchymal tissue makes differentiation between odontogenic and non-odontogenic stromal elements impossible. Calretinin staining was observed in the alveolar epithelial cells directly overlying the developing tooth germ, proposed as the oral epithelium where the dental lamina takes origin from, as well as the dental laminae and Serres rests. No staining was observed in the rest of the oral epithelium and it can therefore be proposed that calretinin could be a useful marker to distinguish between odontogenic and non-odontogenic epithelial cells. In light of the results found in this study on foetal tissue, the expression profile may be different in adult tissue. Odontogenic tumours in adult dogs may originate from remnants of odontogenic tissue like Serres rests and Malassez rests. It is therefore proposed that this study be repeated on adult dog tissue with specific reference to Serres rests, Malassez rests and the associated gingiva Copyright / Dissertation (MSc)--University of Pretoria, 2008. / Oral Pathology and Oral Biology / unrestricted