Introdução: O hipotireoidismo congênito possui prevalência de 1/4000 crianças nascidas vivas e pode ser causado por disgenesia tireoideana (80% dos casos) ou por defeitos de síntese hormonal (20% restantes). A disormonogênese tem sido associada a mutações nos genes: tireoglobulina, simportador sódio/iodo, tireoperoxidase, dual oxidase 2, e pendrina. A tireoglobulina é uma glicoproteína de 660KDa e funciona como matriz para a síntese dos hormônios tireoideanos. Até o momento 38 mutações inativantes, associadas a bócio e hipotireoidismo, foram identificadas no gene da tireoglobulina. Objetivos: Este estudo visou caracterizar mutações no gene da tireoglobulina em 13 pacientes brasileiros com bócio e hipotireoidismo congênito e verificar o efeito funcional da mutação A2215D identificada neste estudo. Casuística e Métodos: Foram estudados 13 pacientes com hipotireoidismo congênito por possível defeito de síntese de tireoglobulina. Foi utilizado DNA de sangue periférico de todos os pacientes e amostra de tecido da paciente portadora da mutação A2215D. Os métodos utilizados foram: amplificação e sequenciamento dos 48 exons e das junções exon/intron, transfecção de células de mamífero com plasmídeos contendo o cDNA da tireoglobulina mutada e não mutada, eletroforese de proteínas e hibridização com anticorpo específico, dosagem de tireoglobulina por fluoroimunoensaio indireto, quantificação de RNAm por PCR quantitativo em tempo real, imunohistoquímica, microscopia por coloração de hematoxilina/eosina e microscopia eletrônica e imunoeletrónica. Resultados: O defeito de síntese de tireoglobulina nos pacientes foi confirmado pela ausência de elevação do valor da tireoglobulina sérica 24 e 48hs após a aplicação de 0,45mg de TSH humano recombinante. No gene da tireoglobulina foram identificadas cinco mutações, sendo duas novas (Q2142X e IVS46-1G>A) e três já descritas na literatura (R277X, IVS30+1G>T e A2215D); 19 polimorfismos e 13 alterações em introns. Doze dos treze pacientes apresentaram mutações bialélicas (homozigose ou em heterozigose composta). O estudo funcional em células de mamíferos revelou diminuição da secreção da proteína mutada e no tecido do paciente portador da mutação A2215D mostrou: localização intracelular da tireoglobulina com ausência quase total no colóide; retículo endoplasmático rugoso de volume extremamente aumentado, presença de tireoglobulina dentro do retículo endoplasmático rugoso e baixo conteúdo de RNAm de tireoglobulina e da própria proteína. Também foram verificados baixo conteúdo de RNAm de genes tireoideanos (TPO, TTF1, PAX-8, NIS, receptor de TSH). Conclusão: Todas as mutações identificadas neste estudo explicaram o hipotireoidismo congênito diagnosticado nos pacientes. A mutação A2215D promoveu a retenção da proteína mutada dentro do retículo endoplasmático rugoso e diminuiu sua secreção para o colóide, ocasionando grave defeito de síntese hormonal e hipotireoidismo. A tireoglobulina endógena portadora da mutação A2215D poderia atuar como regulador da função tireoideana via supressão da expressão dos fatores de transcrição importantes na fisiologia da tireóide. / Introduction: Congenital hypothyroidism is one of the most common hereditary endocrine disorders, which affects 1:4000 newborns. Congenital hypothyroidism is caused by thyroid gland dysgenesis (80%) or inborn errors of thyroid hormone synthesis (20%). Genetic defects in thyroglobulin, pendrin, thyroperoxidase, dual oxidase 2, simporter sodium/iodine have been associated to dyshormonogenesis. Thyroglobulin is a large glycoprotein that functions as the matrix for thyroid hormone synthesis. At least 38 mutations in the TG gene have been identified in patients with thyroid dyshormonogenesis. Objectives: The aims of this study were to identify thyroglobulin gene mutations associated with congenital hypothyroidism in 13 Brazilian patients, and to determine the functional effect of the mutation A2215D identified in this study. Patients and methods: Thirteen patients with congenital hypothyroidism due to defective thyroglobulin synthesis were included. Peripheral blood DNA from all the patients and one thyroid tissue sample from a patient with the A2215D mutation were collected. Thyroglobulin exons and exons/introns borders were amplified by PCR and sequenced. Mammalian cells were transfected with expression vectors encoding mutated and non-mutated thyroglobulin cDNA. Immunoblots, determination of thyroglobulin concentrations, real time PCR to quantify mRNA expression, immunohistochemical analysis, hematoxilyn-eosin staining and electronic and immunogold microscopy were performed. Results Abnormal thyroglobulin synthesis and secretion was confirmed by the absence of a serum thyroglobulin elevation 24 and 48 hours after the stimulation with recombinant human TSH (0.45 mg). Molecular analysis revealed five mutations in the thyroglobulin gene, 2 novel (Q2142X and IVS46-1G>A) and three previously described mutations (R277X, IVS30+1G>T and A2215D); 19 polymorphisms and 13 intronic alterations. Biallelic mutations (homozygous or compound heterozygous) in the thyroglobulin gene were identified in twelve of thirteen patients. Functional studies in mammallian cells showed low secretion of the mutated thyroglobulin (A2215D). The complete analysis of the thyroid tissue from a patient with the A2215D mutation revealed: mutant thyroglobulin in the follicular cell but not in the lumen, marked dilatation of the endoplasmic reticulum, thyroglobulin immunopositivity in the endoplasmic reticulum and low concentration of thyroglobulin protein and mRNA. Furthermore, low mRNA levels of thyroid genes (TPO, TTF1, PAX-8, NIS, receptor de TSH) were detected. Conclusions: All the identified thyroglobulin gene mutations explained the congenital goiter hypothyroidism of the patients. The mutation A2215D promoted the retention of the mutant thyroglobulin in the endoplasmic reticulum, decreased the thyroglobulin secretion to the colloid resulting in an impairment of thyroid hormone synthesis and congenital hypothyroidism. The endogenous A2215D mutant thyroglobulin could be considered a regulator of follicular function mediated by the transcriptional suppression of thyroid genes.

O câncer colorretal é um dos tipos de tumores mais frequentes no mundo. A atual dificuldade na avaliação correta da resposta ao tratamento torna necessário o desenvolvimento de novas abordagens de detecção tumoral. Atualmente, o sequenciamento genômico em larga escala permite um estudo mais compreensivo das alterações estruturais e de sequência presentes no tumor. A aplicação destas abordagens de maneira personalizada permite o desenvolvimento de biomarcadores tumor específicos que podem facilitar a avaliação de resposta ao tratamento e a presença de doença residual, bem como revelar alterações de sequência em genes capazes de servir de novos alvos terapêuticos. Neste estudo foi desenvolvida uma metodologia eficiente para a identificação de biomarcadores baseados na existência de variações estruturais em genomas de tumores de reto, eliminando a necessidade de sequenciamento do genoma normal do mesmo paciente e diminuindo portanto o custo da abordagem. Os biomarcadores encontrados para cada um dos seis pacientes foram utilizados para avaliar a presença de doença residual após o tratamento através da detecção de DNA tumoral circulante nas amostras de plasma coletadas em momentos diferentes do tratamento. O sequenciamento em baixa cobertura personalizado é portanto uma alternativa viável e promissora para avaliar a resposta ao tratamento em pacientes com tumores de reto. Na segunda parte do estudo, a análise de linhagens celulares de tumores colorretais revelou uma grande quantidade de mutações pontuais somáticas (SNVs e InDels) em genes codificadores para proteínas de superfície celular (surfaceoma). Estas alterações no surfaceoma indicam potenciais novos alvos para drogas e vias regulatórias alteradas neste tipo de tumor. Além disso, estas mutações pontuais também são responsáveis pela geração de epítopos com potencial imunogênico e estes novos epítopos podem ser aplicados como vacinas antitumorais personalizadas e já haviam sido propostos como uma alternativa terapêutica. A presença de novos epítopos, principalmente nas linhagens com elevadas taxas de mutação (resultante da instabilidade de microssatélites e mutações em genes de reparo de DNA tipo mismatch ou POLE), sugerem também um potencial uso de drogas moduladoras do sistema imune em pacientes com tumores que apresentam estas mesmas características. Portanto, o estudo de alterações genômicas em tumores primários e linhagens de câncer colorretal permitiu a detecção de variações estruturais que foram utilizadas como biomarcadores personalizados em pacientes com tumores de reto assim como a identificação de genes contendo mutações pontuais em linhagens celulares de câncer colorretal, que revelam potenciais novos alvos terapêuticos a serem explorados na clínica / Colorectal cancer is one of the more frequent tumor types in the world. To select the appropriate treatment course, it is necessary to develop more precise diagnostic approaches. The current availability of high throughput genome sequencing methods allows for a comprehensive characterization of the structural and sequence alterations present in each tumor. The use of tumor genome sequencing in a personalized setting can result in tumor specific biomarkers that help evaluate response to treatment and the presence of residual disease, improving the clinical management of these patients, and also reveal sequence alterations in genes capable of serving as new therapeutic targets. In this study we developed an efficient bioinformatics pipeline to identify biomarkers based on the existing structural alterations in rectal tumor genomes, eliminating the need to sequence the matched normal genome and therefore reducing the cost for this approach. The biomarkers found for each of the six patients were used to evaluate the presence of residual disease after treatment through the detection of circulating tumor DNA in plasma samples collected at different points during the treatment. Sequencing tumor genomes with low coverage is therefore a viable and promising alternative to follow up rectal cancer patient\'s response to treatment. In the second part of this study, the analysis of colorectal cancer cell lines revealed a large quantity of point mutations (SNVs and InDels) in genes coding for proteins located in the cell surface (surfaceome). These alterations in the surfaceome indicate potential new drug targets and altered pathways in this type of tumor. Furthermore, these point mutations are also responsible for the generation of new epitopes with immunogenic potential and these new epitopes can be applied as personalized tumor vaccines and had previously been proposed as a therapeutic alternative. The presence of new epitopes, especially in the cell lines with elevated mutation rates (resulting from MSI and mutations in DNA mismatch-repair genes or POLE), suggests a potential use of immune checkpoint target drugs in patients with tumors that share these genetic characteristics. With a large-scale bioinformatics approach, we detected new tumor epitopes resulting from point mutations, present in most of the cell lines used. The analysis of gene expression data puts into perspective both the somatic mutations found and which targets are promising as well as the development of therapies based on vaccines derived from tumor epitopes. In conclusion, the study of genomic alterations in primary tumors and colorectal cancer cell lines allowed the detection of structural variations that were used as personalized biomarkers in patients with rectal tumors as well as the identification of genes containing point mutations in colorectal cancer cell lines, that reveal potential new therapeutic targets to be explored in the clinical setting.

Different mutations within the NALP3 gene are thought to be associated with development of several types of hereditary auto inflammatory disorders such as neonatal onset multisystem inflammatory disorder (NOMID) and muckle-wells syndrome (MWS). In this work two separate mutations E688K and G569R were supposed to be constructed by site-directed mutagenesis in the cloned wild type NALP3 genes and further expressed in bacterial and mammalian host cells for functional studies in protein -protein interaction models.

A database, AmyProt, was developed that collated details of 32 human amyloid proteins associated with disease and 488 associated mutations and polymorphisms, of which 316 are classified as amyloid. A detailed profile of the mutations was developed in terms of location within domains and secondary structures of the proteins and functional effects of the mutations. The data was used to test the hypothesis that mutations enhance amyloidosis in human amyloid proteins have distinctive characteristics, in terms of specific location within proteins and physico-chemical characteristics, which differentiate them from non-amyloid forming polymorphisms in amyloid proteins and from disease mutations and polymorphisms in non-amyloid disease linked proteins. The aim was to use these characteristics to train a prediction algorithm for amyloid mutations that will provide a more accurate prediction than current general disease prediction tools and amyloid prediction tools that focus on aggregating regions. 66 location specific features and changes upon mutation of 366 amino acids propensities, derived from the amino acid index database AAindex, were analysed. A significant proportion of mutations were located with aggregating regions, however the majority of mutations were not associated with these regions. An analysis of motifs showed that amyloid mutations had a significant association with transmembrane helix motifs such as GxxxG. Statistical analysis of substitutions mutations, using substitution matrices, showed that amyloid mutations have a decrease in α-helix propensity and overall secondary structure propensity compared to the disease mutations and disease and amyloid polymorphisms. Machine learning was used to reduce the large set of features to a set of 18 features. These included location near transmembrane helices, secondary structure features; transmembrane and extracellular domains and 4 amino acid propensities: knowledge-based membrane propensity scale from 3D helix; α-helix propensity; partition coefficient; normalized frequency of coil. The AmyProt mutations and non-amyloid polymorphisms were used to train and test the novel amyloid mutation prediction tool, AmyPred, the first tool developed purely to predict amyloid mutations. AmyPred predicts the amyloidogenicity of mutations as a consensus by majority vote (CMV) and mean probability (CMP) of 5 classifiers. Validation of AmyPred with 27 amyloid mutations and 20 non-amyloid mutations from APP, Tau and TTR proteins, gave classification accuracies of 0.7/0.71 (CMV/CMP) and with an MCC of 0.4 (CMV) and 0.41 (CMP). AmyPred out performed other tools such as SIFT (0.37) and PolyPhen (0.36) and the amyloid consensus prediction tool, MetAmyl (0.13). Finally, AmyPred was used to analyse p53 mutations to characterize amyloid and non-amyloid mutations within this protein.

Le gène BAG3 a été identifié comme étant un nouveau gène responsable de cardiomyopathie dilatée (CMD), première cause d'insuffisance cardiaque (IC). La protéine BAG3 est une co-chaperonne qui participe au contrôle de l'homéostasie protéique via son rôle dans l'autophagie, protégeant ainsi les cellules contre la protéotoxicité induite par les protéines dégradées ou mal repliées. L'hypothèse qu'une inactivation de la voie autophagique contrôlée par BAG3 induirait une protéotoxicité cardiomyocytaire à l'origine de la CMD apparait particulièrement attractive et constitue l'hypothèse centrale de ce travail. Nos résultats indiquent que les mutations de BAG3 abolissent l'interaction avec la chaperonne HSP70, une protéine centrale du contrôle qualité des protéines. Nous avons observé une cytotoxicité des mutants BAG3, une altération de la fonction chaperonne HSP70-dépendante et une absence de réponse autophagique en condition de stress (jeun, choc thermique, expression d'une protéine pro-agrégante). In vivo (modèle poisson-zèbre) l'extinction de l'expression de BAG3 ou la surexpression des mutants conduisent à l'apparition d'un phénotype d'insuffisance cardiaque (¿dème péricardique) chez les embryons injectés. Par édition génomique, nous développons également un modèle de cardiomyocytes dérivés de cellules iPS porteurs ou non de la mutation afin d'explorer plus en avant la fonction contractile de ces cellules. Nos résultats confirment donc le rôle de BAG3 dans la CMD et indiquent que l'altération de la fonction protéostasique serait à l'origine de la maladie. Cette nouvelle voie physiopathologique dans la CMD pourrait s'avérer être, plus généralement, une voie centrale dans l'IC. / The BAG3 gene was identified as a novel gene responsible for dilated cardiomyopathy (DCM), a major cause of heart failure (HF). The BAG3 protein is a co-chaperone that participates in the control of protein homeostasis via its role in autophagy, protecting cells against the proteotoxicity induced by degraded or misfolded proteins. The hypothesis that inactivation of the autophagic pathway controlled by BAG3 would induce cardiomyocyte proteotoxicity behind the CMD appears particularly attractive and is the central hypothesis of this work. Our results indicate that BAG3 mutations abolish the interaction with the chaperone HSP70, a central actor of the protein quality control. We observed cytotoxicity of BAG3 mutants, an impaired HSP70-dependent chaperone function and absence of autophagic response under stress conditions (starvation, heat shock, expression of a pro-aggregating protein). In vivo (zebrafish model) the extinction of BAG3 expression or mutants overexpression lead to the occurrence of a heart failure phenotype (pericardial edema) in injected embryos. Through genomic edition, we also develop a model of iPS-derived cardiomyocytes carrying or not the mutation in order to further explore the contractile function of these cells. Our results confirm the role of BAG3 in DCM and indicate that the alteration of the proteostasis function is the cause of the disease. This new pathophysiological pathway in DCM may prove to be more generally, a central line in the IC.

Breast cancer is the most common cancer in South African women. Approximately 5¬10% of all breast cancer cases are due to an inherited predisposition, resulting from mutations in tumour suppressor genes. The BRCA1 gene on chromosome 17q is one such tumour suppressor gene, that when mutated confers an increased risk of breast/ovarian cancer in carriers. To date, more than 500 different BRCA 1 mutations have been reported worldwide. Some of these mutations are frequently reported and others occur commonly in certain population groups. These population-specific differences in mutations represent founder effects, whereby a single ancestral mutation accounts for the majority of breast cancer cases. This study was undertaken as the nature and frequency of BRCA1 mutations in South African breast/ovarian cancer families is unknown. Fifty-one breast/ovarian cancer families were screened for three commonly occurring mutations (185deIAG, 4184del4 and 5382insC) using polymerase chain reaction (PCR) and allele-specific oligonucleotide (ASO) hybridisation. The protein truncation test (PTT) was utilised to detect truncating mutations in the large exon 11, and the remaining coding exons were screened for mutations using exon-by-exon PCR single strand conformation polymorphism/heteroduplex analysis (SSCP/HA). Seven disease-causing mutations were identified in 15 families, consisting of five different frameshift mutations and two different nonsense mutations. Four Ashkenazi Jewish families were found to harbour the 185delAG mutation; the 5382insC mutation was identified in two Afrikaner families and one Ashkenazi Jewish family. Haplotype analysis revealed that the four Ashkenazi families share the common Ashkenazi Jewish haplotype, suggesting a common ancestor for these families. Similarly, the two Afrikaner families share the same haplotype as families of north and east European ancestry with the 5382insC mutation. The haplotype of the Ashkenazi Jewish family with this mutation was however different to the linked haplotype, indicating a recombination event or an independent mutation. Both these mutations are thought to have occurred in or before the medieval period. Furthermore, four Afrikaner families were found to carry the novel E881X nonsense mutation, which has not been previously described. Haplotype analysis of these families suggested that these patients share a common ancestor, and genealogic studies have identified the founding couple for this mutation, who both arrived in the Cape from France in the late 1600s. Four additional families were found to harbour BRCA1 mutations by SSCP/HA. Three of these mutations have not been previously reported - the S451X nonsense mutation (identified in a family of Scottish origin), the 1493delC mutation identified in an Afrikaner family, and the 4957insC mutation identified in an Indian family. The 448insA mutation was identified in a family of German origin, where the patient had cancer of the fallopian tubes. A number of different described polymorphisms and variants of unknown functional significance were also identified. This is the first study to show that BRCA1 is involved in South African breast/ovarian cancer families, to the extent that 29.4% (15/51) of families have BRCA1 mutations. Furthermore, minor founder effects in the Afrikaner population have been demonstrated. These results enable improved genetic counselling and clinical management of mutation positive families as well as subsequent testing of family members. / Dissertation (MSc (Human Genetics))--University of Pretoria, 2005. / Genetics / unrestricted

La mise au point en 2005 d’un modèle de propagation sur lignée d’hépatocarcinome basé sur la souche hautement réplicative JFH-1 fut une formidable opportunité d’étudier les différentes étapes du cycle infectieux du VHC. Nous avons souhaité étudier les étapes de morphogenèse et de sécrétion du virus, des phases du cycle viral qui sont largement mal connues encore aujourd’hui, mais ou la protéine de capside joue probablement un rôle majeur. Des études comparatives des séquences de capsides de différentes souches du VHC nous ont permis de mettre en évidence 10 résidus spécifiques à la souche JFH-1 qui pourraient expliquer les déficits fonctionnels connus de cette protéine. En effet, le remplacement de ces 10 résidus par ceux plus communément retrouvés dans les souches de génotype 1 et 2 a permis une amélioration significative de l’assemblage et de la sécrétion des particules infectieuses produites. La mise au point de cette souche optimisée pour la production de virus pourrait par ailleurs permettre constituer un atout pour mieux comprendre la structure du virus par des techniques de microscopie électronique ; ce type d’étude n’ayant pas pu être véritablement menée jusqu’à présent, en raison des titres infectieux insuffisants obtenus avec la souche JFH-1 sauvage. / Development and cloning in 2005 of the highly replicative strain JFH-1 was a great opportunity to study the different stages of the infectious cycle of HCV as this strain easily propagate in the hepatocellular carcinoma cell line. Until now, these lates phases of particles assembly remain poorly understood, although the core protein is thought to probably play a major role in initiation of these mechanisms. Comparative studies of the capsid sequences of different strains of hepatitis C have allowed us to identify 10 specific residues in the JFH-1 strain that could explain the functional deficits of this protein. Indeed, the replacement in JFH-1 strain of these 10 residues by those most commonly found in strains of genotype 1 and 2 showed improvement of the assembly and secretion of new infectious particles and new subcellular localization of core. In addition, replacement of these ten residues by most common amino acid found in patients show a great enhancement of in vitro virus production and secretion. As a perspective, development of this optimized virus could also represent a valuable model to better purify and determine viral structure, and true viral assembly site; HCV fields that remain till now largely unknown.

A ?-talassemia, uma hemoglobinopatia, é caracterizada como um distúrbio hereditário monogênico onde a síntese das cadeias globínicas ? está alterada. Devido desiquilíbrio na relação entre as cadeias ? e ? produzidas, observa-se um excesso de cadeias ? livres, determinante da fisiopatologia da doença. As manifestações observadas são eritropoese ineficaz, hemólise extramedular, anemia, expansão medular, esplenomegalia, deformidades ósseas e acúmulo de ferro. Clinicamente classifica-se como ?-talassemia major (BTM) a forma mais grave da doença, devido à ausência de cadeias ? (?0) ou redução acentuada (?+) acarretando em dependência de transfusões sanguíneas periódicas, para sobrevivência. O traço ?-talassêmico (BTT) antes vistos como assintomáticos, também apresentam alterações, inclusive acúmulo de ferro e eritropoese ineficaz, mas não são dependentes de transfusão e tampouco passam por acompanhamento médico. Extremamente heterogênea, apresenta diversos fenótipos e mais de 300 alterações moleculares causadoras de ?-talassemia já foram descritas em todo mundo. O objetivo deste estudo foi identificar as mutações de ?-talassemia em Ribeirão Preto-SP e procurar associar tais alterações à avaliação hematológica e do status férrico, na tentativa de estabelecer uma relação genótipo-fenótipo. Para tanto, 27 BTM, 23 BTT e 28 controles foram recrutados no Ambulatório de Hemoglobinopatias, do HC/FMRP-USP de Ribeirão Preto. Por meio de PCR-Alelo Específico, pesquisamos as quatro mutações mais comuns no Brasil: CD39 (CAG->TAG), IVS1-110 (G->A), IVS1-6 (T->C) e IVS1-1 (G ->A). A distribuição foi 64% CD39, 26% IVS1-110 e 4% IVS1-6. A análise de covariância e comparação múltiplas, entre os grupos formados e o controle, revelou alterações hematológicas e no status férrico. Os pacientes BTM com a mutação CD39, em sua forma heterozigota ou homozigota, e heterozigotos para a IVS1-110, revelaram anemia grave e intensa sobrecarga de ferro. Os BTT heterozigotos para CD39 demonstraram comprometimento do metabolismo ferro e/ou eritropoese. A adoção de medidas paliativas e de monitoramento aos BTT faz-se necessária, uma vez que, alterações apresentadas associam-se a desordens graves, mas quando não negligenciadas podem ser facilmente prevenidas. A metodologia adotada demonstrou-se eficaz para a pesquisa das mutações estudadas. Embora tenhamos conseguido observar uma relação genótipo-fenótipo, um estudo multicêntrico da população brasileira proporcionará a identificação de mais relações, principalmente nos fenótipos menos prevalentes em nossa região, contribuindo para a compreensão da heterogeneidade da ?-talassemia. / The ?-thalassemia, one haemoglobinopathies, is characterized as a monogenic hereditary disorder where the synthesis of ? globin chains is modified. Due to imbalance in the relationship between production of ? and ? chains, there is an excess of free ? chain that determines the pathophysiology of the disease. Manifestations observed are ineffective erythropoiesis, extra medullary hemolysis, anemia, bone marrow expansion, splenomegaly, bone deformities and iron accumulation. Clinically is classified as ?-thalassemia major (BTM), the most severe form of the disease, as a result of the absence of ? chains (?0) or very large reduction of these (?+) resulting in dependence on regular blood transfusions to survive. The ?-thalassemia trait (BTT) before seen as asymptomatic, also show changes, including iron accumulation and ineffective erythropoiesis, despite of that they aren\'t dependent on transfusion nor undergo medical care. It is extremely heterogeneous, presents several phenotypes and more than 300 molecular changes that causing ?-thalassemia have been described worldwide. The purpose of this study was to identify ?-thalassemia mutations in Ribeirao Preto-Brazil and to explore changes in hematological evaluation and iron status in an attempt to establish a genotype-phenotype relationship. Therefore, a group of 27 BTM, 23 BTT and 28 controls were recruited from the outpatient clinic of hemoglobinopathies, from The Clinical Hospital of Medical School of Ribeirao Preto (HC / FMRP-USP), Brazil. Adopting the technique ARMS (Amplification Refractory Mutation System), we searched for the four most common mutations in Brazil: CD39 (CAG -> TAG), IVS1-110 (G -> A), IVS1-6 (T -> C) and IVS1-1 (G -> A). The distribution was 64% presents CD39 mutation, followed by IVS1-110 e IVS1-6, with 26% and 4% respectively. Covariance Analysis and multiple comparison between the studies groups and control, showed differences in hematological parameters and in iron status either. The BTM heterozygous or homozygous for CD39 mutation and heterozygous for IVS1-110 revealed severe anemia and iron overload. The BTT heterozygous for CD39 showed impairment of iron metabolism and / or erythropoiesis. It is necessary the monitorization of the BTT patients is necessary, since changes presented by them are associated with serious disorders, the adoption of mitigation measures which when are not neglected can be easily prevented. The methodology proved to be effective for the investigation of mutations studied. While we were able to observe a genotype-phenotype relationship, a multicenter study of the Brazilian population will provide the identification of more relations, especially in less prevalent phenotypes in our region, contributing to the understanding of the heterogeneity of ?-thalassemia.

This thesis focuses mainly on the consequences that single mutations have on structural, functional and energetic aspects of the protein cyanovirin-N. In order to estimate the free energy of single mutations, we have applied thermodynamics integration and Bennett acceptance ratio techniques. Replica exchange molecular dynamics has been applied to accelerate simulations for complicated scenarios. Our studies suggest that certain single mutations may be promising to improve binding affinity to Manα1→2Manα but we also learned that the simplistic view of a strong hydrogen bond correlating to a high binding affinity may not always be correct. Finally, we explored in detail the widely used mutation P51G for its impact on protein rigidity at the very important hinge region as well as for its possible effect on glycan binding.

Elevated plasma cholesterol is a risk factor for cardiovascular disease. Proprotein convertase subtilisin/kexin type 9 (PCSK9) hinders the uptake of low-density lipoprotein cholesterol (LDL-c) by mediating degradation of LDL receptors (LDLRs) in the liver. Gain-of-function (GOF) mutations in PCSK9 cause familial hypercholesterolemia (FH). In normolipidemic human plasma, 30-40% of PCSK9 is bound to LDL particles, and this association with LDL inhibits PCSK9’s ability to mediate LDLR degradation in cultured cells. To further investigate the physiological relevance of this interaction, we analyzed natural GOF mutations in PCSK9 and assessed their effects in vitro on LDL binding, LDLR binding and LDLR degradation. Our results indicate that several GOF mutations severely inhibit LDL binding compared to wild type (WT) PCSK9, and only modestly affect LDLR affinity and LDLR degradation. These findings shed light on the potential physiological relevance of the PCSK9-LDL interaction, which may have an inhibitory effect on PCSK9 activity in vivo.