Cette thèse se découpe en trois parties. Les deux premières portent sur le développement de méthodes pour la construction de modèles géométriques moyens et pour la comparaison de modèles. Plusieurs problématiques sont abordées, telles que la construction d'une cornée moyenne et la comparaison de cornées. Il existe à ce jour peu d'études ayant ces objectifs car la mise en correspondance de surfaces cornéennes est une problématique non triviale. En plus d'aider à développer la connaissance de l'anatomie cornéenne, la modélisation de la cornée normale permet de détecter tout écart significatif par rapport à la normale permettant un diagnostic précoce de pathologies. La seconde partie a pour objectif de développer une méthode pour reconnaître une surface parmi un groupe de surfaces à l’aide de leurs acquisitions pour une application de biométrie. L’idée est de quantifier la différence entre chaque surface et une surface donnée, et de déterminer un seuil permettant la reconnaissance. Deux méthodes sont proposées et une méthodologie en cascade utilisant ces deux méthodes afin de combiner les avantages de chacune est aussi proposée. La troisième et dernière partie porte sur une nouvelle méthode de décomposition en graphes de maillages 3D triangulés. Nous utilisons des cartes de courbures discrètes comme descripteur de forme afin de découper le maillage en différentes catégorie de carreaux. Ensuite un graphe d'adjacence est construit avec un nœud pour chaque carreau. Ces graphes sont utilisés pour extraire des caractéristiques géométriques décrites par des motifs (ou patterns), ce qui permet de détecter des régions spécifiques dans un modèle 3D, ou des motifs récurrents. / This thesis comprises three parts. The first two parts concern the development of methods for the construction of mean geometric models and for model comparison. Several issues are addressed, such as the construction of an average cornea and the comparison of corneas. Currently, there are few studies with these objectives because the matching of corneal surfaces is a non-trivial problem. In addition to help to develop a better understanding of the corneal anatomy, 3D models of normal corneas can be used to detect any significant deviation from the norm, thereby allowing for an early diagnosis of diseases or abnormalities using the shape of the cornea. The second part of this thesis aims to develop a method for recognizing a surface from a group of surfaces using their 3D acquisitions in a biometric application pertinent to the cornea. The concept behind this method is to quantify the difference between each surface and a given surface and to determine the threshold for recognition. Two complementary methods are proposed. A cascading methodology using both methods to combine the advantages of each method is also proposed. The third and final part of this thesis focuses on a new method for decomposing 3D triangulated meshes into graphs. We use discrete curvature maps as the shape descriptor to split the mesh in eight different categories. Next, an adjacency graph is built with a node for each patch. These graphs are used to extract geometric characteristics described by patterns that allow for the detection of specific regions in a 3D model or recurrent characteristics.

No description available.

Introduction: Relation of diabetes mellitus (DM) to the diabetic keratopathy and various stages of corneal nerve fiber damage has been well accepted. A possible association between changes in the cornea of diabetic patients and diabetic retinopathy (DR), DM duration, and age at the time of DM diagnosis were evaluated. Neuropathies are among the most common long-term complications of diabetes mellitus. Good glycemic control is essential in prevention of this complication. DM patients with similar mean glucose levels or glycated hemoglobin (HbA1c) levels often exhibit differences in evaluation of diabetic complications. One reason for these differences may be the differences in glucose variability. DM patients with similar mean glucose levels or HbA1c levels often exhibit differences in glucose variability Hypothesis: Diabetes mellitus damages the subbasal nerve fibers of the corneal and affects the density of epithelial, endothelial and stromal cells. Corneal changes in patients with DM are dependent on the degree of diabetic retinopathy (DR), age at diagnosis, duration of DM, and compensation parameters. Purpose: To compare changes in cell density in individual layers of cornea and status of subbasal nerve fibers in patients with type 1 DM (DM 1) and in healthy subjects. To evaluate the dependence...

No description available.

La mucopolysaccharidose de type VII (MPS VII) est une maladie génétique de surcharge lysosomale provoquée par une déficience en β-D-glucuronidase (β-glu). β-glu est impliquée dans la cascade de dégradation et de recyclage des glycosaminoglycannes (GAGs). Sa déficience provoque une accumulation vésiculaire de GAGs non dégradés engendrant in fine la mort cellulaire. Notre but est de développer et de tester la pertinence des vecteurs adénoviraux canins helper-dépendant (HD-CAV-2) pour traiter la neurodégénération provoquée par la MPS VII dans le cadre d'une thérapie génique dans le SNC de chien. Parce que les vecteurs CAV-2 transduisent préférentiellement les neurones et qu'ils sont transportés de manière rétrograde le long des axones, leurs distributions dans tout le SNC permettraient de délivrer largement la β-glu dans tout le parenchyme cérébral. Nous avons alors étudié la sureté, la durée d'expression, l'efficacité et la possible réversion du phénotype après injections dans le SNC de chiens MPS VII d'un HD-CAV-2 exprimant le gène humain de la GUSB : HD-RIGIE. Des études préliminaires ont montré la faisabilité du transfert des vecteurs HD-CAV-2 dans le SNC, qu'ils induisaient une réponse immunitaire minimale et qu'ils transduisaient préférentiellement, efficacement et largement les neurones.Nous avons produit un HD-RIGIE de qualité pour les injections intracérébrales et nous avons analysé son efficacité sur l'accumulation de GAGs non dégradés. Les injections de HD-RIGIE montrent une augmentation générale de l'activité β-glu dans tout le SNC des chiens MPS VII (sites d'injections et structures éloignées comme le cortex) et ce 1 mois ou 4 mois après les injections. L'analyse de la GFP confirme une distribution globale de HD-RIGIE dans le SNC d'animaux de grande taille. De plus, grâce aux propriétés intrinsèques de la β-glu (transport rétrograde et phénomène de libération/recapture), nous avons observé une diminution générale de l'accumulation vésiculaire neuronale des GAGs non dégradés dans tout le parenchyme cérébral. D'autre part grâce à la stratégie d'isolement et de non vaccination des chiens MPS VII, nous n'observons ni de réponse immunitaire humorale, ni d'aggravation de l'inflammation du parenchyme. / Mucopolysaccharidosis type VII (MPS VII) is a rare inherited lysosomal storage disease caused by β-D-glucuronidase (β-glu) deficiency. β-glu is involved in the physiological turnover of glycosaminoglycans (GAGs). Its deficiency causes accumulation of undegraded GAGs inside vesicles leading to cell death. Our goals are to develop and test the clinical relevance of helper-dependant (HD) canine adenovirus type 2 (CAV-2) vectors to treat neural degeneration caused by MPS VII in a dog model. Because CAV-2 targets preferentially neurons and traffics via axons, the distribution of the transgene throughout the CNS will allow widespread delivery of the missing lysosomal enzyme in these disorders with a minimum number of injections. We tested a HD-CAV-2 vector expressing the human GUS gene in the canine model of MPS VII for their safety, efficacy, duration of expression and possible reversion of the MPS VII induced symptoms.A previous study based on HD-CAV-EGFP vector injections in MPS VII-/- and healthy dogs showed that we are now able to inject HD-CAV-2 in the dog brain, have a minimal induction of the immune response, an efficient transduction of the neurons and an efficient biodistribution of transduced cells. After the production of a suitable vector (HD-RIGIE) for injections in the CNS of MPS VII dogs we analysed its efficiency on GAGS storage in neurons.Injections of HD-RIGIE showed after 1 month or 4 months post injections a widespread increase in general level of β-glu activity, in the sites of injections and in distant areas such as cortex. Analysis of GFP, also permit to observe a widespread biodistribution of the vector. Because of β-glu property of cross-correction we observed a global decreased in GAGs storage in the entire MPS VII brains. Finally, the dogs did not present humoral immune response and no aggravation of inflammation

Hintergrund: Die Excimer Laser PRK zur Korrektur der geringen bis moderaten Myopie wird als präzises Verfahren der refraktiven Chirurgie angesehen und weltweit angewandt. Die Genauigkeit der PRK nimmt jedoch mit steigender Korrektur ab. Das Ziel dieser Untersuchung bestand darin, den Einfluß unterschiedlicher Laserstrahlapplikations- und -formungssysteme auf die korneale Oberflächenstruktur, das Hornhautprofil und die Zielrefraktion zu untersuchen. Materialien und Methoden: Es wurden mit dem Meditec Mel 60 und dem Schwind Keratom, zwei Excimer Laser der Wellenlänge 193 nm, an jeweils 10 enukleierten Schweinehornhäuten eine PRK mit einer Zielkorrektur von -3, -6, -9 D mit einem Ablationsdurchmesser von 6 mm (5 mm - 9D) durchgeführt, ein Silikonabdruck von der Hornhautoberfläche angefertigt und mit einem dynamisch fokussierenden Topometrie System UBM Microfocus vermessen. Hornhautradius, Brechkraft der stromalen Oberfläche, Profilabweichungen von der idealen sphärischen Form sowie Rauheitsparameter der verschiedenen Zonen wurden ermittelt. Ergebnisse: Epithelfreie Hornhäute besitzen ein sphärisches Profil. Der Meditec Mel60 Laser erzielte nach PRK von -3, -6 und -9 D sphärische Profile mit einer durchschnittlichen Refraktionsänderung von -3.4, -6.7 und -8.7 D. Das Schwind Keratom erzeugte eine mittlere Refraktionsänderung von -3.5, -5.8 und 8.4 D, wobei das korneale Ablationsprofil in allen Korrekturgruppen erhebliche Profilabweichungen in Form von zentralen Profilkuppen aufwies. Die durchschnittliche Höhe der zentralen Profilkuppen betrug nach der -3 D PRK 7.39 (±0.34) µm und stieg auf 16.31 (±1.06) µm bzw. 15.06 (±0.96) µm in der -6 und in der -9 D Serie. Die Relation zwischen der Profilkuppenhöhe und der Abtragtiefe lag zwischen 21 - 25% und konnte durch eine Anti-Central-Island Programm (ACI 100%) nur um 4% auf 18-20 % der Abtragtiefe reduziert werden. Die stromale Oberfläche der unbehandelten, epithelfreien Kontrollgruppe hat eine glatte, homogene Struktur. Die Rauheit der stromalen Oberfläche nach Ablation mit dem Meditec Laser war um 50 % stärker ausgeprägt als beim Schwind Keratom. Diskussion: Die Beschaffenheit der stromalen Oberfläche nach der Excimer Laser PRK zur Korrektur der Myopie, wird durch die Rauheitsparameter quantitativ beschrieben und ermöglicht den direkten Vergleich zwischen den Lasersystemen. Die Rauheit ist positiv mit der Ablationstiefe und dem Ablationsdurchmesser korreliert. Je höher eine myope Korrektur angestrebt wird, desto rauher ist die stromale Oberfläche und damit das Risiko, dass sich eine epitheliale Hyperplasie und subepitheliale Trübungen entwickeln, die ursächlich mit den klinisch beobachteten Phänomenen der myopen Regression, der Abnahme der Kontrastsehschärfe, dem Verlust der bestkorrigierten Sehschärfe und monokularer Doppelbilder in Zusammenhang stehen. Ob der Unterschied der Rauheit von durchschnittlich 50% sich in der Inzidenz der klinischen Komplikationen widerspiegelt, können nur vergleichende Studien belegen. Sicher ist, das die Vorhersagbarkeit des refraktiven Ergebnisses durch die Ablation mit dem Aesculap Meditec MEL 60 Laser besser einzuschätzen ist, als das mit zentralen Profilkuppen komplizierte Ablationsprofil des Schwind Keratoms. / Purpose: To evaluate the predictability of refractive outcome, sphericitiy of corneal profiles and surface roughness parameters after myopic PRK with different, commercially available excimer laser beam delivery and beam shaping systems. Materials and Methods: Myopic Excimer Laser PRK of -3, -6 and -9 D in 6mm ablation zone (5 mm in -9 D) on performed on porcine eyes was performed with the Aesculap Meditec Mel 60, a slit scanning Laser and the Schwind Keratom I, a broad area laser with band mask beam shaping. A silicone replica was obtained to conserve the corneal profile and measured with a dynamic focusing topometry system (UBM Microfocus) to obtain radius, corrected corneal refraction and corneal surface roughness parameters. Results: Untreated corneas of the control group displayed spherical profiles. PRK of intended -3, -6 and -9 D correction with the slit scanning Aesculap Meditec Mel60 excimer laser achieved a refractive change of an average -3.4, -6.7 and -8.7 D respectively without major profile deviations. PRK with the Schwind Keratom, a broad area beam excimer laser resulted a refractive change of -3.5, -5.8 and -8.4 D respectively. The Ablation created considerable central profile deviations representing central islands of 7.39 (±0.34) µm after -3 D, 16.31 (±1.06) µm and 15.06 (±0.96) µm height after - 6 and -9 D PRK, respectively. Mean central island height was 21 - 25% of ablation depth and was reduced by anti-central-island-program to 18-20 % of ablation depth. Stromal surface roughness increased with ablation depth and was significantly rougher after scanning beam ablation compared to broad area ablation. Conclusions: Profile deviations increase with higher corrections and lessen the predictability of the refractive results. The Aesculap Meditec MEL60 Slit scanning system creates predictable spherical corneal profiles. The Schwind Keratom broad area laser create with band mask beam shaping central islands increasing with higher corrections. The application of an Anti-Central-Island Program does not eliminate the central profile elevations sufficiently. Stromal surface was rougher after scanning beam compared to broad area beam ablation.

INTRODUÇÃO: O endotélio corneal exerce papel primordial para a fisiologia da córnea. Seus dados morfológicos gerados pelo microscópio especular (MEC) como densidade endotelial (DE), área celular média (ACM), coeficiente de variação (CV) e porcentagem de células hexagonais (HEX) são importantes para avaliar sua vitalidade. Para interpretar estes dados de forma padronizada e reprodutível, foi utilizado um programa estatístico de análise amostral, Cells Analyzer PAT. REQ.(CA). OBJETIVO: Demonstrar valores de referência para DE, ACM, CV e HEX. Demonstrar o percentual de células endoteliais marcadas e desconsideradas no exame ao marcar-se 40, 100 e 150 células em uma única imagem do mosaico endotelial e o perfil do intervalo de confiança (IC) das variáveis estudadas ao se considerar 40, 100, 150 e tantas células quantas indicadas pelo CA. Demonstrar o erro amostral de cada grupo estudado. MÉTODOS: Estudo transversal. Os exames de MEC foram realizados com o aparelho Konan NONCON ROBO® SP-8000, nos 122 olhos de 61 portadores de catarata (63,97 ± 8,15 anos de idade). As imagens endoteliais caracterizaram se pelo número de células marcadas e consideradas para cálculo dos seguintes dados: DE, ACM, CV e HEX. Os grupos foram formados de 40, 100, 150 células marcadas numa única imagem endotelial e Grupo CA em que foram marcadas tantas células quanto necessárias em diferentes imagens, para obter o erro relativo calculado inferior ao planejado (0,05), conforme orientação do programa CA. Estudou-se o efeito do número de células sobre IC para as variáveis endoteliais utilizadas. RESULTADOS: A média dos valores de referência encontrados para DE foi 2395,37 ± 294,34 cel/mm2; ACM 423,64 ± 51,09 m2; CV 0,40 ± 0,04 e HEX 54,77 ± 4,19%. O percentual de células endoteliais desconsideradas no Grupo 40 foi 51,20%; no Grupo 100, 35,07% e no Grupo 150, 29,83%. O número médio de células calculado inicialmente pelo CA foi 247,48 ± 51,61 e o número médio de células efetivamente incluídas no final do processo amostral foi 425,25 ± 102,24. O erro amostral dos exames no Grupo 40 foi 0,157 ± 0,031; Grupo 100, 0,093 ± 0,024; Grupo 150, 0,075 ± 0,010 e Grupo CA, 0,037 ± 0,005. O aumento do número de células diminuiu a amplitude do IC nos olhos direito e esquerdo para a DE em 75,79% e 77,39%; ACM em 75,95% e 77,37%; CV em 72,72% e 76,92%; HEX em 75,93% e 76,71%. CONCLUSÃO: Os valores de referência da DE foi 2395,37 ± 294,34 cel/mm2; ACM foi 423,64 ± 51,09 m2; CV foi 0,40 ± 0,04 e HEX foi 54,77 ± 4,19%. O percentual de células endoteliais desconsideradas no Grupo 40 foi 51,20%; no Grupo 100 foi 35,07% e no Grupo 150 foi 29,83%. O programa CA considerou correto os exames nos quais 425,25 ± 102,24 células foram marcadas entre duas e cinco imagens (erro relativo calculado de 0,037 ± 0,005). O aumento do número de células diminuiu a amplitude do IC para todas as variáveis endoteliais avaliadas pela MEC / INTRODUCTION: Corneal endothelium plays an important role in physiology of the cornea. Morphological data generated from specular microscope such as endothelial cell density (CD), average cell area (ACA), coefficient of variance (CV) and percentage of hexagonal cells (HEX) are important to analyze corneal status. For a standard and reproducible analysis of the morphological data, a sampling statistical software called Cells Analyzer PAT. REC (CA) was used. PURPOSE: To determine normal reference values of CD, ACA, CV and HEX. To analyze the percentage of marked and excluded cells when the examiner counted 40, 100, 150 cells in one endothelial image. To analyze the percentage of marked and excluded cells according to the statistical software. To determine the confidence interval of these morphological data. METHODS: Transversal study of 122 endothelial specular microscope image (Konan, non-contact NONCON ROBO® SP- 8000 Specular Microscope) of 61 human individuals with cataract (63.97 ± 8.15 years old) was analyzed statistically using CA. Each image was submitted to standard cell counting. 40 cells were counted in study Group 40; 100 cells were counted in study Group 100; and 150 cells were counted in study Group 150. In study group CA, the number of counted cells was determined by the statistical analysis software in order to achieve the most reliable clinical information (relative error < 0,05). Relative error of the morphological data generated by the specular microscope were then analyzed by statistical analysis using CA software. For Group CA, relative planned error was set as 0.05. RESULTS: The average normal reference value of CD was 2395.37 ± 294.34 cells/mm2, ACA was 423.64 ± 51.09 m2, CV was 0.40 ± 0.04 and HEX was 54.77 ± 4.19%. The percentage of cells excluded for analysis was 51.20% in Group 40; 35.07% in Group 100; and 29.83% in Group 150. The average number of cells calculated initially by the statistical software was 247.48 ± 51.61 cells and the average number of cells included in the final sampling process was 425.25 ± 102.24 cells. The average relative error was 0.157 ± 0.031 for Group 40; 0.093 ± 0.024 for Group 100; 0.075 ± 0.010 for Group 150 and 0.037 ± 0.005 for Group CA. The increase of the marked cells decreases the amplitude of confidence interval (right and left eyes respectively) in 75.79% and 77.39% for CD; 75.95% and 77.37% for ACA; 72.72% and 76.92% for CV; 75.93% and 76.71% for HEX. CONCLUSION: The average normal reference value of CD was 2395.37 ± 294.34 cells/mm2, ACA was 423.64 ± 51.09 m2, CV was 0.40 ± 0.04 and HEX was 54.77 ± 4.19%. The percentage of excluded cells for analysis was 51.20% in Group 40; 35.07% in Group 100 and 29.83% in Group 150. CA software has considered reliable data when 425.25 ± 102.24 cells were marked by the examiner in two to five specular images (calculated relative error of 0.037 ± 0.005). The increase of the marked cells decreases the amplitude of confidence interval for all morphological data generated by the specular microscope

By performing microRNA microarrays to globally detect any novel miRNAs in the limbus, eleven microRNAs (hsa-miR-136, hsa-miR-373*, hsa-miR-150, hsa-miR-143, hsa-miR-455, hsa-miR-145, hsa-miR-381, hsa-miR-224, hsa-miR-338, hsa-miR-154, hsa-miR-377) were found to be upregulated while two microRNAs (hsa-miR-122a and hsa-miR-425-3p) were identified as downregulated by more than 2 folds. Among these, hsa-miR-143 and hsa-miR-145 were distingushed to be the most significantly up-regulated limbal miRNAs. Individual assessement of the microarray results of a recently reported stem cell specific microRNA, hsa-miR-21, were also upregulated by more than two thousand fold when comparing limbus and cornea. miR-21, miR-143 and miR-145 were therefore selected as the most likely microRNA candidates in the present study. The expression level of these miRNA candidates were validated and confirmed by quantitative reverse transcription polymerase chain reaction (qRT-PCR). To localize these candidates, we performed in situ hybridization on frozen corneal rim sections using locked nucleic acid (LNA)-modified oligonucleotide probes. Results showed that miR-2I, 143 and 145 were confined in the limbal region with gradation of expression level along the basal-suprabasal line. / Functional roles of these microRNAs were then deciphered by overexpressing human corneal epithelial cell line (HCE) with precursor microRNAs (pre-miRs) through lipophilic transfection. Results showed that high endogenous level of miR-145 could inhibit cell proliferation by 3.5 fold as shown from MTT proliferation assay at day 5, and could generate discrete spherical colonies that resembles the morphology of holoclones at day 8, but not the other two candidate miRNAs. / In conclusion, 1 have identified three novel microRNAs (hsa-miR-21, 143, 145) which were precisely upregulated in the limbus region, while miR-145 was being the most limbal specific. In addition, the functions of miR-145 were found to be inhibitory on cell proliferation, possibly through the indirect regulation of IFNB1. These unprecedented results may suggest a therapeutic potential of miR-145 on limbal stem cell deficiency and limbal tumors because miR-145 can affect cell survival and proliferation. / MicroRNAs is a family of small non-coding RNAs that, in human, binds imperfectly to the 3' untranslated region (UTR) of target mRNAs for translational repression or negative regulation. Recent studies have shown that such negative regulatory pathways may play pivotal roles in the maintenance of asymmetric cell division in embryonic and tissue specific stem cells. Human corneal epithelial progenitor cells (CEPC), a tissue specific stem cell lineage residing between cornea and conjunctiva in the Palisade of Vogt of the limbus region, is known to maintain corneal homeostasis throughout human life. They respond to injury and normal wearing by rapid proliferation and differentiation into transit amplifying cells (TACs) and eventually corneal epithelial cells, though the biological factors controlling this homeostatic switch are still largely unknown. Here I hypothesized that microRNAs can participate in CEPC regulation. Experiments elucidating the anatomical distribution and functional roles of microRNAs on the human cornea rims were performed to testify this proposition. / Protocols aim at enriching the CEPC population were then devised. For the first time a four parameter cell sorting system utilizing ABCG2, Connexin 43, Notch 1 and pyronin Y as markers was established for the prospective in vitro study. Nevertheless, manual microdissection isolating the limbus region and the cornea region was employed for the present study of microRNAs. / This study begins with the phenotypic validation of human cornea rims recruited from the Chinese Hong Kong population using immunohistochemistry. Conventional CEPC markers (p63, EGFR, cytochrome oxidase and cytokeratin 15), embryonic stem cell marker (stat1) and cancer stem cell markers (p73, MDM2 and pStat1) were expressed in the limbus region, suggesting that these specimens contained a source of CEPC for attesting our hypothesis. / To determine the mRNA targets of candidate microRNAs in HCE cells, Whole Human Genome Oligo Microarray Kits (Agilent Technologies) which contained 41K human genes and transcripts were employed. When compared to the scrambled control, HCE cells over-expressed with hsa-miR-21, 143 or 145 revealed differential expression of genes that participate in cell activation, motility and proliferation. Of note, interferon beta 1 fibroblast (IFNB1), a gene that is often deleted or rearranged in cancers, was significantly upregulated by a medium of 1093 fold in pre-miR-145 treated cells as confirmed by real time PCR assays. / Lee, Sharon Ka-wai. / "December 2009." / Advisers: Calvin Chi-Pui Pang; Gary Hin Fai Yam. / Source: Dissertation Abstracts International, Volume: 72-01, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 216-252). / 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 Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Objetivos: Os objetivos deste estudo foram: 1) determinar a eficácia da transferência gênica do vetor de adenovírus sorotipo 5, carreando o gene do receptor do fator de crescimento endotelial vascular (VEGF) solúvel humano (sVEGFR1) para a glândula lacrimal (GL); 2) investigar se a expressão de sVEGFR1 interfere na neovascularização da córnea (NVC), induzida por queimadura alcalina; 3) avaliar a segurança do procedimento. Métodos: Trinta e dois ratos Wistar foram submetidos à queimadura central da córnea direita com solução de hidróxido de sódio (NaOH) 1 M. Os animais foram divididos em três grupos e injetados diretamente em sua GL direita 25 ?l de vetores virais AdVEGFR1 (1x1010 pfu) (12 animais), 25 ?l do vetor AdNull (1x1010pfu) (10 animais), ou 25 ?l de solução salina (Controle). Após sete dias, a NVC foi observada e fotografada na lâmpada de fenda. A secreção lacrimal foi medida com fenol. A presença do sVEGFR1 na GL foi testada por qPCR (quantitative polymerase chain reaction) e a coloração, por imunofluorescência. O qPCR foi também utilizado para comparar o RNA mensageiro (RNAm) de ilterleucina-1beta (IL-1?), ilterleucina-6 (IL-6) e fator de necrose tumoral alfa (TNF-?) na GL e no gânglio do trigêmeo (GT). Resultados: O vetor AdVEGFR1 transfectou 83% dos ratos. O sVEGFR1 estava presente nas células acinares da GL. A NVC foi prevenida em nove de doze animais do grupo AdVEGFR1, em comparação com o grupo Ad-Null (3:10) e o grupo Controle (1:10) (p=0,0317). A secreção lacrimal e o RNAm das citocinas na GL e no GT foram semelhantes nos três grupos (p>0,05). Conclusões: A transferência gênica do vetor adenoviral para a GL demonstrou expressão local do sVEGFR1 humano, e evitou a NVC na maioria dos olhos expostos a queimaduras alcalinas, se mostrando seguro para a estrutura e função da GL. / Purpose: The aims of this study were: 1) to determine the efficacy of adenovirus vector serotype 5 (Ad) encoding human soluble VEGF receptor 1 (sVEGFR1) gene transfer to the lacrimal gland (LG); 2) to investigate whether expression of sVEGFR1 acts in corneal neovascularization (CNV), induced by alkali burn and; 3) to evaluate the safety of the procedure. Methods: AdVEGFR1viral vectors (25 ?l, 1x1010pfu) were injected in the right LG of rats and compared with AdNull vector (25 ?l, 1x1010pfu) or 25?l saline (Control) before cornea alkali burn with 1 M NaOH. After seven days, CNV was observed and photographed in the slit lamp. Tear secretion was measured with phenol red thread. The animals were tested for human VEGFR1 mRNA and protein in the LG by qPCR and immunofluorescence staining, respectively. qPCR was also used to compare the mRNA of IL-1?, IL-6, and TNF-? in LG and ipsilateral trigeminal ganglion (TG). Results: Ad-VEGFR1 transfected 83% of the rats. VEGFR1 was present in LG acinar cells. CNV was prevented in 9 of 12 animals of Ad-VEGFR1 group, compared to Ad-Null (3:10) and Control (1:10) (p=0.0317). The tear secretion and the cytokines mRNA in LG and TG were similar all three groups (p>0.05). Conclusion: Adenoviral vector gene transfer to LG as the has shown local expression of human sVEGFR1, as It prevented CNV in most of the eyes exposed to alkali burn and was safe for LG structure and function.

Marine turtle numbers are in a state of flux around the world. Six of the seven remaining species of these long-lived animals are threatened; with the seventh being listed data deficient. Reasons for these fluctuations are speculated to be due to human related impacts (direct) and increase in disease occurrence caused by changes in the natural environment (indirect). Most direct impacts have been identified and strategies implemented to mitigate their effects with varying degrees of success; however the indirect effects on marine animals remain an understudied area. This thesis outlined the development of ante- and post-mortem diagnostic techniques to identify prevalent diseases affecting two marine turtle species in southern Queensland over a four year (2006-2009) period. This data was used to determine the impact of disease on turtle survivorship. Two-hundred and ninety green turtles (Chelonia mydas) from Moreton and Shoalwater Bays were captured, clinically assessed and blood sampled. Clinically healthy animals (n = 211) were used to derive biochemical and haematological reference intervals using two methods. Comparisons with clinically unhealthy animals (n = 25) indicated all unhealthy animals had at least some plasma biochemical and haematological values outside the derived intervals (albumin, 48% of unhealthy animals; alkaline phosphatase (ALP), 35%; aspartate transaminase (AST), 13%; creatinine, 30%; globulin, 3%; glucose, 34%; lactic dehydrogenase (LDH), 26%; phosphorus, 22%; sodium, 13%; thrombocytes, 57%; and monocytes, 5%). Amongst small immature animals, those with Chelonibia testudinaria plastron barnacle counts of at least 20 were approximately three times more likely to be unhealthy than turtles with no barnacles. In addition, small immature and mature turtles were more likely to be unhealthy than large immature turtles (Chapter 2). By the same method, 101 loggerhead turtles (Caretta caretta) in Moreton Bay were assessed and bled. Clinically healthy animals (n = 63) were used to derive intervals. Comparisons with clinically unhealthy animals (n = 23) indicated 82% and 45% had at least one biochemical and hematological result, respectively, outside of at least one of the calculated intervals. Neither sex nor maturity (mature versus large immature) influenced the risk of being clinically unhealthy (Chapter 3). A standardised approach to post-mortem examination of marine turtles for veterinary clinicians with a concurrent descriptive review of gross and microscopic pathological lesions commonly seen during examination in Australia (Chapter 4) was used to accurately determine diseases and causes of death in 100 green turtles submitted from various regions of southern Queensland for examination. Spirorchiid parasitism was found to be the most frequently occurring cause of mortality (41.8%), followed by gastrointestinal impaction (11.8%), microbiological infectious diseases (5.2%) and trauma (5.2%). Spirorchiid parasitism with associated inflammation (75%) was the most frequently occurring disease followed by gastrointestinal impaction (5.1%). Season and turtle age had limited influences on disease. Severity of spirorchiidiasis in the brain was independent of severity in other organs (Chapter 5). From these examinations, the most prevalent disease syndrome (spirorchiidiasis) and a previously unreported finding in Australian waters (corneal fibropapillomatosis) were selected to be examined in greater detail. Spirorchiid parasites from four organs in five green turtles were identified by established morphological and molecular techniques. Morphological study of adults identified Carettacola sp. in the serosal wall of the gastrointestinal tract, Hapalotrema mehrai in the heart and Learedius learedi in the spleen. Worms from the brain probably belonged to the genus Neospirorchis. DNA sequences from a portion of the 28S ribosomal RNA gene were obtained; but only matches for Hapalotrema mehrai and Learedius learedi were made. The prevalence and severity of this disease warrants further investigation into development of molecular techniques for use as a prognostic tool for turtles entering rehabilitation (Chapter 6). Chelonid corneal fibropapillomatosis, a previously unreported disease manifestation in Australia, was identified in 0.5% of 787 examined green turtles in 2008 (Chapter 7). This novel syndrome was shown to reduce visibility, potentially negatively affecting turtle survivorship and should be monitored for further spread. Findings from this thesis and the published literature were used to derive a mathematical model to determine the effects of identified diseases on Moreton Bay green turtle survivorship. This model demonstrated diseases at current prevalence will not negatively affect survivorship but an adverse environmental disruption or an increase in current disease frequency may threaten these animals (Chapter 8). Information presented in this thesis was used to test the general hypothesis ‘Differences in disease and health between stranded and functional populations of marine turtles will indicate major and currently unmeasured causes of population decline.’ This hypothesis was partially upheld. Differences in disease and health status between stranded and functional populations were demonstrated, but more work is required to comprehensively examine these statuses. Diagnostics and continued environmental assessment should become the focus of future investigations. These findings should be incorporated in future management strategies.