Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2017-04-03T15:39:07Z No. of bitstreams: 2 Tese - Francisco Weliton Rodrigues - 2016.pdf: 2820619 bytes, checksum: af037c3015b0edc5e8691d768f2d6194 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-04-03T15:39:26Z (GMT) No. of bitstreams: 2 Tese - Francisco Weliton Rodrigues - 2016.pdf: 2820619 bytes, checksum: af037c3015b0edc5e8691d768f2d6194 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-04-03T15:39:26Z (GMT). No. of bitstreams: 2 Tese - Francisco Weliton Rodrigues - 2016.pdf: 2820619 bytes, checksum: af037c3015b0edc5e8691d768f2d6194 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-07-19 / Keratoconus is a chronic non-inflammatory ocular disorder characterized by central thinning, protrusion and conical shape of the cornea. The progression of this disorder cause a significant decrease in visual acuity. It has been suggested that the development of keratoconus has a genetic component. Therefore, the aim of this study was to evaluate the frequency of single nucleotide polymorphisms (SNP) mutations associated with keratoconus in unrelated Brazilian patients compared to healthy subjects. This was a case-control clinical study with 108 participants, 46 patients with keratoconus and 62 healthy subjects (controls). Peripheral blood, collected from all participants, was used to extract DNA samples. Subsequently, genotyping of three single nucleotide polymorphisms, TGFBI rs4669 and rs2072239 and VSX1 rs6138482, was performed through real-time polymerase chain reactions (qPCR). Single nucleotide polymorphisms were observed in both keratoconus patients and healthy subjects. For the VSX1 gene, allelic frequency and discrimination was similar for keratoconus patients and controls. Conversely, the frequency of the mutant allele was significantly higher for two SNPs on the TGFBI gene in patients with keratoconus. For the SNP rs4669, the patients with keratoconus had 15 % higher frequency of the mutated allele, while for the SNP rs2072239 the patients had 11 % higher frequency of the mutated allele compared to the controls. Individuals carrying the mutant allele had two-times more risk in developing the disease. The allelic discrimination of genotypes (homozygous and heterozygous) was also significantly different for both SNPs on the TGFBI gene. This study has demonstrated, for the first time, an association of SNP mutations and the development of keratoconus in Brazilian patients. The frequency of the mutant and potentially pathogenic allele on the TGFBI gene was significantly higher in patients compared to controls. Finally, these findings contribute to the advance of molecular knowledge of the pathogenesis, development of early diagnostic tools and therapeutics options for patients with keratoconus. / O ceratocone é uma desordem ocular caracterizada pelo afinamento central, protrusão e formato cônico da córnea. A progressão desta desordem causa diminuição significativa da acuidade visual. Existem evidências de que o ceratocone apresenta componente genético. Assim, o objetivo desse estudo foi avaliar a frequência de mutações de polimorfismo de nucleotídeo único (SNP) associados a ocorrência do ceratocone em pacientes brasileiros sem parentesco conehcido em comparação à voluntários saudáveis. Este foi um estudo clínico do tipo caso-controle com um total de 108 participantes, 46 pacientes com ceratocone e 62 voluntários saudáveis (controles). Amostras de sangue periférico foram coletadas e utilizadas para extração do DNA. Subsequentemente, o genótipo de três polimorfismos de nucleotídeo único, TGFBI rs4669 e rs2072239 e VSX1 rs6138482, foram determinados através de reações em cadeia polimerizada em tempo real (qPCR). Polimorfismos de nucleotídeo único foram observados em ambos os pacientes e controles. Para o gene VSX1 (SNP rs6138482) a frequência e discriminação alélica não houve diferenças estatisticamente significantes (p<0,005) em pacientes e controles. No entanto, para o gene TGFBI, existiram diferenças significativas em relação a frequência e discriminação alélica. Para o SNP rs4669 a frequência do alelo mutante foi 15 % maior e para o SNP rs2072239 uma frequência 11 % maior em pacientes com ceratocone comparado aos controles. Além disso, os indivíduos que apresentaram o alelo mutante têm um risco (razão de probabilidade) duas vezes maior de desenvolver a doença. Este estudo demostrou, pela primeira vez, que existe uma associação entre dois SNP e o desenvolvimento do ceratocone em pacientes brasileiros. A frequência do alelo mutante e potencialmente patogênico no gene TGFBI foi significativamente maior pacientes comparado aos controles. Concluindo, os resultados deste estudo contribuem para o conhecimento molecular da patogênese, o desenvolvimento de técnicas de diagnóstico precoce e consequentemente mais opções de tratamento para pacientes com ceratocone.

Ceratite ulcerativa é uma das enfermidades oculares mais comuns em cães e requer tratamento clínico-cirúrgico urgente. Neste contexto, o tratamento cirúrgico com implante de membrana biológica é opção benéfica. Estudou-se o implante de membrana amniótica (MA) e membrana alantoamniótica (MAAM) alógenas preservadas em glicerina a 98% em vinte córneas de cães submetidas previamente à ceratectomia lamelar superficial. Dividiram-se os cães em dois grupos para o estudo aos 2, 7, 15, 30 e 60 dias de pós-operatório. Avaliaram-se, segundo parâmetros clínico-oftalmológicos e histopatológicos. Clinicamente detectou-se fotofobia e blefarospasmo, tipos e graus de secreção, edema e neovascularização corneana e transparência da córnea no local do implante. Nos períodos pré-determinados as córneas foram submetidas à avaliação histológica. Foram constatados, com base no exame ocular, graus leves a moderado de fotofobia e blefarospasmo; predominou o tipo de secreção mucoso com grau moderado e o edema corneano permaneceu leve no grupo que recebeu MA, e moderada a leve no grupo que recebeu MAAM. A neovascularização intensificou-se no 15º dia de pós-operatório em ambos os grupos. Com base nos achados histopatológicos os grupos evoluíram de forma semelhante; constatou-se o desenvolvimento do processo de cicatrização dentro dos parâmetros normais para a córnea. Concluiu-se que a utilização da MA comparada com a MAAM de cães preservadas em glicerina a 98% em ceratoplastias não diferem quanto sua evolução segundo os parâmetros adotados para análise. Ambas as membranas são igualmente efetivas como membrana biológica alógena na reparação de defeito corneano. / Ulcerative ceratitis is one of the most frequent ocular diseases in dogs and it requires prompt surgical and clinical therapy. In this context, surgical treatment using biological membrane implants is a benefic option. In this study alogenous 98% glycerin-preserved amniotic (MA) and alantoamniotic (MAAM) membranes were implanted on twenty corneas from dogs previously submitted to superficial lamellar keratectomy. Clinical, ophthalmologic and histological parameters were investigated. The animals were divided in two groups and pos-operative corneal histological evaluation was taken on days 2, 7, 15, 30 and 60. Photophobia, blefarospasm, different types and severity of ocular secretion, corneal edema and neovascularization and corneal transparency at the implant site. Ocular examination revealed mild to severe photophobia and blefarospasm, moderate mucous secretion was the most frequent. Corneal edema was mild in the MA group and mild to moderate in the MAAM group. From the 15th pos-operative day neovascularization was more pronounced for both groups. Based on the histological findings the groups showed similar evolution and the healing process was found to be within normal parameters for the corneal tissue. We concluded that in dogs 98% glycerin-preserved MA and MAAM applied to keratoplasty do not differ among the parameters evaluated. Both membranes are equally effective as allogenous biological membrane for the repair of corneal defects.

O objetivo do presente trabalho foi avaliar a influência da aplicação de bevacizumab (Avastin®) sobre a angiogênese corneal. Para tanto ratos Wistar, machos, com idade entre 8 e 10 semanas, pesando 300 a 350g, foram submetidos a cauterização química com nitrato de prata por 10 segundos. Após realização da lesão, cada grupo de 5 animais foi tratado com injeções subconjuntivais de 0,02 ml de bevacizumab (Avastin ®) pela via subconjuntival no momento da lesão, no 3° e 5° dia e submetidos à eutanásia ao 7° dia após cauterização corneal. A rede vascular neoformada foi quantificada após preenchimento do leito vascular com Tinta da China e análise das imagens em sistema computadorizado (Image Pro-Plus®). Para a segunda etapa do experimento, avaliou-se a atividade das metaloproteinases MMP-2 e MMP-9 utilizando-se do mesmo modelo de angiogênese e dos mesmos tempos de tratamento. Transcorridos 7 dias de lesão corneal os animais foram eutanasiados e suas córneas submetidas a zimografia. Os resultados mostraram haver uma inibição da angiogênese quando se compara o grupo controle aos grupos tratados nos diferentes períodos de tempo. Quando a densidade vascular é comparada entre os tempos de aplicação, não se observa diferença estatisticamente significante. Estes resultados em conjunto indicam que o tempo de aplicação não influencia a inibição da angiogênese e que o bevacizumab foi eficiente na redução da formação de vasos quando se compara a densidade vascular do grupo controle. O modelo experimental produziu considerável aumento das metaloproteinases MMP-2 e MMP-9. Houve uma tendência a diminuição da atividade das MMPs quando bevacizumab foi aplicado no momento da lesão e quando foi aplicado ao 3° dia após cauterização. A atividade das MMPs mostrou-se aumentada quando o tratamento foi realizado no 5° dia. Desta forma pode-se concluir que o bevacizumab foi capaz de inibir a angiogênese corneal, independentemente do período de tratamento e ainda que houve uma tendência a redução da atividade das MMPs carecendo, por enquanto, de mais investigações que possam elucidar a interação deste fármaco com a inibição de sua atividade. / The purpose of this study was to evaluate the effects of the use of the subconjunctival injection of bevacizumab (Avastin®) on angiogenesis and metalloproteinases activities in the rat cornea after cauterization. Wistar male rats, aging 8 to 10 weeks, were used. The animals were divided in four groups: control group (GC) that received subconjunctivally 0,02 ml of 0,9% saline solution; group GO that received subconjunctivally 0,02 ml of bevacizumab just after the lesion; group G3 that received bevacizumab at day 3 after lesion and G5 that received bevacizumab at day 5 after lesion. The animals were euthanized at day 7 after lesion. The new formed vessels were quantified after the China Ink perfusion and photographs were obtained and analyzed in computadorized system (Image Pro-Plus®). The activities of metalloproteinases MMP-2 and MMP-9 were evaluated by zimography in a pool of corneas obtained from animals submitted to the same model and treatment schedule. The results showed an inhibition of angiogesenis when the control group were compared with all treated groups. There were no statistic differences when vascular density was compared between the treated groups. These results may indicate that bevacizumab was able to inhibit corneal angiogenesis and that there was no influence of the injection day. This angiogenesis model promoted an increase of matrix metalloproteinases MMP-2 and MMP-9 activity. Zymography also showed a slight reduction in matrix metalloproteinases activity when bevacizumab were administrated at the injury moment and most significant at day 3. At day 5 zymography showed an increase of the activity of these metalloproteinases. Together these results may indicate that the subconjunctival injection of bevacizumab may have a therapeutic potential as an antiangiogenic agent and that matrix metalloproteinases activity decreased, but not at significant levels, demanding more investigations regarding to the interaction of bevacizumab and activity of matrix metalloproteinases.

Submitted by isabela.moljf@hotmail.com (isabela.moljf@hotmail.com) on 2016-08-05T12:09:31Z No. of bitstreams: 1 robertateixeiraprado.pdf: 1563717 bytes, checksum: 7af32473ba9fb882aead951cc29eb6cd (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-08-05T16:11:10Z (GMT) No. of bitstreams: 1 robertateixeiraprado.pdf: 1563717 bytes, checksum: 7af32473ba9fb882aead951cc29eb6cd (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-08-05T16:11:17Z (GMT) No. of bitstreams: 1 robertateixeiraprado.pdf: 1563717 bytes, checksum: 7af32473ba9fb882aead951cc29eb6cd (MD5) / Made available in DSpace on 2016-08-05T16:11:17Z (GMT). No. of bitstreams: 1 robertateixeiraprado.pdf: 1563717 bytes, checksum: 7af32473ba9fb882aead951cc29eb6cd (MD5) Previous issue date: 2013-05-10 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O estudo busca analisar as especificidades do trabalho dos enfermeiros em Banco de Tecidos Oculares Humanos tendo por base as etapas do processo de captação de órgãos e tecidos estabelecidas pelo Ministério da Saúde. Trata-se de um estudo exploratório e descritivo, com abordagem qualitativa. Os dados foram coletados por meio de entrevista semiestruturada, com sete enfermeiros que atuam neste serviço no estado de Minas Gerais e foram analisados com o suporte da análise de conteúdo. As categorias identificadas nos depoimentos dos enfermeiros, quais sejam, conhecimentos e habilidades necessários aos enfermeiros dos Bancos de Olhos; facilidades e dificuldades enfrentadas pelos enfermeiros; gerência de enfermagem e lidando com a morte, ofereceram subsídios relevantes para a análise da atuação dos enfermeiros neste cenário de prática. Foi apontada a necessidade de conhecimentos sobre anatomia humana, fisiologia e patologia, conhecimento de diluição de amostra sanguínea em pacientes hemotransfundidos que são potenciais doadores de córneas e dos diferentes eventos patológicos que contraindicam a doação de córneas. Além do conhecimento da legislação sobre a temática, dentre outros. Dentre as habilidades requeridas para o trabalho nos Bancos de Olhos sob a ótica dos enfermeiros dos serviços destacam-se o equilíbrio emocional, habilidades relacionadas à educação, habilidade de liderança para manter a união e a integração da equipe, habilidade para lidar com o cadáver, assim como a destreza nos procedimentos técnicos envolvidos no serviço. Ao analisar as falas dos sujeitos desta pesquisa verifica-se que a dimensão gerencial recebeu relevância nas falas dos sujeitos. Ficou evidente que os enfermeiros precisam dedicar maior atenção às dimensões de ensino e pesquisa. Verificou-se que as facilidades mais citadas para a realização do trabalho dos enfermeiros estão associadas ao relacionamento desenvolvido com a equipe multidisciplinar, a aspectos de infraestrutura disponíveis para uso no serviço e à formação profissional. Os conflitos no exercício do trabalho do enfermeiro apareceram sob diferentes manifestações e envolvem muitas vezes relações hierárquicas conflituosas. Os discursos mostram que os enfermeiros consideram-se membros responsáveis pelo serviço de enfermagem e pela integração da equipe, mas se sentem tolhidos diante das estruturas políticas presentes. Constatou-se que o enfermeiro é o grande responsável pelo gerenciamento do serviço da equipe de enfermagem e acaba gerenciando os processos de trabalho no setor. Constatou-se que algumas determinações e princípios que regem a atuação dos profissionais da enfermagem muitas vezes são desrespeitados e que a atuação dos enfermeiros nos diferentes Bancos de Olhos precisa ser revista e transformada. Observou-se que os enfermeiros dos Bancos de Olhos lidam de formas diferentes com a morte. Há mortes que geram maior desgaste e sofrimento ao profissional, a formação dos profissionais não permite um olhar amplo sobre a temática e que mesmo diante da exposição a situações de morte e desgastes relacionados ao trabalho, o serviço no Banco de Olhos é considerado algo nobre e recompensador pelos enfermeiros entrevistados. / The study seeks to examine the specifics of the work of nurses in Human Ocular tissue bank based on the stages of the process of obtaining organs and tissues set out by the Ministry of health. This is an exploratory and descriptive study with a qualitative approach. The data were collected through semi-structured interview with seven nurses working in this service in the State of Minas Gerais in the Southeast region and were analyzed with the support of content analysis. The categories identified in the affidavits of nurses, namely, knowledge and necessary skills to nurses of Eyes; facilities and difficulties faced by nurses; nursing management and dealing with death, offered subsidies that are relevant to the analysis of the role of nurses in this practical scenario. Was pointed to the need for knowledge about human anatomy, Physiology and pathology, knowledge of blood sample dilution in transfused patients who are potential donors of corneas and pathological events that contraindicate the donation of corneas. In addition to the knowledge of the legislation on the subject, among others. One of the skills required to work in banks of Eyes from the perspective of the nurses of the services include the emotional balance, education-related skills, leadership ability to keep the Union and the team integration, ability to deal with the corpse, as well as the dexterity in the technical procedures involved in the service. By analyzing the speeches of the subjects of this research it turns out that the managerial dimension relevance in speeches of the subjects received. It was evident that nurses need to devote greater attention to the dimensions of teaching and research. It was found that the most cited facilities to carry out the work of nurses are associated with the relationship developed with the multidisciplinary team, the aspects of infrastructure available for use in the service and vocational training. The conflicts in the work of the nurses appeared under different forms and often involve conflicting hierarchical relationships. The speeches show that nurses are members responsible for nursing service and integration team, but feel restrained in the face of political structures present. It was found that the nurse is responsible for the management of the team of nursing service and ends up managing the work processes in the industry. It was noted that some regulations and principles governing the actions of nursing professionals are often not respected and that the work of nurses in different Banks of Eyes needs to be reviewed and processed. It was observed that the nurses of eye Banks deal in different ways with the death. There are deaths that lead to increased wear and suffering to the professional, the training of professionals does not allow a broad look on the subject and that even in the face of death situations exposure and work-related damage, the service at the eye Bank is considered something noble and rewarding by the nurses interviewed. Keywords: Eye Banks. Nursing. Cornea.

Orientador: Jose Paulo Cabral de Vasconcellos / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-25T23:02:33Z (GMT). No. of bitstreams: 1 Okanobo_Andre_M.pdf: 2897473 bytes, checksum: 5bad5928ff037c8396c72781f5829cc1 (MD5) Previous issue date: 2014 / Resumo: Avaliação da qualidade de Vida dos Pacientes Antes e Após Realização de Transplante Penetrante de Córnea. OBJETIVO Verificar o impacto do transplante de córnea sobre a qualidade de vida dos indivíduos submetidos à cirurgia no serviço de Oftalmologia HC-UNICAMP no período de outubro de 2005 a outubro de 2006. METODO: O estudo foi aprovado pelo comitê de ética e pesquisa da FCM-UNICAMP. Foram inclusos indivíduos com indicação de transplante de córnea do ambulatório de Doenças Externas da Oftalmologia do HC UNICAMP no período de outubro de 2005 a outubro de 2006 e que aceitaram em participar do estudo. Foram incluídos 32 indivíduos. Os critérios de exclusão foram transplante tectônico, indivíduos com déficit visual importante causado por outra afecção ocular além das alterações corneanas. Realizou-se exame oftalmológico assim como aspectos demográficos e história ocular dos pacientes incluídos no estudo. Métodos objetivos como claridade do transplante e melhor acuidade visual corrigida e método subjetivo, a qualidade de vida, através do questionário SF-36 adaptado. RESULTADOS: Houve diferença significativa de satisfação entre gêneros com maior escore no sexo masculino (p = 0.0319). No método objetivo como a acuidade visual corrigida teve aumento significativo do olho transplantado (p<0.0001); A AV média antes do transplante era de 0,98 ± 0,1 logMAR e após 0,48 ± 0.38 logMAR. Quanto o escore de qualidade de vida (SF-36), método subjetivo, após o transplante houve um aumento significativo (p<0.0001). O escore do SF-36 antes do transplante foi de 49,11% ± 19,28 (média ± DP) e após o transplante de 71, 98% ± 24,28 (média ± DP) com uma melhora de 22, 87% (p<0.0001).Os indivíduos mais satisfeitos foram aqueles que tiveram a pontuação mais alta no SF-36 (r = 0.60; P = 0.0002). A satisfação parece não estar correlacionada com a melhora da AVCC no olho transplantado (r = 0.3186; P = 0.0755). Ocorreu uma fraca correlação entre a melhora da AVCC no olho transplantado e a melhora do SF-36 (r=0.28382; p=0.1154). Não houve diferença significativa do SF-36 nos indivíduos com visão baixa nos dois olhos quando comparado com os pacientes com visão boa em um dos olhos (p=0.2998 ) CONCLUSÃO: O transplante penetrante de córnea proporcionou melhora na pontuação dos métodos subjetivo e objetivos. Os indivíduos com maior satisfação foram melhor avaliados através do método subjetivo (SF-36). Não houve diferença significativa na qualidade de vida dos indivíduos com pior acuidade visual no olho contralateral quando comparado com os indivíduos com boa visão no olho contralateral, mas análise de um maior número de indivíduos é necessária / Abstract: Purpose: To assess and measures the patient satisfaction before and after penetrating keratoplasty. Methods: The study of approved by the ethics committee of FCM-UNICAMP. Data were collected from 32 patients who underwent penetrating keratoplasty (PK) between October 2005 and October 2006. Demographic, ocular history, objective treatment outcome measures such as clarity of the graft and best-corrected visual acuity (BCVA) of both eyes were collected prospectively. In addition to assessment of quality of life were obtained by an interview before the PK and after at least one year of postoperative. Results: The average age of subjects was 42 ± 22 (mean ± SD) years and 68.75 % were women. On average, men were most satisfied. BCVA was 0,98 ± 0,1 logMAR before and 0,48 ± 0.38 logMAR after PK showing a significant improvement on BCVA (P < 0.0001). SF-36 score was 49.11% ± 19.28% before and 71.98% ± 24.28% after PK showing a significant improvement after the PK (p< 0.0001). The most satisfied patients where those who had a better score on SF-36 (r = 0.60; p = 0.0002). Satisfaction seems not to be correlated to improvement in BCVA at the transplanted eye (r = 0.3186; p = 0.0755). There was weak correlation between improvement in BCVA on the transplanted eye and improvement of SF-36 score (r = 0.28382; p=0.1154). There was no significant improvement in SF-36 in patients with worse BCVA in the other eye (p = 0.2998). Conclusions: Penetrating keratoplasty has a positive effect on objective and subjective outcome measures. Patient satisfaction is better predicted by subjective outcomes. There is no significant difference in quality of life in patients with worse BCVA in the contralateral eye when compared to eye with good BCVA in the contralateral eye, but analysis of a larger number of patients is necessary to confirm that / Mestrado / Oftalmologia / Mestre em Ciências Médicas

Les cellules endothéliales cornéennes (CEC) jouent un rôle essentiel pour le maintien de la transparence de la cornée. Cependant, chez l’homme, elles sont incapables de proliférer en raison d’un arrêt de leur cycle cellulaire en phase G1, ce qui rend la couche endothéliale cornéenne particulièrement vulnérable. La délivrance de molécules thérapeutiques (gènes ou médicaments) représente une solution prometteuse pour maintenir la viabilité des CEC. Néanmoins, la difficulté majeure de cette technique repose sur le fait de traverser la membrane cellulaire, normalement imperméable aux molécules de grande taille. Plusieurs techniques de délivrance de molécules ont déjà été testées sur le tissu cornéen mais aucune d’entre elles ne donnent de résultats suffisamment probants pour être utilisée en applications cliniques. L’objectif de cette thèse est d’adapter et de développer une nouvelle technique de délivrance intracellulaire de molécules, basé sur une perforation cellulaire via un phénomène photoacoustique induite par l’activation de nanoparticules de carbone par laser femtoseconde, sur un modèle d’endothélium cornéen in vitro et ex vivo / Corneal endothelial cells (CEC) are essential for corneal transparency. However, on humans, they are unable of proliferation owing to its arrest of G1 phase of the cell cycle, making corneal endothelial monolayer particularly vulnerable. The gene and drug delivery represents a promising solution to maintain CEC viability. Unfortunately, the major difficulty of this technique is the transport across the cell membrane, normally impermeable to high-size molecules. Several techniques of molecules delivery have already been tested on corneal tissue but none of them gives results sufficiently convincing to be used in clinical applications. The aim of this thesis is to adapt and develop a new technique of intracellular molecules delivery, based on cell perforation via photoacoustic effect induced by the activation of carbon nanoparticles by femtosecond laser, on in vitro and ex vivo models of corneal endothelium

It is widely known that Central Corneal Thickness (CCT) and Radius of Curvature (RoC) in uence the estimated IntraOcular Pressure (IOP) obtained from Goldmann Applanation Tonometry (GAT). However, not much is known about the in uence of corneal material properties, especially in a clinical setting. Several numerical studies have been conducted in an attempt to quantify the in uence of corneal material properties on the IOP. These studies agree that corneal material properties do in uence the estimated IOP, which contradict the initial premise on which GAT was designed, namely that material properties do not in uence the obtained GAT readings. Also, there is no consensus among these studies with respect to corneal material properties, thus a wide range of proposed properties exist. A possible explanation for this range of available corneal properties is the numerical modi elling assumptions used, which seem to be quite different. Different sets of experimental in ation test data were used to calibrate the constitutive models and different limbal boundary conditions were applied to simulate the experimental setup as well as in vivo conditions during GAT simulations. Therefore the purpose of this study is to determine whether these modelling assumptions in uence the obtained IOP and ultimately the overall conclusions. A Finite Element (FE) model of the human cornea is developed, implementing a constitutive model to represent the complex corneal structure and two limbal boundary conditions. This model is then calibrated using two different sets of experimental in ation test data. During calibration of the fibre reinforced elastic constitutive model it is found that independent of the assumptions made regarding the material coe cients, that the numerical in ation data compare well with the experimental data for all cases. Using this model a GAT simulation is conducted to estimate the IOP and the in uence of the modelling assumptions, cornea geometry and material properties are then investigated. The results indicate that the modelling assumptions, cornea geometry and material properties do infuence the estimated IOP. However, when assuming the cornea ground substance stiffness to be constant, it is found that the in uence on IOP due to material properties is not as significant. A correction equation is also proposed to account for the corneal geometric properties by calibrating the numerical model for a numerically normal cornea. This is done by utilising the various data sets which are obtained during the calibration of the constitutive model with the experimental inflation test data. It is concluded that using only inflation data to calibrate the constitutive model is not sufficient to uniquely describe the corneal material. This is evident as different material data sets are obtained, even though the experimental inflation data is matched well for a variety of considered cases. Each of these material data sets, in conjunction with geometric properties, yield different estimates for IOP during GAT simulations. This study therefore recommends the use of additional experimental data, such as strip extensometry, along with inflation test data to adequately calibrate a numerical model. It should also be noted that when modelling GAT care should be taken when considering the choice of limbal boundary condition, experimental data for calibration and assumptions made with regards to material coe cients, as these choices could potentially influence the outcomes and conclusions of a study. / Dissertation (MEng)--University of Pretoria, 2014. / gm2014 / Mechanical and Aeronautical Engineering / unrestricted

L'objectif du projet est la synthèse d'une cornée artificielle biocompatible à base de collagène de type I extrait et purifié à partir de tendons de queues de rats. La synthèse utilise les propriétés mésogènes (cristal-liquides) de la molécule de collagène ainsi qu'une transition sol-gel mimant l'étape de fibrillogenèse qui se déroule in vivo. Des solutions acides de collagène (500 mM en acide acétique) sont dyalisées contre des solutions de diverses concentrations en acide acétique et en acide chlorhydrique puis concentrées jusqu'à 90 mg/mL. Les phases cristal-liquides données par les différentes conditions physico-chimiques sont analysées par microscopie à lumière polarisée et par génération de seconde harmonique. L'une des conditions permet d’obtenir une phase dite en contreplaqué, ce qui est l’organisation des lamelles de fibrilles de collagène dans la cornée.Analysée par microscopie électronique à transmission, la structure des matrices obtenues après fibrillogénèse présente des domaines en contreplaqué indiquant une conservation et une stabilisation de l’organisation cristal-liquide d'origine. L’organisation obtenue est proche de celle du stroma cornéen. Par une optimisation des conditions physico-chimiques, les matrices synthétisées présentent une transparence proche de 90 % et possèdent de bonnes propriétés mécaniques avec un module d’Young proche de 1 MPa. Des cultures cellulaires effectuées sur les matrices transparentes montrent qu’elles sont un très bon support pour la culture de cellules cornéennes, en particulier des cellules épithéliales. Tous ses résultats confortent la méthode utilisée et les essais in vivo constituent l’étape suivante. / In view to generate artificial corneas, dense transparent collagen type-I scaffolds were synthesized exploiting the intrinsic liquid crystals properties of collagen molecules. 3 mg/mL collagen solutions in 500 mM acetic acid were dialyzed against a solution of precise concentrations in acetic and hydrochloric acid. When concentrated, solution provided a liquid-crystal organization resembling plywood, which is the organization of the collagen fibrils in the cornea. This was verified by polarized light and second harmonic generation microscopy experiments. In parallel these collagen solutions were also concentrated by centrifugation-filtration up to 90 mg/mL. The concentrated solutions were pressed into cornea-like shape and submitted to ammonia vapor in order to induce the fibrillogenesis of collagen. The result is a transparent dense fibrillated collagen matrix (transparency 90 %). Transmission electron microscopy revealed that fibrils kept the organization of the concentrated solution. Using a custom made device, mechanical tests showed that the Young modulus reached 900kPa. Human donor limbal explants were sewed on top of the scaffolds and cultured for 14 days. Optical microscopy and immunocytochemical analysis showed the development of an epithelium with characteristics of corneal epithelial cells. Preliminary experiments showed that keratocytes could be successfully inserted during the synthesis process. Thus, the results show the viability of the process of fabrication, and the following step is the in vivo experiment.

La cornée est le tissu le plus densément innervé du corps humain. Cette innervation joue un rôle dans la régulation de la sécrétion du film lacrymal et exerce un rôle trophique direct sur l'épithélium cornéen. Les axones cornéens expriment différents types de récepteurs et répondent à des fonctions de mécano-nocicepteurs, de récepteurs au froid ou de récepteurs polymodaux. Nous avons pu identifier de nouvelles lignées de souris transgéniques et les utiliser pour caractériser ces différentes populations axonales. L'innervation cornéenne débute à E12.5 chez la souris et est régulée par les molécules de Slits et de Sémaphorines et leurs récepteurs Robo et Plexines/Neuropilines respectivement. Nous avons pu étudier le rôle de ces deux familles dans le développement de l'innervation cornéenne. Les mutants Slits et Robos présentent une réduction du nombre et de la taille des terminaisons axonales épithéliales cornéennes. A l'âge adulte, les mutants Robos présentent une dégénérescence précoce des terminaisons épithéliales. Les mutants plexine-A4 et Neuropiline-1 présentent à l'inverse une augmentation du nombre de divisions des troncs stromaux cornéens. A l'âge adulte les mutants Plexine-A4 retrouvent une organisation classique de l'innervation alors que les mutants Neuropiline-1 conservent la désorganisation de l'innervation cornéenne. Nous avons également étudié la régénération de l'innervation après lésions de grattage de l'épithélium cornéen. Nos résultats préliminaires semblent en faveur d'une augmentation de la régénération de l'innervation cornéenne chez les mutants Neuropiline-1. / The cornea is the most densely innervated tissue in the entire body. Corneal innervation plays a role in regulating the secretion of lacrimal film and exerts a direct trophic role on the corneal epithelium. Corneal axons express different types of sensory receptors ranging between mechano-, thermo-, and polymodal nociceptors. We identified transgenic mouse lines to characterize these different axonal populations. Corneal innervation begins at E12.5 in mice and is regulated by a range of axon guidance cues such as Slits and Semaphorins, which respond to their receptors Robo and Plexin/Neuropillin respectively. We studied the role of these two families in the development of corneal innervation. The Slits and Robos mutants show a reduction in the number and size of the corneal epithelial nerves endings. In adult, Robos mutants exhibit early degeneration of the epithelial nerves endings. Plexin-A4 and Neuropilin-1 mutants, on the other hand, show an increase in the number of divisions of the corneal stromal nerve trunks. In adult, Plexin-A4 mutants regain a classical organization of innervation whereas Neuropilin-1 mutants retain the disorganization of corneal innervation. Following a lesion, corneal innervation is able to regenerate, however the axons never regain their initial morphology or complexity. Due to the increased corneal innervation observed in the Neuropilin-1 mutants, we wondered whether the regeneration of innervation after scrapping lesions of the corneal epithelium could be enhanced in Neuropilin-1 loss of function. Our preliminary results support an increase in corneal innervation regeneration in Neuropilin-1 mutants.

La greffe lamellaire postérieure, ou greffe endothéliale, consiste à remplacer l’endothélium défectueux d’une cornée par un endothélium sain prélevé sur un donneur décédé, en laissant en place l’épithélium et le stroma du patient. La découpe du greffon lamellaire endothéliale est l’une des étapes critiques de la greffe de cornée postérieure. Supérieur au microkeratome pour la réalisation de capots cornéens peu profonds, le laser femtoseconde montre cependant des résultats plutôt décevant concernant les découpes cornéennes profondes. L’objectif de cette thèse est d’étudier et d’optimiser les découpes lamellaires endothéliales cornéennes, réalisées au microkeratome et au laser femtoseconde. Ces optimisations passent par le développement d’un bioréacteur cornéen, ainsi que par l’amélioration des techniques d’estimation de la viabilité endothéliale cornéenne / Posterior lamellar graft, also named endothelial graft, consist in a replacement of a defective corneal endothelium by a healthy one take on a deceased donor. The epithelial and stromal layers of the patient stay untouched. A critical step of this technic is the preparation of the lamellar graft. Femtosecond lasers are better in comparison with microkeratome for low depth lamellar cut. However, femtosecond high depth corneal lamellar cuts show disappointing results. The aim of this thesis is to study and optimize corneal endothelial lamellar cuts, realized by microkeratome and femtosecond laser. Development of a corneal bioreactor and improve of corneal endothelial viability assessment are necessary for the realization of these objectives