Submitted by Cláudia Bueno (claudiamoura18@gmail.com) on 2016-01-20T13:43:03Z No. of bitstreams: 2 Dissertação - Bruno Moraes Assis - 2015.pdf: 6677163 bytes, checksum: a2704584ebce8e9d25969d79a0fc639e (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-01-21T07:11:50Z (GMT) No. of bitstreams: 2 Dissertação - Bruno Moraes Assis - 2015.pdf: 6677163 bytes, checksum: a2704584ebce8e9d25969d79a0fc639e (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2016-01-21T07:11:50Z (GMT). No. of bitstreams: 2 Dissertação - Bruno Moraes Assis - 2015.pdf: 6677163 bytes, checksum: a2704584ebce8e9d25969d79a0fc639e (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2015-10-29 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Despite not being a common subject of studies about buffalo, the incidence of hulls diseases has important scientific aspects as these animals, as they are breeded in adverse conditions, have different resistance for the occurrence of podais diseases when compared to cattle, especially dairy cattle. The knowledge about microstructural structures of buffalo’s hoof generated important information for understanding of the intrinsic resistance of buffaloes foot diseases. This study aimed to investigate the microstructure of adult Jafarabadi buffaloes, through histomorphometric ratings, two-dimensional microtomography, three-dimensional, nanohardness test and biochemical composition. Histomorphometric ratings revealed important features not yet investigated by other researchers, highlighting the structural behavior of epidermal papillae in different regions of corneal layer, the morphology and disposition of the horn tubules, the architecture of extratubular and intratubular keratin and even organization of melanosomes . It was observed that the average length of buds, thickness and spacing in pelvic and thoracic digit did not show statistically significant differences. However, an increase in thickness of the laminar corium papillae of the wall and spacing was observed when comparing the average of the coronary corium and wall and sole laminar corium. When compared to the findings of other researchers, this study have shown that the epidermal papillae on the hulls of these animals are presented larger than the epidermal papillae of Holstein and Gir animals, suggesting that the buffalo’s has greater keratin production and better epidermis and horny case fixing. It can be conjectured that such characteristics are associate with greater resistance to foot diseases of this animals, although additional studies are needed. Analysis of microtomography and nanohardness test showed increased medial digit of forelimbs and side-digit of hindlimbs exhibit increased horn tubules with larger diameters. But the abaxial wall of forelimbs digit showed higher values than hindlimbs digit. Even there was not significant difference between the digits analyzed, this study could clarify important aspects of the nanohardness of different regions of the corneal case of buffalo’s digits. 3D microtomography analysis revealed that the dorsal wall presents a greater hardness compared to the abaxial wall of intermediate hardness, and with the sole of the hoof, that showed the lowest hardness among the evaluated areas. It was inferred that tridimensional microtomography testing is more specific than Vickers nanohardness test for this structure. However, further research are required. Biochemical tests generated parameters about structural elements of the corneal case, which showed different concentrations in relation to the region and anatomical position of the hull. These differences are related to the digits wich recive higher load of body weight and impact. This study generated knowledge and parameters to be used in further researches on different bovine species and breeds. / As enfermidades de cascos, apesar de ser alvo de poucos estudos na espécie bubalina, apresentam aspectos científicos importantes uma vez que estes animais, mesmo criados em condições adversas, apresentam resistência diferenciada quanto à ocorrência de doenças podais, quando comparados aos bovinos, com destaque para os de aptidão leiteira. Acredita-se que o conhecimento microestrutural do casco de bubalinos gerou informações importantes para melhor entendimento da resistência podal intrínseca dos bubalinos às enfermidades podais. O presente estudo teve por finalidade pesquisar a microestrutura do estojo córneo de búfalas adultas, raça Jafarabadi, por meio de avaliações histomorfométricas, microtomografia bidimensional, tridimensional, teste de nanodureza e composição bioquímica. As avaliações histomorfométricas em bubalinos revelaram características importantes ainda não investigadas por outros pesquisadores, destacando o comportamento estrutural das papilas epidérmicas nas diferentes regiões do estojo córneo, a morfologia e disposição dos túbulos córneos, a arquitetura da queratina extratubular e intratubular e ainda organização dos melanossomas. Pode-se observar nessas avaliações que as médias de comprimento das papilas, espessura e espaçamento nos dígitos dos membros pélvicos e torácicos não apresentaram diferenças estatísticas significativas entre si. Todavia, ao comparar as médias do córion coronário, córion laminar da muralha e sola, observou-se entre as regiões do estojo córneo, um aumento da espessura das papilas do córion laminar da muralha e do espaçamento comparando-se as demais regiões. Os achados dessa pesquisa quando comparados aos achados de outros pesquisadores, demonstraram que as papilas epidérmicas nos cascos desses animais, apresentam-se maiores que as papilas epidérmicas de bovinos das raças, Holandesa e Gir, sugerindo que possuem maior capacidade de produção de queratina e melhor fixação da epiderme ao estojo córneo. Contudo, pôde-se conjecturar tais características com a maior resistência desses animais as enfermidades podais, embora haja a necessidade de estudos adicionais. Por meio das análises de microtomografia e teste de nanodureza, pode-se verificar que os dígitos mediais dos membros torácicos e os dígitos laterais dos membros pélvicos apresentaram maior quantidade de túbulos córneos com diâmetros maiores. Porém, a muralha abaxial dos dígitos dos membros torácicos apresentaram valores superiores aos dígitos dos membros pélvicos. Apesar de não ter sido observada diferença significativa entre os dígitos estudados, o presente estudo pôde esclarecer, ainda, aspectos importantes sobre a nanodureza das diferentes regiões do estojo córneo dos dígitos de bubalinos. A análise de microtomografia 3D revelou que a muralha dorsal apresenta maior dureza quando comparada com a muralha abaxial, de dureza intermediária, bem como, com a sola do casco, que apresentou menor dureza entre as regiões avaliadas. Inferiu-se assim que o teste de microtomografia tridimensional seja mais específico que o teste de nanodureza de Vickers para a estrutura considerada. No entanto, tal afirmação requer pesquisas adicionais. Os testes bioquímicos geraram parâmetros quanto aos elementos estruturais do estojo córneo, que demonstraram diferentes concentrações em relação à região e posição anatômica do casco, sendo essas diferenças associadas aos dígitos que recebem maior sobrecarga de massa corporal e impacto. O presente estudo gerou conhecimento e parâmetros a serem utilizados em outras investigações científicas, em outras espécies e raças de bovídeos.

OBJETIVOS: Validar o uso de questionários multidimensionais, como o Inventário Resumido da Dor (BPI) e o Questionário de Dor de McGill (MPQ) no pós-operatório da ceratectomia fotorefrativa (PRK). Comparar o perfil da dor no pós-operatório da PRK entre os dois olhos operados sob as mesmas condições e verificar preditores de dor como sexo, estado de ansiedade, conhecimento prévio da cirurgia e equivalente esférico do erro refrativo (EEER). MÉTODOS: Oitenta e seis olhos de 43 pacientes submeteram-se à PRK com intervalo de 14 dias entre cada olho. Uma hora antes da cirurgia, os pacientes responderam ao Inventário de Estado de Ansiedade (IDEA). No pós-operatório os pacientes receberam tratamento usual para dor e responderam aos questionários Escala Visual Analógica (EVA), BPI e MPQ após uma, 24, 48, 72 e 96 horas. Estudaram-se a consistência interna e as correlações de cada questionário. Compararam-se as pontuações de dor e a ansiedade entre primeiros e segundos olhos operados usando o teste de Wald, pareados através do teste t de Student. Utilizou-se o teste de Wald para comparar o comportamento da dor de acordo com sexo e EEER. RESULTADOS: Os questionários MPQ e BPI demonstraram alta consistência interna. Os questionários apresentaram pontuações mais elevadas na primeira mensuração da EVA (4.93 ± 2.38), MPQ - Índice de Estimativa de Dor (PRI) (26.95 ± 10.58), BPI - Índice de Intensidade de Dor (IID) (14.53 ± 7.36) e o BPI - índice de Interferência Funcional de Dor (IIFD) (22.30 ± 15.13), reduzindo-se gradativamente a cada momento subseqüente de avaliação. O MPQ-PRI na subescala subjetiva, apresentou curva de dor com redução lentificada. Todas as escalas apresentaram redução média estatisticamente significativa de um momento para o outro (p < 0.05) no pósoperatório, exceto no MPQ-PRI Subjetivo. Observaram-se correlações positivas entre as subescalas BPI e MPQ com a EVA (p < 0.05). Não houve diferença estatisticamente significativa nas pontuações de dor da EVA, BPI e MPQ-PRI entre ambos os olhos para todos os momentos avaliados. Os pacientes estavam menos ansiosos antes da PRK do segundo olho (p < 0.001), mas isto não apresentou correlação com níveis de dor após a cirurgia. O sexo e o conhecimento prévio do procedimento cirúrgico não influenciou significativamente em qualquer das escalas de dor. O EEER entre -3D to -5D correlacionou-se (p=0.035) com o BPI. CONCLUSÃO: O BPI e o MPQ apresentaram boas propriedades psicométricas em relação a confiabilidade e validade. Questionários multidimensionais fornecem uma avaliação mais abrangente sobre o perfil de dor após a PRK, se comparados à EVA, principalmente nos aspectos afetivos e cognitivos. O perfil da dor pósoperatória da PRK apresentou-se similar em ambos os olhos sob as mesmas condições. O EEER entre -3D to -5D foi o único fator preditor deste estudo para elevado nível de dor pós-operatória / PURPOSE: to validate the use of multidimensional questionnaires, such as the Brief Pain Inventory (BPI) and the McGill Pain Questionnaire (MPQ) in the postoperative photorefractive keratectomy (PRK). To compare the profiles of postoperative PRK pain between both eyes operated under the same conditions and to verify the preoperative predictors of pain such as gender, anxiety, knowledge of the procedure, and spherical equivalent refractive error (SERE). METHODS: eighty-six eyes of 43 patients with myopia underwent PRK in both eyes at an interval of 14 days between the procedures. One hour before surgery, subjects answered the State Anxiety Inventory (SAI). After surgery, usual PRK pain treatment was given and subjects answered to the Visual Analogue Scale (VAS), BPI and MPQ pain questionnaires at one, 24, 48, 72 and 96 hours intervals. The internal consistency was evaluated and convergent validity of each questionnaire was assessed using correlation testing. Pain scores and anxiety were compared between each eye using the Wald test and paired Student t test. Wald test was also used to test gender and SERE for each eye separately. RESULTS: both BPI and MPQ questionnaires showed internal consistency higher than 0.70. Subjects reported higher postoperative pain scores at the first measurement of the VAS (4.93 ± 2.38), MPQ - Pain Rating Index (26.95 6 10.58), BPI - Pain Severity Index (14.53 ± 7.36), and BPI- Pain Interference Index (22.30 ± 15.13) with decreasing scores at each subsequent observation period in all scales. All scales showed statistically significant (p < 0.05) pain reduction from one measurement to the next postoperatively, except the MPQ-PRI Evaluative. The majority of the scales and subscales showed a statistically significant (p < 0.05) direct correlation with the VAS at all of the evaluation periods. There were no statistically significant differences between the two eyes at all examination intervals regarding the VAS, BPI, and MPQ scores. Subjects were less anxious on average before the second surgery compared to the first surgery (p < 0.001), but this finding was not related to pain ratings after surgery. Gender and knowledge of the procedure did not significantly interfere with any scale of pain. The SERE between -3 D (diopters) and -5 D (p=0.035) revealed interference on the BPI. CONCLUSION: the BPI and the MPQ showed good psychometric properties regarding reliability and validity. The multidimensional questionnaires expanded the assessment of the PRK postoperative pain profile, compared to VAS, mainly in cognitive and affective aspects. The profiles of postoperative pain after PRK were similar between both eyes under the same conditions. In this study, a high SERE was the only predictor for increased pain after PRK

Le cross-linking du collagène cornéen (CXL) est une cornéoplastie mini-invasive reposant surun concept biomécanique difficile à objectiver physiquement et dont les preuves del’efficacité thérapeutique sont d’interprétation complexe. Les principes, les nuances et lesrésultats du CXL sont colligés dans cette thèse afin de valider l’intérêt du modèleexpérimental choisi pour tester la pertinence de notre travail de recherche sur l’élastographiecornéenne par ondes de cisaillement.Notre cheminement expérimental a abouti au choix du modèle de CXL trans-épithélial (TCXL)assisté par iontophorèse (I-CXL), réalisé in vivo, sur oeil de lapin. Les mesuresélastographiques obtenues après euthanasie ont ainsi pu démontrer une modificationsignificative du profil d’élasticité de la cornée après CXL, testé successivement de manièredynamique et statique.Nos résultats confirment donc l’efficacité biomécanique instantanée du I-CXL et donnent uneidée plus précise de la valeur de la photo-polymérisation du tissu cornéen isolée desphénomènes liés à la cicatrisation. Les enjeux technologiques de l’élastographe cornéen paranalyse des ondes de cisaillement ont pu être définis afin de développer une stratégie de miseen oeuvre d’un système pertinent pour la pratique clinique. / Corneal collagen cross-linking (CXL) is a kind of minimaly invasive corneoplasty mainlybased on a biomechanical concept, which is very difficult to measure physically, and whichthe therapeutic efficacy understanding is complex.Principles, different protocols and resultsare summarized in this thesis in order to illustrate the usefulness of the experimental modelchosen in our experimentations about elastographic corneal shear wave imaging.The pathway of our experimental work have led to the choice of trans-epithelial CXL (TCXL)assisted by iontophoresis (I-CXL), performed in vivo, on rabbits eyes. Elastographicmeasurements we obtained after animals euthanasia have shown a significant change of thecorneal elasticity profile after CXL, successively tested in a dynamic and in a static fashion.Our results do confirm the biomechanical efficacy of the I-CXL procedure and give a moreprecise idea of the sole photo-polymerization effect by avoiding any confounding healingconcern. Technological issues for corneal elastography with shear wave imaging have beenraised in this thesis to develop a realistic strategy for the launch of a clinically useful device.

Purpose In ophthalmology, data from both eyes of a person are frequently included in the statistical evaluation. This violates the requirement of data independence for classical statistical tests (e.g. t-Test or analysis of variance (ANOVA)) because it is correlated data. Linear mixed models (LMM) were used as a possibility to include the data of both eyes in the statistical evaluation. Methods The LMM is available for a variety of statistical software such as SPSS or R. The application was applied to a retrospective longitudinal analysis of an accelerated corneal cross-linking (ACXL (9*10)) treatment in progressive keratoconus (KC) with a follow-up period of 36 months. Forty eyes of 20 patients were included, whereas sequential bilateral CXL treatment was performed within 12 months. LMM and ANOVA for repeated measurements were used for statistical evaluation of topographical and tomographical data measured by Pentacam (Oculus, Wetzlar, Germany). Results Both eyes were classified into a worse and better eye concerning corneal topography. Visual acuity, keratometric values and minimal corneal thickness were statistically significant between them at baseline (p < 0.05). A significant correlation between worse and better eye was shown (p < 0.05). Therefore, analyzing the data at each follow-up visit using ANOVA partially led to an overestimation of the statistical effect that could be avoided by using LMM. After 36 months, ACXL has significantly improved BCVA and flattened the cornea. Conclusion The evaluation of data of both eyes without considering their correlation using classical statistical tests leads to an overestimation of the statistical effect, which can be avoided by using the LMM.

Indiana University-Purdue University Indianapolis (IUPUI) / A hierarchy of endothelial colony forming cells (ECFCs) with different levels of proliferative potential has been identified in human circulating blood and blood vessels. High proliferative potential ECFCs (HPP-ECFCs) display properties (robust proliferative potential in vitro and vessel-forming ability in vivo) consistent with stem/progenitor cells for the endothelial lineage. Corneal endothelial cells (CECs) are different from circulating and resident vascular endothelial cells (ECs). Whereas systemic vascular endothelium slowly proliferates throughout life, CECs fail to proliferate in situ and merely expand in size to accommodate areas of CEC loss due to injury or senescence. However, we have identified an entire hierarchy of ECFC resident in bovine CECs. Thus, this study provides a new conceptual framework for defining corneal endothelial progenitor cell potential. The identification of persistent corneal HPP-ECFCs in adult subjects might contribute to regenerative medicine in corneal transplantation. While human cord blood derived ECFCs are able to form vessels in vivo, it is unknown whether they are committed to an arterial or venous fate. We have demonstrated that human cord blood derived ECFCs heterogeneously express gene transcripts normally restricted to arterial or venous endothelium. They can be induced to display an arterial gene expression pattern after vascular endothelial growth factor 165 (VEGF165) or Notch ligand Dll1 (Delta1ext-IgG) stimulation in vitro. However, the in vitro Dll1 primed ECFCs fail to display significant skewing toward arterial EC phenotype and function in vivo upon implantation, suggesting that in vitro priming is not sufficient for in vivo specification. Future studies will determine whether ECFCs are amenable to specification in vivo by altering the properties of the implantation microenvironment. There is emerging evidence suggesting that the concentration of circulating ECFCs is closely related to the adverse progression of cardiovascular disorders. In a pig model of acute myocardial ischemia (AMI), we have demonstrated that AMI rapidly mobilizes ECFCs into the circulation, with a significant shift toward HPP-ECFCs. The exact role of the mobilized HPP-ECFCs in homing and participation in repair of the ischemic tissue remains unknown. In summary, these studies contribute to an improved understanding of ECFCs and suggest several possible therapeutic applications of ECFCs.

Humane corneale Endothelzellen (HCEC) bilden einen Monolayer aus differenzierten Zellen an der posterioren Oberfläche der Cornea und sind essenziell für den Erhalt der cornealen Transparenz. HCEC zeigen nahezu keine proliferative Aktivität in vivo und nur eine begrenzte Proliferationsfähigkeit in vitro. Bei übermäßigem Zellverlust aufgrund von Traumata, Erkrankungen oder des Alters kann die Transparenz der Cornea irreversibel beeinträchtigt werden und die Transplantation einer Spenderhornhaut erforderlich sein, um die Hornhauttransparenz und damit die Sehfähigkeit wiederherzustellen. Dabei ist die weltweite Begrenzung der medizinischen Versorgung mit hochwertigen Spenderhornhäuten das derzeit größte Problem für die Therapie von Cornea-assoziierten Erkrankungen. Zellersatzstrategien mit in vitro kultivierten, quantitativ und qualitativ ausreichenden Spenderzellen sollen die weitestgehend ausgereizten logistischen Ansätze zur Verringerung des Spendermangels ergänzen. Die Entwicklung einer abschaltbaren bzw. transienten Methode zur in vitro- und in situ-Vervielfältigung primärer HCEC ohne Verlust ihrer typischen morphologischen Merkmale würde die Herstellung sowie eine detaillierte und umfassende Charakterisierung von Transplantaten aus primären HCEC ermöglichen. In dieser Arbeit sollten daher zunächst verschiedene proliferationsfördernde Faktoren (PF) identifiziert werden, die nach stabilem retroviralen Gentransfer mit Integrations-kompetenten lentiviralen Vektoren (ICLV) in primären HCEC ein starkes proliferationsförderndes Signal provozieren, das eine Immortalisierung der Zellen zur Folge hat. Dabei sollte die Pseudotypisierung der ICLV-Partikel mit alternativen viralen Glykoproteinen zytopathische Effekte verringern und die Transduktionseffizienz steigern. Nachfolgend sollten die identifizierten PF auf ihre Fähigkeit, die Proliferation primärer HCEC transient zu stimulieren, ohne die Zellen dabei zu transformieren, getestet werden. Mit Hilfe verschiedener retroviraler Expressionssysteme sollte ein klinisch anwendbares System entwickelt werden, das eine kontrollierte, zeitlich begrenzte Stimulierung der Proliferation bei gleichzeitiger Unterdrückung eines tumorartigen Zellwachstums ermöglichte. Hierzu dienten 1) Integrase-defiziente lentivirale Vektoren (IDLV), die eine transiente Transgenexpression durch direkte Transkription des episomalen DNA-Vektorgenoms erlauben, und 2) das transiente Foamyvirus-Vektorsystem (TraFo-VS), dass auf der Enkapsidierung und dem Transfer nicht-viraler mRNA in permissiven Zielzellen basiert. Es konnte gezeigt werden, dass ICLV-Pseudotypen, die entweder eine SFVmcy-Glykoproteinvariante (ICLVSFV) oder das VSV-G-Protein enthielten (ICLVVSV), eine signifikante Transduktionseffizienz aufwiesen und dabei keine zytopathischen Effekte in den Zielzellen auslösten, weshalb beide Glykoproteine für weiterführende Experiment genutzt wurden. Unter Verwendung des optimierten ICLV-Systems konnten drei PF identifiziert werden, die eine reproduzierbare Immortalisierung primärer HCEC infolge stabiler Expression durch Transduktion mit den ICLV-Pseudotypen ermöglichten. Dazu zählten der Cyclin D1/CDK4-Proteinkomplex (4D), die SV40 T-Antigene (SV40T) sowie die transformierenden Proteine E6 und E7 (E6/E7) des HPV-16. Es konnte auch gezeigt werden, dass die Proliferation transduzierter primärer HCEC nach stabiler Transduktion mit PF-codierenden ICLV-Partikeln in einer dosisabhängigen Weise signifikant erhöht werden konnte. Untersuchungen mit IDLV-Varianten haben jedoch gezeigt, dass transduzierte HCEC ein vergleichbares proliferatives Verhalten wie ihre stabil transduzierten Äquivalente aufwiesen. Dies demonstrierte die restliche, geringgradige, nicht-kanonische Integrationskapazität von IDLV-Partikeln besonders im Zusammenhang mit der Expression von potenten PF. Nach erfolgter Transduktion mit TraFo-VP konnten die transferierten PF-codierenden mRNA in den Primärzellen nachgewiesen werden. Die Anwendung dieses Systems resultierte jedoch weder in einer nachweisbaren PF-Expression noch konnte eine proliferationsfördernde Wirkung in transduzierten Zellen festgestellt werden. Auch durch sequenzielle Transduktion der Zielzellen konnte keine Steigerung der Proliferationsrate induziert werden. Durch Verwendung von 50 fach konzentrierten SV40T-codierenden TraFo-VP konnte der mRNA-Transfer erhöht werden, wodurch dann auch die SV40T-Proteinexpression in den transduzierten Zellen nachweisbar wurde. Zudem konnte erstmalig gezeigt werden, dass sich im zeitlichen Verlauf sowohl die zellassoziierte SV40T-mRNA als auch die SV40T-Proteinkonzentration verringerte, bis sie nicht mehr nachweisbar war. Dabei konnte jedoch auch mit den konzentrierten TraFo VP keine nachweisbare transiente Immortalisierung primärer HCEC erreicht werden. Zusammenfassend kann festgestellt werden, dass eine permanente genetische Manipulation mit den viralen PF und dem 4D-Komplex eine Verlängerung der replikativen Lebensdauer ermöglichte und damit einhergehend die Immortalisierung primärer HCEC. Obgleich eine transiente Immortalisierung primärer HCEC mit den getesteten Systemen in dieser Arbeit nicht möglich war, ist eine klinische Anwendung des TraFo-VS, nicht aber des IDLV-Systems, in der angewandten Form, vielversprechend, um die Verfügbarkeit von qualitativ geeignetem Spendergewebe für die Transplantation bzw. Zellen für das Bioengineering des Hornhautendothels zu erhöhen. Daneben könnte das TraFo-VS ebenfalls genutzt werden, um andere zelluläre Funktionen in HCEC oder auch anderen Zielzellen transient zu modifizieren, z. B. Ionenfluss, replikative Seneszenz, Phagozytose oder Apoptose. / Human corneal endothelial cells (HCEC) form a monolayer of differentiated cells on the posterior surface of the cornea and are essential for maintaining corneal transparency. HCECs show almost no proliferative activity in vivo and only limited proliferative capacity in vitro. With excessive cell loss due to trauma, disease, or age-related degeneration, corneal transparency may be irreversibly compromised, and donor cornea transplantation may be required to restore vision. In this context, the global limitations in the medical supply of high-quality donor corneas are currently the most significant obstacle to the treatment of cornea-associated diseases. Cell replacement strategies using in vitro cultured donor cells of sufficient quantity and quality could complement the largely exhausted logistic approaches to alleviate donor shortage. The development of a method for strictly transient in vitro and in situ replication of primary HCECs without loss of their natural morphological characteristics would allow the production of well-characterized grafts derived from primary HCECs. To this end, I first aimed to identify different proliferation factors (PF) that provoke a robust proliferation-promoting signal in primary HCECs through stable retroviral gene transfer of candidate PF genes with integration-competent lentiviral vectors (ICLVs). Additionally, the pseudotyping of ICLV particles with alternative viral glycoproteins should reduce cytopathic effects and increase transduction efficiency. Subsequently, it should be clarified to what extent the identified PFs are capable of stimulating the proliferation of primary HCEC for a limited duration in a non-transformed context. Using different retroviral expression systems, I attempted to develop a clinically applicable system that allowed controlled, time-limited stimulation of proliferation while circumventing tumor-like cell growth. For this purpose, 1) integrase-deficient lentiviral vectors (IDLV), which allow transient transgene expression by direct transcription of the episomal DNA vector genome, and 2) the transient foamy virus vector system (TraFo-VS), which is based on encapsidation and transfer of non-viral mRNA in permissive target cells, were used. It was shown that ICLV pseudotypes containing either an SFVmcy glycoprotein variant (ICLVSFV) or the VSV-G protein (ICLVVSV) exhibited significant transduction efficiency without eliciting cytotoxic effects in target cells, highlighting both as viable candidates. Employing the optimized ICLV system, three PFs were identified that enabled reproducible immortalization of primary HCECs through stable expression after transduction with the ICLV pseudotypes. These included the cyclin D1/CDK4 protein complex (4D), the SV40 T antigens (SV40T), and the transforming proteins E6 and E7 (E6/E7) of HPV16. It was also shown that proliferation of transduced primary HCEC could be significantly increased in a dose-dependent manner following stable transduction with PF encoding ICLV particles. However, studies conducted using IDLV variants showed that PF-transduced HCEC exhibited a comparable proliferative behavior to their stably transduced equivalents. This demonstrated the residual, non-canonical integration capacity of IDLV particles especially in the context of potent PF expression. After successful transduction with TraFo-VP, the transferred PF-encoding mRNA could be detected in primary cells. However, application of this system did not result in detectable PF protein expression, nor could a proliferation-promoting phenotype be detected in transduced cells. Sequential transduction of target cells also failed to induce an increased proliferation rate. By using 50-fold concentrated SV40T-encoding TraFo-VPs, mRNA transfer could be increased, enabling detectable SV40T protein expression in transduced cells. In addition, it was shown for the first time that both cell-associated SV40T mRNA and SV40T protein levels decreased over time until they were no longer detectable. No observable transient immortalization of primary HCEC could be achieved even with the concentrated SV40T-encoding TraFo-VP. In conclusion, permanent genetic manipulation with the viral PFs and 4D protein complex allowed the prolonging of the cellular replicative lifespan in vitro and concomitant immortalization of primary HCEC. Although transient immortalization of primary HCECs was not possible with the systems tested in this investigation, clinical application of the TraFo-VS, but not the IDLV system as applied, remains a promising approach to increase the availability of suitable donor tissue for transplantation or cells for corneal endothelial bioengineering. Additionally, the TraFo-VS could also be used to transiently modify other cellular functions in HCEC or other target cells, e.g., ion flux, replicative senescence, phagocytosis, or apoptosis, for further cell biological research approaches.

Dans le circuit de la greffe de cornée, l'endothélium de chaque greffon est observé en microscopie optique conventionnelle afin de vérifier que sa densité cellulaire est suffisante pour maintenir une bonne transparence après l'opération. Les greffons étant conservés dans un milieu spécifique, ils sont imprégnés de liquide et présentent donc des plis qui perturbent l'observation et le comptage des cellules. Ce problème pratique est à l'origine d’une étude théorique sur les concepts de profondeur de champ étendue et de shape-from-focus. A partir d'une séquence d'images acquise par sectionnement optique, les informations les plus nettes permettent d'une part d'accéder à la topographie de la surface observée et d'autre part de restaurer l'image de sa texture. Une reconstruction surfacique 3-D est alors obtenue en projetant la texture sur la topographie. Cette thèse considère essentiellement l’étape fondamentale de mesure de netteté du processus de reconstruction. Des nouvelles mesures génériques offrant une haute sensibilité à la netteté sont introduites. De par une stratégie 3-D originale au travers de la séquence d'images, une autre mesure très robuste au bruit est proposée. Toutes ces mesures sont testées sur des données simulées puis diverses acquisitions réelles en microscopie optique conventionnelle et comparées aux méthodes de la littérature. Par ailleurs, la mesure 3-D améliore nettement les reconstructions d'endothéliums cornéens à partir de leurs acquisitions particulièrement perturbées (inversions de contraste). Un processus itératif complet de reconstruction 3-D d’endothéliums cornéens est finalement décrit, aboutissant à des résultats solides et exploitables. / In the cornea transplant process, each graft endothelium is observed by conventional optical microscopy to check that its cell density is sufficient to maintain a proper transparency after the transplantation. The grafts are stored in a specific preservation medium, they are thus impregnated with fluid and therefore exhibit folds which make cell observation and counting difficult. This practical issue led to the following theoretical study about the so-called concepts: extended-depth-of-field and shape-from-focus. Throughout a sequence of images acquired by optical sectioning, the in-focus information allows on the one hand to recover the topography of the observed surface and on the other hand to restore the image of its texture. A 3-D reconstruction is then obtained by mapping the texture onto the topography. This thesis basically considers the fundamental step of the reconstruction process that is the focus measurement. New generic focus measurements exhibiting high sharpness sensitivity are introduced. Another one offering high noise robustness is proposed, due to an original 3-D strategy through the image sequence, unlike traditional methods that operate in 2-D. All of them are tested on simulated data and various real acquisitions, and compared to the state-of-the-art methods. Furthermore, the aforementioned 3-D focus measurement clearly improves the 3-D surface reconstructions of the corneal endotheliums from their particularly disturbed acquisitions (contrast reversals). A complete iterative process of 3-D reconstruction of the corneal endothelial surfaces is finally described, resulting in solid results that can already be transferred to cornea banks.

OBJECTIVE: Ocular function is fundamental for environmental adaptation and survival capacity. Growth factors are necessary for a mature eyeball, needed for adequate vision. However, the consequences of the deficiency of circulating growth hormone (GH) and its effector insulin-like growth factor I (IGF-I) on the physical aspects of the human eye are still debated. A model of untreated isolated GH deficiency (IGHD), with low but measurable serum GH, may clarify this issue. The aim of this study was to assess the ocular aspects of adult IGHD individuals who have never received GH therapy. DESIGN: Cross sectional study. METHODS: Setting University Hospital, Federal University of Sergipe, Brazil. Patients: Twenty-five adult (13 males, mean age 50.1 years, range 26 to 70 years old) IGHD subjects homozygous for a null mutation (c.57+1G>A) in the GHRH receptor gene, and 28 (15 males, mean age 51.1 years, range 26 to 67 years old) controls were submitted to an endocrine and ophthalmological assessment. Forty-six IGHD and 50 control eyes were studied. Main outcome measures: Visual acuity, intraocular pressure (IOP), refraction (spherical equivalent), ocular axial length (AL), anterior chamber depth (ACD),lens thickness (LT), vitreous depth (VD), mean corneal curvature (CC) and central corneal thickness (CCT). RESULTS: IGHD subjects exhibited unmeasurable serum IGF-I levels, similar visual acuity, intraocular pressure and LT, higher values of spherical equivalent and CC, and lower measures of AL, ACD, VD and CCT in comparison to controls, but within their respective normal ranges. While mean stature in IGHD group was 78 % of the control group, mean head circumference was 92 % and axial AL was 96 %. CONCLUSIONS: These observations suggest mild ocular effects in adult subjects with severe IGF-I deficiency due to non-treated IGHD. / OBJETIVO: A função ocular é fundamental para a adaptação ambiental e a capacidade de sobrevivência. Fatores de crescimento são julgados necessáriospara alcançar um globo ocular maduro, e conseqüente visão adequada. No entanto, as consequências da deficiência isoladadohormônio de crescimento circulante (GH) edo seu efetor, o fator de crescimento semelhante à insulina I (IGF-I) nos aspectos físicos do olho humano ainda são debatidas. Um modelo de deficiência isolada de GH não tratada (DIGH) pode esclarecer esta questão. O objetivo deste estudo foi avaliar os aspectos físicos do globo ocular de indivíduos adultos com DIGH que nunca receberam terapia com GH. DESENHO: Estudo transversal. MÉTODOS: Ambiente: Hospital Universitário, Universidade Federal de Sergipe, Brasil. Pacientes: 25 indivíduosadultos (13 homens,com média de idade de 50,1 anos, entre 26 e 70 anos), com DIGH homozigotos para uma mutação nula (c.57 + 1G> A) no gene do receptorGHRH do grupo DIGH e 28 controles (15 homens, com média de idade de 51,1 anos, entre 26 e 67 anos), pareados, foram submetidos à avaliação endócrina e oftalmológica. Principais medidas: acuidade visual(AV), pressão intraocular(PIO),refração (equivalente esférico, EE), comprimento axial ocular (CA), profundidade da câmara anterior(PCA), medida da espessura do cristalino(EC), profundidade do vítreo(PV), curvatura corneana média(CCM) e espessura central corneana(ECC). RESULTADOS:Indivíduos com DIGH apresentaram IGF-I sérico não mensurável, similarAV, PIO e EC, valores mais altos doEEe CCM, e menores valores do CA, PCA, PV e ECC em comparação com os controles, mas dentro das respectivas faixas normais. Enquanto a estaturamédia no grupo DIGH foi de 78% do grupo de controle, a média da circunferência da cabeça foi de 92% e a média docomprimento axial foi de 96%. CONCLUSÃO: Essas observações sugerem efeitos oculares discretosem indivíduos adultos com grave deficiência de IGF-I devido à DIGH não tratada.

OBJECTIVE: Ocular function is fundamental for environmental adaptation and survival capacity. Growth factors are necessary for a mature eyeball, needed for adequate vision. However, the consequences of the deficiency of circulating growth hormone (GH) and its effector insulin-like growth factor I (IGF-I) on the physical aspects of the human eye are still debated. A model of untreated isolated GH deficiency (IGHD), with low but measurable serum GH, may clarify this issue. The aim of this study was to assess the ocular aspects of adult IGHD individuals who have never received GH therapy. DESIGN: Cross sectional study. METHODS: Setting University Hospital, Federal University of Sergipe, Brazil. Patients: Twenty-five adult (13 males, mean age 50.1 years, range 26 to 70 years old) IGHD subjects homozygous for a null mutation (c.57+1G>A) in the GHRH receptor gene, and 28 (15 males, mean age 51.1 years, range 26 to 67 years old) controls were submitted to an endocrine and ophthalmological assessment. Forty-six IGHD and 50 control eyes were studied. Main outcome measures: Visual acuity, intraocular pressure (IOP), refraction (spherical equivalent), ocular axial length (AL), anterior chamber depth (ACD),lens thickness (LT), vitreous depth (VD), mean corneal curvature (CC) and central corneal thickness (CCT). RESULTS: IGHD subjects exhibited unmeasurable serum IGF-I levels, similar visual acuity, intraocular pressure and LT, higher values of spherical equivalent and CC, and lower measures of AL, ACD, VD and CCT in comparison to controls, but within their respective normal ranges. While mean stature in IGHD group was 78 % of the control group, mean head circumference was 92 % and axial AL was 96 %. CONCLUSIONS: These observations suggest mild ocular effects in adult subjects with severe IGF-I deficiency due to non-treated IGHD. / OBJETIVO: A função ocular é fundamental para a adaptação ambiental e a capacidade de sobrevivência. Fatores de crescimento são julgados necessáriospara alcançar um globo ocular maduro, e conseqüente visão adequada. No entanto, as consequências da deficiência isoladadohormônio de crescimento circulante (GH) edo seu efetor, o fator de crescimento semelhante à insulina I (IGF-I) nos aspectos físicos do olho humano ainda são debatidas. Um modelo de deficiência isolada de GH não tratada (DIGH) pode esclarecer esta questão. O objetivo deste estudo foi avaliar os aspectos físicos do globo ocular de indivíduos adultos com DIGH que nunca receberam terapia com GH. DESENHO: Estudo transversal. MÉTODOS: Ambiente: Hospital Universitário, Universidade Federal de Sergipe, Brasil. Pacientes: 25 indivíduosadultos (13 homens,com média de idade de 50,1 anos, entre 26 e 70 anos), com DIGH homozigotos para uma mutação nula (c.57 + 1G> A) no gene do receptorGHRH do grupo DIGH e 28 controles (15 homens, com média de idade de 51,1 anos, entre 26 e 67 anos), pareados, foram submetidos à avaliação endócrina e oftalmológica. Principais medidas: acuidade visual(AV), pressão intraocular(PIO),refração (equivalente esférico, EE), comprimento axial ocular (CA), profundidade da câmara anterior(PCA), medida da espessura do cristalino(EC), profundidade do vítreo(PV), curvatura corneana média(CCM) e espessura central corneana(ECC). RESULTADOS:Indivíduos com DIGH apresentaram IGF-I sérico não mensurável, similarAV, PIO e EC, valores mais altos doEEe CCM, e menores valores do CA, PCA, PV e ECC em comparação com os controles, mas dentro das respectivas faixas normais. Enquanto a estaturamédia no grupo DIGH foi de 78% do grupo de controle, a média da circunferência da cabeça foi de 92% e a média docomprimento axial foi de 96%. CONCLUSÃO: Essas observações sugerem efeitos oculares discretosem indivíduos adultos com grave deficiência de IGF-I devido à DIGH não tratada.

Les tessellations, aussi appelées mosaïques, permettent de modéliser de nombreuses structures, comme des assemblages de cellules en biologie ou de grains en science des matériaux. La tessellation aléatoire la plus connue est le diagramme de Voronoï qui à partir d'un ensemble de points, appelés germes, partitionne le plan. L'approche innovante de cette thèse est d'utiliser des champs aléatoires gaussiens pour générer des germes et des distances aléatoires, qui vont permettre de simuler une grande variété de tessellations en termes de formes et de tailles des cellules.Pour connaître les propriétés des tessellations simulées à partir de champs aléatoires gaussiens, celles-ci vont être caractérisées et comparées à d'autres tessellations. Tout d'abord par une approche ponctuelle en étudiant les germes, dont leur distribution spatiale. Puis par une approche par région, en étudiant la géométrie et la morphométrie des cellules.L'endothélium cornéen humain est une monocouche de cellules formant un pavage hexagonal régulier à la naissance, et perdant de sa régularité ensuite. La qualité du greffon cornéen est donnée par certaines observations, comme la densité, l'homogénéité de la forme et des tailles des cellules endothéliales.L'évolution avec l'âge de cette mosaïque cornéenne va être caractérisée à partir d’une base d’images de l’endothélium. L'originalité est ensuite d'effectuer une estimation de l'âge d’un endothélium à partir des différentes mesures permettant de caractériser les tessellations, et enfin de mettre en place une méthode prometteuse afin de savoir si une cornée a une évolution normale. / Tessellations, also called mosaics, are used to model many structures, for example cellular arrangements in biology or grains in material science. The most known tessellation is the Voronoï diagram which partitions the space from a set of points, called germs. The innovative approach of this thesis is to use Gaussian random fields to generate germs and random distances. The use of random fields allows to simulate a great variety of tessellations in terms of cells forms and sizes.To study the properties of each type of tessellation, they are characterized: first, by studying the germs, including their spatial distribution, and then by analyzing the cells geometry and morphometry. These tessellations are also compared to other known tessellations.The human corneal endothelium is a mono-layer of cells forming a regular hexagonal mosaic at birth, and losing his regularity later. The corneal graft quality is given by some observations made on the endothelial mosaic (cells density, the homogeneity of cells sizes and shapes).A database of endothelium images allows to characterize the evolution with age of the corneal mosaic. The originality is to estimate the age of an endothelium based on the measures computed to characterize the tessellations, and finally to set up a promising method to evaluate if a corneal evolution is normal.