Introdução- As Leucemias Linfocíticas Agudas (LLA) constituem-se na mais comum das neoplasias em crianças. Alguns estudos epidemiológicos identificaram riscos aumentados de LLA em crianças expostas a campos magnéticos gerados por linhas de força de alta tensão, porém, essa associação não foi confirmada por outros estudos. Objetivo- Verificar a associação entre exposição a campos magnéticos e a incidência da LLA em crianças residentes na Região Metropolitana de São Paulo, considerando-se a distância das residências das crianças de linhas de transmissão de energia (88, 138, 230, 345 e 440 kV). Métodos- Estudo casocontrole de base populacional. Os casos foram selecionados em cinco hospitais na capital do município de São Paulo, que concentram o atendimento a crianças com LLA. Quatro controles populacionais foram selecionados para cada caso, emparelhados por sexo, idade e cidade de nascimento. Casos e controles foram entrevistados utilizando-se questionário similar para obtenção de informações sobre as variáveis de interesse e potenciais variáveis de confusão. Os domicílios foram avaliados em relação às distâncias de linhas de transmissão de energia mais próxima utilizando-se o Global Positioning System (GPS). Na análise da associação entre campos magnéticos e LLA foi utilizada regressão logística condicional, incluindo o controle de potenciais variáveis de confusão. Foram calculados os odds ratios (OR) e os respectivos intervalos com 95 por cento de confiança (IC95 por cento). Resultados- A associação entre a distância de linhas de transmissão e LLA foi ajustada pela variável escolaridade da pessoa entrevistada, resultando em OR de 2,91 (IC95 por cento 0,92-9,22). Conclusão- Concluiu-se que, crianças residentes a menos de 160 metros de linhas de transmissão de energia na RMSP apresentam risco maior, porém não estatisticamente significativo de desenvolver LLA quando comparadas com as que residem a mais que 160 metros. / Introduction-The Acute Lymphocytic Leukemia (ALL) is the most common cancers in children. Some epidemiological studies have identified increased risk of ALL in children exposed to magnetic fields generated by high voltage power lines, however, this association was not confirmed by other studies. Purpose- Check the association between exposure to magnetic fields and the incidence of ALL in children residing in the Metropolitan Region of São Paulo, considering the distance from childrens home and transmission lines (88, 138, 230, 345 and 440 kV). Methods- A population-based case-control study. The cases were selected in five hospitals in the city of Sao Paulo, which account for the care of children with ALL. Four population controls were selected for each case, matched by sex, age and city of birth. Cases and controls were interviewed using a similar questionnaire to obtain information on the interest variables and potential confounding variables. The homes were evaluated by distances of the nearest transmission lines using the Global Positioning System (GPS). To examine the association between magnetic fields and ALL was used conditional logistic regression, including the control of potential confounding variables. We calculated the odds ratios (OR) and their 95per cent confidence interval (95 per cent CI). Results-The association between the distance of transmission lines and ALL was adjusted by education of the person interviewed, and the OR was 2.91 (95 per cent CI 0.92-9.22). Conclusion- Children living at less than 160 m of power transmission lines in RMSP have higher risk, though not statistically significant, when compared with those living in more than 160 m.

The presence of TP53 mutations has been associated with inferior outcome in diffuse large B-cell lymphoma (DLBCL) and chronic lymphocytic leukemia (CLL). In DLBCL, the impact of the TP53 codon 72 polymorphism and MDM2 SNP309 has not been clearly elucidated, whereas MDM2 SNP309 was suggested as a poor-prognostic marker in CLL. In addition, p16INK4a promoter hypermethylation has been implicated as a negative prognostic factor in DLBCL. The aim of this thesis was to further evaluate these molecular markers in well-characterised materials of DLBCL and CLL. In paper I, we investigated the prognostic role of TP53 mutation, codon 72 polymorphism and MDM2 SNP309 in DLBCL (n=102). The presence of TP53 mutations (12.7%) correlated with a poor lymphoma-specific and progression-free survival, and a particularly pronounced effect was observed in the germinal center subtype. Neither the MDM2 SNP309 nor the TP53 codon 72 polymorphism had an impact on age of onset or survival. In paper II, we applied pyrosequencing to measure the level of p16INK4a methylation in DLBCL (n=113). Thirty-seven percent of cases displayed p16INK4a methylation; however, no clear association could be observed between degree of methylation and clinical characteristics or lymphoma-specific survival. In papers III–IV, we investigated the prognostic role of MDM2 SNP309 (n=418) and TP53 mutation (n=268) in CLL. No correlation was observed between any particular MDM2 SNP309 genotype and time to treatment and overall survival. Furthermore, no association was found between the different MDM2 SNP309 genotypes and established CLL prognostic markers. TP53 mutations were detected in 3.7% of CLL patients; where the majority showed a concomitant 17p-deletion and only three carried TP53 mutations without 17p-deletion. We confirmed a significantly shorter overall survival and time to treatment in patients with both TP53 mutation and 17p-deletion. Altogether, our studies could confirm the negative prognostic impact of TP53 mutations in DLBCL, whereas MDM2 SNP309 and TP53 codon 72 polymorphisms appear to lack clinical relevance. We also question the role of p16INKa methylation as a poor-prognostic factor in DLBCL. Finally, the presence of TP53 mutation in CLL appears to be rare at disease onset and instead arise during disease progression.

Chronic lymphocytic leukemia (CLL) is a heterogeneous malignancy displaying varying clinical outcome, where molecular markers today can divide patients into prognostic subgroups. Despite the introduction of new agents for treatment, remissions are usually not sustained in CLL and resistance towards treatment can partly be explained by aberrant apoptosis. The aim of this thesis was to find new drugs for CLL patients resistant to conventional therapy and to analyze genes involved in apoptosis within different prognostic subgroups. In paper I-II, the in vitro activity of substances was investigated using the fluorometric microculture cytotoxicity assay (FMCA). When evaluating rapamycin (paper I), an inhibitor of mTOR, in 97 tumor samples from different entities, CLL was found to be one of the most sensitive tumor types. Combination experiments on patient CLL cells indicated that rapamycin acted synergistically with the CLL drugs vincristine and chlorambucil. An investigation of 20 anti-cancer agents in cells from 40 CLL patients (paper II) revealed that prednisolone and rolipram displayed high activity in poor-prognostic patients, in particular IGHV unmutated CLL. Furthermore, when used in combination these agents were found to produce a synergistic effect. In paper III, the anti-apoptotic BCL2 family member BFL1 was evaluated in 37 CLL cases. Levels of BFL1 were higher in fludarabine-resistant patients compared to fludarabine-sensitive patients. In addition, the high expression of BFL1 inversely correlated to fludarabine-induced apoptosis in CLL cells. A single nucleotide polymorphism in the anti-apoptotic BCL2 gene (-938C>A) has been suggested as a novel poor-prognostic marker in CLL. In paper IV, we investigated this BCL2 polymorphism in 268 CLL patients and correlated genotypes to clinical data. However, no association could be confirmed between this polymorphism and clinical outcome or established prognostic markers. In conclusion, this thesis has shown that rapamycin is a potential drug for treatment in CLL. Furthermore, prednisolone and rolipram were identified as interesting candidates for treatment of poor-prognostic patients. Finally, the anti-apoptotic protein BFL1 may contribute to chemoresistance and hence represents a potential therapeutic target in CLL, whereas from our data, the BCL2 -938C>A polymorphism does not appear to have any prognostic significance.

Η παρούσα διδακτορική διατριβή έχει ως στόχο τη δημιουργία ενός αποδοτικού μοντέλου για το Λειτουργικό Συνδυασμό Βιο-Ιατρικών δεδομένων (BioMedical data integration). Ξεκινώντας από τη σχεδιαστική ανάλυση της ιατρικής γνώσης και των προβλημάτων που προκύπτουν από τον τρόπο παραγωγής των ιατρικών δεδομένων, προχωρεί στην επίλυση των επιμέρους θεμάτων Λειτουργικού Συνδυασμού εντός ενός συγκεκριμένου ιατρικού πεδίου και καταλήγει στον ολοκληρωμένο Λειτουργικό Συνδυασμό ιατρικών δεδομένων προερχόμενων από διαφορετικές πηγές και πεδία γνώσης. Συνεχίζει με τη σχεδίαση ενός μοντέλου βάσεων δεδομένων που ακολουθεί «οριζόντια» λογική και είναι αρκετά αποδοτικό ώστε να αποκρίνεται σε πολύπλοκα και ευρείας κλίμακας ερωτήματα σε πραγματικό χρόνο. Καταλήγει με την παρουσίαση μίας ολοκληρωμένης εφαρμογής η οποία εκμεταλλευόμενη τα πλεονεκτήματα του Λειτουργικού Συνδυασμού και της οριζόντιας δομής των δεδομένων είναι σε θέση να διαχειριστεί εξετάσεις προερχόμενες από κάθε κυτταρομετρητή ροής και συνδυάζοντάς αυτές με τις υπόλοιπες αιματολογικές κλινικοεργαστηριακές εξετάσεις να απαντά σε καθημερινά και σύνθετα ερευνητικά, ιατρικά ερωτήματα. Τα πρωτότυπα ερευνητικά αποτελέσματα που προέκυψαν στα πλαίσια της παρούσης εργασίας δημοσιεύτηκαν σε έγκυρα διεθνή περιοδικά και σε διεθνή και ελληνικά συνέδρια με κριτές. / Current dissertation focuses on the creation of an efficient model for Bio-medical data integration. Starting with an analytical approach of the medical knowledge and the problems that may occur cause of the way that medical data are produced, continues with the necessary solutions for single domain data integration and concludes with the proposal of a working framework for mass data integration, originating from multiple medical domains. The proposed integration model is based on the “horizontal” logic of a database design and it’s efficient enough to produce query results in real time, even for complex real-life medical questions. The proof of concept of the working framework and its goals for mass data integration is achieved through the presentation of a medical information system. The presented system, by taking advantage of the “horizontal” database design, is able to manage Flow Cytometry measurements, originating for any available hardware and by integrating the cytometric data with other types of hematological data is able to give answers to everyday and research medical questions. All original research results that produced within the scope of this dissertation were published in international research journals and medical conferences.

The clinical course of chronic lymphocytic leukemia (CLL) is highly heterogeneous, which has prompted the search for biomarkers that can predict prognosis in this disease. The IGHV gene mutation status and certain genomic aberrations have been identified as reliable prognostic markers of clinical outcome for this disorder. However, the search for more feasible prognostic markers in CLL is still being pursued. Recently, certain single nucleotide polymorphisms (SNPs) in the GNAS1, BCL2 and MDM2 genes and the RNA expression levels of the LPL, ZAP70, TCL1, CLLU1 and MCL1 genes were suggested as novel prognostic markers in CLL. In papers I-III, we performed genotyping analyses of the GNAS1 T393C, BCL2 -938C>A and MDM2 SNP309 polymorphisms in 268-418 CLL patients and related the genotypes with clinical data. Association studies between the polymorphisms and established prognostic markers (i.e. IGHV mutation status, genomic aberrations, CD38 expression) were also performed. Our studies did not find any significant relationship between these SNPs with either clinical outcome or other known prognostic markers in CLL. In paper IV, we measured the RNA expression levels of LPL, ZAP70, TCL1, CLLU1 and MCL1 in 252 CLL cases and correlated these levels with clinical outcome. Here, we verified that high expression of all these RNA-based markers, except MCL1, were associated with an unfavourable prognosis. We also confirmed a close relationship between IGHV mutation status and the RNA-based markers, especially for LPL and CLLU1 expression. Among the RNA-based markers, multivariate analysis revealed LPL expression as the strongest independent prognostic marker for overall survival and time to treatment. Furthermore, the RNA-based markers could add further prognostic information to established markers in subgroups of patients, with LPL expression status giving the most significant results. In summary, data from papers I-III could not verify the GNAS1 T393C, BCL2 -938C>A and MDM2 SNP309 polymorphisms as prognostic markers in CLL. Future SNP markers must hence be confirmed in large, independent cohorts before being proposed as prognostic marker in CLL. In paper IV, we conclude that LPL expression appears to be the strongest among the RNA-based markers for CLL prognostication. Further efforts to standardize LPL quantification are required before it can be applied in the clinical laboratory to predict clinical outcome in this disease.

Η Β-χρόνια Λεμφογενής Λευχαιμία (Β-ΧΛΛ) αποτελεί τον πιο κοινό τύπο λευχαιμίας στο Δυτικό κόσμο. Η πρόγνωσή της θεωρείται ως ένα από τα πιο ενδιαφέροντα προβλήματα απόφασης στην κλινική έρευνα και πρακτική. Για διάφορους κλινικούς και εργαστηριακούς δείκτες είναι γνωστό ότι σχετίζονται με την εξέλιξη της νόσου. Για τις παραμέτρους, όμως, που εξάγονται με ανάλυση κυτταρομετρίας ροής, οι οποίες αποτελούν τον ακρογωνιαίο λίθο της διαδικασίας διάγνωσης της νόσου, το αν προσφέρουν επιπρόσθετη προγνωστική πληροφορία αποτελεί ανοιχτό πρόβλημα. Στη διατριβή αυτή προτείνουμε ένα σύστημα υποβοήθησης για τις αποφάσεις των ειδικών του πεδίου, το οποίο πραγματοποιεί πολυπαραμετρική πρόγνωση ασθενών με Β-ΧΛΛ, συνδυάζοντας τη χρήση ποικίλων ετερογενών προγνωστικών δεικτών (κλινικών, εργαστηριακών και κυτταρομετρίας ροής) που σχετίζονται με τη νόσο. Η διάγνωση της Β-ΧΛΛ βασίζεται κυρίως στη μελέτη του αντιγονικού φαινότυπου των κυττάρων των ασθενών, η οποία διενεργείται με κυτταρομετρία ροής. Αν και η διαδικασία που ακολουθείται κατά την ανάλυση αυτή είναι σαφώς ορισμένη, ο τρόπος με τον οποίο οι εργαστηριακοί υπεύθυνοι την πραγματοποιούν παραδοσιακά χαρακτηρίζεται από ανακρίβεια και υποκειμενικότητα. Καθώς η τεχνολογία της κυτταρομετρίας ροής εξελίσσεται ραγδαία, γίνεται όλο και πιο επιτακτική η ανάγκη για την ανάπτυξη αυτοματοποιημένων μεθόδων ανάλυσης των δεδομένων που παράγει. Σε αυτά τα πλαίσια, παρουσιάζουμε ένα χρήσιμο παράδειγμα αυτοματοποιημένης ανάλυσης κυτταρομετρικών δεδομένων, η οποία δεν απαιτεί την άμεση επίβλεψη των ειδικών, για τη διάγνωση ασθενών με Β-ΧΛΛ. Οι τιμές των χαρακτηριστικών παραμέτρων που εξάγονται με εφαρμογή της προτεινόμενης μεθοδολογίας, ενσωματώνονται κατόπιν στο προαναφερθέν προγνωστικό σύστημα. Ανάγοντας το πρόβλημα της πρόγνωσης της Β-ΧΛΛ σε ένα στιγμιότυπο ταξινόμησης προτύπων, καθώς και προσομοιώνοντας κάθε ένα από τα βήματα της διαδικασίας της διάγνωσης της νόσου με ένα στιγμιότυπο συσταδοποίησης δεδομένων, αντιμετωπίσαμε τα δύο προβλήματα εφαρμόζοντας τεχνικές στατιστικής μάθησης. Εστιάσαμε σε μεθοδολογίες δικτύων πεποίθησης, χρησιμοποιώντας συγκεκριμένα το naïve-Bayes μοντέλο και για τις δύο περιπτώσεις, στην επιβλεπόμενη και στη μη επιβλεπόμενη εκδοχή του, αντίστοιχα. Τα χαρακτηριστικά και η φύση των δεδομένων (κυρίως των κυτταρομετρικών) που παράγονται από έναν παθολογικό υποκείμενο μηχανισμό, όπως αυτός της νόσου, δεν ευνοούν την απευθείας εφαρμογή του παραπάνω μοντέλου στο εκάστοτε στιγμιότυπο. Για το λόγο αυτό, συνδυάσαμε την εφαρμογή του naïve-Bayes μοντέλου με κατάλληλες ευρετικές αλγοριθμικές διαδικασίες, για την επίτευξη καλύτερων αποτελεσμάτων, με κριτήριο βέλτιστου όχι μόνο κάποιες συχνά χρησιμοποιούμενες μετρικές αποτίμησης αλγόριθμων, αλλά και τη γνώμη των αιματολόγων. Χάρη στην ιδιότητά τους να ενσωματώνουν την έμπειρη γνώση των ειδικών ως εκ των προτέρων πληροφορία αρχικοποίησης των μεθόδων μάθησής τους, οι Bayesian μεθοδολογίες κρίνονται ως οι πλέον κατάλληλες για την εφαρμογή τους σε τέτοιου τύπου προβλήματα. / B-Chronic Lymphocytic Leukemia (B-CLL) is known to be the most common type of leukemia in the Western world. Its prognosis remains one of the most interesting decision problems in clinical research and practice. Various clinical and laboratory factors are known to be associated with the evolution of the disease. However, for the parameters obtained by flow cytometry analysis, that are traditionally utilized as the cornerstone during the diagnosis procedure of the disease, whether they offer additional prognostic information is an open issue. In this dissertation, we propose a decision support system to the hematologists, that provides multiparametric B-CLL patients’ prognosis, combining the usage of diverse heterogeneous factors (clinical, laboratory and flow cytometry) associated with the disease. B-CLL diagnosis is primarily derived from the study of the antigenic phenotype of the patients’ blood cells, which is held with flow cytometry analysis. Despite the fact that the method of the analysis is well defined, the process traditionally followed by the laboratory experts is characterized by amounts of inexactness and subjectivity. As flow cytometry technology advances rapidly, the need for adequate automated (computer-assisted) analysis methodologies on the data it produces is accordingly increasing. In this context, we present a useful paradigm of automated analysis of flow cytometry data, that does not require the direct supervision of the expert, for B-CLL patients’ diagnosis. The values of the flow cytometry characteristic parameters extracted by applying the proposed methodology are afterward incorporated to the prognostic system for B-CLL mentioned above. By reducing the B-CLL prognosis problem to an instance of the pattern classification problem, as well as by simulating each step of the B-CLL diagnosis procedure with an instance of the data classification problem, we proceeded with applying statistical learning techniques. We focused on Bayesian network methodologies and utilized the naïve-Bayes model for both cases, in its supervised and unsupervised version, respectively. The characteristics of the data (especially of the flow cytometry ones) generated by a pathological underlying mechanism, like the disease’s one, did not encourage the direct use of the above model. Therefore, we combined the naïve-Bayes model with a set of suitable heuristic algorithmic procedures to obtain better results, not only with respect to some commonly used algorithmic optimality metrics, but also by considering the experts’ opinion. Due to their ability of incorporating the expert knowledge as a priori initial information to their learning methods, Bayesian methodologies are considered as the most appropriate ones to make use of in such types of applications.

Chronic Lymphocytic Leukemia is the primary lymphoid neoplasm in adults and and it is especially manifested in the elderly. Because it is a heterogeneous disease it awakens great interest regarding its prognosis. Rai and Binet developed staging systems to predict the evolution of the disease and currently, the analysis of expression of CD38 and Zap-70 has been investigated as a prognostic factor for indicating presence or absence of the mutation in the gene IgVH, so, the objective of this study was to analyze the clinical and laboratory profiles of patients with Chronic Lymphocytic Leukemia taking as reference the clinical staging of Rai and Binet and quantification of CD38 and Zap-70 expression as prognosis factors. We searched the medical records of 64 patients treated at University Hospital of Santa Maria and the variables considered were swollen lymph nodes, presence or absence of hepatomegaly and / or splenomegaly, hematological evaluation of peripheral blood and immunophenotype. The data obtained were correlated with the staging of Rai (1975) and Binet (1981), the expression of CD38 and Zap-70 and clinical stage. The results showed no association between ataging Rai and Binet and the expression of CD38, Zap-70 and Binet clinical staging. / A Leucemia Linfocítica Crônica é a principal neoplasia linfóide em adultos e se manifeta principalmente em indivíduos idosos. Por ser uma doença heterogênea, desperta grande interesse quanto ao seu prognóstico. Rai e Binet desenvolveram sistemas de estadiamento capazes de prever a evolução da doença e atualmente, a análise da expressão de CD38 e Zap- 70 tem sido investigada como fator prognóstico por indicar presença ou ausência da mutação no gene IgVH, assim, o objetivo deste estudo foi analisar o perfil clínico-laboratorial dos pacientes com Leucema Linfocítica Crônica, tomando como referência os estadiamentos clínicos de Rai e Binet e a quantificação da expressão de CD38 e Zap-70 como fatores prognóstico. Foram pesquisados 64 prontuários médicos de pacientes atendidos no Hospital Universitário de Santa Maria e as variáveis consideradas foram aumento de linfonodos, presença ou ausência de hepatomegalia e/ou esplenomegalia, avaliação hematológica de sangue periférico e imunofenótipo. Os dados obtidos foram correlacionados com o estadiamento de Rai (1975) e Binet (1981), a expressão de CD38 e Zap-70 com o estádio clínico de Binet. Os resultados demonstraram que não há associação entre o estadiamento de Raí e Binet e a expressão de CD38, Zap-70 com o estadiamento clínico de Binet.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Hematogones are B-lineage lymphoid precursors cells normally found in bone marrow. The B-cell Acute Lymphocytic Leukemia (B-ALL) is a lymphocytic precursors neoplasia with consequent accumulation of immature cells called lymphoblasts in bone marrow. The increase of hematogones after treatment for B-ALL or marrow transplantation may cause doubt to the prognosis due to morphological and immunophenotypic similarities between these normal and malignant precursors cells. The aim of this study was to differentiate subpopulations of hematogones and B lymphoblasts by immunophenotyping using flow cytometry, attempting to identify the hematological laboratory profile at diagnosis and the environmental factors that characterize patients with B-ALL. Group 1 consisted of healthy controls free of hematologic malignancy and group 2 consisted of pediatric patients recently diagnosed with B-ALL before the start of chemotherapy, both in bone marrow samples. Antibodies directed against CD45, CD10, CD19, CD34 and mIgM (membrane) were used, which enabled us to distinguish the degree of cell maturation. Despite of the morphological and immunophenotypic similarities, hematogones and B lymphoblasts were differentiated by flow cytometry through distribution and continuous expression of antibodies on the association of CD45 x CD10 and mIgM x CD34 in B population. This technique allowed a better characterization of hematogones in healthy patients, with a progressive maturational pattern within the same cell population, with immature, intermediate and mature cells. The B lymphoblasts exhibited an incomplete maturation spectrum represented by a single population with immaturity characteristics. Among leukemia patients evaluated, 100% were white, with a predominance of children under 10 years (73%) and female sex (64%). These patients had lower hemoglobin levels (91% of cases), platelets (82%) and leukocytes (45.45%), with elevated leukocyte count in 27.3% of subjects. Predominant signs and symptoms were pallor, weakness, petechiae or bruising presence and fever. The same proportion of leukemic patients resided in both rural and urban zones. Patients living in rural area had features that can justify the etiology of ALL, as the professional connection of their parents to contact with toxic products. Morphological revision combined with immunophenotyping has allowed the correct cell identification and distinction between phenotypic subpopulations of hematogones and B lymphoblasts, contributing to an accurate laboratorial prognostic. Due to number of patients with B-ALL of this study, further researches are suggested to correlate the exposure of parents and leukemia patients to potentially oncogenic environmental factors, with the objective of preventing this neoplasia. / As hematogônias são células jovens linfoides de linhagem B normalmente encontradas na medula óssea. A leucemia linfocítica aguda de células B (LLA-B) é uma neoplasia de precursores linfocíticos com consequente acúmulo de células imaturas denominadas linfoblastos na medula. O aumento das hematogônias após o tratamento para LLA-B ou um transplante medular pode causar dúvidas quanto ao prognóstico, devido às semelhanças morfológicas e imunofenotípicas entre essas células jovens normais e malignas. O objetivo deste trabalho foi diferenciar subpopulações de hematogônias e linfoblastos B por imunofenotipagem através da citometria de fluxo, buscando identificar o perfil laboratorial hematológico ao diagnóstico e os fatores ambientais que caracterizam os pacientes de LLA-B. O grupo 1 foi constituído de controles sadios isentos de neoplasia hematológica e o grupo 2 constou de pacientes pediátricos recém-diagnosticados com LLA-B antes do início do tratamento quimioterápico, ambos em amostras de medula óssea. Foi utilizado um painel de anticorpos monoclonais composto por: CD45, CD10, CD19, CD34 e mIgM (membrana), que possibilitaram a distinção do grau de maturação das células. Apesar das semelhanças morfológicas e imunofenotípicas, hematogônias e linfoblastos B foram diferenciados por citometria de fluxo através da distribuição e expressão contínua dos anticorpos diante da associação de CD45 x CD10 e mIgM x CD34 na população B. Esta técnica permitiu uma melhor caracterização das hematogônias nos pacientes sadios, apresentando um padrão maturacional progressivo dentro da mesma população celular, com células imaturas, intermediárias e maduras. Os linfoblastos B exibiram um espectro maturacional incompleto representado por única população com características de imaturidade. Entre os pacientes com leucemia avaliados, 100% eram de raça branca, com predomínio de crianças menores de 10 anos (73%) e do sexo feminino (64%). Estes pacientes apresentaram valores reduzidos de hemoglobina (91% dos casos), plaquetas (82%) e leucócitos (45,45%), com leucometria elevada em 27,3% dos indivíduos. Os sinais e sintomas predominantes foram: palidez, astenia, presença de petéquias ou equimoses e febre. A mesma proporção de pacientes leucêmicos residia nas zonas rural e urbana. Os pacientes que moram na área rural apresentaram características que podem justificar a etiologia da LLA, como a ligação profissional dos pais ao contato com produtos tóxicos. A revisão morfológica combinada com a imunofenotipagem permitiu a correta identificação celular e a distinção entre as subpopulações fenotípicas das hematogônias e dos linfoblastos B, contribuindo para um preciso prognóstico laboratorial. Devido ao número de portadores de LLA-B deste estudo, pesquisas adicionais são sugeridas para correlacionar a exposição de pais e pacientes com leucemia aos fatores ambientais potencialmente oncogênicos, com o objetivo de prevenir essa neoplasia.

Introdução- As Leucemias Linfocíticas Agudas (LLA) constituem-se na mais comum das neoplasias em crianças. Alguns estudos epidemiológicos identificaram riscos aumentados de LLA em crianças expostas a campos magnéticos gerados por linhas de força de alta tensão, porém, essa associação não foi confirmada por outros estudos. Objetivo- Verificar a associação entre exposição a campos magnéticos e a incidência da LLA em crianças residentes na Região Metropolitana de São Paulo, considerando-se a distância das residências das crianças de linhas de transmissão de energia (88, 138, 230, 345 e 440 kV). Métodos- Estudo casocontrole de base populacional. Os casos foram selecionados em cinco hospitais na capital do município de São Paulo, que concentram o atendimento a crianças com LLA. Quatro controles populacionais foram selecionados para cada caso, emparelhados por sexo, idade e cidade de nascimento. Casos e controles foram entrevistados utilizando-se questionário similar para obtenção de informações sobre as variáveis de interesse e potenciais variáveis de confusão. Os domicílios foram avaliados em relação às distâncias de linhas de transmissão de energia mais próxima utilizando-se o Global Positioning System (GPS). Na análise da associação entre campos magnéticos e LLA foi utilizada regressão logística condicional, incluindo o controle de potenciais variáveis de confusão. Foram calculados os odds ratios (OR) e os respectivos intervalos com 95 por cento de confiança (IC95 por cento). Resultados- A associação entre a distância de linhas de transmissão e LLA foi ajustada pela variável escolaridade da pessoa entrevistada, resultando em OR de 2,91 (IC95 por cento 0,92-9,22). Conclusão- Concluiu-se que, crianças residentes a menos de 160 metros de linhas de transmissão de energia na RMSP apresentam risco maior, porém não estatisticamente significativo de desenvolver LLA quando comparadas com as que residem a mais que 160 metros. / Introduction-The Acute Lymphocytic Leukemia (ALL) is the most common cancers in children. Some epidemiological studies have identified increased risk of ALL in children exposed to magnetic fields generated by high voltage power lines, however, this association was not confirmed by other studies. Purpose- Check the association between exposure to magnetic fields and the incidence of ALL in children residing in the Metropolitan Region of São Paulo, considering the distance from childrens home and transmission lines (88, 138, 230, 345 and 440 kV). Methods- A population-based case-control study. The cases were selected in five hospitals in the city of Sao Paulo, which account for the care of children with ALL. Four population controls were selected for each case, matched by sex, age and city of birth. Cases and controls were interviewed using a similar questionnaire to obtain information on the interest variables and potential confounding variables. The homes were evaluated by distances of the nearest transmission lines using the Global Positioning System (GPS). To examine the association between magnetic fields and ALL was used conditional logistic regression, including the control of potential confounding variables. We calculated the odds ratios (OR) and their 95per cent confidence interval (95 per cent CI). Results-The association between the distance of transmission lines and ALL was adjusted by education of the person interviewed, and the OR was 2.91 (95 per cent CI 0.92-9.22). Conclusion- Children living at less than 160 m of power transmission lines in RMSP have higher risk, though not statistically significant, when compared with those living in more than 160 m.

Orientadores: Jorg Kobarg, Jose Andres Yunes / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-13T22:20:50Z (GMT). No. of bitstreams: 1 Trindade_DanielMaragno_D.pdf: 7352494 bytes, checksum: 5b03d143205e747b05e3d4b6a8ed8e34 (MD5) Previous issue date: 2009 / Resumo: Staniocalcinas (STCs) representam uma pequena família de hormônios glicoprotéicos, encontrados em todos os vertebrados e composta por STC1 e STC2, que foram inicialmente implicados na homeostase de cálcio e recentemente também foram implicados em vários outros processos. Stanniocalcina-1 (STC1), o primeiro membro encontrado, foi originalmente descoberto em peixes ósseos e posteriormente identificado em humanos onde parece ter um papel, embora ainda obscuro, na carcinogênese e angiogênese. Nos seres humanos STC1 pode ser encontrado em duas formas: um dímero ou um grupo de variantes de alto peso molecular coletivamente chamados bigSTC. Uma vez que ambas as células leucêmicas e os tumores sólidos dependem vascularização, a angiogênese é um passo fundamental na interação tumorhospedeiro e essencial para a progressão do câncer. Análises prévias de microarray resultaram na identificação de vários genes ativados em células endoteliais da medula óssea (BMEC) modulados pela presença de células leucêmicas. Apresentamos aqui STC1 como um marcador microambiente de medula óssea (BMM) durante leucemia linfoblástica aguda (LLA) pela validação dos dados prévios de microarray através de PCR em tempo real quantitativos. Em vista da falta de informações funcionais e estruturais sobre STC1 realizamos: (1) um screen no sistema de duplo híbrido em levedura, a fim de encontrar algumas das proteínas que interagem com a STC1 humana, e (2) uma caracterização estrutural inicial à baixa resolução desta proteína. Fomos capazes de construir um mapa interação proteína-proteína, obtido a partir dos 22 interactantes encontrados em screen de duplo híbrido em levedura. As proteínas encontradas apresentam-se em vários compartimentos celulares nos quais STC1 já foi demonstrada para estar presente, e essas novas informações podem ajudar a esclarecer como e qual o papel STC1 desempenha nesses locais. A fim de fornecer informação estrutural sobre STC1, realizamos análises bioquímicas e estruturais da proteína recombinante STC1 com 6xHis tag produzida em células de inseto utilizando o sistema baculovírus. A análise de dicroísmo circular confirmou a predição in silico do elevado conteúdo de alfa-helices. A análise por espectrometria de massas forneceu os dados experimentais que confirmaram o padrão conservado de pontes dissulfeto anteriormente descrito para STC1 de peixes. Finalmente, os dados de espalhamento de raios-X a baixo ângulo demonstraram que, STC1 adota uma estrutura dimérica, ligeiramente alongada em solução fornecendo deste modo os primeiros dados estruturais à baixa resolução desta família de proteínas. Além disso, foi possível obter proteína suficiente e de elevado grau de pureza para realizar ensaios cristalização que resultaram em cristais os quais difrataram a boa resolução. / Abstract: Staniocalcins (STCs) represent a small family of glycoprotein hormones found in all vertebrates composed by STC1 and STC2, which have been initially implicated in calcium homeostasis and also recently implicated in several other processes. Stanniocalcin-1 (STC1), the first member found, was originally discovered in bony fishes and later identified in humans were it seems to have a role, although still unclear, in carcinogenesis and angiogenesis. In humans STC1 can be found in two forms: a dimer or a group of higher molecular weigh variants collectively called bigSTC. Since both leukemic cells and solid tumors depend on vascularization, angiogenesis is a fundamental step in tumor-host interaction and essential to the cancer progression. Previous microarray analysis resulted in the identification of several activated genes in bone marrow (BM) endothelial cells (BMEC) modulated by presence of leukemic cells. Here we present STC1 as a BM microenvironment marker during acute lymphoblastic leukemia (ALL) by validating previous microarray data by quantitative RealTime-PCR. In view of the lack of functional and structural information on STC1 we performed (1) a yeast two hybrid screen in order to find some of the human STC1 interacting proteins and (2) a initial structural lowresolution characterization of this protein. We were able to construct a protein-protein interaction map, derived from the 22 interactants found in our yeast two-hybrid screen. The proteins found are located in several cellular compartments in which STC1 has already been shown to be present, and the new information might help to clarify how and which role it performs at these sites. As a means to provide structural information about STC1, we performed biochemical and structural analyses of recombinant STC1 6xHis tagged protein produced in insect cells by using the baculovirus system. Circular dichroism analysis confirmed the in silico predicted high alpha-helical content. By mass spectroscopy analysis we provided experimental data that confirmed the conserved disulfide pattern previously described for fish STC1. Finally Small Angle X-ray Scattering data demonstrated that STC1 adopts a dimeric, slightly elongated structure in solution, providing there by the first low resolution structural data of this family of proteins. Additionally, we could obtain enough protein of high purity to perform crystallization trials that resulted in crystals diffracting at good resolution. / Doutorado / Bioquimica / Doutor em Biologia Funcional e Molecular