Orientador: Adriana Camargo Ferrasi / Resumo: Os Astrocitomas são tumores de Sistema Nervoso Central (SNC) caracterizado, quanto à sua nomenclatura e grau de malignidade, conforme seu tipo histológico e seu potencial proliferativo, presença de necrose e invasão cortical. Os interferons (IFNs) foram inicialmente associados à resposta antiviral, contudo, estudos posteriores evidenciaram o envolvimento dessas citocinas na regulação do crescimento celular e no efeito imunomodulatório. A Interleucina 28B (IL28B), membro da família dos IFNs do tipo III, parece estar envolvida na resposta imune antiviral e antitumoral. Sendo assim, o objetivo do presente estudo foi avaliar a frequência de variações genéticas em IL28B em pacientes acometidos por Astrocitomas. Cinquenta e sete pacientes tratados pelo serviço de Neurocirurgia da UNESP de Botucatu/SP foram incluídos neste estudo. A análise do polimorfismo rs12979860 C/T não teve significância estatística quando comparou-se a frequência genotípica da população brasileira em relação a presente casuística. Foi descrito a detecção de novas variações genéticas (missense e silent) no gene IL28B ao comparar com a sequência referência disponível em banco de dados (NCBI). Estes dados sugerem uma importante relação destas variações genéticas em relação a estrutura e função das proteínas envolvidas, entretanto um estudo mais aprofundado das consequências dessas alterações na gênese e/ou progressão dos astrocitomas devem ser considerado. / Abstract: The astrocytomas are tumors of the central nervous system (CNS) characterized as to its nomenclature and degree of malignancy, according to their histological type and their proliferative potential, presence of necrosis and cortical invasion. Interferons (IFNs) were originally associated with the antiviral response, however, subsequent studies revealed the involvement of these cytokines in the regulation of cell growth and immunomodulatory effect. Interleukin 28B (IL28B), member of the family of IFNs type III appears to be involved in the antiviral and anti-tumor immune response. Thus, the aim of this study was to evaluate the frequency of genetic variation in IL28B in patients suffering from astrocytomas. Fifty-seven patients treated by our service at UNESP in Botucatu / SP were included in this study. Rs12979860 C / T polymorphism analysis was not statistically significant when compared to genotypic frequency of the population in relation to this study. It described the detection of new genetic variants (missense and silent) in IL28B gene to compare with the reference sequence available in the database (NCBI). These data suggest an important relationship between these genetic variations regarding the structure and function of the proteins involved, however further study of the consequences of these changes in the genesis and / or progression of astrocytomas should be considered. / Mestre

nÃo hà / Introdução: O cÃncer Ãum dos principais problemas mundiais de saÃde e uma das causas mais importantes de morbidade e mortalidade em saÃde pÃblica. Segundo a OrganizaÃÃo Mundial de SaÃde (OMS) Ãa segunda causa de morte no mundo;perdendo apenas para as doenÃas cardiovasculares.Temos alta incidÃncia nacional de pacientes com cÃncer de pulmÃo avanÃado. Hà imprevisibilidade de perfis epidemiolÃgicos e respostas terapÃuticas. A biologia molecular tem sido importante na caracterizaÃÃo sobre a diferenciaÃÃo tumoral e prognÃstico da doenÃa.Novos tratamentos sÃo direcionados por caracterÃsticas farmacogenÃmicas. Pouco se sabe sobre a prevalÃncia desses genes no CearÃe na AmÃrica Latina, bem como sobre caracterÃsticas clÃnicas e desfechos relacionados. Objetivo: Identificar as caracterÃsticas e distribuiÃÃo do perfil epidemiolÃgico dos pacientes portadores de neoplasia de pulmÃo avanÃado, atendidos no ambulatÃrio de oncologia do Hospital de Messejana - Dr. Carlos Alberto Studart. Material e Métodos: Estudo observacional, prospectivo e analítico com inclusão de 135 pacientes portadores de neoplasia de pulmÃo avanÃado, entre 08/2012 a 12/2014; sendo analisados quanto a distribuiÃÃo de frequÃncia por procedÃncia, sexo, raÃa, escolaridade, queixas clinicas, tabagismo e perfil molecular. Resultados: Houve predominÃncia na procedÃncia de pacientes oriundos de cidades do interior do Cearà (69%); pacientes do sexo feminino (51,1%), baixa escolaridade (1o grau ou menos -19%), agricultores (22,4%), Pardos (47,4%) e tabagistas.(79%)O subtipo histolÃgico adenocarcinoma foi predominante (32,5%). DispnÃia foi a principal queixa clinica. O atendimento inicial predominante se deu no perÃodo de internamento (75,5%). Conclusão: Neste grupo de pacientes identificou-se um perfil de caracterÃsticas epidemiolÃgicas prÃprias. Criou-se um registro de cÃncer do hospital.Hà proporcionalidade de resultados com a literatura. / Introduction: Cancer is a leading global health problems and one of the most important causes of morbidity and mortality in public health. According to the World Health Organization (WHO) is the second leading cause of death worldwide, second only to the diseases cardiovasculares.Temos national high incidence of patients with advanced lung cancer. There unpredictability of epidemiological profiles and therapeutic responses. Molecular biology has been important in the characterization of tumor differentiation and prognosis of the disease. New treatments are directed by pharmacogenomic characteristics. Little is known about the prevalence of these genes in CearÃand Latin America as well as on clinical characteristics and outcomes related. Objective: To identify the characteristics and distribution of the epidemiological profile of patients with advanced lung cancer, treated at the oncology clinic at the Messejana Hospital - Dr. Carlos Alberto Studart. Material and Methods: An observational, prospective and analytical study with inclusion of 135 patients with advanced lung cancer, between 08/2012 to 12/2014; being analyzed for the distribution of origin by frequency, sex, race, education, clinical complaints, smoking and molecular profile. Results: There was a predominance in the success of patients from small towns of CearÃ(69%); female patients (51.1%), low education (first degree or less -19%), farmers (22.4%), Pardos (47.4%) and smokers. (79%) The histological subtype was adenocarcinoma predominantly (32.5%). Dyspnea was the main complaint clinica.O predominant initial treatment occurred in hospital stay (75.5%). Conclusion: In this group of patients identified a profile of epidemiological characteristics. Created a hospital cancer registry. There proportionality results with the literature.

Abstract Collagens IX, a non-fibrillar collagen, and XI, a fibrillar collagen, are minor components of cartilage collagen fibrils, which form a supportive meshwork in the cartilage extracellular matrix (ECM). Collagens IX and XI are known to be present also in other tissues, including the vitreous body of the eye, the intervertebral disc, the inner ear, and various tissues during embryonic development. Collagen IX is suggested to act as a macromolecular bridge between collagen fibrils and other ECM molecules, and it may be important for the cohesive and compressive properties of cartilage, as well as the long-term stability of articular cartilage. Collagen XI is speculated to have a role in regulating the fibril diameter, and it may participate in interactions with other ECM components. However, the role of neither collagen IX nor XI has been confirmed yet. As important but minor components of the cartilage ECM, collagens IX and XI are excellent candidates for relatively mild chondrodysplasias and even milder disease phenotypes involving cartilaginous tissues, such as non-syndromic hearing loss. There are in fact many reports describing defects in the genes for collagens IX and XI in patients with a variety of chondrodysplasias, including multiple epiphyseal dysplasia, Stickler syndrome, Marshall syndrome and otospondylomegaepiphyseal dysplasia. In order to screen the minor cartilage collagen genes for mutations, it is essential to know their gene structures. Therefore, the complete structures of the human COL9A1, COL9A2 and COL11A2 genes were characterized in this study. Also, to facilitate the analysis of the 5' region of the COL11A2 gene, the cDNA and partial genomic structure of the mouse Col11a2 gene were defined. The information obtained in this study was utilized in the mutation analysis of a family with non-ocular Stickler syndrome. The COL11A2 gene was analyzed with conformation sensitive gel electrophoresis (CSGE) and sequencing, and a heterozygous single-nucleotide mutation causing a premature termination codon was found in the affected family members. Studying the effect of the mutation on the RNA revealed that the nonsense mutation caused the skipping of a 54-bp exon, presumably through a pathway called nonsense-associated altered splicing.

Abstract The nucleotide sequence of the entire COL9A3 gene, coding for the human α3(IX) chain, was determined. The gene was approximately 23 kb in length and consisted of 32 exons. The polymerase chain reaction (PCR)-based procedure of conformation-sensitive gel electrophoresis (CSGE) was used to screen the gene for sequence variations and mutations in 83 unrelated patients with generalized primary osteoarthritis (OA), 31 with rheumatoid arthritis (RA), 171 with intervertebral disc disease (IDD), and 50 with various osteochondrodysplasias. The frequencies of certain sequence variations in healthy individuals were also determined. The COL9A3 gene was analyzed for mutations in two unrelated families with multiple epiphyseal dysplasia (MED). The analysis revealed a splice site mutation leading to skipping of exon 3 and an in-frame loss of 12 amino acid residues in the COL3 domain, the first diseasecausing mutation to be identified in the COL9A3 gene. Sequencing also indicated a 9 bp deletion in one allele in the second MED family that removed a Gly-Pro-Pro triplet. Surprisingly, the deletion did not co-segregate with the MED phenotype in the family. A similar 9 bp deletion, was also found in an unrelated family with no obvious phenotype, suggesting that the two 9 bp deletions represent neutral sequence variants. A construct with the deletion was then made in order to produce a recombinant protein, and the mutant type IX collagen was analyzed under reducing conditions by SDS-PAGE. The results indicated that the recombinant type IX collagen proteins consisted of three α chains, α1(IX), α2(JX), α3(IX), in a 1:1:1 ratio. To study the triple helix stability, pepsin treatment followed by SDS-PAGE was performed on normally folded and denatured recombinant type IX collagen samples. The results demonstrated that the recombinant type IX collagen containing the Gly-X-Y deletion in the a3(IX) chain is secreted as a correctly folded triple-helical molecule. CSGE analysis of exon 5 of the COL9A3 gene identified two nucleotide variations in the same codon, and thus three alleles: CGG (Arg), CAG (Gln), and TGG (Trp). The frequency of the Trp for Arg substitution, the Trp3 allele, was 0.244 among the probands with the IDD, while its overall frequency in the combined group of all non-IDD cases was 0.093. This difference was significant, with a p-value of 0.000013. The Trp3 allele increases the relative risk of IDD by a factor of 2.6 (95 percent confidence interval, 1.6 to 4.3). COL9A3 mutations are shown to be associated with mild cartilage and intervertebral disc diseases.

Currently, the most widely used in vivo mutation monitoring system in humans is the hypoxanthine-guanine phosphoribosyltransferase (hprt) T-cell clonal assay. This dissertation examines the current state of the hprt monitoring system and the usefulness of hprt mutational spectra in revealing environmental exposures. The nature of spontaneous mutational spectra recovered through the implementation of this system is detailed. An examination of hprt mutation frequencies obtained from a set of monozygotic twins revealed a striking influence of genetic factors. As age increases, the influence of genetic factors controlling mutation frequency appears to be modified by environmental factors. Mutational spectra obtained from Russian individuals living in Moscow were distinct from the spectrum of mutation observed in age-matched Western controls. Analysis of the relationship between mutation frequency and subject age clearly demonstrated the lack of any relationship for subjects after the age of 55. This finding contradicts many previously published reports on the relationship between mutation frequency and age. Finally, the influence of tobacco smoking on mutational frequency is clear, however, no change in the mutational spectrum of smokers was revealed. Changes in mutational spectrum are analyzed in the context of the T-cell biology and reveal that the dynamics of this tissue are likely responsible for the observations made in this dissertation. Although the hprt gene is a highly robust and suitable target for the analysis of mutation, the target has not yet been saturated, and new single base-pair substitutions are still being characterized. The data clearly suggest that the T-cell clonal assay in its current state may not be a suitable mutational monitoring system for human populations. This dissertation concludes that new mutational assays need to be developed for monitoring mutations in human populations. / Graduate

No description available.

Note:

The expression of the adenovirus (Ad) genome is co-ordinately regulated and the various early regions of the genome are known to exert feedback controls on each other. The region E1B is known to encode functions which are required for oncogenic transformation by adenovirus yet its potential regulatory role is poorly understood. In this study the regulatory role(s) of the region E1B was investigated at the level of RNA accumulation. The Northern blot technique using various cloned early regions as probes and RNAs from E1B mutants Ad 12, Ad 2 and Ad 5 were used. The levels of region E1A specific RNAs were found to be aberrant. Thus for Ad 12 cyt 68 and Ad 2 dl 250 the levels were higher than wild type ones at late times whereas for Ad 5 dl 313 hr-6 the levels were consistently lower than Ad 5. This implies that the region E1B normally encodes functions which are involved either directly or indirectly in the efficient accumulation of region E1A specific RNA. Other regions also seemed to be affected in these mutants. In cyt 68, region E1B RNA was higher than wild ones at late time, indicating an auto-regulatory mode of control for this region. The expression of region E3, E4 and LS RNA were all perturbed by the E1B mutation as some of the mutants either accumulated higher or lower than wild type levels of RNA. / Thesis / Master of Science (MS)

It is well known that ionizing radiation is genotoxic, and can trigger heritable mutations in the germ cells of an animal. Recently, researchers have used hypervariable expanded simple tandem repeat (ESTR) regions of DNA to explore this phenomenon. ESTRs facilitate the examination of induced genetic mutations using relatively low radiation doses and fewer mice than more traditional approaches. Numerous studies have examined the responses of ESTRs to radiation in the germ line; however the mechanism behind germ line mutations at ESTR loci is poorly understood. Current hypotheses propose that error-prone DNA repair, which allows for misalignment of DNA strands through replication slippage produces in changes in ESTR size. P53 is involved in DNA replication as well as repair of DNA damage, apoptosis and other cancer-related processes. We use p53-deficient heterozygous male mice to examine the role of p53 in germ line mutations at ESTR loci. Males were irradiated with a variety of dose combinations both prior to and post-meiosis, and were mated to unirradiated wildtype females. DNA from the adults and offspring was analyzed for mutations at ESTR loci using DNA fingerprinting. Surprisingly, the study found no significant differences in germ line mutation rate between any treatment groups, including the 0Gy and 1Gy treatments. I discuss the possibility that these results are due to the p53 deficiency of the males, and that p53 homozygosity is necessary for radiation-induced germ line mutations at ESTR loci to occur. I conclude that further studies need to be done, including a control study using wildtype males of the same background strain as that of the p53 deficient line in order to verify our results. / Thesis / Master of Science (MS)

In this thesis, we present a new register-transfer level (RTL) test generation method that makes use of two coverage metrics, Branch Coverage, and Mutation Coverage across two stages, to cover hard-to-reach points previously unreached. We start with a preprocessing stage by converting the RTL source to a C++ equivalent using a modified Verilator, which also automatically creates mutants and the corresponding mutated C++ design, based on arithmetic, logical and relational operators during conversion. With the help of extracted Data Dependency and Control Flow Graphs, in every <golden, mutation> pair, branches containing variables dependent on the mutated statement are instrumented to track them. The first stage uses Evolutionary algorithms with Ant Colony Optimization to generate test vectors with mutation coverage as the metric. Two new filtering techniques are also proposed which optimize the first stage by eliminating the need for generating tests for redundant mutants. The next stage is the original BEACON which now takes the generated mutation test vectors as the initial population unlike random vectors, and output final test vectors. These test vectors succeed in improving the coverage up to 70%, compared to the previous approaches for most of the ITC99 benchmarks. With the application of filtering techniques, we also observed a speedup by 85% in the test generation runtime and also up to 78% reduction in test vector size when compared with those generated by the previous techniques. / MS / In the recent years, Verification has become one of the major bottlenecks in integrated circuit design process, which is exacerbated by the increasing design complexities today. Today designers start the design process by abstracting the initial design in a manner similar to software programming language using a higher abstraction language called Hardware Descriptive Language(HDL). Hence, an HDL based design also contains a number of case statements and if-else statements, also called branches, similar to a software design. Branches indicate decision points in the design and high branch coverage based tests can give us an assurance that the design is properly exercised as compared to those given by randomly generated tests. In this thesis, we introduce a new test generation methodology which generates tests using the help of user introduced mutants to ensure higher branch coverage. Mutation testing is similar to a fault testing method, in which an error or a fault is deliberately introduced into the design and we check if the tests generated are able to detect the fault. An important property of a mutant is that: when a mutant is applied and if the mutated part of the design is exercised by the given test suite, then the following data and control flow path taken can be different from that taken on the original design. This important property along with proper guidance is used in our work to reach some branches which are difficult to cover by random test vectors, and this is the main basis of this thesis. Applying this method, we observed that the branch coverage increased with a decrease in test generation runtime and test vector length when compared to previously proposed techniques.