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Nowdays there is a plethora of evidence for H₂O₂-mediated oxidative stress in the epidermis as well as in the system in patients with vitiligo (for review see (Schallreuter, Bahadoran et al. 2008). Xanthine dehydrogenase/xanthine oxidase (XDH/XO) catalyses the oxidative hydroxylation of hypoxanthine to xanthine followed by xanthine to uric acid, the last two steps in purine degradation pathway. Under oxidative conditions, XDH is converted to XO. The reactions catalysed by this enzyme generate H₂O₂ and O₂̇⁻, yielding in the presence of ROS accumulation, allantoin from uric acid. Therefore XO has been considered a major biologic source of oxygen-derived free radicals in many organs. The presence of XO in the human epidermis has not been shown so far. In this study several techniques were utilised to nail the presence and activity of XO in epidermal melanocytes and keratinocytes. The enzyme is regulated by H₂O₂ in a concentration dependent manner, where concentrations of 10-6M upregulate activity. Importantly, the results showed that the activity of XO is little affected by H₂O₂ in the mM range. H₂O₂-mediated oxidation of tryptophan and methionine residues in the sequence of XO yields only subtle alterations in the enzyme active site. These findings are in agreement with enzyme kinetics in the presence of 10-3M H₂O₂. Since uric acid is the end product of XO activity and this can be oxidised to allantoin by H₂O₂, we wanted to know whether allantoin is formed in the epidermis of patients with vitiligo. In order to address this issue, we utilised HPLC/mass spectrometry analysis. Analysis of epidermal cell extracts from suction blister tissue identified the presence of allantoin in patients with acute vitiligo, while this product was absent in healthy controls. In conclusion, our results provide evidence for functioning epidermal XO in the human epidermis which 4 can be a major source for the production of H₂O₂ contributing to oxidative stress in vitiligo. In addition, this thesis also demonstrates for the first time the presence of XO in melanosomes, and we showed that both 7BH4 and 7-biopterin inhibit XO activity in a concentration dependent manner. Moreover, XO has the potential to bind to 6/7BH4 and 6/7-biopterin from the pterin/tyrosinase inhibitor complex. This discovery adds another receptor independent mechanism for regulation of tyrosinase within the melanocyte similar to α/ß-MSH as shown earlier (Moore, Wood et al. 1999; Spencer, Chavan et al. 2005). Since the entire epidermis of patients with vitiligo is under H₂O₂-mediated oxidative stress, oxidative DNA damage would be highly expected. This thesis shows for the first time that epidermal 8-oxoG levels as well as plasma level of this oxidised DNA base are significantly increased in patients compared to healthy controls. We have shown that epidermal cells from patients with vitiligo respond to oxidative DNA damage via the overexpression of p21 and Gadd45α leading to a functioning increased short-patch base-excision repair (BER), while increased apoptosis can be ruled out due to lower caspase 3 and cytochrome c response compared to healthy controls. Our results show that patients develop effective DNA repair machinery via hOgg1, APE1 and DNA polymeraseß. Taking into consideration that these patients do not have an increased prevalence for solar-induced skin cancers, our data suggest that BER is a major player in the hierarchy to combat H₂O₂-mediated oxidative stress preventing ROS-induced tumourigenesis in the epidermis of these patients.

Nowdays there is a plethora of evidence for H2O2-mediated oxidative stress in the epidermis as well as in the system in patients with vitiligo (for review see (Schallreuter, Bahadoran et al. 2008). Xanthine dehydrogenase / xanthine oxidase (XDH / XO) catalyses the oxidative hydroxylation of hypoxanthine to xanthine followed by xanthine to uric acid, the last two steps in purine degradation pathway. Under oxidative conditions, XDH is converted to XO. The reactions catalysed by this enzyme generate H2O2 and O2 ¿- , yielding in the presence of ROS accumulation, allantoin from uric acid. Therefore XO has been considered a major biologic source of oxygen-derived free radicals in many organs. The presence of XO in the human epidermis has not been shown so far. In this study several techniques were utilised to nail the presence and activity of XO in epidermal melanocytes and keratinocytes. The enzyme is regulated by H2O2 in a concentration dependent manner, where concentrations of 10-6M upregulate activity. Importantly, the results showed that the activity of XO is little affected by H2O2 in the mM range. H2O2-mediated oxidation of tryptophan and methionine residues in the sequence of XO yields only subtle alterations in the enzyme active site. These findings are in agreement with enzyme kinetics in the presence of 10-3M H2O2. Since uric acid is the end product of XO activity and this can be oxidised to allantoin by H2O2, we wanted to know whether allantoin is formed in the epidermis of patients with vitiligo. In order to address this issue, we utilised HPLC/mass spectrometry analysis. Analysis of epidermal cell extracts from suction blister tissue identified the presence of allantoin in patients with acute vitiligo, while this product was absent in healthy controls. In conclusion, our results provide evidence for functioning epidermal XO in the human epidermis which 4 can be a major source for the production of H2O2 contributing to oxidative stress in vitiligo. In addition, this thesis also demonstrates for the first time the presence of XO in melanosomes, and we showed that both 7BH4 and 7-biopterin inhibit XO activity in a concentration dependent manner. Moreover, XO has the potential to bind to 6/7BH4 and 6/7-biopterin from the pterin/tyrosinase inhibitor complex. This discovery adds another receptor independent mechanism for regulation of tyrosinase within the melanocyte similar to ¿/ß-MSH as shown earlier (Moore, Wood et al. 1999; Spencer, Chavan et al. 2005). Since the entire epidermis of patients with vitiligo is under H2O2-mediated oxidative stress, oxidative DNA damage would be highly expected. This thesis shows for the first time that epidermal 8-oxoG levels as well as plasma level of this oxidised DNA base are significantly increased in patients compared to healthy controls. We have shown that epidermal cells from patients with vitiligo respond to oxidative DNA damage via the overexpression of p21 and Gadd45¿ leading to a functioning increased short-patch base-excision repair (BER), while increased apoptosis can be ruled out due to lower caspase 3 and cytochrome c response compared to healthy controls. Our results show that patients develop effective DNA repair machinery via hOgg1, APE1 and DNA polymeraseß. Taking into consideration that these patients do not have an increased prevalence for solar-induced skin cancers, our data suggest that BER is a major player in the hierarchy to combat H2O2-mediated oxidative stress preventing ROS-induced tumourigenesis in the epidermis of these patients.

Submitted by IGOR HENRIQUE SENA DA SILVA null (igor.sena@outlook.com.br) on 2017-09-05T13:59:07Z No. of bitstreams: 1 DISSERTAÇÃO_Igor_Henrique_Sena_Silva.pdf: 1536012 bytes, checksum: da24e2e008037696caee4c8842fc8f05 (MD5) / Approved for entry into archive by Luiz Galeffi (luizgaleffi@gmail.com) on 2017-09-06T13:13:51Z (GMT) No. of bitstreams: 1 silva_ihs_me_jabo.pdf: 1536012 bytes, checksum: da24e2e008037696caee4c8842fc8f05 (MD5) / Made available in DSpace on 2017-09-06T13:13:51Z (GMT). No. of bitstreams: 1 silva_ihs_me_jabo.pdf: 1536012 bytes, checksum: da24e2e008037696caee4c8842fc8f05 (MD5) Previous issue date: 2017-07-26 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Helicoverpa armigera é uma praga altamente polífaga e ataca culturas de grande importância agrícola em diversos países do mundo. O controle desta praga é realizado primariamente por inseticidas químicos. Porém, o uso indiscriminado do controle químico levou a resistência de populações desta praga a maioria dos inseticidas químicos usados para seu controle, dificultando o seu manejo no campo. Além do controle químico, o controle de H. armigera tem sido realizado com uso de plantas transgênicas que expressam a proteína Cry1Ac de Bacillus thuringiensis (Bt) ou por bioinseticidas que contem esta e outras proteínas. No entanto, estudos têm demonstrado uma diminuição significativa na susceptibilidade de H. armigera às proteínas Cry com o aumento de seu desenvolvimento larval. O mecanismo de resistência mais comum dos insetos às proteínas Cry é a redução de ligação da proteína aos receptores presentes na membrana, levando a uma menor afinidade de ligação da proteína aos receptores intestinais. Desta forma, o objetivo deste trabalho foi avaliar a susceptibilidade de lagartas de diferentes ínstares de H. armigera à Cry1Ac e correlacionar com a capacidade de ligação da proteína Cry1Ac às microvilosidades apicais das células colunares do intestino médio (BBMVs) isoladas de todos ínstares larvais. Além disso, por meio de ensaios de imunoprecipitação e análise por cromatografia líquida acoplada a espectrofotometria de massa, identificar as proteínas envolvidas na interação com a proteína Cry1Ac no segundo e quinto ínstares de H. armigera. Foi observada uma redução significativa na susceptibilidade dos últimos ínstares larvais de H. armigera à proteína Cry1Ac comparado aos ínstares iniciais. Os valores estimados de CL50 variaram de 31,1 a 2525,7 ng de proteína/cm² de dieta, em lagartas de primeiro e sexto ínstar, respectivamente. Estes resultados evidenciam uma diferença de 80 vezes na susceptibilidade à proteína Cry1Ac do último para o primeiro ínstar. Nos testes de ligação de ELISA da proteína Cry1Ac às BBMVs dos diferentes ínstares, foi constatada uma diminuição total na capacidade de ligação da proteína Cry1Ac as BBMVs dos estádios mais tardios comparados aos iniciais, com afinidade de ligação aparente de 3,88 vezes menor no último ínstar comparado ao primeiro. Assim, uma clara correlação direta entre toxicidade de Cry1Ac e a afinidade de ligação da proteína às BBMVs de H. armigera foi demonstrada. Os resultados dos ensaios de imunoprecipitação demonstraram um padrão diferenciado de interação com a proteína Cry1Ac no segundo e quinto ínstar. A proteína fosfatase alcalina (ALP) foi identificada apenas no segundo ínstar, bem como, outras proteínas de membrana, como proibitina e uma proteína de canal iônico, que podem estar envolvidas para a alta toxicidade de Cry1Ac em ínstares iniciais de H. armigera. A identificação e o papel funcional das proteínas de ligação nos diferentes estádios de desenvolvimento de H. armigera, facilitará a elucidação do mecanismo de ação da proteína Cry1Ac e poderá ajudar a propor estratégias que retardem a evolução da resistência dos insetos às cultivares transgênicas que expressam esta proteína. / Helicoverpa armigera is a highly polyphagous pest and attacks important crops worldwide. The control of this pest is carried out primarily by chemical insecticides. However, the indiscriminate use of chemical control, led to pest populations to develop resistance to most of the chemical insecticides used for their control, making it difficult to management in the field. In addition to chemical control, H. armigera has been done by transgenic crops expressing Cry1Ac toxin from Bacillus thuringiensis (Bt) or by biopesticides that contains Cry1Ac or other toxins. However, studies have demonstrated a susceptibility decrease of H. armigera to Cry toxins with their larval development increase. The most common mechanism of resistance used by insects against Cry toxins is the reduced toxin binding to receptors present on the membrane, leading to a lower binding affinity of the toxin to intestinal receptors. Thus, the objective of this work was to evaluate the susceptibility of different instar larvae of H. armigera to Cry1Ac toxin and to correlate with the Cry1Ac toxin binding capacity to BBMV isolated from all larval instar. Furthermore, by pull-down techniques and liquid chromatography coupled to mass spectrometry analysis, to identify the proteins involved in the Cry1Ac toxin interaction in the second and fifth instars of H. armigera. A significant reduction in the susceptibility of the late instars of H. armigera to Cry1Ac toxin was observed compared to early instars. LC50 estimated values ranged from 31.1 to 2525.7 ng of toxin/cm2 of diet in first and sixth instar larvae, respectively. These results point a difference of 80-fold in the susceptibility to Cry1Ac toxin from late to first larval instar. In the ELISA binding assays results to BBMV of the different instars was found a total decrease in the binding capacity of Cry1Ac toxin to BBMVs from late instars compared to BBMV from early instars, presenting an apparent binding affinity of 3.88 times lower in the last instars than the first. Thus, a clearly correlation between Cry1Ac toxicity and binding toxin affinity to H. armigera BBMV has been demonstrated. The pull-down assays demonstrated a different pattern of the interaction between Cry1Ac toxin with the second and fifth instars. The protein phosphatase alkaline (ALP) was identified only in the second instar, as well as, other membrane proteins, as prohibitin and an ion channel protein, which may be involved for higher toxicity of Cry1Ac in early instars of H. armigera. The identification and functional role of binding proteins in the different stages of development of H. armigera will facilitate the elucidation of the Cry1Ac toxin mechanism of action and will may help to propose strategies that delay the insect resistance evolution to transgenic crops that express this protein. / FAPESP: 2015/24330-5

Medical imagery is increasingly evolving towards higher resolution and throughput. The increasing volume of data and the usage of multiple and often novel imaging modalities necessitates the use of mathematical and computational techniques for quicker, more accurate and more robust analysis of medical imagery. The fields of computer vision and machine learning provide a rich set of techniques that are useful in medical image analysis, in tasks ranging from segmentation to classification and population analysis, notably by integrating the qualitative knowledge of experts in anatomy and the pathologies of various disorders and making it applicable to the analysis of medical imagery going forward. The object of the proposed research is exactly to explore various computer vision and machine learning methods with a view to the improved analysis of multiple modalities of brain and cardiac imagery, towards enabling the clinical goals of studying schizophrenia, brain tumors (meningiomas and gliomas in particular) and cardiovascular disorders. In the first project, a framework is proposed for the segmentation of tubular, branched anatomical structures. The framework uses the tubular surface model which yields computational advantages and further incorporates a novel automatic branch detection algorithm. It is successfully applied to the segmentation of neural fiber bundles and blood vessels. In the second project, a novel population analysis framework is built using the shape model proposed as part of the first project. This framework is applied to the analysis of neural fiber bundles towards the detection and understanding of schizophrenia. In the third and final project, the use of mass spectrometry imaging for the analysis of brain tumors is motivated on two fronts, towards the offline classification analysis of the data, as well as the end application of intraoperative detection of tumor boundaries. SVMs are applied for the classification of gliomas into one of four subtypes towards application in building appropriate treatment plans, and multiple statistical measures are studied with a view to feature extraction (or biomarker detection). The problem of intraoperative tumor boundary detection is formulated as a detection of local minima of the spatial map of tumor cell concentration which in turn is modeled as a function of the mass spectra, via regression techniques.

L'α-synucléine est reconnue comme une protéine clé dans la physiopathologie de la maladie de Parkinson ainsi que d'autres troubles neurodégénératifs appelés synucléinopathies. Dans ces maladies, la surexpression de l’α-synucléine entraîne la formation d'agrégats toxiques dans les neurones dopaminergiques (DA). Dans cette thèse, nous avons exploré l’effet de la régulation de microARN (miARN) sur l’expression de l’α-synucléine. Pour se faire, des études ont été menées avec la lignée cellulaire humaine SH-SY5Y qui peut être différenciée pour créer un modèle de neurones DA et ensuite traitée avec une neurotoxine pour induire des caractéristiques cellulaires de la maladie de Parkinson. Des observations importantes ont été supportées dans des modèles cellulaires plus avancés, notamment les neurones induits par reprogrammation directe de fibroblastes humains (iNs) et les neurones DA primaires de souris purifiés. Le premier objectif était de mieux comprendre comment la surexpression aberrante de l'α synucléine dans les synucléinopathies pourrait être due à une dérégulation de la maturation des miARN qui ciblent son ARN messager. Tout d’abord, nous avons sélectionné les miARN les plus susceptibles d'avoir un effet régulateur sur l’expression de l’α-synucléine à partir de recherche de la littérature et d’analyse de bases de données spécialisées. Nous avons observé que l’augmentation de l'expression de l'α-synucléine associée à l’ajout de neurotoxine est accompagnée d’une diminution concomitante de l'expression de plusieurs miARN sélectionnés. Sur la base de ces résultats, l'impact de ces miARN sur l'expression de l'α-synucléine a été évalué dans plusieurs types de cellules humaines, notamment les HEK 293T, les SH-SY5Y différenciées et les iNs. À cette fin, nous avons utilisé des cibles de miARN exogènes pour réprimer l'activité régulatrice des miARN et avons mesuré leur effet sur l'expression de l'α synucléine. Ainsi, nous avons démontré que la répression de miR-7, miR-93, miR-140, miR 153 et miR 214 mène systématiquement à la surexpression de l’α-synucléine dans les différents types de cellules. De plus, nous avons démontré que certains miARN sont régulés de manière post-transcriptionnelle en mesurant les niveaux des formes immatures et matures des miARN dans différents contextes cellulaires. Le deuxième objectif était d’identifier des protéines potentiellement aptes à réguler la maturation post-transcriptionnelle de miARN. Des études de purification par affinité et de spectrométrie de masse ont permis d'identifier les protéines qui s’associent avec la tige-boucle des formes immatures des miARN et régulent potentiellement leur maturation. Quelques protéines candidates ont été sélectionnées sur la base d’analyse informatique pour examiner l’effet de leur surexpression dans différents essais cellulaires. À ce jour, nous avons identifié quatre protéines (MIF, PCBP2, Prohibitin-2, and Tfr1) qui, en plus de répondre à certains critères de bases (lient l’ARN, sont présentes dans le cerveau et impliquées dans des maladies associées au système nerveux), ont un effet sur l’activité et l’expression de miR-153 ainsi que sur l’expression de l’α-synucléine. Ces travaux ont permis d’établir de solides bases dans notre compréhension de la régulation de l'α-synucléine par les miARN et d’ouvrir la voie à des études plus élaborées qui permettront d’établir les mécanismes de régulation des niveaux de miARN qui ciblent l’α-synucléine. À plus long terme, cet axe de recherche pourrait fournir des pistes pour le développement d'outils diagnostiques et thérapeutiques pour les synucléinopathies. / Alpha-synuclein is a key protein in the pathophysiology of Parkinson's disease and other neurodegenerative disorders called synucleinopathies. In these diseases, overexpression of α-synuclein leads to the formation of toxic aggregates in dopaminergic (DA) neurons. In this thesis, we explored the effect of microRNA (miRNA) regulation on α-synuclein expression. To do so, studies were conducted with the human SH-SY5Y cell line, which can be differentiated to create a model of DA neurons and then treated with a neurotoxin to induce cellular features of Parkinson's disease. Important observations were supported in more advanced cell models, including neurons induced by direct reprogramming of human fibroblasts (iNs) and purified primary mouse DA neurons. The first objective was to better understand how aberrant overexpression of α-synuclein in synucleinopathies results in the deregulation of the maturation of miRNAs that target its messenger RNA. First, we selected the miRNAs most likely to have a regulatory effect on α-synuclein expression based on literature searches and specialized database analyses. We observed that the increase in α-synuclein expression associated with neurotoxin addition is accompanied by a concomitant decrease in the expression level of several selected miRNAs. Based on these results, the impact of these miRNAs on αsynuclein expression was evaluated in several human cell types, including HEK 293T, differentiated SHSY5Y, and iNs. To this end, we used exogenous miRNA targets to repress miRNA regulatory activity and measured their effect on α-synuclein expression. Thus, we demonstrated that repression of miR-7, miR-93, miR-140, miR-153, and miR-214 consistently leads to overexpression of α-synuclein in different cell types. In addition, we demonstrated that some miRNAs are regulated in a posttranscriptional manner by measuring the levels of immature and mature forms of miRNAs in different cellular contexts. The second objective was to identify proteins potentially able to regulate the post-transcriptional maturation of miRNAs. Affinity purification and mass spectrometry studies were used to identify proteins that associate with the stem-loop of immature forms of miRNAs and potentially regulate their maturation. A few candidate proteins were selected based on computational analysis to examine the effect of their overexpression in different cell-based assays. To date, we have identified four proteins (MIF, PCBP2, Prohibitin-2, and Tfr1) that, in addition, to fitting basic criteria (known to bind RNA, are present in the brain and associated with nervous system-related diseases) affect miR-153 activity and expression as well as α-synuclein expression. This work has established a solid foundation in our understanding of the regulation of α-synuclein by miRNAs and has paved the way for more elaborate studies that will establish the mechanisms of regulation of miRNA levels that target α-synuclein. In the longer term, this line of research could provide avenues for the development of diagnostic and therapeutic tools for synucleinopathies.