La rétinopathie du prématuré, modélisée par les modèles de rétinopathie induite par l’oxygène (OIR), est l’une des principales causes de cécité dans l’enfance. Elle est constituée d’une première phase de vaso-oblitération rétinienne et choroïdienne suivie d’une phase de néovascularisation post-ischmémique rétinienne. La phase de dégénérescence vasculaire est entre autres liée d’une part à une baisse de l’expression des facteurs pro-angiogéniques et d’autre part à une inflammation rétinienne excessive. Toutefois, les mécanismes post-transcriptionnels à l’origine de ces phénomènes demeurent peu connus. La dérégulation des microARNs (miRs), des ARNs non codants régulant négativement l'expression des gènes, est impliquée dans la modulation de multiples processus physiologiques et pathologiques dont l’angiogenèse et l'inflammation. Cependant, le rôle des miRs dans l’angiogenèse et l’inflammation au cours de l’OIR reste à explorer. Basé sur l’établissement préalable d’un profil de modulation de l’expression des miRs au cours de l’OIR, nous avons sélectionné et caractérisé dans cette thèse le rôle d’un miR sur la fonction angiogénique puis d’un miR sur la fonction inflammatoire dans l’OIR. Nous avons caractérisé dans un premier temps, le miR-96. L’expression du miR-96 était significativement diminuée in vivo dans la rétine et la choroïde lors de la phase de vaso-oblitération du modèle murin de l’OIR. In vitro, le miR-96 était régulé négativement par l’hyperoxie dans les cellules endothéliales rétiniennes. La supplémentation en miR-96 avait un effet pro-angiogénique sur les cellules endothéliales rétiniennes soumises à l’hyperoxie par la préservation de la signalisation de facteurs angiogéniques, tels que VEGF et Ang2, leur permettant de maintenir leur capacité de migration et de tubulogenèse. In vivo, la supplémentation intravitréenne en miR-96 exerçait également ces fonctions vaso-protectives et permettait de préserver la microvascularisation rétinienne et choroïdienne par le maintien du niveau d’expression physiologique de VEGF et Ang2. Dans un second temps, nous avons caractérisé le miR-125a. L’expression du miR-125a était significativement diminuée in vivo dans la rétine lors de la phase de vaso-oblitération du modèle murin de l’OIR, mais également in vitro dans les cellules microgliales soumises à l’inflammation par hyperoxie ou LPS, ce qui était inversement corrélé à une augmentation de cytokines pro-inflammatoires telles que TNF-a, IL-6 et IL-16. Le miR-125a a été caractérisé comme anti-inflammatoire et sa supplémentation dans les cellules microgliales activées diminuait significativement l’expression de ces marqueurs pro-inflammatoires. La modification du sécrétome des cellules microgliales permettait une récupération des capacités angiogéniques des cellules endothéliales rétiniennes avec amélioration de leur prolifération et de leur tubulogenèse. In vivo, la supplémentation intravitréenne en mir-125a permettait de maintenir une expression physiologique de TNF-a, IL-6 et IL-16, ce qui était associé à une diminution de la vaso-oblitération rétinienne. Collectivement, ces travaux ont permis d’identifier et de caractériser le rôle du miR-96 dans la dysfonction angiogénique et du miR-125a dans la dysfonction inflammatoire lors de l’OIR. Cette thèse démontre pour la première fois qu’une thérapie basée sur la modulation de miRs spécifiques permettait de prévenir la dégénérescence vasculaire de l’OIR. Ces résultats pourraient constituer la base de nouvelles stratégies thérapeutiques dans le traitement précoce des rétinopathies ischémiques comme la rétinopathie du prématuré. / Retinopathy of prematurity is one of the leading causes of blindness in childhood and is represented by oxygen-induced retinopathy (OIR) models. It’s characterized by a first phase of retinal and choroidal vasoobliteration followed by a retinal neovascularization. The vascular degeneration is partly linked to a decrease in the expression of pro-angiogenic factors and to an excessive retinal inflammation. However, the post-transcriptional mechanisms implicated remain poorly understood. microRNAs (miRs) are non-coding RNAs that negatively regulate gene expression. Dysregulation of miRs is involved in the modulation of multiple physiological and pathological processes including angiogenesis and inflammation. However, the role of miRs in angiogenesis and inflammation during OIR remains to be explored. Based on the prior establishment of the modulation profile of miRs expression during OIR, we selected and characterized in this thesis the role of a miR on the angiogenic function then another miR on the inflammatory function in OIR. We first characterized the miR-96. In vivo, miR-96 expression was significantly downregulated in the retina and choroid during the vaso-obliteration phase of OIR rat. In vitro, miR-96 was downregulated by hyperoxia in retinal endothelial cells. miR-96 overexpression had a pro-angiogenic effect on retinal endothelial cells subjected to hyperoxia by preserving the signaling of angiogenic factors including VEGF and Ang2. This allowed to maintain their capacity for migration and tubulogenesis. In vivo, intravitreal supplementation with miR-96 also exerted these vasoprotective functions and preserved retinal and choroidal microvasculature by maintaining the physiological expression level of VEGF and Ang2. Secondly, we characterized the miR-125a. In vivo, the expression of miR-125a was significantly reduced in the retina during the vaso-obliteration phase of the OIR rat. In vitro, miR-125a was downregulated in microglial cells subjected to inflammation by hyperoxia or LPS, which was inversely correlated with an increase in pro-inflammatory cytokines such as TNF-a, IL-6 and IL-16. miR-125a was characterized as anti-inflammatory and its overexpression in activated microglial cells significantly decreased the expression of these pro-inflammatory markers. The modification of the secretome of the microglial cells allowed a recovery of the angiogenic capacities of the retinal endothelial cells with improvement of their proliferation and their tubulogenesis. In vivo, intravitreal supplementation with mir-125a maintained a physiological expression of TNF-a, IL-6 and IL-16, which was associated with a decrease in retinal vaso-obliteration. Collectively, these tasks identified and characterized the role of miR-96 in angiogenic dysfunction and miR-125a in inflammatory dysfunction during OIR. This thesis demonstrates for the first time that a therapy based on the modulation of specific miRs can prevent the OIR vascular degeneration. These results could form the basis of new therapeutic strategies in the early treatment of ischemic retinopathies such as retinopathy of prematurity.

La dégénérescence vasculaire et l’incapacité l’organisme à produire des vaisseaux sanguins de façon adéquate lors d’une condition ischémique est un fait saillant des rétinopathies ischémiques telles que la rétinopathie du prématuré (ROP). La ROP demeure la principale cause de défaillance visuelle et dans les cas extrêmes, de cécité chez les nourrissons prématurés. Elle présente deux phases distinctes soit une phase initiale clef de vasooblitération (VO) rétinienne et choroïdale qui entraînent la deuxième phase de néovascularisation (NV) rétinienne désorganisée et excessive. Au cours du développement normal, la NV oculaire a recours au phénomène d’angiogenèse qui consiste en la formation de nouveaux capillaires à partir de vaisseaux préexistants et de vasculogenèse qui consiste en la formation de nouveaux capillaires à partir de cellules endothéliales progénitrices dérivées de la moelle osseuse (BM-EPCs). Cette vasculogenèse implique la mobilisation des EPCs de la moelle osseuse vers la circulation afin d’être recrutées au site de NV pour contribuer de façon directe, soit en intégrant directement les structures vasculaires pour former des néovaisseaux, ou bien de façon indirecte par leur activité paracrine en libérant différents facteurs de croissance vasculaires. Toutefois, les mécanismes moléculaires impliqués dans la dysfonction des EPCs lors de la ROP sont encore mal compris. Au cours de mon mémoire, mes travaux ont ciblé la première phase de VO rétinienne afin de promouvoir la revascularisation par une thérapie basée sur une supplémentation d’EPCs natives ou reprogrammées. Compte tenu du rôle capital des EPCs dans la NV, mon mémoire s’est d’abord intéressé au rôle de Nogo-A (une protéine de la famille de réticulon), connue pour son action anti-angiogénique, sur l'activité fonctionnelle des EPCs en condition de ROP. Pour ce faire, nous avons utilisé un modèle de rétinopathie induite par l’oxygène (OIR) simulant la ROP. L’objectif global de ce projet consiste à évaluer l’interrelation entre l’effet de l’hyperoxie (une condition clef de la ROP) sur la voie de signalisation Nogo-A et de son récepteur NgR1 sur la fonction des EPCs. Premièrement, les résultats obtenus montrent une augmentation de l’expression de Nogo-A et NgR1 chez les BM-EPCs soumis ex vivo à l’hyperoxie, mais aussi dans les EPCs extraites des rats OIR. En addition, l’augmentation de l’expression de Nogo-A/NgR1 par l’hyperoxie corrèle avec la dysfonction angiogénique des EPCs caractérisées par une diminution de leurs capacités de migration et de tubulogenèse. De façon intéressante, l’inhibition de Nogo-A (par un peptide neutralisant) améliore la capacité migratoire et tubulogénique des EPCs, et protège leur fonction contre l’hyperoxie. Également, l’inhibition de Nogo-A induit l’expression du facteur angiogénique et mobilisateur d’EPCs, SDF-1, suggérant que NgR1 régule négativement l’expression de SDF-1. Par ailleurs, nous avions également pour objectif final d’évaluer l’efficacité protectrice d’une supplémentation d’EPCs natives ou reprogrammées (Nogo-/-) pour améliorer la revascularisation rétinienne dans un modèle de rat OIR. Les résultats montrent qu’une supplémentation intrapéritonéale d’EPCs natives diminue significativement la VO rétinienne, mais que cet effet pro-angiogénique devient plus prononcé par le traitement d’EPCs préconditionnées (reprogrammées par l’inhibition de Nogo-A) chez les rats OIR. Collectivement, nos résultats démontrent que : 1) l’hyperoxie cause une dysfonction angiogénique des BM-EPCs en induisant Nogo-A ce qui contribue à la VO rétinienne chez les rats OIR, et que 2) une supplémentation d’EPCs conditionnées (reprogrammées par l’inhibition de Nogo-A) est plus efficace qu’une supplémentation d’EPCs natives pour améliorer la réparation vasculaire rétinienne. Pour conclure, nous mettons donc en évidence une cible potentielle qui est la protéine Nogo-A afin de préserver l’activité biologique des EPCs et ultimement, l’intégrité vasculaire chez les rats OIR. / Vascular degeneration and the inability of the body to produce adequate blood vessels during an ischemic condition is a salient feature of ischemic retinopathies such as retinopathy of prematurity (ROP). ROP remains the leading cause of visual impairment and in extreme cases, blindness in premature infants. It presents two distinct phases: a key initial phase of retinal and choroidal vasoobliteration (VO) which leads to the second phase of disorganized and excessive retinal neovascularization (NV). During normal development, ocular NV uses the phenomenon of angiogenesis which consists of the formation of new capillaries from pre-existing vessels and vasculogenesis which consists of the formation of new capillaries from progenitor endothelial cells derived from the marrow bone (BM-EPCs). This vasculogenesis involves the mobilization of EPCs from the bone marrow to the circulation in order to be recruited at the NV site to contribute directly, either by directly integrating the vascular structures to form new vessels, or indirectly by their paracrine activity by releasing different vascular growth factors. However, the molecular mechanisms involved in the dysfunction of EPCs during ROP are still poorly understood. During my thesis, my work targeted the first phase of retinal VO in order to promote revascularization by therapy based on supplementation of native or reprogrammed EPCs. Given the capital role of EPCs in NV, my thesis was first interested in the role of Nogo-A (a protein of the reticulon family), known for its anti-angiogenic action, on the functional activity of EPCs in ROP condition. To do this, we used an oxygen-induced retinopathy (OIR) model simulating ROP. The overall objective of this project is to assess the interrelationship between the effect of hyperoxia (a key condition of ROP) on the Nogo-A signaling pathway and its NgR1 receptor on the function of EPCs. First, the results obtained show an increase in the expression of Nogo-A and NgR1 in BM-EPCs subjected to hyperoxia ex vivo, but also in EPCs extracted from OIR rats. In addition, the increase in the expression of Nogo-A / NgR1 by hyperoxia correlates with the angiogenic dysfunction of EPCs characterized by a decrease in their capacity for migration and tubulogenesis. Interestingly, inhibition of Nogo-A (by a neutralizing peptide) improves the migratory and tubulogenic capacity of EPCs, and protects their function against hyperoxia. Also, inhibition of Nogo-A induces expression of the angiogenic and mobilizing factor of EPCs, SDF-1, suggesting that NgR1 negatively regulates the expression of SDF-1. In addition, our final objective was also to evaluate the protective efficacy of supplementation of native or reprogrammed EPCs (Nogo - / -) to improve retinal revascularization in an OIR rat model. The results show that intraperitoneal supplementation of native EPCs significantly decreases retinal VO, but that this pro-angiogenic effect becomes more pronounced by treatment of preconditioned EPCs (reprogrammed by inhibition of Nogo-A) in OIR rats. Collectively, our results demonstrate that: 1) hyperoxia causes angiogenic dysfunction of BM-EPCs by inducing Nogo-A which contributes to retinal VO in OIR rats, and that 2) supplementation of conditioned (reprogrammed by inhibition of Nogo-A) is more effective than supplementation of native EPCs in improving retinal vascular repairs. To conclude, we therefore highlight a potential target which is the Nogo-A protein in order to preserve the biological activity of EPCs and ultimately, vascular integrity in OIR rats.

No description available.

While architects think and work in a visual way, people who are visually impaired may pay more attention to other senses and are able to appreciate other spatial qualities. In my thesis project, I have explored architecture for the visually impaired. The idea is rooted in a frustration of that architecture is almost always valued visually. All other senses are mostly ignored - or at least never prioritised. What is architecture based on other senses than sight? If architecture is experienced by all senses - mustn’t architects involve all senses in their design process? In this project, the aim has been to create architecture that improves the visitors’ spatial perception by activating all senses as an experience and a source of information.

The aim of this study was to determine the prevalence and causes as well as the risk factors of visual impairment (VI) and blindness among Black South Africans with diabetes mellitus (DM) aged 40 years and older in Mopani District, Limpopo province, South Africa. This was a cross-sectional study in which Black South Africans with DM aged ≥40 years old were examined for VI and blindness. In addition, anthropometric as well as risk factors for VI and blindness were studied. A total of 225 participants were selected from seven Public Health Facilities in Mopani District. Data was collected using standard optometric instruments, anthropometric instruments and structured interviews. Data analysis was done using the Statistical Analysis System (SAS) and Microsoft Excel software packages. The ages of the participants ranged from 40 to 90 years with a mean of 61.5±10.49 years. There were more females (71.5%) than males (28.4%). The prevalence of uncorrected VI and blindness in the right eyes of the participants was 70.7% and 3.6%, respectively. In the left eyes, it was 72% and 3.1%, respectively. However, following optical correction, the prevalence in right eyes was 41.3% and 3.6%, respectively. In the left eyes, it was 42.2% and 3.1%, respectively. Risk factors that were individually associated with VI and blindness include age, educational qualification, monthly income, knowledge of DM types, oral DM treatment (pills), losing weight, compliance to losing weight, family history of DM, physical activity, and date of last eye examination .When logistic regression was used, knowledge of DM types, pills, and compliance to losing weight, family history of DM, monthly income and physical activity remained associated with VI and blindness. The high prevalence of VI in this diabetes population was not primarily due to DM itself, but due to refractive error and cataract, conditions which have effective and easy treatments. A total of 84% of the participants were visually impaired due to either refractive error or cataract or both and only 3.8% due to diabetes retinopathy. It is therefore recommended that appropriate and affordable refraction and cataract surgical services be made available and accessible to this population / Health Studies / D. Litt. et Phil. (Health Studies)

Diabetic vasculopathy (DV) is the most important consequence of chronic hyperglycemia in patients with diabetes mellitus (DM). This thesis explores the interaction of blood, bone and kidney in the pathogenesis of DV by i) reviewing the current understanding of pathogenesis of macrovascular and microvascular diseases in DM to identify gaps in literature and generate hypotheses relating to various facets of DV ii) undertaking a series of prospective studies to examine these hypotheses iii) analysing the findings and integrating any new information obtained from the clinical studies into the current knowledge base and iv) generating hypotheses upon which future work might be based. The literature search was carried out with the aim of understanding current concepts of pathogenesis of DV and its potential modulators. The original reviews resulting from this process are presented in chapters 2 to 4. A series of pilot studies reported in chapters 7 to 11, were then carried out to interrogate hypotheses originating from this process. The first study was carried out in healthy individuals to define the biological variation of potential modulators of DV, namely erythropoietin (EPO), parathyroid hormone, 25 hydroxyvitamin D and 1, 25-dihydroxyvitamin D to facilitate the design and interpretation of subsequent studies. It revealed a wide biological variation of these modulators in the healthy population thus,emphasizing the need to have a control group in the subsequent study population. To examine whether tubulointerstitial dysfunction occurs before the onset of microalbuminuria, a measurement of the above mentioned parameters was carried out along with markers of tubulointerstitial injury in patients with type 1 and type 2 DM without microalbuminuria and in non-diabetic controls. It was found that tubulointerstitial dysfunction with low levels of EPO and 1, 25-dihydroxyvitamin D and higher excretion of tubular injury markers, occurs before the onset of microalbuminuria. Subsequently, diabetic and nondiabetic chronic kidney disease (CKD) patients with EPO deficiency anaemia were examined to study the effects of EPO therapy on the excretion of tubular injury markers. However, in these patient groups, we were unable to demonstrate an effect of EPO therapy on the markers of tubular injury in spite of a beneficial haematological response. To examine whether vascular calcification (VC) and bone mineral density (BMD) were linked in patients with diabetes mellitus and to explore their relationship to modulators of DV, an assessment of VC and BMD was undertaken in patients with type 2 DM with different degrees of proteinuria and normoalbuminuria. VC was assessed by CT scan and BMD by a DEXA scan. Modulators of DV were measured including serum Osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-b-ligand (RANKL). The findings were i) a high prevalence of VC and osteopenia in normoalbuminuric type 2 DM patients with normal serum creatinine ii) a weak inverse relationship between VC and osteopenia iii) proteinuric patients had worse VC but not osteopenia iv) weak relationships between OPG levels and both VC and osteopenia, masked by age in multivariate analysis. The final study examined the relationship between modulators of DV, including OPG and RANKL, and the degree of CKD. It was found that abnormalities of OPG and RANKL occur before the onset of microalbuminuria and progress with deterioration of renal function. Compared to nondiabetics, DM patients have higher OPG levels in the predialysis phase and lower levels in haemodialysis phase, a phenomenon that might indicate endothelial exhaustion in dialysis patients with DM. The derangements associated with DV seem to occur earlier than previously thought. Further work is required to untangle these complexities and to define the contribution of factors such as the adverse blood milieu, the vasculature, abnormal bone and mineral metabolism, and early tubulointerstitial damage. The findings from the studies reported here may help in the formulation of new hypotheses, which might contribute to future work in this area.

Dans les pays industrialisés, les rétinopathies ischémiques proliférantes telles que la rétinopathie diabétique et la rétinopathie du prématuré sont les principales causes de cécité chez les individus en âge de travailler et la population pédiatrique. Ces pathologies sont caractérisées par une dégénérescence microvasculaire initiale suivie d’une hyper-vascularisaton compensatoire disproportionnée et pathologique. Les sirtuines constituent une importante famille de protéines impliquées dans le métabolisme et la réponse au stress. Plus particulièrement, sirtuine 3 (SIRT3) est une déacétylase mitochondriale primordiale qui agit au cœur du métabolisme énergétique et de l’activation de nombreuses voies métaboliques oxydatives. Nos résultats démontrent pour la première fois qu’une déficience en SIRT3 diminue la sévérité des lésions vasculaires dans le modèle murin de rétinopathie induite par l’oxygène (OIR). En plus de stimuler l’angiogénèse, l’absence de SIRT3 est aussi associée à une augmentation de la glycolyse, possiblement en activant la famille de gènes 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB). Nous suggérons que le manque de SIRT3 est impliqué dans l’effet Warburg et procure ainsi un avantage prolifératif et protecteur dans l’OIR. La présente étude propose SIRT3 comme nouvelle cible thérapeutique potentielle dans la rétinopathie du prématuré, une maladie dont les complications désastreuses persistent tout au long de la vie. / Proliferative ischemic retinopathies such as proliferative diabetic retinopathy and retinopathy of prematurity (ROP) are the leading causes of blindness in working age and pediatric populations in industrialized countries. These pathologies are characterized by an initial microvascular degeneration followed by a disproportionate compensatory but pathological hyper-vascularization mounted by the hypoxic and energy deficient retina in an attempt to reinstate metabolic equilibrium. Sirtuins are an important family of protein involved in metabolism and stress response. Sirtuin 3 (SIRT3) in particular is a major mitochondrial deacetylase central to energy metabolism and the regulation of many oxidative pathways. For the first time, our results show that a lack of SIRT3 decreases the severity of vascular lesions in the oxygen-induced retinopathy (OIR) mouse model. Deficiency in SIRT3 not only stimulates angiogenesis, but also increases glycolysis, possibly through indirect activation of the gene family 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB). We suggest that a lack of SIRT3 is involved in the Warburg effect and therefore confers a proliferative advantage that is protective in OIR. The present study puts forward SIRT3 as a new potential therapeutic target for ROP, a disease leading to life-long vision complications.

The physical complications of Type 1 diabetes mellitus (T1DM) have been understood as an accelerated ageing process (Morley, 2008). Do people with T1DM also experience accelerated cognitive and brain ageing? Using findings from research of the normal cognitive and brain ageing process and conceptualized in theories of the functional brain changes in cognitive ageing, a combination of cognitive testing and functional magnetic resonance imaging (fMRI) techniques were used to evaluate evidence of accelerated cognitive and brain ageing in middle-aged adults with T1DM. The first part of this thesis comprises a cognitive study of 94 adults (≥ 45 years of age) with long duration (≥ 10 years) of T1DM. Participants completed cognitive assessment and questionnaires on general mood and feelings about living with diabetes. Findings highlighted the importance of microvascular disease (specifically retinopathy) as an independent predictor of cognitive function. The incidence and predictors of mild cognitive impairment (MCI) were then explored. Results indicate a higher percentage of the group met criteria for MCI than expected based on incidence rates in the general population, providing initial evidence of accelerated cognitive ageing. Psychological factors were explored next. The relationship between the measures of well-being, diabetes health, and cognitive function highlighted the need for attention to patient's psychological well-being in diabetes care. Finally, a subgroup of 30 participants between the ages of 45 and 65 who differed on severity of retinopathy were selected to take part in an fMRI study. Blood oxygen level dependent (BOLD) activity was evaluated while participants were engaged in cognitive tasks and during rest. The findings provided evidence that the pattern of BOLD activation and functional connectivity for those with high severity of retinopathy are similar to patterns found in adults over the age of 65. In line with the theories of cognitive ageing, functional brain changes appear to maintain a level of cognitive function. Evidence of accelerated brain ageing in this primarily middle-aged group, emphasizes the importance of treatments and regimens to prevent or minimize microvascular complications.

INTRODUÇÃO: Fatores associados à etiopatogenia das complicações diabéticas, incluindo hiperglicemia e estresse oxidativo, podem causar alterações epigenéticas que modificam a expressão de genes em células-alvo, sem alterar sua sequência de DNA. São considerados mecanismos epigenéticos: (1) as modificações pós-traducionais das histonas; (2) a metilação do DNA e (3) a ação dos micro-RNAs (miRNAs); todos já foram reconhecidos na patogênese da \"memória metabólica\", situação na qual a hiperglicemia continua a exercer efeitos deletérios prolongados mesmo depois de ser normalizada. A sirtuína-1 é uma enzima que causa modificações pós-traducionais das histonas por sua atividade de histona desacetilase, silenciando a transcrição gênica. O silenciamento gênico também pode ocorrer pela ação da DNA metiltransferase 1 (DNMT1), enzima que adiciona um grupamento metil (CH3) na posição 5 de resíduos de citosina localizadas em ilhas CpG presentes nas regiões promotoras dos genes. Os miRNAs constituem uma classe de pequenos RNAs não codificadores com cerca de 19 a 25 nucleotídeos que controlam a expressão gênica por meio da repressão da tradução ou da degradação do RNA mensageiro-alvo. As hipóteses do presente estudo são (1) que exista um perfil sérico de miRNAs associado à presença ou ausência de complicações crônicas e (2) que existam variantes em genes relacionados à desacetilação das histonas e à metilação de citosinas que poderiam predispor ao aparecimento das complicações diabéticas, o que se constituiria na \"genética da epigenética\". OBJETIVOS: (1) caracterizar e comparar o perfil de miRNAs sérico de pacientes com diabetes mellitus tipo 1 (DM1) sem nenhuma complicação microvascular versus aqueles com três complicações microvasculares: retinopatia diabética (RD), doença renal diabética (DRD) e neuropatia diabética, para identificar vias epigeneticamente moduladas nesses dois grupos de pacientes e (2) avaliar a frequência de polimorfismos de um único nucleotídeo nos genes que codificam as enzimas DNMT1 e sirtuína-1 e suas associações com cada uma das complicações microvasculares em pacientes com DM1. MÉTODOS: O perfil sérico de 381 miRNAs foi avaliado com o uso do estojo comercial Taqman® Human MicroRNA Array A em 10 pacientes bem caracterizados clínica e laboratorialmente divididos em dois grupos: Pacientes com DM1 sem complicações [sem DRD (Clearance de creatinina > 90 ml/min/1,73 m2 e excreção urinária de albumina < 20 mg/g de creatinina), sem polineuropatia sensitivo-motora distal (ausência de sintomas sugestivos de neuropatia, sensibilidade térmica e dolorosa e reflexo aquileu normais), sem neuropatia autonômica cardiovascular (NAC) e sem RD] e Pacientes com DM1 com complicações [DRD (Clearance de creatinina < 60 ml/min/1,73 m2 e excreção urinária de albumina > 200 mg/g de creatinina), com polineuropatia sensitiva-motora distal, com NAC instalada e RD moderada ou grave]. Os cinco miRNAs mais diferencialmente expressos foram validados em uma casuística bem caracterizada de 20 pacientes com DM1 sem nenhuma complicação e 27 com todas as complicações microvasculares, com o emprego do estojo comercial TaqMan(TM) Advanced miRNA cDNA Synthesis. A avaliação da frequência de polimorfismos de um único nucleotídeo nos genes que codificam as enzimas DNMT1 (rs8112895, rs7254567, rs11085721, rs17291414, rs10854076) e sirtuína-1 (rs10997870; rs12766485) foi realizada após a genotipagem por reação em cadeia da polimerase em tempo real, em uma casuística composta por 466 pacientes com DM1. RESULTADOS: Do total de 377 miRNAs-alvo avaliados no soro dos pacientes com DM1, um total de 21 miRNAs estava superexpresso no grupo com complicações. Dos 5 miRNAs para os quais foi realizada a validação na casuística de 47 pacientes com DM1, dois foram confirmados como superexpressos na população com complicações (hsa-miR-518d-3p e hsa-miR-618). O polimorfismo rs11085721 no gene que codifica a DNMT1 associou-se à presença de NAC no sexo feminino, sendo o alelo raro C considerado de risco e conferindo um odds ratio (intervalo de confiança de 95%) de 2,44 (1,26-5,28). Nenhum polimorfismo da sirtuína-1 associou-se às complicações microvasculares avaliadas. CONCLUSÃO: o perfil de miRNAs séricos difere entre pacientes com DM1 com e sem complicações. O achado de uma variante em um gene que codifica a enzima de uma via epigenética conferir suscetibilidade a uma complicação crônica sugere que também exista a \"genética da epigenética\" modulando o desenvolvimento das complicações / INTRODUCTION: Factors associated with the etiopathogenesis of diabetic complications, including hyperglycemia and oxidative stress, may cause epigenetic changes that modify the expression of genes in target cells without altering their DNA sequence. The following mechanisms are considered epigenetics: (1) post-translational modifications of histones; (2) methylation of DNA and (3) action of micro-RNAs (miRNAs); all have already been recognized in the pathogenesis of \"metabolic memory\", a situation in which hyperglycemia exerts prolonged deleterious effects even after its normalization. Sirtuin-1 is an enzyme that causes post-translational modifications of histones by their histone deacetylase activity, silencing gene transcription. Gene silencing may also occur through the action of DNA methyltransferase 1 (DNMT1), an enzyme that adds a methyl group (CH3) at position 5 of cytosine residues located in CpG islands from gene-promoter regions. miRNAs are a class of small non-coding RNAs with about 19 to 25 nucleotides that control gene expression by promoting translation repression or degradation of target messenger RNAs. The hypotheses of the present study are (1) there is a serum profile of miRNAs associated with the presence or absence of chronic complications and (2) there are variants in genes related to histone deacetylation and cytosine methylation that could predispose to diabetes complications, which would constitute the \"genetics of epigenetics\". OBJECTIVES: (1) to characterize and compare the serum miRNA profile of patients with type 1 diabetes mellitus (T1D) without any microvascular complications versus those with three microvascular complications: diabetic retinopathy (DR), diabetic kidney disease (DKD) and diabetic neuropathy to identify signaling pathways epigenetically modulated in these two groups of patients and (2) to assess the frequency of single nucleotide polymorphisms in the genes encoding DNMT1 and sirtuin-1 and their associations with each of the microvascular complications in T1D patients. METHODS: The serum profile of 381 miRNAs was evaluated using the Taqman® Human MicroRNA Array A kit in 10 clinical and laboratory well-characterized patients divided into two groups: Patients without microvascular complications: without DKD (creatinine clearance> 90 ml/min/1.73 m2 and urinary albumin excretion < 20 mg / g creatinine), without distal sensory-motor polyneuropathy (absence of symptoms suggestive of neuropathy and normal thermal and pain sensitivity and Achilles reflex), without cardiovascular autonomic neuropathy (CAN) and without DR; and T1D patients with complications: with DKD (creatinine clearance < 60 ml / min / 1.73 m2 and urinary albumin excretion> 200 mg / g creatinine), with distal sensory-motor polyneuropathy, with CAN and with DR moderate or severe. The five most differentially expressed miRNAs were validated in a well-characterized case series of 20 patients with no complications and 27 patients with all microvascular complications using the TaqMan (TM) Advanced miRNA cDNA Synthesis kit. The evaluation of the frequency of single nucleotide polymorphisms in genes encoding the DNMT1 (rs8112895, rs7254567, rs11085721, rs1729414, rs10854076) and sirtuin-1 (rs10997870; rs12766485) was performed by genotyping using real-time polymerase chain reaction in a sample of 466 T1D patients. RESULTS: Of the total of 377 target miRNAs evaluated in the serum of T1D patients, 21 miRNAs were overexpressed in the group with complications. Of the 5 miRNAs for which validation was performed in 47 patients, two were confirmed as overexpressed in the group with complications (hsa-miR-518d-3p and hsa-miR-618). The polymorphism rs11085721 in the gene encoding DNMT1 was associated with the presence of CAN in female patients, with the minor allele C being considered of risk and conferring an odds ratio (95% confidence interval) of 2.44 (1.26 - 5.28). Polymorphisms in the gene encoding Sirtuin-1 did not associate with microvascular complications. CONCLUSION: the serum miRNA profile differs between patients with and without microvascular complications. A variant in a gene encoding a enzyme of an epigenetic pathway conferring susceptibility to a chronic complication suggests that there is also the \"genetics of epigenetics\" modulating the development of complications

O presente texto descreve métodos e apresenta resultados do projeto de pesquisa de mestrado intitulado \"Dois Problemas em Análise de Formas de Estruturas de Ramificação\". Ambos os problemas abordados estão relacionados às sub-áreas da Análise de Formas denominadas Caracterização e Descrição de Formas. O primeiro problema consiste na investigação de um conjunto de características propostas para distingüir, primeiramente, entre estruturas de ramificação de vasos sangüíneos em imagens de retina segmentadas manualmente e automaticamente. A seguir, as mesmas características são aplicadas para discernir entre estruturas de ramificação de vasos sangüíneos em imagens de retina com e sem retinopatia diabética proliferativa (Proliferative Diabetic Retinopathy - PDR). A PDR é uma das patologias associadas à diabetes, que pode culminar na cegueira do indivíduo. Diagnósticos são possíveis por meio de imagens de fundo de olho e, quando efetuados precocemente, viabilizam intervenções oportunas evitando a perda da visão. Neste trabalho, 27 imagens digitais de fundo de olho foram segmentadas por dois processos distintos, isto é, segmentação manual por um especialista e a segmentação automática, mediante a transformada contínua Wavelet - CWT e classificadores estatísticos. Visando à caracterização destas formas, um conjunto de 08 características foi proposto. Este conjunto foi formado por três grupos, a saber: descritores tradicionais geométricos (Área, Perímetro e Circularidade), descritores associados à transformada wavelet ( 2o momento estatístico da distribuição de módulos da CWT, Entropia de Orientação da distribuição de fases da CWT e Curvatura) e um descritor fractal (Dimensão de Correlação - Global e Mediana). Uma Análise Discriminante Linear LDA revelou que as características geométricas tradicionais não detectam o início da retinopatia diabética proliferativa. A maior capacidade discriminante individual foi exibida pela Curvatura, com Área sob a curva ROC de 0.76. Um subconjunto com 6 características apresentou grande capacidade discriminante com Área sob a curva ROC de 0.90. O segundo problema diz respeito à extração de contorno de estruturas de ramificação bidimensionais de neurônios tridimensionais. Este trabalho contribui originalmente com uma solução para este problema, propondo dois algoritmos desenvolvidos para Rastreamento de Ramos e Extração do Contorno Paramétrico de estruturas de ramificação, capazes de transpor regiões críticas formadas por cruzamentos ocasionados pela projeção de estruturas 3D no plano das imagens 2D. Grande parte dos métodos baseados em contorno para análise de formas de estruturas de ramificação de células neuronais não produz representações corretas destas formas, devido à presença de sobreposições entre processos neuronais, levando os algoritmos tradicionais de extração de contorno a ignorar as regiões mais internas destas estruturas, gerando representações incompletas. O sistema proposto neste trabalho foi desenvolvido objetivando a solução do problema de extração de contorno, mesmo na presença de múltiplas sobreposições. Inicialmente, a imagem de entrada é pré-processada, gerando um esqueleto 8-conexo com ramos de um pixel de largura, um conjunto de sementes de sub-árvores dendríticas e um conjunto de regiões críticas (bifurcações e cruzamentos). Para cada sub-árvore, o algoritmo de rastreamento rotula todos os pixels válidos de um ramo, até chegar em uma região crítica, onde o algoritmo decide a direção em que deve continuar o rastreamento. Nosso algoritmo mostrou-se robusto, mesmo quando aplicado a imagens com segmentos paralelos muito próximos. Resultados obtidos com imagens reais (neurônios) são apresentados. / This document describes methods and presents results from the Master of Science\'s research project in computer science entitled \"Two Problems in Shape Analysis of Branching Structures\". Both tackled problems herein are related to Shape Analysis sub-fields, namely Characterization and Description of shapes. The former problem consists of an investigation on a proposed set of features aimed at discriminating, firstly, between blood vessels branching structures manually and automatically segmented. In the sequel, the same features are used to assess their discriminative capability in distinguishing between blood vessels branching structures with and withoud proliferative diabetic retinopathy (PDR). The PDR is a pathology related to diabetes, which may lead to the blindness. Diagnosis is possible through optic fundus image analysis, which may allow timely interventions preventing vision loss. In this work, 27 digital optic fundus images were segmented by two distinct segmentation processes, i.e. manual segmentation carried out by an especialist and automated segmentation, through the CWT (Continuous Wavelet Transform) and statistical classifiers. In order to characterize such a shapes, a set of 8 features has been proposed. The aforementioned set was comprised of three features groups, that is: traditional geometric descriptors (Area, Perimeter and Circularity), wavelet-based descriptors (2nd statistical moment from the CWT Modulus distribution, Orientation Entropy from the CWT Phase distribution and Curvature) and a fractal descriptor (Correlation Dimension - global and median). Linear Discriminant Analysis LDA revelead that the traditional geometric features are not able to detect early proliferative diabetic retinopathy. The largest singular discriminant capability was shown by the Curvature, with area under the ROC curve of 0.76. A subset of 6 features presented a good discriminating power with area under the curve of 0.90. The second problem concerns contour extraction from 2D branching structures of 3D neurons. This work contributes with an original solution for such a problem, proposing two algorithms devised for Branches Tracking and Branching Structures Contour Extraction. The proposed algorithms are able to traverse critical regions implied by the projection of 3D structures onto a 2D image plane. Most of contour-based methods intended to shape analysis of neuronal branching structures fall short of yielding proper shape representations, owing to the presence of overlapings among neuronal processes, causing the traditional algorithms for contour following to ignore the innermost regions, thus generating incomplete representations. The proposed framework system was developed aiming at the solution of the contour extraction problem, even in the presence of multiple overlapings. The input image is pre-processed, so as to obtain an 8-connected skeleton with one-pixel wide branches, a set of seeds of dendritic sub-trees and a set of critical regions (bifurcations, crossings and superpositions). For each sub-tree, the Branches Tracking Algorithm labels all valid pixels of a branch, until reaching a critical region, where the algorithm decides about the direction to go on with the tracking. Our algorithm has shown robustness, even in images plenty of very close parallel segments. Results with real images (neurons) are presented.