L’insuffisance rénale aiguë (IRA) représente une complication fréquente de lachirurgie cardiaque pédiatrique sous circulation extracorporelle (CEC). Son traitement resteessentiellement conservateur, visant à prévenir l’aggravation de l’atteinte rénale, ousubstitutif, par épuration extrarénale (EER). L’EER précoce améliore le pronostic des adultesen défaillance multiviscérale, mais ceci n’a pas été montré chez l’enfant après chirurgiecardiaque. Le diagnostic de l’IRA repose actuellement sur une diminution du débit defiltration glomérulaire (DFG), reflété par la hausse de la créatinine sérique (sCr), et laréduction de la diurèse. Ces critères traduisent une perte de la fonction glomérulaire ; orl’atteinte initiale dans l’IRA post-chirurgicale se situe au niveau tubulaire. Ces critères sontpeu spécifiques et tardifs, et risquent de retarder le diagnostic et la prise en charge de l’IRA.Des nouveaux biomarqueurs rénaux, traduisant la présence d’une lésion tubulaire, dont leNeutrophil Gelatinase-Associated Lipocaline urinaire (NGALu) est le plus populaire,permettraient un diagnostic plus précoce.Objectif. L’objectif de ce travail est triple : (i) explorer l’association entre le délai de la miseen route de l’EER et le pronostic à court et moyen terme chez le patient < 1an qui développeune IRA post-chirurgie cardiaque ; (ii) explorer les performances diagnostiques desvariations précoces de la sCr pour l’IRA sévère ; et (iii) explorer les performancesdiagnostiques de l’élévation de NGALu pour l’IRA sévère dans des populations similaires depatients < 1an.Méthode. Une cohorte monocentrique rétrospective de patients < 1an constituée sur 10 ansa servi pour étudier l’association entre le délai de la mise en route de l’EER et la survie àcourt et moyen terme. La technique de pondération inverse par le score de propension a étéutilisée afin de réduire le biais associé aux changements des pratiques inhérents à la longuepériode d’inclusion. Une deuxième cohorte monocentrique rétrospective de patients < 1anconstituée sur 3 ½ ans a servi pour étudier les performances diagnostiques des variationsprécoces de la sCr pour l’IRA sévère. Enfin, une troisième cohorte monocentriqueprospective de patients < 1an constituée sur 18 mois a servi pour étudier les performancesdiagnostiques de NGALu pour l’IRA sévère. Pour l’étude des deux biomarqueurs, sCr etNGALu, la méthodologie utilisée a été similaire : partition des trajectoires individuelles devariation, puis analyse de l’association avec un critère composite (recours à l’EER et/oudécès postopératoire) et définition du profil à faible risque d’IRA; enfin, utilisation de laméthodologie des courbes ROC et des tables de reclassification pour quantifier leursperformances diagnostiques respectives.Résultats. La mise en route de l’EER le jour de la chirurgie ou le lendemain a été associéeavec une augmentation d’environ 45% de la survie à 30 et 90 jours. La variation de la sCrdans les 2 jours suivant la chirurgie a été spécifique mais peu sensible et peu discriminantepour le diagnostic de l’IRA sévère ; le profil à faible risque, rencontré chez près de 50% despatients a été une diminution durable d’environ 25% de la sCr par rapport à la valeur basale.NGALu a été discriminant et prédictif pour le critère composite ; la concentration de NGALu aaugmenté dans les 2 heures suivant la chirurgie, et est restée élevée chez les patientsprésentant le critère composite.Discussion et conclusions. Si la prise en charge précoce par EER de l’IRA sévère est unepriorité en termes de pronostic chez le patient < 1an, alors il faut se munir de moyensdiagnostiques précoces et performants. La variation précoce de la sCr est peu sensible etpeu discriminante. En revanche, l’élévation précoce de l’NGALu présente d’excellentesperformances diagnostiques pour l’IRA sévère, faisant de NGALu un marqueur rénalprometteur dans la population < 1an bénéficiant d’une chirurgie cardiaque. / Acute kidney injury (AKI) is common following congenital cardiac surgery withcardiopulmonary bypass (CPB). To date, no prophylactic intervention has proved to beuseful for the prevention of postoperative AKI. When AKI occurs, treatment is mainlysupportive and, when severe, requires renal replacement therapy (RRT). Several reportshave shown better outcome with early RRT in adults with multiorgan failure. No such data isavailable in children undergoing cardiac surgery, and criteria for RRT vary among centres.The definition of AKI is a reduction in the glomerular filtration rate (GFR), and the diagnosis isbased on an increase in serum creatinine (sCr) and a reduction in urine output; these arefunctional criteria, translating the consequences of glomerular injury. However, it iscommonly admitted that the first pathophysiologic finding in AKI following cardiac surgery istubular injury. Besides, the functional criteria are late, are not specific, and may delay thediagnosis of AKI. Novel AKI biomarkers, specific of tubular injury are available nowadays,with urine Neutrophil Gelatinase-Associated Lipocaline (uNGAL) being the most popular –they may allow for an early diagnosis of AKI.Objectifs. The aim of this work was: (i) explore associations between the delay to RRT, earlyand mid-term outcome in patients younger than 1 year of age who develop AKI followingcardiac surgery; (ii) assess the accuracy of early sCr variations and (iii) of uNGAL for severeAKI in two similar populations aged < 1 year.Methods. A single centre retrospective cohort of patients aged < 1 year undergoing surgeryover 10 years was used to asses the association between the delay to RRT et short and midtermsurvival. Inverse probability of treatment weighting was used to reduce bias due tochanges in practices that occurred during the long study period. A second retrospectivecohort of patients aged < 1 year undergoing surgery over 3 ½ years was used to asses theaccuracy of early sCr variations for the diagnosis of severe AKI. Finally, a third prospectivecohort of patients aged < 1 year undergoing surgery over 18 month was used to asses theaccuracy of uNGAL for the diagnosis of severe AKI. The study of both sCr and uNGAL useda similar methodology: first clustering of all individual trajectories of variation, enablingassessment of the association with a composite outcome (need for RRT and/or death) andidentification of the « normally expected » postoperative evolution of both sCr an uNGAL,associated with the best outcome; second, use of ROC curves and reclassification tables toassess the accuracy of each biomarker for the diagnosis of AKI.Results. Early RRT, initiated on the day of surgery or on day 1 following surgery, wasassociated with a 45% increase in 30-days and 90-days survival. Early sCr variation, within 2days of surgery, had a good specificity but was lacking sensitivity and discrimination for thediagnosis of severe AKI; the « expected » sCr evolution was a persistent 25% postoperativereduction relative to baseline. uNGAL had good discrimination and predictive ability for thecomposite outcome; uNGAL concentration increased within 2 hours of surgery, andremained high in patients with the composite outcome.Discussion and conclusions. If early RRT improves outcome in patients aged < 1 yearswith AKI following cardiac surgery, then it becomes important to perform an early diagnosisof severe AKI. To date, diagnosis of AKI is based on early sCr variations, but such variationslack sensitivity and discrimination for the diagnosis of severe AK. On the other hand, theincrease in uNGAL within hours of surgery has excellent accuracy for the diagnosis of severeAKI, making uNGAL a promising AKI biomarker in patients aged < 1 year undergoing cardiacsurgery with cardiopulmonary bypass.

Glioblastoma multiforme (G BM), ou astrocitoma grau IV, é o tumor mais comum e letal do sistema nervoso central. Uma de suas características mais marcantes é seu alto potencial invasivo do tecido normal adjacente. Neste processo, o remodelamento da matriz extracelular, modulado por enzimas que degradam seus componentes e por inibidores destas enzimas, é crucial. Foi descrito que a expressão de MMP-2 e MMP-9, membros da família das metaloproteinases de matriz, aumentam conforme a progressão de astrocitomas. A variante canônica de RECK suprime a invasão tumoral e metástase através da inibição da atividade de, pelo menos, três MMPs: MMP-2, MMP-9 e MMP-14. Uma correlação positiva tem sido observada entre a abundância da expressão de RECK em amostras tumorais e um prognóstico mais favorável para pacientes com diversos tipos de tumores. Neste estudo, variantes de splicing do gene supressor de tumor RECK foram identificadas através da análise de Expressed sequenced Tags (ESTs), isoladas por RT-PCR, sequenciadas e clonadas. Três novas variantes de splicing do gene RECK foram identificadas e caracterizadas. O perfil de expressão dos transcritos de RECK foi determinado através de ensaios de RT-PCR quantitativo em um painel de tecidos normais e, também, durante a progressão de astrocitomas. Foram utilizadas, para esta análise, amostras macro dissecadas de tumores de pacientes com astrocitomas grau I (n=15), II (n=15), III (n=15) e GBMs (n=30). Os resultados mostram que maior expressão de RECK canônico, acompanhada de maior razão de expressão da variante canônica em relação às variantes de splicing alternativo, correlaciona positivamente com maior sobrevida global de pacientes com GBM, sugerindo seu papel como potenciais biomarcadores para o prognóstico destes pacientes. Análise funcional das isoform as de RECK em células U87 MG revelou que as células superexpressando as isoformas não apresentam inibição do processo de invasão celular, como observado para superexpressão da proteína canônica. Dentre as isoformas analisadas, destaca-se RECK-B, isoforma potencialmente ancorada à membrana plasmática por GPI, como a proteína canônica RECK, sugerindo uma possível colocalização destas variantes. Observa-se que células superexpressando RECK-B apresentam maior capacidade tumorigênica. Os resultados indicam que as variantes de RECK e o balanço entre a expressão destas variantes, apresentam um papel importante no comportamento e na agressividade de GBMs, tendo potencial valor na clínica. Além disso, para abrir perspectivas para o estudo das variantes de RECK, o balanço de expressão dos transcritos canônico e alternativos deste gene foi explorado durante os processos de diferenciação osteogênica e adipogênica. Os resultados indicam que a expressão da variante canônica é mais abundante em relação à expressão de suas isoformas em estágios tardios da adipogênese, sendo que o perfil inverso é observado em relação à isoforma B durante a osteogênese, sugerindo que o balanço entre os níveis de expressão das isformas de RECK possui um potencial papel biológico que deve ser explorado durante esses processos. Em conjunto, os resultados demonstram a existência de, pelo menos, três variantes de splicing do gene supressor de tumor RECK com envolvimento na tumorigênese e na diferenciação celular, abrindo novas perspectivas para o estudo e a aplicação do gene RECK na clínica. / Glioblastoma multiforme (GBM) or grade IV astrocytoma is the most common and lethal tumor of the central nervous system. One of the most striking features of GBMs is their invasive potential of the normal surrounding brain tissue. It has been described that MMP-2 and MMP-9 expression levels increase during astrocytoma progression. Canonical RECK suppresses tumor invasion and metastasis by negatively regulating at least three matrix metalloproteinases, namely: MMP-9, MMP-2 and MT1-MMP. A positive correlation has been observed between the abundance of RECK express ion in tumor samples and a more favorable prognosis for patients with several types of tumors. In this study, splice variants of the RECK tumor suppressor gene were identified by Expressed Sequence Tag (EST) analysis, isolated by RT-PCR, sequenced and cloned. Three novel alternatively spliced variants of the RECK tumor suppressor gene were identified and characterized. The RECK transcripts expression profiles were investigated using quantitative RT-PCR assays in a normal tissue RNA panel and, also, during astrocytoma progression in macrodissected tumor samples of patients with astrocytoma grades I (n=15), II (n=15), III (n=15) and IV/GBM (n=30). The results show that higher canonical RECK expression, accompanied by a higher ratio of canonical to alternative transcript expression, positively correlated with higher overall survival rate after chemotherapeutic treatment of GBM patients. Our findings suggest that these RECK transcript variants may potentially be used as biomarkers for prognosis of GBM patients. U87 MG cells overexpressing each RECK alternative variant were generated and found to lack the supressive role of cellular invasion processes found upon overexpressing the canonical protein. Among the characterized isoforms, RECK-B stands out, since this isoform is potentially anchored to the cell membrane by a GPI anchor, exactly as the canonical RECK and, also, since cells overexpressing RECK-B display greater tumorigenic capacity. The results indicate that RECK variants and the balance between the expressions of these variants, play an important role in the behavior and aggressiviness of GBMs, therefore have a potential translational application. In addition, in order to investigate new perspectives for the analysis of these isoforms, the expression balance of RECK transcripts was assessed during osteogenesis and adipogenesis, by qRT - PCR. The results show that the expression of the canonical RECK variant is more abundant that that of its alternative isoforms in later stages of adipogenic differentiation. The opposite profile is found regarding RECK-B during osteogenesis, suggesting that the balance between the expressions of these transcripts may have a potential role during these processes. Taken together, the results show the existence of, at least, three alternatively spliced variants of the RECK tumor suppressor gene, which are involved in tumogigenesis and cellular differentiation, o pening new perspectives for studies and clinical application of the RECK gene.

En médecine personnalisée, les mesures et les descriptions radiologiques jouent un rôle important. En particulier, elles facilitent aux cliniciens l’établissement du diagnostic, la prise de décision thérapeutique ainsi que le suivi de la réponse au traitement. On peut citer à titre d’exemple, les critères d’évaluation RECIST (en anglais Response Evaluation Criteria in Solid Tumors). De nombreuses études de corrélation en radiologie-pathologie montrent que les caractéristiques d'imagerie quantitative et qualitative sont associées aux altérations génétiques et à l'expression des gènes. Par conséquent, une gestion appropriée des phénotypes d'imagerie est nécessaire pour faciliter leur utilisation et leur réutilisation dans de multiples études concernant les mesures radiologiques. En littérature, les mesures radiologiques qui caractérisent les processus biologiques des sujets imagés sont appelées biomarqueurs d'imagerie. L'objectif principal de cette thèse est de proposer une conceptualisation ontologique des biomarqueurs d'imagerie pour rendre leur sens explicite et formel, améliorer le reporting structuré des images. La première partie de la thèse présente une ontologie générique qui définit les aspects fondamentaux du concept de biomarqueur d'imagerie, à savoir : les caractéristiques biologiques mesurées, les protocoles de mesure et les rôles des biomarqueurs imagerie dans la prise de décision. La deuxième partie de la thèse traite des problèmes de modélisation sémantique liés à la description des données d’observation en neuro-imagerie en utilisant les connaissances biomédicales existantes. Ainsi, elle propose des solutions ''pertinentes'' aux situations les plus typiques qui doivent être modélisées dans le glioblastome. / In personalized medicine, radiological measurements and observations play an important role; in particular they help clinicians in making their diagnosis, selecting the appropriate treatment and monitoring the therapeutic response to an intervention as for example the Response Evaluation Criteria in Solid Tumors (RECIST). Many radiology-pathology correlation studies show that quantitative and qualitative imaging features are associated to genetic alterations and gene expression. Therefore, suitable management of imaging phenotypes is needed to facilitate their use and reuse in multiple studies regarding radiological measurements. In litterature, radiological measurements that characterize biological processes of imaged subjects are called imaging biomarkers. The main objective of this thesis is to propose an ontological conceptualisation of imaging biomarkers to make their meaning explicit and formal, improve structured reporting of images. The first part of the thesis presents a generic ontology that defines basic aspects of the imaging biomarker concept, namely; measured biological characteristic, measurement protocols and role in decision making application. The second part of the thesis adresses important semantic modeling challenges related to the description of neuro-imaging data using existing biomedical knowledge, as well as it proposes some “relevant” solutions to the most typical situations that need to be modeled in glioblastoma.

Radiological imaging already has a key role in the detection and management of patients with metastatic colorectal cancer (mCRC). With the evolution of personalised medicine there is a need for non-invasive imaging biomarkers that can detect early tumour response to targeted therapies. Translation from bench to bedside requires a multicentre approach that follows an agreed development roadmap to ensure that the proposed biomarker is precise (reproducible/ repeatable) and accurate in its characterisation of a meaningful physiological, pathological or post treatment response. The following thesis (organized in the alternative format with experimental studies written as individual complete manuscripts) investigates methods to improve precision and accuracy of the Apparent Diffusion Coefficient (ADC), a proposed quantitative imaging biomarker with a potential role in characterisation of post treatment responses in mCRC. The first objective was to establish baseline multicentre reproducibility (n=20) for ADC. A change in ADC greater than 21.1% was required to determine a post treatment response. Using a statistical error model, the dominating factors that influenced reproducibility were motion artefact and tumour volume. In the second study these factors were addressed using a single centre cohort with pre and post treatment data. Correcting for errors due to motion and tumour volume improved sensitivity from 30.3% to 1.7%, so a post treatment response was detected in 6/12 tumours compared to 0/12 using the baseline approach. In the third study, motion correction was implemented and the statistical error model was applied successfully to a multicentre cohort of 15 patients (1.9% sensitivity). The results of this thesis highlights that with careful consideration and correction of factors that negatively influence sensitivity, ADC is a potential imaging biomarker for use in post treatment response for patients with mCRC.

Deux grandes classes de cellules composent le pool de lymphocytes T (LT) CD8 mémoires. D'une part, les LT CD8 mémoires conventionnels sont générés via la reconnaissance spécifique d'antigènes dérivés de pathogènes ou de tumeurs. D'autre part, les LT CD8 mémoires innés sont générés via différents mécanismes impliquant de fortes stimulations par des cytokines γc indépendamment de la reconnaissance d'antigènes du non soi. Le phénotype extrêmement similaire de ces deux populations cellulaires ne permet pas de les distinguer in vivo. En conséquence, la population de LT CD8 mémoires innés est relativement peu caractérisée. Mon travail de thèse comportait donc deux objectifs majeurs : 1 / Identifier des marqueurs permettant de distinguer in vivo ces deux classes de LT CD8 mémoires. 2/ Caractériser la population de LT CD8 mémoires innés. Dans cette étude, nous démontrons qu'au sein du pool de LT CD8 mémoires, seules les cellules conventionnelles expriment la chimiokine CCL5 et le récepteur NKG2D. Ces deux biomarqueurs permettent ainsi pour la première fois de distinguer les LT CD8 mémoires innés et conventionnels in vivo, à la fois chez la souris et chez l'homme. Grâce à l'expression de NKG2D, nous démontrons que ces LT CD8 mémoires innés possèdent des caractéristiques typiques de cellules mémoires, notamment une réactivité augmentée ainsi qu'un programme génétique comparable à celui des LT CD8 mémoires conventionnels. Néanmoins, cette population cellulaire conserve certaines caractéristiques de cellules naïves. Ainsi, le répertoire TCR diversifié de cette population cellulaire permet à ces cellules de participer à des réponses immunitaires primaires contre différents pathogènes. Enfin, dans un contexte inflammatoire, les LT CD8 mémoires innés présentent un défaut d'accès au tissu pulmonaire comparé aux LT CD8 mémoires conventionnels. Ceci corrèle avec un déficit d'expression de certaines intégrines par les LT CD8 mémoires innés. L'ensemble de nos résultats démontre que les LT CD8 mémoires innés, caractérisés par l'absence d'expression de CCL5 et NKG2D, constituent une population cellulaire hybride, à la frontière entre cellules naïves et cellules mémoires conventionnelles / The pool of memory CD8 T cells is composed of two major cell classes. On one hand, conventional memory CD8 T cells are generated consequently to the specific recognition of pathogen or tumor derived antigens. On the other hand, innate memory CD8 T cells are generated through several mechanisms involving strong yc cytokine stimulation in the absence of cognate antigen recognition. However, these cell classes harbor a very similar phenotype. As a consequence, innate memory CD8 T cell population remains poorly characterized. This PhD has two main objectives : 1 / Identify new biomarkers that enable the discrimination between memory CD8 T cell classes 2/ Characterize the population of innate memory CD8 T cells in physiological condition Our results show that among the pool of memory CD8 T cells, only the conventional ones express the chemokine CCL5 and the NK receptor NKG2D. These two biomarkers enable for the first time the discrimination of memory CD8 T cell classes in physiological settings, in both mouse and human. Thanks to these new tools, we show that innate memory CD8 T cells hold typical memory features, such as an increased reactivity compared to naïve cells and a genetic program similar to the one of conventional memory cells. Nevertheless, this cell population also retains some features typical of naïve cells. The diversified TCR repertoire of this cell population allows it to participate to primary immune responses against various intracellular pathogens. Moreover, like naïve cells, innate memory CD8 T cells fail to access peripheral tissues upon local inflammation, which correlate with an absence of expression of some integrins. Altogether, these results demonstrate that innate memory CD8 T cells, characterized by the absence of expression of CCL5 and NKG2D, represent a hybrid cell population, at the boundary between naïve cells and conventional memory cells

Submitted by Maria Aparecida (cidazen@gmail.com) on 2017-11-27T17:43:41Z No. of bitstreams: 1 Marta Belfort.pdf: 1659970 bytes, checksum: 7aca8a1f671f32a3e25959a2840b065f (MD5) / Made available in DSpace on 2017-11-27T17:43:41Z (GMT). No. of bitstreams: 1 Marta Belfort.pdf: 1659970 bytes, checksum: 7aca8a1f671f32a3e25959a2840b065f (MD5) Previous issue date: 2017-08-28 / FAPEMA,CNPQ. / Penile cancer (PeCa) is a rare neoplasm in developed countries; however, its incidence is high in underdeveloped countries. In Brazil, regions North and Northeast are those with the higher number of cases for the disease. Among the factors associated with this neoplasm we highlight: poor hygiene, phimosis, chronic inflammation and infection by human papilloma virus (HPV). Changes in the PI3K/AKT/PTEN cell signaling pathway have been reported for several malign neoplasms, but little is known about the involvement of this pathway in penile tumors. Thus, the aim of this study was to verify the role of HPV infection and the occurrence of copy number alterations (CNA) in genes from the signaling pathway mediated by receptors of growth factors and PI3K in a population characterized by advanced tumors and high frequency of high risk HPV. To achieve that, we collected tumor tissue samples (both fresh and in formalin-fixed paraffin-embedded tissue-FFPE) from 34 patients from two reference hospitals: Instituto Maranhense de Oncologia Aldenora Belo (IMOAB) and the Hospital Universitário Presidente Dutra from the Universidade Federal do Maranhão (HU-UFMA). Fresh tumors were submitted to detection and genotyping of HPV by Nested PCR (Polymerase Chain Reaction) and direct sequencing. CNA analyzes were carried out in FFPE tissue from a subgroup HPV positive (91.2%), of which 88.2% were at high oncogenic risk. TaqMan® Copy Number Assays (Life TechnologiesTM) and CopyCaller software version v2.0 were performed to determine copy number for EGFR, HER3, HER4, AKT1, AKT2, PI3KCA and PTEN. Increase of 3 and 4 copies was considered gains, while increase of 5 or more copies was considered amplifications. The presence of a single gene copy was referred to as loss, while the absence of two copies was named deletion. Clinical and histopathological parameters were analyzed as to the presence of HPV and to CNAs. Our data showed that EGFR/PI3K/AKT/PTEN signaling pathway is highly altered in PeCa. The results showed that 100% of the tumors presented an increase of the number of copies for HER3; out of those, 93.9% were amplified, with 84.4% having 10 or more copies. EGFR also showed an increase of copies in 87.8% of tumor samples, out of which 65.6% were amplifications, with 48.2% having more than 10 copies. Furthermore, HER4 and AKT1 also presented an increase in the number of copies of 20.6% and 15%, respectively. AKT1 had a higher frequency of tumors with a regular number of copies (78.8%). On the other hand, PI3KCA, HER4, PTEN and AKT2 presented a higher frequency of samples with deletions, presenting 56%, 52.9%, 39% and 36%, respectively. Loss of copies was also frequent on tumors, so that genes AKT2, PTEN and PI3KCA appeared in heterozygosis in 60.6%, 54.5% and 37.5%, respectively. Our data show the occurrence of genetic alterations that may justify the differential expression of growth factor receptors and the downstream genes of the PI3K/ AKT pathway in penile carcinoma. However, in this study there was no association between CNAs and clinical and histopathological variables. On the other hand, taking into consideration the high frequency of HPV in the evaluated tumors, we suggest that CNAs are related to HPV integration into genome host. Finally, we highlight the high frequency of tumors with amplifications in HER3 and EGFR, reinforcing these markers as targets for specific therapies in CaPe. / Câncer de pênis (CaPe) é uma neoplasia rara em países desenvolvidos, entretanto, sua incidência é elevada em países subdesenvolvidos. No Brasil, as regiões Norte e Nordeste são as regiões com os maiores números de casos da doença. Dentre os fatores de risco, destaca-se a má higiene do órgão genital, fimose, inflamação crônica e infecção pelo papilomavírus-humano (HPV). Tem sido reportada alterações na via de sinalização celular PI3K/AKT/PTEN em diversas neoplasias malignas, entretanto, pouco se sabe sobre o envolvimento dessa via nos tumores de pênis. Assim, buscou-se neste trabalho verificar o papel da infecção por HPV e a ocorrência de alteração no número de cópias (CNAs) em genes da via de sinalização controlada por receptores dos fatores de crescimento e PI3K, em uma amostra caracterizada por tumor avançado e alta frequência de HPV de alto risco. Para isso, foram coletadas amostras de tecido tumoral (fresco e de tecido fixado com formalina e embebido em parafina - FFPE) de 34 pacientes provenientes de dois hospitais de referência, Instituto Maranhense de Oncologia Aldenora Belo (IMOAB) e o Hospital Universitário Presidente Dutra da Universidade Federal do Maranhão (HU-UFMA). As amostras de tumor fresco foram submetidas a detecção e genotipagem de HPV por Nested PCR (Polymerase Chain Reaction) e sequenciamento direto. As análises de CNAs foram realizadas em amostras de FFPE, HPV positivas (91,2%), das quais 88,2% eram de alto risco oncogênico. Utilizou-se o ensaio TaqMan® Copy Number Assays (Life TechnologiesTM) e o software CopyCaller versão 2.0 para determinação do número de cópias dos genes EGFR, HER3, HER4, AKT1, AKT2, PIK3CA e PTEN. Aumento de 3 e 4 cópias foi considerado ganho, enquanto aumento de 5 ou mais cópias foi considerado amplificação. A presença de uma única cópia gênica foi nomeada perda, enquanto a ausência de duas cópias foi nomeada deleção. Os parâmetros clínicos e histopatológicos foram analisados quanto a presença de HPV e também quanto a CNAs. A via de sinalização celular EGFR/PI3K/AKT/PTEN revelou-se altamente alterada nos tumores de pênis. Observou-se que 100% dos tumores apresentaram aumento de número de cópias do gene HER3. Destes, 93,9% apresentaram-se amplificados, sendo que 84,4% apresentavam 10 ou mais cópias. O gene EGFR apresentou aumento de cópias em 87,8% das amostras, das quais 65,6% eram amplificações, sendo que 48,2% apresentavam mais de 10 cópias. Além desses, os genes HER4 e AKT1 também apresentaram aumento de cópias gênicas, em 20,6% e 15%, respectivamente. AKT1 apresentou maior porcentagem de tumores com número normal de cópias (78,8%). Por outro lado, os genes PI3KCA, HER4, PTEN e AKT2 apresentaram maiores frequências de amostras com deleções, com 56%, 52,9%, 39% e 36%, respectivamente. As perdas também foram frequentes nos tumores, de modo que os genes AKT2, PTEN e PI3KCA apresentaram-se em heterozigose em 60,6%, 54,5% e 37,5%, respectivamente. Nossos dados mostram a ocorrência de alterações genéticas que podem justificar a expressão diferencial dos receptores de fatores de crescimento e de genes downstream da via PI3K/AKT em carcinoma peniano. No entanto, não foi encontrada associação entre CNAs e as variáveis clínicas e histopatológicas. Por outro lado, considerando-se a alta frequência de HPV nos tumores avaliados, levanta-se a possibilidade de haver uma relação entre a integração do HPV no genoma do hospedeiro e a ocorrência de CNAs em CaPe. Finalmente, destacamos a alta frequência de tumores com amplificações em HER3 e EGFR, abrindo a possibilidade desses marcadores serem utilizados como alvos para terapias específicas em CaPe.

É grande a preocupação na identificação de compostos envolvidos no desenvolvimento de processos deletérios, como o câncer. O tetraidrofurano (THF) é um solvente amplamente utilizado em indústria e pesquisa e estudos recentes indicaram uma atividade carcinogênica em animais experimentais, incentivando a investigação da sua possível interação com biomoléculas. Adutos de DNA servem como marcadores para a identificação da atividade genotóxica de substâncias e para o monitoramento da exposição humana aos agentes genotóxicos. Estudos realizados in vitro, em células em cultura, bem como em animais experimentais, permitem que mecanismos sejam desvendados e metodologias sejam desenvolvidas para uma posterior avaliação da exposição humana. O contínuo desenvolvimento de técnicas de análise de adutos em DNA para monitoramento biológico constitui importante preocupação para pesquisadores envolvidos com a saúde ocupacional e a medicina preventiva. No presente trabalho foi investigada a reação de produtos de oxidação do THF com os nucleosídeos 2\'-desoxiadenosina (dAdo) e 2\'-desoxicitidina (dCyd). As estruturas dos adutos formados foram elucidadas por análises de espectros de massas (ESI/MS-MS) e ressonância magnética nuclear (RMN) após purificação de quantidade suficiente dos produtos por cromatografia líquida de alta eficiência (HPLC). Com isso, foi aberta a possibilidade de validação de novos marcadores de exposição a THF. Tais lesões foram detectadas em DNA incubado com THF oxidado in vitro, após o desenvolvimento de uma metodologia sensível e seletiva baseada em HPLC/ESI/MS-MS. Além disso, camundongos foram expostos a vapor de THF e foi feita análise de dano oxidativo (8-oxo-2-desoxiguanosina) no DNA de fígado e rim. Um aumento de aproximadamente oito vezes no nível de 8-oxo-2-desoxiguanosina foi observado no DNA dos órgãos dos camundongos expostos ao THF. Nossos estudos in vitro apontam para a possibilidade de ação genotóxica do THF oxidado (formação de adutos com o DNA). A ocorrência dessa via in vivo será investigada em estudos posteriores. Adicionalmente, há aumento de dano oxidativo em órgãos alvos de carcinogênese induzida por esse solvente. / There is a huge concern to identify compounds involved in the development of harmful processes, like cancer. Tetrahydrofuran (THF) is a solvent widely used in industries and recent researches and studies indicated a carcinogenic activity in experimental animals, instigating the investigation of its possible interaction with biomolecules. DNA adducts are used as markers for the identification of genotoxic activity of substances and monitoring the human exposition to genotoxic agents. Studies carried out in vitro, in culture cells and in experimental animals allow to disclose mechanisms and develop methodologies for a posterior evaluation of the human exposition. The continuous development of techniques for the analysis of DNA adducts for biological monitoring constitutes a remarkable concern for the researchers involved with occupational health and preventive medicine. In the present paper, the reaction of THF oxidation products with the nucleosides 2\'-deoxyadenosine (dAdo) and 2\'-deoxycitidine (dCyd) were investigated. The structures of the formed adducts were elucidated by mass spectra analysis (ESI/MS-MS) and nuclear magnetic resonance (RMN) after purification of sufficient quantities of the products by high-performance liquid chromatography (HPLC). Thus, the validation of new markers of exposition to THF was made possible. Such damages were detected in DNA incubated with oxidized THF in vitro, after the development of a sensible and selective methodology based on HPLC/ESI/MS-MS. Moreover, mice were exposed to THF vapors and an oxidative damage analysis (8-oxo-2\'-deoxyguanosine) was carried out on DNA from liver and kidney. An approximate 8-fold increase in the level of 8-oxo-2\'-deoxyguanosine was observed on DNA from the mices organs exposed to THF. Our in vitro studies indicate the possibility of oxidized THF genotoxic action (formation of adducts with DNA). The occurrence of this path in vivo will be investigated in future studies. In addition, the oxidative damage in target organs of carcinogenesis induced by this solvent was increased.

Made available in DSpace on 2017-07-10T14:57:34Z (GMT). No. of bitstreams: 1 Jardel_ Nimet.pdf: 1924881 bytes, checksum: 4755b41b1458253b217679d76b6eeead (MD5) Previous issue date: 2016-02-24 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Abstract: This study evaluated the use of gill and liver of Astyanax bifasciatus as histological biomarkers for biomonitoring of streams with different uses and land occupation. The fish were collected by electric fishing technique, in six streams classified as afforested, rural and urban, in the basin of the lower Iguaçu River, west region of Paraná State, in December 2014. It was tested the hypothesis that in streams that are influenced by effluents from urban and rural areas occur greater number and histopathological severity in gill and liver of A. Bifasciatus. In this context, the objective of this study was to correlate the presence and degree of histopathology of gill and liver with the environmental variables among streams with different uses and land occupation. The environments considered afforested showed higher values of dissolved Oxygen (DO) and lower conductivity (CO) when compared to the rural and urban environments. The gills of fish from the afforested streams indicated normal functioning of the organ, while in the rural and urban streams, the histopathology in the gills suggested light to moderate damage to the organ. In relation to the liver, the histopathology indicated light to moderate damage in rural streams and moderate to severe damage in urban streams. When considering the abiotic variables and frequencies of histopathology of gill and liver, the afforested streams were separated from rural and urban areas. The histopathology in gills and livers were significantly more severe, corroborating our hypothesis. It was possible to distinguish streams with different uses and land occupation, therefore, these histopatologias showed favorable biomarkers for studies of biomonitoring / Resumo: Este estudo avaliou o uso de brânquia e fígado de Astyanax bifasciatus como biomarcadores histológicos para biomonitoramento de riachos com diferentes usos e ocupação do solo. Os peixes foram coletados por meio da técnica de pesca elétrica, em seis riachos classificados em florestados, rurais e urbanos, na bacia do baixo Rio Iguaçu, região Oeste do Estado do Paraná, em dezembro de 2014. Testou-se a hipótese de que em riachos que sofrem influências de efluentes oriundos de áreas rurais e urbanas ocorrem maior número e severidade histopatológicas em brânquia e fígado de A. bifasciatus. Nesse contexto, este estudo teve como objetivo correlacionar a presença e o grau de histopatologias de brânquia e fígado com as variáveis ambientais entre riachos com diferentes usos e ocupação do solo. Os ambientes considerados florestados apresentaram maiores valores de Oxigênio dissolvido (OD) e menores de condutividade (CO) quando comparados com os ambientes rurais e urbanos. As brânquias dos peixes dos riachos florestados indicaram funcionamento normal do órgão, enquanto que nos riachos rurais e urbanos, as histopatologias nas brânquias sugeriram danos leves para moderados ao órgão. Em relação ao fígado as histopatologias indicaram danos leves para moderados nos riachos rurais e danos moderados para severos nos riachos urbanos. Ao considerar as variáveis abióticas e as frequências das histopatologias de brânquia e fígado, os riachos florestados foram separados dos rurais e urbanos. As histopatologias nas brânquias e fígados foram significativamente mais severas, corroborando a nossa hipótese. Foi possível distinguir os riachos com diferentes usos e ocupação do solo, portanto, essas histopatologias se mostraram biomarcadores favoráveis para estudos de biomonitoramento

A tuberculose (TB) é uma doença infecciosa bacteriana causada por Mycobacterium tuberculosis (Mtb), acomete seres humanos em todo mundo, e é considerada uma das principais causas de morbidade e mortalidade dentre as doenças infecciosas. Nesse cenário, a TB ainda é vista como um grande desafio para a ciência e para medicina. Com o estudo de novos alvos moleculares para terapia e diagnóstico, por exemplo, a sinalização Notch tem sido descrita como uma via importante para a manutenção da resposta imunológica durante a infecção por Mtb. Assim, o objetivo deste trabalho foi avaliar a participação da via Notch na modulação da resposta imune durante a infecção por Mtb em humanos. Neste trabalho foram incluidos 13 pacientes com tuberculose ativa e 13 indivíduos saudáveis. Células mononucleares de sangue periférico (PBMCs) foram isoladas e avaliadas quanto à expressão de receptores Notch 1 - 2 em células T e ligantes Notch, DLL1 - 4 em subpopulações de monócitos por citometria de fluxo e expressão de genes relacionados à via Notch e resposta imune. Além disso, o plasma foi utilizado para avaliar níveis de citocinas, CD14s e CD163s. Ainda, avaliamos a função celular dependente da ativação da via Notch durante a infecção de células mononucleares de sangue periférico de indivíduos saudáveis com Mtb H37Rv in vitro. Observamos que pacientes com TB ativa apresentaram aumento na expressão de DLL4 em monócitos intermediários e não-clássicos e diminuição na expressão de Notch 2 em células T CD8+. In vitro, observamos nas culturas de PBMCs, aumento da expressão de DLL4 em monócitos intermediários e Notch 2 em células T CD4+. A expressão do ligante DLL4 em monócitos intermediários e a expressão do Notch 1 em células T CD4+ em pacientes com TB ativa se correlacionaram positivamente com o grau de lesão pulmonar. Além disso, pacientes com quadros de lesão pulmonar moderado e avançado têm maior expressão de Notch 1 em células T CD4+ quando comparados a pacientes com grau mínimo de lesão pulmonar. Pacientes com TB ativa apresentam aumento significativo nos níveis plasmáticos de IL-6, IP- 10, CD14s e CD163s e diminuição nos níveis de IFN-?, IL-17A, IL-4, IL-2, IL-1? e RANTES. Os níveis plasmáticos de IFN-?, TNF-?, IL-17A, IL-4, IL-2, IL-12, IL-1?, RANTES e IL-10 se correlacionaram positivamente com o maior grau de lesão pulmonar.Além disso, demonstramos que os níveis de IFN-?, IL-17A, IL-1?, IL-4 e IL-2 se relacionam com maior expressão do receptor Notch 1 em células T CD4+ , enquanto níveis plasmáticos de IP-10, CD163s e de procalcitonina se correlacionam com maior expressão de Notch 1 em células T CD8+. Evidenciamos maior tendência na expressão de HES1 em pacientes com TB ativa e células infectadas in vitro, indicando que a via Notch está sendo ativada durante a infecção por Mtb. De forma interessante, in vitro as culturas de PBMCs tratadas com inibidor farmacológico da via Notch (GSI), reduziram a produção de IL-17A, IL-2 e IL-10 e aumentaram IL-8, enquanto o tratamento com anti-hDLL4 promoveu o aumento significativo nos níveis de TNF-? e atividade fagocítica de monócitos. Em conclusão, a expressão de receptores e ligantes Notch é alterada durante a TB, em especial o ligante DLL4 em subpopulações de monócitos. Além disso, a via Notch parece ser importante na modulação de respostas pró e anti-inflamatórias. Nesse trabalho, sugerimos que a expressão de Notch 1 em células T e DLL4 em subpopulações de monócitos estão associados com a gravidade da TB. Assim, a detecção desses constituintes da via Notch em PBMCs de pacientes com TB ativa são potenciais biomarcadores para avaliar a progressão da doença. / Tuberculosis (TB) is a bacterial infectious disease caused by Mycobacterium tuberculosis (Mtb), affects humans worldwide, and is considered a major cause of morbidity and mortality among infectious diseases. In this scenario, TB is still seen as a major challenge for science and medicine. With the study of new molecular targets for therapy and diagnosis, for example, Notch signaling has been described as an important pathway for the maintenance of immune response during Mtb infection. Thus, the objective of this work was to evaluate the participation of the Notch pathway in the modulation of the immune response during Mtb infection in humans. In this study, 13 patients with active tuberculosis and 13 healthy individuals were included. Peripheral blood mononuclear cells (PBMCs) were isolated and evaluated for Notch 1 - 2 receptor expression in T cells and Notch, DLL1 - 4 ligands in monocyte subpopulations by flow cytometry and expression of Notch - pathway related genes and immune response. In addition, plasma was used to assess levels of cytokines, CD14s and CD163s. Furthermore, we evaluated the cellular function dependent on the activation of the Notch pathway during the infection of peripheral blood mononuclear cells of healthy subjects with Mtb H37Rv in vitro. We observed that patients with active TB had increased DLL4 expression in intermediate and nonclassic monocytes and decreased expression of Notch 2 in CD8+ T cells. In vitro, we observed in the cultures of PBMCs, increased expression of DLL4 in intermediate monocytes and Notch 2 in CD4+ T cells. The expression of the DLL4 linker in intermediate monocytes and the expression of Notch 1 in CD4+ T cells in patients with active TB correlated positively with the degree of lung injury. In addition, patients with moderate and advanced lung injury have higher Notch 1 expression in CD4+ T cells when compared to patients with a minimal degree of lung injury. Patients with active TB showed a significant increase in plasma levels of IL-6, IP-10, CD14s and CD163s, and decreased levels of IFN-?, IL-17A, IL-4, IL-2, IL-1? and RANTES. The plasma levels of IFN-?, TNF-?, IL-17A, IL-4, IL-2, IL-12, IL-1?, RANTES and IL-10 correlated positively with the highest degree of lung injury. In addition, we demonstrated that levels of IFN-?, IL-17A, IL-1?, IL-4 and IL-2 are related to higher Notch 1 receptor expression in CD4+ T cells, whereas plasma levels of IP-10,CD163s and procalcitonin correlate with increased Notch 1 expression in CD8+ T cells. We have shown a greater trend in HES1 expression in patients with active TB and in vitro infected cells, indicating that the Notch pathway is being activated during Mtb infection. Interestingly, in vitro cultures of PBMCs treated with the Notch pharmacological inhibitor (GSI), reduced IL-17A, IL-2 and IL-10 production and increased IL-8, while anti-hDLL4 treatment promoted the significant increase in TNF-? levels and monocyte phagocytic activity. In conclusion, the expression of Notch receptors and ligands is altered during TB, especially the DLL4 linker in monocyte subpopulations. In addition, the Notch pathway appears to be important in modulating pro and anti-inflammatory responses. In this work, we suggest that Notch 1 expression in T cells and DLL4 in monocyte subpopulations is associated with TB severity. Thus, detection of these constituents of the Notch pathway in PBMCs of patients with active TB are potential biomarkers to assess disease progression.

abstract: Alzheimer’s Disease (AD) and Frontotemporal Dementia (FTD) are the leading causes of early onset dementia. There are currently no ways to slow down progression, to prevent or cure AD and FTD. Both AD and FTD share a lot of the symptoms and pathology. Initial symptoms such as confusion, memory loss, mood swings and behavioral changes are common in both these dementia subtypes. Neurofibrillary tau tangles and intraneuronal aggregates of TAR DNA Binding Protein 43 (TDP-43) are also observed in both AD and FTD. Hence, FTD cases are often misdiagnosed as AD due to a lack of accurate diagnostics. Prior to the formation of tau tangles and TDP-43 aggregates, tau and TDP-43 exist as intermediate protein variants which correlate with cognitive decline and progression of these neurodegenerative diseases. Effective diagnostic and therapeutic agents would selectively recognize these toxic, disease-specific variants. Antibodies or antibody fragments such as single chain antibody variable domain fragments (scFvs), with their diverse binding capabilities, can aid in developing reagents that can selectively bind these protein variants. A combination of phage display library and Atomic Force Microscopy (AFM)-based panning was employed to identify antibody fragments against immunoprecipitated tau and immunoprecipitated TDP-43 from human postmortem AD and FTD brain tissue respectively. Five anti-TDP scFvs and five anti-tau scFvs were selected for characterization by Enzyme Linked Immunosorbent Assays (ELISAs) and Immunohistochemistry (IHC). The panel of scFvs, together, were able to identify distinct protein variants present in AD but not in FTD, and vice versa. Generating protein variant profiles for individuals, using the panel of scFvs, aids in developing targeted diagnostic and therapeutic plans, gearing towards personalized medicine. / Dissertation/Thesis / Doctoral Dissertation Neuroscience 2018