L’injection de cellules souches provenant de la moelle osseuse est reconnue pour améliorer la fonction ventriculaire ainsi que le remodelage cicatriciel après un infarctus du myocarde (IM). Le Stromal Cell-derived factor-1 alpha (SDF-1 alpha), une chimiokine induite par l’ischémie cardiaque, représente une grande importance en raison de son rôle dans le recrutement de cellules inflammatoires et de cellules souches de la moelle osseuse vers les sites endommagés. Quoique les recherches sur le rôle de la chimiokine SDF-1 alpha dans le remodelage ventriculaire se multiplient, son implication dans la phase aiguë du remodelage reste inexplorée. Le but de la présente étude est de déterminer l’effet du SDF-1 alpha sur la taille de la cicatrice, l’hypertrophie cardiaque ainsi que la fonction ventriculaire chez des rats et des souris une semaine après un IM. La stratégie utilisée implique l’administration de l’AMD3100 (1 mg/kg, 24 heures après l’IM, pendant 6 jours), l’antagoniste sélectif du récepteur du SDF-1 alpha, le CXCR4. Ce récepteur est couplé à une protéine G alpha i et induit la migration et la prolifération cellulaire. Chez les rats du groupe IM, l’expression de la chimiokine a été détectée surtout dans les cellules musculaires lisses et les cellules endothéliales des vaisseaux cicatriciels. Le profil d’expression de la chimiokine dans le cœur infarci indique un gradient de concentration vers la cicatrice. Une semaine après l’IM, le traitement avec l’AMD3100 a diminué la taille de la cicatrice, résultant en une amélioration de la fonction ventriculaire et une diminution de l’élévation de l’expression de l’ARNm de l’ANP dans le ventricule gauche non infarci (VGNI). Chez les souris, le traitement avec l’AMD3100 a engendré les mêmes effets, soit une diminution de la taille de la cicatrice ainsi qu’une amélioration de la fonction ventriculaire. La réduction de la taille de la région infarcie chez les souris traitées avec l’AMD3100 est associée avec une atténuation de l’infiltration des neutrophiles dans la région ischémique. Ces résultats suggèrent que le blocage pharmacologique de l’axe SDF-1 alpha/CXCR4 lors de la phase aiguë du remodelage ventriculaire après un IM diminue la taille de la cicatrice et améliore la fonction ventriculaire, en partie, par la diminution de la réaction inflammatoire. / The injection of bone marrow stem cells in the infarcted heart was shown to improve ventricular function and scar remodelling. The chemokine Stromal Cell-derived factor-1 alpha (SDF-1 alpha) is implicated in the migration of inflammatory and bone marrow derived stem cells to damaged region. Despite a local increase of SDF-1 alpha expression in the damaged myocardium, the biological impact of the chemokine during the acute phase of remodelling in the ischemic heart remains undefined. Therefore, the present study examined the role of SDF-1 alpha on scar expansion, cardiac hypertrophy and ventricular function in rats following myocardial infarction (MI) via administration of AMD3100 (1 mg/kg, 24 hours post-MI and continued for 6 days) a selective antagonist of the SDF-1 alpha receptor, CXCR4. This receptor is coupled to a G alpha i protein and induced migration and proliferation of cells. In 1-week post-MI rats, chemokine expression was detected in smooth muscle and endothelial cells of blood vessels residing in the infarcted region. An SDF-1 alpha gradient towards the infarcted region was detected in the post-MI rat heart. In 1-week post-MI rats, AMD3100 therapy reduced scar size, concomitantly improved left ventricular function and partially supressed the elevated expression of ANP mRNA in the non-infarcted left ventricule. Preliminary studies on mice showed that the reduced infarct size in AMD3100-treated post-MI mice was associated with an attenuation of neutrophil infiltration in the ischemic region. These data highlight the novel observation that pharmacological antagonism of the SDF-1 alpha/CXCR4 axis during the acute phase of repartive fibrosis post-MI attenuated scar expansion and improved ventricular function in part via attenuation of the inflammatory response.

Resumo: A seleção assistida por marcadores moleculares têm contribuído com os estudos para o desenvolvimento de cultivares resistentes. Os ganhos mais evidentes podem ocorrer em doenças como a ferrugem asiática da soja, onde a alta variabilidade do patógeno e a busca por novas fontes de resistência têm dificultado o sucesso dos melhoristas. Assim, os objetivos deste trabalho foram estudar a herança da resistência à ferrugem em diferentes fontes e desenvolver marcadores SCAR ligados a um loco de resistência para seleção assistida. Populações F2 oriundas dos cruzamentos PI 459025 x CD 208 (1), PI 200526 x CD 205 (2), PI 200456 x Conquista (3) e GC 84058-21-4 x IAC Foscarin 31 (4) foram submetidas à inoculação com a ferrugem e avaliadas quanto ao tipo de lesão (RB - resistente ou TAN - suscetível). O teste de Qui-quadrado indicou a presença de um gene dominante para os cruzamentos 1 e 2, enquanto no 3 e 4 observou-se a presença de um gene recessivo. A análise multivariada agrupou os genótipos mais similares, com base nos caracteres severidade da doença, número de vagens, número de sementes e ciclo, avaliados nos cruzamentos 2 a 4. Diferenças significativas foram encontradas em número de lesões e esporulação para genótipos RB e TAN. Dois bulks de DNA foram obtidos de plantas homozigotas resistentes e suscetíveis no cruzamento 1, para a análise de BSA. Dos 600 iniciadores RAPD testados, três foram discriminativos e localizam-se a 4,5, 6,2 e 10,2 cM do loco de resistência, em fase de repulsão. Um desses marcadores foi convertido a SCAR e se manteve discriminativo, podendo ser indicado para seleção assistida de genótipos resistentes com a mesma fonte. / Abstract: Assisted selection by molecular markers has contributed to development studies of resistant cultivars. The most evident gains may occur for diseases like the Asian soybean rust, where the high variability of pathogen and the search for new resistance sources has difficult the breeder success. Thus, the aims of this work were to study the rust resistance inheritance in different sources and develop SCAR markers linked to a rust resistance locus for assisted selection. F2 populations from crosses PI 459025 x CD 208 (1), PI 200526 x CD 205 (2), PI 200456 x Conquista (3) and GC 84058-21-4 x IAC Foscarin 31 (4) were submitted to rust inoculation and evaluated by the lesion type reaction (RB - resistant or TAN - susceptible). Chi-square test indicated the presence of a single dominant gene for crosses 1 and 2, whereas the 3 and 4 crosses showed a single recessive gene. Multivariate analysis has grouped the most similar genotypes based on disease severity, number of pods, number of seeds and cycle for 2 to 4 crosses. Significant differences were observed in lesions number and sporulation rate for RB and TAN genotypes. Two DNA bulks were obtained, for BSA analysis, on cross 1, from resistant and susceptible homozygous plants. From the 600 tested RAPD primers, three of them were discriminative and located in repulsion phase at 4.5, 6.2 and 10.2 cM from the resistance locus. One of the RAPD markers was converted to SCAR and confirmed its discriminative nature. So it can be indicated for assisted selection of genotypes with the same resistance source. / Orientador: Antonio Orlando Di Mauro / Coorientador: José Baldin Pinheiro / Coorientadora: Sandra Helena Unêda Trevisoli / Banca: Eduardo Antonio Gavioli / Banca: Natal Antonio Vello / Banca: Janete Apparecida Desidério Sena / Banca: Rinaldo César de Paula / Doutor

L'objectif de ce travail de recherche est le développement, la mise au point et la validation d'une méthode de spectroscopie multi-modalités en diffusion élastique et autofluorescence pour caractériser des tissus cutanés cicatriciels hypertrophiques in vivo. Ces travaux sont reposés sur trois axes. La première partie des travaux présente l'instrumentation : développement d'un système spectroscopique qui permet de réaliser des mesures de multimodalités in vivo de manière automatique et efficace. Des procédures métrologiques sont mise en place pour caractériser le système développé et assurer la repétabilité les résultats de mesure. La deuxième partie présente une étude préclinique. Un modèle animal et un protocole expérimental ont été mises en place pour créer des cicatrices hypertrophiques sur lesquelles nous pouvons recueillir des spectres à analyser. La troisième partie porte sur la classification des spectres obtenus. Elle propose des méthodes algorithmiques pour débruiter et corriger les spectres mesurés, pour extraire automatiquement des caractéristiques spectrales interprétables et pour sélectionner un sous-ensemble de caractéristiques "optimales" en vue d'une classification efficace. Les résultats de classification réalisée respectivement par trois méthodes (k-ppv, ADL et RNA) montrent que la faisabilité d'utiliser la spectroscopie bimodale pour la caractérisation de ce type de lésion cutané. Par ailleurs, les caractéristiques sélectionnées par notre méthode montrent que la cicatrisation hypertrophique implique un changement de structure tissulaire et une variation de concentration de porphyrine / This research activity aims at developing and validating a multimodal system combining diffuse reflectance spectroscopy and autofluorescence spectroscopy in characterizing hypertrophic scar tissues in vivo. The work relies on three axes. The first part concerns the development of an automatic system which is suitable for multimodal spectroscopic measurement. A series of calibration procedures are carried out for ensuring the reliability of the measurement result. The second part presents a preclinical study on an animal model (rabbit ear). An experimental protocol was implemented in order to create hypertrophic scars on which we can collect spectra to analyze. The third part deals with the classification problem on the spectra obtained. It provides a series of algorithmic methods for denoising and correcting the measured spectra, for automatically extracting some interpretable spectral features and for selecting an optimal subset for classification. The classification results arched using respectively 3 different classifiers (knn, LDA and ANN) show the ability of bimodal spectroscopy in characterization of the topic skin lesion. Furthermore, the features selected my selection method indicate that the hypertrophic scarring may involve a change in tissue structure and in the concentration of porphyrins embedded in the epidermis

Este projeto se propõe a analisar as obras A Cicatriz é a Flor e Agreste, ambas do dramaturgo brasileiro Newton Moreno, com o propósito de melhor compreender os limites do drama relacionado à forma épica e lírica no teatro, tendo como foco principal o envolvimento deste último com o gênero lírico. Paralelamente a isso, pretende-se analisar o estofo político dessas obras, identificando os assuntos sociais de que tratam por meio do lirismo e do olhar subjetivo do autor, que empresta o olhar de mulheres homossexuais em embate com nosso contexto social homofóbico e machista. As citadas análises, em paralelo, visam a pesquisa dos possíveis embricamentos que podem ocorrer entre o teatro lírico e o teatro político. / This project proposes to analyze the written works The Scar is the Flower and Agreste, both of the Brazilian playwright Newton Moreno, with the purpose of better understanding the limits of the drama related to the epic and lyrical form in the theater, having as main focus the theater itself with the lyrical genre. Parallel to this, we intend to analyze the political reasoning of these plays, identifying the social issues they deal with through lyricism and the subjective view of the author, which lends the look of homosexual women in conflict with our homophobic and male chauvinist social context. The aforementioned analyzes, in parallel, seek to investigate the possible nexus that may occur between lyrical theater and political theater.

Este projeto se propõe a analisar as obras A Cicatriz é a Flor e Agreste, ambas do dramaturgo brasileiro Newton Moreno, com o propósito de melhor compreender os limites do drama relacionado à forma épica e lírica no teatro, tendo como foco principal o envolvimento deste último com o gênero lírico. Paralelamente a isso, pretende-se analisar o estofo político dessas obras, identificando os assuntos sociais de que tratam por meio do lirismo e do olhar subjetivo do autor, que empresta o olhar de mulheres homossexuais em embate com nosso contexto social homofóbico e machista. As citadas análises, em paralelo, visam a pesquisa dos possíveis embricamentos que podem ocorrer entre o teatro lírico e o teatro político. / This project proposes to analyze the written works The Scar is the Flower and Agreste, both of the Brazilian playwright Newton Moreno, with the purpose of better understanding the limits of the drama related to the epic and lyrical form in the theater, having as main focus the theater itself with the lyrical genre. Parallel to this, we intend to analyze the political reasoning of these plays, identifying the social issues they deal with through lyricism and the subjective view of the author, which lends the look of homosexual women in conflict with our homophobic and male chauvinist social context. The aforementioned analyzes, in parallel, seek to investigate the possible nexus that may occur between lyrical theater and political theater.

Doctor of Philosophy (PhD) / BACKGROUND: The accumulation of abnormal extracellular deposits beneath the retinal pigment epithelium characterises the pathology of early age-related macular degeneration. However, the histopathological threshold at which age-related changes become early AMD is not defined, and the effect of each of the deposits (basal laminar deposit and membranous debris) on disease progression is poorly understood. Evidence suggests that macrophages play a key role in the development of AMD lesions, but the influence of basal laminar deposit (BLamD) and membranous debris on the recruitment and programming of local macrophages has not been explored. Although evidence also suggests that inflammation and innate immunity are involved in AMD, the significance of anti-retinal autoantibodies to disesase pathogenesis is not known. AIMS: (i) To determine the histopathological threshold that distinguishes normal ageing from early AMD; (ii) to determine the influence of BLamD and membranous debris on disease progression; (iii) to examine whether distinct early AMD phenotypes exist based on clinicopathological evidence; (iv) to determine the histopathological context in which Bruch’s membrane macrophages first found; (v) to examine the relationship between Bruch’s membrane macrophages and subclinical neovascularisation; (vi) to determine if the progressive accumulation of BLamD and membranous debris alters the immunophenotype of Bruch’s membrane macrophages and/or resident choroidal macrophages; (vii) to determine if the anti-retinal autoantibody profile differs significantly between normal individuals and those with early AMD, neovascular AMD or geographic atrophy; (viii) to examine whether baseline anti-retinal autoantibodies can predict progression to advanced AMD in individuals with early AMD; and (ix) to examine whether baseline anti-retinal autoantibodies can predict vision loss in individuals with neovascular AMD. METHODS:Clinicopathological studies were performed to correlate progressive accumulation of BLamD and membranous debris to fundus characteristics and visual acuity, as well as to sub-macular Bruch’s membrane macrophage count. Immunohistochemical studies were perfomed to determine whether the presence of BLamD and membranous debris altered the programming of Bruch’s membrane or resident choroidal macrophages. The presence of serum anti-retinal autoantibodies was determined by western blotting, and the association with disease progression examined in early and neovascular AMD. RESULTS: The presence of both basal linear deposit (BLinD) and a continuous layer of BLamD represents threshold early AMD histopathologically, which was seen clinically as a normal fundus in the majority of cases. Membranous debris accumulation appeared to influence the pathway of progression from early AMD to advanced AMD. Bruch’s membrane macrophages were first noted when a continuous layer of BLamD and clinical evidence of early AMD were present, and increased with the amount of membranous debris in eyes with thin BLamD. Eyes with subclinical CNV had high macrophage counts and there was some evidence of altered resident choroidal macrophage programming in the presence of BLamD and membranous debris. Serum anti-retinal autoantibodies were found in a higher proportion of early AMD participants compared with both controls and participants with neovascular AMD, and in a higher proportion of individuals with atrophic AMD compared to those with neovascular AMD. The presence of baseline anti-retinal autoantibodies in participants with early AMD was not associated with progression to advanced AMD. Participants with neovascular AMD lost more vision over 24 months if they had IgG autoantibodies at baseline compared to autoantibody negative participants. CONCLUSIONS: The finding that eyes with threshold early AMD appear clinically normal underscores the need to utilise more sophisticated tests to enable earlier disease detection. Clinicopathological evidence suggests two distinct early AMD phenotypes, which follow two pathways of AMD progression. Macrophage recruitment and programming may be altered by the presence of BLamD and membranous debris, highlighting the need to further characterise the biology of human resident choroidal macropahges. Anti-retinal autoantibodies can be found in both control and AMD sera, and future approaches that allow the examination of subtle changes in complex repertoires will determine whether they are involved in AMD disease pathogenesis.

Stable single-unit recordings from the nervous system using microelectrode arrays can have significant implications for the treatment of a wide variety of sensory and movement disorders. However, the long-term performance of the implanted neural electrodes is compromised by the formation of glial scar around these devices, which is a typical consequence of the inflammatory tissue reaction to implantation-induced injury in the CNS. The glial scar is inhibitory to neurons and forms a barrier between the electrode and neurons in the surrounding brain tissue. Therefore, to maintain long-term recording stability, reactive gliosis and other inflammatory processes around the electrode need to be minimized. This work has succeeded in the development of neural electrode coatings that are capable of sustained release of anti-inflammatory agents while not adversely affecting the electrical performance of the electrodes. The effects of coating methods, initial drug loadings on release kinetics were investigated to optimize the coatings. The physical properties of the coatings and the bioactivity of released anti-inflammatory agents were characterized. The effect of the coatings on the electrical property of the electrodes was tested. Two candidate anti-inflammatory agents were screened by evaluating their anti-inflammatory potency in vitro. Finally, neural electrodes coated with the anti-inflammatory coatings were implanted into rat brains to assess the anti-inflammatory potential of the coatings in vivo. This work represents a promising approach to attenuate astroglial scar around the implanted silicon neural electrodes, and may provide a promising strategy to improve the long-term recording stability of silicon neural electrodes.

This thesis describes the electrical and physiological characterization of cardiac tissue with myocardial infarction (MI) responsible for abnormal cardiac rhythms such as ventricular tachycardia (VT), using a newly-developed magnetic resonance imaging (MRI) electrophysiology system. In electrophysiology (EP), radiofrequency (RF) catheter ablation combined with cardioverter-defibrillator implantation is a first-line action to manage ventricular VT. Unfortunately, this therapy is known to have sub-optimal success rates in a large number of patients because of difficulties to accurately identifying the arrhythmic target regions. Currently, characterization of post-MI scars is performed by using catheters to measure electrical signals of the endocardial tissue (electroanatomical mapping), under x-ray fluoroscopy guidance. Prolonged radiation exposure to both the cardiologist and the patient have made the use of MRI extremely attractive; further, unlike x-ray imaging, MRI provides post-MI scars with direct visualization, characterization in three dimensions and the ability to visualize ablation lesions. Although recent research has focused on registration between pre-acquired MR images and electroanatomical maps, a potentially more useful approach is to use real-time MRI to directly locate and characterize potential arrhythmogenic regions during the EP procedure. A real-time MR-guided EP system was developed and validated to perform EP diagnostic procedures, such as mapping and pacing. In a series of animal studies, the system demonstrated the ability to use active catheter tracking and intra-procedural MR imaging to navigate to specific regions in the left ventricle and record intracardiac electrical signals. A study correlating myocardial fibrotic scar detected by multicontrast late enhancement (MCLE) MRI and electroanatomical voltage mapping demonstrated that MRI information (transmurality, tissue classification, and relaxation rate) can accurately predict areas of myocardial fibrosis identified with bipolar voltage mapping. Finally, MCLE-derived gray zone was shown to have a high correspondence to regions with a high proportion of abnormal intracardiac signals. The methods described in this thesis help advance the understanding of infarcted tissue responsible for ventricular tachycardia. Further studies are proposed to perform RF ablation lesions and correlate pre- and post-ablation tissue electrophysiological properties with MRI.

L’injection de cellules souches provenant de la moelle osseuse est reconnue pour améliorer la fonction ventriculaire ainsi que le remodelage cicatriciel après un infarctus du myocarde (IM). Le Stromal Cell-derived factor-1 alpha (SDF-1 alpha), une chimiokine induite par l’ischémie cardiaque, représente une grande importance en raison de son rôle dans le recrutement de cellules inflammatoires et de cellules souches de la moelle osseuse vers les sites endommagés. Quoique les recherches sur le rôle de la chimiokine SDF-1 alpha dans le remodelage ventriculaire se multiplient, son implication dans la phase aiguë du remodelage reste inexplorée. Le but de la présente étude est de déterminer l’effet du SDF-1 alpha sur la taille de la cicatrice, l’hypertrophie cardiaque ainsi que la fonction ventriculaire chez des rats et des souris une semaine après un IM. La stratégie utilisée implique l’administration de l’AMD3100 (1 mg/kg, 24 heures après l’IM, pendant 6 jours), l’antagoniste sélectif du récepteur du SDF-1 alpha, le CXCR4. Ce récepteur est couplé à une protéine G alpha i et induit la migration et la prolifération cellulaire. Chez les rats du groupe IM, l’expression de la chimiokine a été détectée surtout dans les cellules musculaires lisses et les cellules endothéliales des vaisseaux cicatriciels. Le profil d’expression de la chimiokine dans le cœur infarci indique un gradient de concentration vers la cicatrice. Une semaine après l’IM, le traitement avec l’AMD3100 a diminué la taille de la cicatrice, résultant en une amélioration de la fonction ventriculaire et une diminution de l’élévation de l’expression de l’ARNm de l’ANP dans le ventricule gauche non infarci (VGNI). Chez les souris, le traitement avec l’AMD3100 a engendré les mêmes effets, soit une diminution de la taille de la cicatrice ainsi qu’une amélioration de la fonction ventriculaire. La réduction de la taille de la région infarcie chez les souris traitées avec l’AMD3100 est associée avec une atténuation de l’infiltration des neutrophiles dans la région ischémique. Ces résultats suggèrent que le blocage pharmacologique de l’axe SDF-1 alpha/CXCR4 lors de la phase aiguë du remodelage ventriculaire après un IM diminue la taille de la cicatrice et améliore la fonction ventriculaire, en partie, par la diminution de la réaction inflammatoire. / The injection of bone marrow stem cells in the infarcted heart was shown to improve ventricular function and scar remodelling. The chemokine Stromal Cell-derived factor-1 alpha (SDF-1 alpha) is implicated in the migration of inflammatory and bone marrow derived stem cells to damaged region. Despite a local increase of SDF-1 alpha expression in the damaged myocardium, the biological impact of the chemokine during the acute phase of remodelling in the ischemic heart remains undefined. Therefore, the present study examined the role of SDF-1 alpha on scar expansion, cardiac hypertrophy and ventricular function in rats following myocardial infarction (MI) via administration of AMD3100 (1 mg/kg, 24 hours post-MI and continued for 6 days) a selective antagonist of the SDF-1 alpha receptor, CXCR4. This receptor is coupled to a G alpha i protein and induced migration and proliferation of cells. In 1-week post-MI rats, chemokine expression was detected in smooth muscle and endothelial cells of blood vessels residing in the infarcted region. An SDF-1 alpha gradient towards the infarcted region was detected in the post-MI rat heart. In 1-week post-MI rats, AMD3100 therapy reduced scar size, concomitantly improved left ventricular function and partially supressed the elevated expression of ANP mRNA in the non-infarcted left ventricule. Preliminary studies on mice showed that the reduced infarct size in AMD3100-treated post-MI mice was associated with an attenuation of neutrophil infiltration in the ischemic region. These data highlight the novel observation that pharmacological antagonism of the SDF-1 alpha/CXCR4 axis during the acute phase of repartive fibrosis post-MI attenuated scar expansion and improved ventricular function in part via attenuation of the inflammatory response.

This thesis describes the electrical and physiological characterization of cardiac tissue with myocardial infarction (MI) responsible for abnormal cardiac rhythms such as ventricular tachycardia (VT), using a newly-developed magnetic resonance imaging (MRI) electrophysiology system. In electrophysiology (EP), radiofrequency (RF) catheter ablation combined with cardioverter-defibrillator implantation is a first-line action to manage ventricular VT. Unfortunately, this therapy is known to have sub-optimal success rates in a large number of patients because of difficulties to accurately identifying the arrhythmic target regions. Currently, characterization of post-MI scars is performed by using catheters to measure electrical signals of the endocardial tissue (electroanatomical mapping), under x-ray fluoroscopy guidance. Prolonged radiation exposure to both the cardiologist and the patient have made the use of MRI extremely attractive; further, unlike x-ray imaging, MRI provides post-MI scars with direct visualization, characterization in three dimensions and the ability to visualize ablation lesions. Although recent research has focused on registration between pre-acquired MR images and electroanatomical maps, a potentially more useful approach is to use real-time MRI to directly locate and characterize potential arrhythmogenic regions during the EP procedure. A real-time MR-guided EP system was developed and validated to perform EP diagnostic procedures, such as mapping and pacing. In a series of animal studies, the system demonstrated the ability to use active catheter tracking and intra-procedural MR imaging to navigate to specific regions in the left ventricle and record intracardiac electrical signals. A study correlating myocardial fibrotic scar detected by multicontrast late enhancement (MCLE) MRI and electroanatomical voltage mapping demonstrated that MRI information (transmurality, tissue classification, and relaxation rate) can accurately predict areas of myocardial fibrosis identified with bipolar voltage mapping. Finally, MCLE-derived gray zone was shown to have a high correspondence to regions with a high proportion of abnormal intracardiac signals. The methods described in this thesis help advance the understanding of infarcted tissue responsible for ventricular tachycardia. Further studies are proposed to perform RF ablation lesions and correlate pre- and post-ablation tissue electrophysiological properties with MRI.