Amyotrophic lateral sclerosis (ALS) is a devastating motor neuron disease which results from the degeneration of upper and lower motor neurons in the brainstem, spinal cord and motor cortex. Tragically there is no treatment to prevent ALS. The drug Riluzole acts to delay progression, but only by a month or so in this disease that has a survival length of three to five years. The identification of genes that are mutated in patients with ALS would help devise novel therapeutic strategies as much remains to be discovered about the genetics of ALS. Familial forms of the disease account for only 5-10% of patients. Among these familial cases, about 15-20% are caused by mutations in the zinc/copper superoxide dismutase gene, but the genetic basis of the remaining familial cases and the many sporadic cases continues to be largely unknown. / Altogether, the results presented in this thesis came from the use of several strategies to establish the genetic cause of ALS and the related motor neuron disorders like hereditary spastic paraplegia (HSP) and primary lateral sclerosis (PLS). A concerted and collaborative effort was put forth to identify the gene causative for ALS3 on chromosome 18. In addition, a recently reported locus has been confirmed on chromosome 9p for patients that present both ALS and frontotemporal dementia. The major finding involves the discovery of eight mutations in the TARDBP gene in nine patients with sporadic and familial ALS. Furthermore, a large association study evaluated the role of common polymorphisms in the paraoxonase gene cluster in susceptibility to the development of ALS. In the analysis of upper motor neuron diseases, mutations in a novel gene, KIAA0196, were identified for the HSP locus SPG8 on chromosome 8. Finally, the first locus for PLS was discovered on the p-arm of chromosome 4 following genome scan analysis of a large Quebec family with PLS. / These genetic discoveries all contributed novel advances to the field of motor neuron disorders. As more is elucidated regarding the biochemical function of these the proteins encoded by these genes, a more comprehensive picture of ALS and other motor neuron disorders will hopefully emerge.

Hereditary myopathy with lactic acidosis (HML, OMIM#255125) is an autosomal recessive disorder which originates from Västerbotten and Ångermanland in the Northern part of Sweden. HML is characterized by severe exercise intolerance which manifests with tachycardia, dyspnea, muscle pain, cramps, elevated lactate and pyruvate levels, weakness and myoglobinuria. The symptoms arise from malfunction of the energy metabolism in skeletal muscles with defects in several important enzymes involved in the TCA cycle and the electron transport chain. All affected proteins contain iron-sulfur (Fe-S) clusters, which led to the suggestion that the disease was caused by malfunctions in either the transportation, assembly or processing of Fe-S clusters. The aim of my thesis was to identify the disease causing gene of HML and to investigate the underlying disease-mechanisms. In paper I we identified a disease-critical region on chromosome 12; a region containing 16 genes. One of the genes coded for the Fe-S cluster assembly protein ISCU and an intronic base pair substitution (g.7044G>C) was identified in the last intron of this gene. The mutation gave rise to the insertion of intron sequence into the mRNA, leading to a protein containing 15 abberant amino acids and a premature stop. In paper II we investigated why a mutation in an evolutionary well conserved protein with a very important cellular role, which in addition is expressed in almost all tissues, gives rise to a muscle-restricted phenotype. Semi-quantitative RT-PCR analysis showed that the mutant transcript constituted almost 80% of total ISCU mRNA in muscle, while in both heart and liver the normal splice form was dominant. We could also show that, in mice, complete absence of Iscu protein was coupled with early embryonic death, further emphasizing the importance of the protein in all tissues. These data strongly suggested that tissue-specific splicing was the main mechanism responsible for the muscle-specific phenotype of HML. In paper III the splicing mechanisms that give rise to the mutant ISCU transcript was further investigated. We identified three proteins; PTBP1, IGF2BP1 and RBM39, that could bind to the region containing the mutation and could affect the splicing pattern of ISCU in an in vitro system. PTBP1 repressed the inclusion of the intronic sequence, while IGF2BP1 and RBM39 repressed the total ISCU mRNA level though the effect was more pronounced for the normal transcript. Moreover, IGF2BP1 and RBM39 were also able to reverse the effect of PTBP1. IGF2BP1, though not a splicing factor, had higher affinity for the mutant sequence. This suggested that the mutation enables IGF2BP1 binding, thereby preventing the PTBP1 induced repression seen in the normal case. In conclusion, we have determined the genetic cause of HML, identifying a base pair substitution in the last intron of the ISCU gene that gives rise to abnormally spliced transcript. The muscle-specific phenotype was also analyzed and tissue-specific splicing was identified as the main disease-mechanism. Furthermore, nuclear factors with ability to affect the splicing pattern of the mutant ISCU gene were identified. This work has thoroughly investigated the fundamental disease mechanisms, thus providing deeper understanding for this hereditary myopathy.

Doctor of Philosophy / Abstract Neurodegenerative disorders are a diverse group of disorders that affect specific subsets of neurons. Motor neuron diseases, neurodegenerative disorders of motor neurons, are seen commonly as sporadic cases and less frequently as familial disease forms. The familial forms show genetic and phenotypic heterogeneity. Clinically motor neuron diseases may be seen as rapidly progressive disorders like amyotrophic lateral sclerosis, ALS or slowly progressive disorders like hereditary motor neuropathies, HMN. The only proven causes for motor neuron diseases are gene mutations that lead to motor neuron degeneration in familial disease forms. Only some of these genes have been identified and have contributed greatly to our understanding of the neurobiology of familial and sporadic disease forms. Identification of additional disease causing genes would help enhance our knowledge of the pathophysiological mechanisms underlying all forms of motor neuron disorders, which would lead to early diagnoses, effective prophylaxis and efficient therapies for these disorders. This study aimed to find gene mutations that cause rapid and slowly progressive familial motor neuron disorders in Australian families and to determine their relevance to sporadic forms of motor neuron disease. The familial forms of ALS show reduced disease penetrance, that is, not all gene mutation carriers manifest the disease. This study examines ALS penetrance in a group of Australian families. The most frequently observed mutations in ALS families are cytosolic superoxide dismutase/SOD1 gene mutations. In a collection of ALS families in our centre, families without the common SOD1 gene mutations were genotyped for other ALS genes and loci and studied using genetic linkage and haplotype analyses. Studies in a large Australian ALS family further confirmed genetic heterogeneity in non-SOD familial ALS, all known autosomal dominant ALS genes and chromosomal loci were excluded as cause of disease in this family. Such families can be studied further to identify additional disease genes and loci mapped in other ALS families. These families represent powerful resources for identification of additional ALS genes. Identifying the pathogenic genes in families with reduced disease penetrance may be more relevant to sporadic forms of disease. dHMN is a chronic neurodegenerative disorder predominantly affecting motor neurons. In a large Australian dHMN family, all the known dHMN genes and chromosomal loci were excluded as cause of disease. A genome wide microsatellite screen was performed in this family and genetic linkage was established to a novel 12.98 Mb locus on chromosome 7q34.2-q36. Candidate genes in this large interval will be screened based on their function and expression profile. Identification of a new dHMN locus provides the basis for future identification of a novel gene involved in motor neuron degeneration. Genes in dHMN have been shown to be pathogenic in ALS and Charcot Marie Tooth syndromes. The new locus for dHMN mapped in this project would lead to identification of a novel dHMN gene, which may elucidate the pathogenesis underlying a wide range of neurodegenerative disorders.

Hereditary non-polyposis colon cancer syndrome (HNPCC) is associated with an increased risk of developing several types of cancer and is the most common cause of hereditary ovarian cancer after BRCA1 and BRCA2 mutations. HNPCC results from a germline mutation in one of the DNA mismatch repair (MMR) genes: MLH1, MSH2, PMS1, PMS2, MSH6, MSH3 and MLH3. While there has been extensive investigation of MMR deficiency in colorectal cancer, MMR in ovarian cancer is relatively under-investigated. The goal of this project was to study MMR deficiency in ovarian cancer at both the clinical and molecular level. The first aim was to examine the frequency of MMR loss in a large patient cohort and investigate the clinical consequences of MMR deficiency. The second aim was to describe the molecular characteristics of MMR deficiency in ovarian cancer cell lines and establish an in vitro cell line model of MMR deficiency in ovarian cancer. The third aim was to identify synthetic lethal strategies for the treatment of ovarian cancer to maximise cytotoxicity in a MMR-deficient background. In order to characterise the clinical consequences of MMR deficiency, a large patient cohort was studied with regard to MMR status. Three tissue microarrays consisting of 581 ovarian tumours were constructed, and expression of the four most frequently lost MMR proteins: MLH1, MSH2, PMS2 and MSH6 were detected by immunohistochemistry. Afterwards, MMR status and histology subtypes were analysed in combination with the associated clinical data. The overall incidence of MMR deficiency (loss of any MMR protein) was 15.7%, with PMS2 being the most frequently lost protein (9.7%). In addition, MMR deficiency tended to appear in a grouped fashion: MLH1 with PMS2; MSH2 with MSH6. Patients with non-serous subtypes of ovarian cancer, clear cell or mucinous especially, had higher incidence of MMR deficiency compared to patients with serous ovarian cancer. Overall MMR deficient patients were more likely to be diagnosed at early stages compared with MMR proficient patients, and this is probably due to the association between MMR deficiency and non-serous histology. However, platinum-based treatment for patients with MMR deficiency gives no advantage over those without MMR deficiency. Therefore better treatments for this subgroup of patients may be needed. The features of MMR deficiency in ovarian cancer were also characterized at the molecular level. After quantifying mRNA and protein expression of MMR genes in 19 ovarian cell lines, three cell lines (SKOV3, TOV21G and IGROV1) were found to have a defect in MLH1 expression at both the mRNA and protein level. Interestingly, the three cell lines also carried a defect in PMS2 expression at the protein level but not at the mRNA level, which is consistent with our clinical data demonstrating that MLH1 protein and PMS2 protein are paired in loss. In addition, across the 19 cell lines, MLH1 and PMS2 showed positive correlation at both the mRNA level (R=0.53, p=0.02) and protein level (R=0.72, p=0.0006). In order to study co-expression of MLH1 and PMS2, a plasmid encoding the cDNA for MLH1 was transfected into the three MLH1 deficient cell lines; and conversely siRNA targeting MLH1 was transfected into the MMR proficient cell line A2780 and expression of MLH1 protein and PMS2 protein was quantified. The results showed that re-introduction of MLH1 into MLH1 deficient cells resulted in increased expression of PMS2 protein, while knocking down MLH1 in MMR proficient cells leads to decreased PMS2 protein expression. This indicates that MLH1 may play a crucial role in regulating PMS2 protein expression. As the three MLH1 and PMS2 protein deficient cell lines all express PMS2 mRNA, the regulation of PMS2 expression by MLH1 is likely to be at the translational or post-translational level. However, the expression of PMS2 protein was not increased in the absence of MLH1, even when the proteasomal and lysosomal protein degradation pathways were blocked (as seen with SKOV3 cells), suggesting decreased PMS2 protein expression is not due to rapid degradation in the absence of MLH1. Therefore MLH1 may play a role in regulating the synthesis of PMS2 protein at the translational level, rather than preventing the degradation of PMS2. Thus, to investigate the mechanism by which PMS2 protein levels are regulated by MLH1, future work should focus on translational regulation of PMS2. In order to identify synthetic lethal strategies to target MMR deficiency in ovarian cancer, an isogenic cell line model of MMR deficiency was established by stable transfection of a plasmid for MLH1 and its corresponding empty vector into SKOV3 cells. The MLH1+ cell line SAC-1 and MLH1- cell line SN-5 were selected for drug screening based on their phenotype and growth rate. The AlamarBlue assay, with z’ above 0.5, was chosen for drug screening and a kinase inhibitor library containing 362 drugs of known target was screened. Two drugs with similar structures that targeted PLK1 showed greater growth inhibition of SN-5 compared with SAC-1. When the two cell lines were treated with another PLK1 inhibitor, BI2536, with different structure, a 2-fold difference in growth inhibition between SAC-1 and SN-5 was also observed, suggesting PLK1 is a potential synthetic lethal target for MLH1 deficiency in ovarian cancer. Together these data demonstrate that clinically, MMR deficiency is associated with non-serous subtypes of ovarian cancer and specific MMR proteins are paired in loss. While current standard therapy offers no selective benefit to ovarian cancer patients with MMR deficiency, inhibiting PLK1 activity may confer selective benefit.

Spontaneous mutations in the human LIS1 gene are responsible for Type I lissencephaly ("smooth brain"). The distribution of neurons within the cerebral cortex of lissencephalic children appears randomized, probably owing to a defect in neuronal migration during early development. LIS1 has been implicated in the dynein pathway by genetic analyses in fungi. We previously reported that the vertebrate LIS1 co-localized with dynein at prometaphase kinetochores, and interference with LIS1 function at kinetochore caused misalignment of chromosomes onto the metaphase plate. This leads to a hypothesis that LIS1 might regulate kinetochore protein targeting. In order to test this hypothesis, I created dominant inhibitory constructs of LIS1. After removal of the endogenous LIS1 from the kinetochore by overexpression of the N-terminal self-association domain of LIS1, dynein and dynactin remained at the kinetochores. This result indicated that LIS1 is not required for dynein to localize at the kinetochore. Next, CLIP-170 was displaced from the kinetochores in the LIS1 full-length and the C-terminal WD-repeat overexpressers, suggesting a role for LIS1 in targeting CLIP-170 onto kinetochores. LIS1 was co-immunoprecipitated with dynein and dynactin. Its association with kinetochores was mediated by dynein and dynactin, suggesting LIS1 might interact directly with subunits of dynein and/or dynactin complexes. I found that LIS1 interacted with the heavy and intermediate chains (HC and IC) of dynein complex, and the dynamitin subunit of dynactin complex. In addition to kinetochore targeting, the LIS1 C-terminal WD-repeat domain was responsible for interactions with dynein and dynactin. Interestingly, LIS 1 interacted with two distinct sites on HC: one in the stem region containing the subunit-binding domain, and the other in the first AAA motif of the motor domain, which is indispensable for the ATPase function of the motor protein. This LIS1-dynein motor domain interaction suggests a role for LIS1 in regulating dynein motor activity. To test this hypothesis, changes of dynein ATPase activity was measured in the presence of LIS1 protein. The ATPase activity of dynein was stimulated by the addition of a recombinant LIS1 protein. Besides kinetochores, others and we have found LIS1 also localized at microtubule plus ends. LIS1 may mediate dynein and dynactin mitotic functions at these ends by interacting with astral microtubules at cortex, and associating with the spindle microtubules at kinetochores. Overexpression of LIS1 displaced dynein and dynactin from the microtubule plus ends, and mitotic progression was severely perturbed in LIS1 overexpressers. These results suggested that the role for LIS1 at microtubule plus ends is to regulate dynein and dynactin interactions with various subcellular structures. Results from my thesis research clearly favored the conclusion that LIS1 activates dynein ATPase activity through its interaction with the motor domain, and this activation is important to establish an interaction between dynein and microtubule plus ends during mitosis. I believe that my thesis work not only has provided ample implications regarding dynein dysfunction in disease formation, but also has laid a significant groundwork for more future studies in regulations of the increasing array of dynein functions.

Introdução: As causas estabelecidas para o desenvolvimento do câncer são multifatoriais e incluem fatores ambientais, endógenos e/ou herança genética. Estimase que 5-10% dos casos de câncer sejam hereditários. Objetivos: Descrever o perfil dos pacientes e atendimentos do ambulatório de Oncogenética do Hospital de Clínicas de Porto Alegre (HCPA) no período de 2006 a 2013; desenvolver e implantar um Sistema de Informação e Assessoramento em Oncogenética para Profissionais de Saúde através de consultorias por e-mail e telefone. Métodos: Utilizaram-se duas estratégias quanto à fonte de informações, sendo uma das etapas uma coorte histórica para revisão de dados em prontuário eletrônico e sistemas de informações gerenciais do HCPA referente a atendimentos realizados no período definido; e uma etapa transversal, através de um questionário, para avaliação do entendimento acerca do encaminhamento para o ambulatório de oncogenética. A implantação do Sistema de Informação em Oncogenética engloba a disponibilização de material informativo (folders e cartazes), linha telefônica e e-mail direcionados aos profissionais de saúde. As consultorias são discutidas em uma reunião da equipe multidisciplinar e posteriormente, é emitido um relatório para o profissional solicitante (retorno no prazo máximo de 7 dias úteis). Resultados: Considerando amostra de 1326 pacientes, observa-se crescimento importante no número de atendimentos ao longo dos anos, da mesma maneira também foram registradas importantes taxas de absenteísmo, porém no decorrer dos anos estas caíram progressivamente. A maior parcela dos pacientes pertence ao sexo feminino (64,7%), com idade maior ou igual a 18 anos (79,9%), solteiros (48,7%), tendo em média 1,2 filhos, com ensino fundamental incompleto (32,7%) e procedentes de regiões metropolitanas (34,2%) e de Porto Alegre (33,9%). Observou-se um número considerável de pacientes com diagnóstico de neoplasia (56,4%) e com diagnóstico de síndrome de predisposição hereditária ao câncer (33,9%). Quanto à origem dos encaminhamentos, a maioria teve sua origem dentro da instituição (38,7%) e por meio de um familiar de paciente já atendido no ambulatório (35,6%). As especialidades que mais encaminharam pacientes foram a oncologia, oncopediatria, coloproctologia e dermatologia respectivamente. Quanto ao entendimento do paciente acerca do seu encaminhamento ao ambulatório (subamostra composta por 44 indivíduos), verificouse que a maioria, no primeiro atendimento, soube dizer o motivo da consulta. Em relação ao Sistema de Informação em Oncogenética foram realizadas 7 consultorias, procedentes do interior do Rio Grande do Sul (4 casos) e de Porto Alegre (3 casos). Todas foram pertinentes e com indicação de encaminhamento para uma avaliação com uma equipe especializada em Oncogenética. Adicionalmente foram recebidas 11 ligações de pacientes interessados em avaliação oncogenética no HCPA e 9 ligações de profissionais ou secretaria de saúde para encaminhamento de pacientes para o ambulatório de genética e câncer. Conclusão: O conhecimento sobre a dinâmica dos atendimentos e perfil dos pacientes é fundamental para o contínuo aprimoramento, crescimento do ambulatório de oncogenética, bem como auxilia a equipe na elaboração de material educativo para os pacientes. O desenvolvimento do Sistema de Informação permite profissionais de saúde reconhecer e discutir casos suspeitos de câncer hereditário onde esses serviços não estão disponíveis. / Introduction: Cancer is a multifactorial disease and risk factors include environmental factors exposures and lifestyle, endogenous risk factors and/or genetic susceptibility or predisposition. An estimated 5-10% of all cancers are hereditary. Objectives: Describe the profile of patients and consultations at the outpatient cancer genetics clinic of Hospital de Clínicas de Porto Alegre (HCPA) from 2006 to 2013; develop and implement a telephone and email based Information and Advisory System of Oncogenetics for health care professionals. Methods: This is a quantitative crosssectional, descriptive study. Two strategies were used as source of information to describe patient and consultation profiles. The first, retrospective, included review of electronic records and management information systems of HCPA and aimed to characterize the consultations. The second strategy, prospective, included patient interviews and questionnaires to assess motivations and understanding about the referral to the outpatient oncogenetic clinic. The implementation of the Information system in Oncogenetics encompassed provision of informative material (brochures and posters), and informative calls and emails returning requests of health care professionals. All consultations were discussed with a multidisciplinary team and a report with recommendations and information regarding the specific question was issued to the requesting professional in up to 7 days. Results: The total number of patients evaluated in the period was 1326. A constant growth in the number of annual consultations was observed over the years. There were important rates of absenteeism, although over the years these numbers have fallen progressively. Most of the patients were female (64.7%), at or above age 18 years (79.9%), single (48.7%) and the average number of offspring was 1.2 children. About one third had incomplete elementary education (32.7%) and most were from the metropolitan region (34.2%) and from Porto Alegre itself (33.9%). There was a considerable number of patients with diagnosed with at least one tumor (56.4%) and in 33.9% of the patients, diagnosis of an hereditary cancer syndrome was confirmed. Regarding source of referrals, the majority had its origin within the institution (38.7%) and through family members that previously consulted at the clinic (35.6%). Medical specialties responsible for most of the referrals were Oncology, Pediatric Oncology, Colorectal Surgery and Dermatology. As for the patient's understanding of their referral (sub-sample composed of 44 individuals), most of them demonstrated clear understanding of the reasons for referral already in the first consultation. Regarding the Information system in Oncogenetics, seven consultations were conducted and originated from the countryside of Rio Grande do Sul (4 cases) and from Porto Alegre (3 cases). All were relevant and resulted in referrals to a specialized team in Oncogenetics. Additionally, 11 calls were received from patients interested in oncogenetics evaluations at HCPA and 9 calls were received from health care professionals requesting referrals of patients to a cancer genetics outpatient clinic. Conclusion: Knowledge about patient profile and dynamics of consultations of cancer genetics outpatient clinics is fundamental to qualify the service provision and stimulate its continuous improvement and growth; it is also helpful to reassess the needs of the patients and act on it, for instance, guiding the elaboration of educational materials for patients and health care professionals. The development an information system for health care professionals enables recognition and discussion of suspected cases in remote areas where such services are not available.

A presente tese tem por objetivo analisar as decisões dos agricultores familiares de Frederico Westphalen/RS com relação à sucessão geracional nas suas propriedades, sob a ótica da teoria da decisão e orientações de valor, elencando os diferentes fatores que influenciam sucessores e sucedidos nesse processo. Para alcançar este propósito, além de análises bibliográficas e documentais acerca do tema e suas nuances, teorias balizadoras e elaboração de uma construção histórica dos sistemas agrários do campo empírico, considerando o papel de pessoas e instituições nessa construção, foi aplicado um questionário a 50 potenciais sucessores e a 50 sucedidos, buscando compreender os direcionamentos decisórios destes atores sobre sucessão. Os dados coletados a campo, entre os meses de julho e setembro de 2015, foram tabulados com o suporte estatístico do programa Statistical Package for the Social Science (SPSS), gerando cruzamentos e informações relevantes acerca do tema e suas relações com o futuro das propriedades rurais familiares. O estudo mostra que a abertura de possibilidades e alternativas de aprimoramento acadêmico dos potenciais sucessores, mesmo em áreas agrícolas, tem oportunizado uma gama de escolhas profissionais que contribui para o constante desinteresse pela sucessão. A postura dos pais nessas situações também é condicionante para que o filho desperte ou não o interesse pela propriedade e tenha uma preparação adequada para assumi-la, sendo o incentivo dos sucedidos visto como um condicionador importante nas decisões dos potenciais sucessores O estudo aponta também para uma necessidade de pensar a sucessão geracional como um processo e não como uma decisão pontual. A participação dos filhos nas decisões cotidianas da propriedade, bem como na divisão dos resultados financeiros e das responsabilidades, mostram-se fatores determinantes para que esse processo aconteça. Além de delinear e analisar fatores influenciadores das decisões dos atores, o estudo classifica potenciais sucessores e sucedidos dentro das orientações de valor de Ruth Gasson (1973) – instrumental, social, expressiva e intrínseca. Comparando as orientações de pais e filhos, tem-se uma importante diferença de percepção. Assim, tem-se nas variáveis que influenciam o processo decisório dos atores e suas percepções e orientações de valor, atreladas à importância da agricultura familiar, como base de sustentação econômica e social local, a justificativa para que se estruturem alternativas viáveis em prol da manutenção do jovem no campo. / The aim of this thesis is to analyze the decisions of family farmers of Frederico Westphalen / RS regarding the generational succession in their properties, from the point of view of decision theory and value orientations, listing the different factors that influence successors and successors in this process. In order to achieve this purpose, besides bibliographical and documentary analyzes about the theme and its nuances, beacon theories and elaboration of a historical construction of the agrarian systems of the empirical field, considering the role of people and institutions in this construction, a questionnaire was applied to 50 potential successors and 50 family farmers, seeking to understand the decision making process direction of these actors over succession. The data collected in the field between July and September 2015 were tabulated with statistical support from the Statistical Package for the Social Science (SPSS), generating crosses and relevant information about the theme and its relations with the future of the properties rural areas. The study shows that the opening of possibilities and alternatives for academic improvement of potential successors, even in agricultural areas, has provided a range of professional choices that contributes to the constant lack of interest in succession. The parents attitude in these situations is also a condition for the child to arouse or not the interest for the property and to have adequate preparation to assume it, being the incentive of the successes seen as an important conditioner in the decisions of the potential successors The study also points to a need to think of generational succession as a process and not as a one off decision. The participation of the children in the daily decisions of the property, as well as in the division of the financial results and the responsibilities, are determining factors for this process to happen. In addition to outlining and analyzing factors influencing actors' decisions, the study ranks potential successors and succeeded within Ruth Gasson (1973) value orientations - instrumental, social, expressive, and intrinsic. Comparing the orientations of parents and children, one has an important difference of perception. Thus, the variables influencing the decision-making process of the actors and their perceptions and value orientations, linked to the importance of family agriculture, as a basis for local economic and social support, are the justification for structuring viable alternatives for maintenance the young man in the field.

Made available in DSpace on 2014-12-17T14:16:20Z (GMT). No. of bitstreams: 1 GiocondaDRL.pdf: 1031402 bytes, checksum: 0016e69e527bddffea93909fa2748a03 (MD5) Previous issue date: 2007-08-29 / Hereditary Hemochromatosis (HH) is a genetic disease caused by high iron absorption and deposition in several organs. This accumulation results in clinical disturbances such as cirrhosis, arthritis, cardiopathies, diabetes, sexual disorders and skin darkening. The H63D and C282Y mutations are well defined in the hemochromatosis etiology. The aim of this paper was that of identifying the H63D and C282Y genetical mutations in the hemochromatosis gene and the frequency assessment of these mutations in the HFE protein gene in patients with hyperferritin which are sent to the DNA Center laboratory in Natal, state of Rio Grande do Norte. This paper also evaluates the HH H63D and C282Y gene mutations genotype correlation with the serum ferritin concentration, glucose, alanine aminotransferasis, aspartato aminotransferasis, gama glutamil transferasis and with the clinical complications and also the interrelation with life habits including alcoholism and iron overload. The biochemical dosages and molecule analyses are done respectively by the enzymatic method and PCR with enzymatic restriction. Out of the 183 patients investigated, 51,4% showed no mutation and 48,6% showed some type of mutation: 5,0% were C282Y heterozygous mutation; 1,1%, C282Y homozygous mutation; 31%, H63D heterozygous mutation; 8,7%, H63D homozygous mutation; and 3,3%, heterozygous for the mutation in both genes. As to gender, we observed a greater percentage of cases with molecular alteration in men in relation to women in the two evaluated mutations. The individuals with negative results showed clinical and lab signs which indicate hemochromatosis that other genes could be involved in the iron metabolism. Due to the high prevalence of hemochromatosis and taking into account that hemochromatosis is considered a public health matter, its gravity being preventable and the loss treatment toxicity, the early genetic diagnosis is indicated, especially in patients with high ferritin, and this way it avoids serious clinical manifestations and increases patients' life expectation. Our findings show the importance of doing such genetic studies in individuals suspected of hereditary hemochromatosis due to the high incidence of such a hereditary disease in our region / A hemocromatose heredit?ria (HH) ? uma doen?a gen?tica causada pela absor??o e deposi??o elevada de ferro em v?rios ?rg?os. Este ac?mulo resulta em complica??es cl?nicas como cirrose, artrite, cardiopatias, diabetes, desordens sexuais e escurecimento da pele. As muta??es H63D e C282Y est?o bem definidas na etiologia da hemocromatose. O objetivo deste trabalho foi a identifica??o das muta??es gen?ticas H63D e C282Y no gene da Hemocromatose e avalia??o da freq??ncia dessas muta??es no gene da prote?na HFE em pacientes com hiperferritinemia que s?o encaminhados ao laborat?rio DNA Center Natal / RN. Al?m disso, avaliar a correla??o dos gen?tipos das muta??es H63D e C282Y do gene da HH com a concentra??o s?rica da ferritina, glicose, alanina aminotransferase, aspartato minotransferase, gt e com as complica??es cl?nicas e ainda a interrela??o com os h?bitos de vida incluindo o etilismo e dieta com sobrecarga de ferro. As dosagens bioqu?micas e an?lises moleculares foram realizadas respectivamente atrav?s do m?todo enzim?tico e PCR com restri??o enzim?tica. Dos 183 pacientes investigados 51,4% apresentaram aus?ncia de muta??o e 48,6% com algum tipo de muta??o: 5,0% C282Y heterozigoto mutado; 1,1% C282Y homozigoto mutado; 31% H63D heterozigoto mutado; 8,7% H63D homozigoto mutado; e 3,3% heterozigoto para a muta??o em ambos os genes. Com rela??o ao sexo, observou-se o maior percentual de casos com altera??o molecular em homens em rela??o a mulheres nas duas muta??es avaliadas. Os indiv?duos com resultados negativos apresentaram sinais cl?nicos e laboratoriais indicativos de hemocromatose sugerindo que outros genes poder?o estar envolvidos no metabolismo do ferro. Devido ? alta preval?ncia da hemocromatose, e tendo em vista que a hemocromatose ? considerada um problema de sa?de p?blica, sua gravidade ser preven?vel e a baixa toxicidade do tratamento, o diagn?stico gen?tico precoce torna-se indicado, principalmente nos pacientes com ferritina elevada, e com isso evitar manifesta??es cl?nicas graves e aumentar a expectativa de vida dos pacientes com esta doen?a. Nossos achados mostram a import?ncia da realiza??o de estudos gen?ticos em indiv?duos com suspeita de hemocromatose heredit?ria em virtude de elevada incid?ncia dessa doen?a de cunho heredit?rio em nossa regi?o

A presente tese tem por objetivo analisar as decisões dos agricultores familiares de Frederico Westphalen/RS com relação à sucessão geracional nas suas propriedades, sob a ótica da teoria da decisão e orientações de valor, elencando os diferentes fatores que influenciam sucessores e sucedidos nesse processo. Para alcançar este propósito, além de análises bibliográficas e documentais acerca do tema e suas nuances, teorias balizadoras e elaboração de uma construção histórica dos sistemas agrários do campo empírico, considerando o papel de pessoas e instituições nessa construção, foi aplicado um questionário a 50 potenciais sucessores e a 50 sucedidos, buscando compreender os direcionamentos decisórios destes atores sobre sucessão. Os dados coletados a campo, entre os meses de julho e setembro de 2015, foram tabulados com o suporte estatístico do programa Statistical Package for the Social Science (SPSS), gerando cruzamentos e informações relevantes acerca do tema e suas relações com o futuro das propriedades rurais familiares. O estudo mostra que a abertura de possibilidades e alternativas de aprimoramento acadêmico dos potenciais sucessores, mesmo em áreas agrícolas, tem oportunizado uma gama de escolhas profissionais que contribui para o constante desinteresse pela sucessão. A postura dos pais nessas situações também é condicionante para que o filho desperte ou não o interesse pela propriedade e tenha uma preparação adequada para assumi-la, sendo o incentivo dos sucedidos visto como um condicionador importante nas decisões dos potenciais sucessores O estudo aponta também para uma necessidade de pensar a sucessão geracional como um processo e não como uma decisão pontual. A participação dos filhos nas decisões cotidianas da propriedade, bem como na divisão dos resultados financeiros e das responsabilidades, mostram-se fatores determinantes para que esse processo aconteça. Além de delinear e analisar fatores influenciadores das decisões dos atores, o estudo classifica potenciais sucessores e sucedidos dentro das orientações de valor de Ruth Gasson (1973) – instrumental, social, expressiva e intrínseca. Comparando as orientações de pais e filhos, tem-se uma importante diferença de percepção. Assim, tem-se nas variáveis que influenciam o processo decisório dos atores e suas percepções e orientações de valor, atreladas à importância da agricultura familiar, como base de sustentação econômica e social local, a justificativa para que se estruturem alternativas viáveis em prol da manutenção do jovem no campo. / The aim of this thesis is to analyze the decisions of family farmers of Frederico Westphalen / RS regarding the generational succession in their properties, from the point of view of decision theory and value orientations, listing the different factors that influence successors and successors in this process. In order to achieve this purpose, besides bibliographical and documentary analyzes about the theme and its nuances, beacon theories and elaboration of a historical construction of the agrarian systems of the empirical field, considering the role of people and institutions in this construction, a questionnaire was applied to 50 potential successors and 50 family farmers, seeking to understand the decision making process direction of these actors over succession. The data collected in the field between July and September 2015 were tabulated with statistical support from the Statistical Package for the Social Science (SPSS), generating crosses and relevant information about the theme and its relations with the future of the properties rural areas. The study shows that the opening of possibilities and alternatives for academic improvement of potential successors, even in agricultural areas, has provided a range of professional choices that contributes to the constant lack of interest in succession. The parents attitude in these situations is also a condition for the child to arouse or not the interest for the property and to have adequate preparation to assume it, being the incentive of the successes seen as an important conditioner in the decisions of the potential successors The study also points to a need to think of generational succession as a process and not as a one off decision. The participation of the children in the daily decisions of the property, as well as in the division of the financial results and the responsibilities, are determining factors for this process to happen. In addition to outlining and analyzing factors influencing actors' decisions, the study ranks potential successors and succeeded within Ruth Gasson (1973) value orientations - instrumental, social, expressive, and intrinsic. Comparing the orientations of parents and children, one has an important difference of perception. Thus, the variables influencing the decision-making process of the actors and their perceptions and value orientations, linked to the importance of family agriculture, as a basis for local economic and social support, are the justification for structuring viable alternatives for maintenance the young man in the field.

Introduction: Spontaneous abortion (SA) is the most common complication in pregnancy. The aim of the study was to investigate the causality of selected genetic factors - Apolipoprotein E (Apo E) gene polymorphisms, factor V Leiden (FVL), Prothrombin (PT G20210A) and nongenetics factors - Thyroid stimulating hormone (TSH), free thyroxine (fT4), antibodies against thyroid peroxidase (a-TPO) in the role of early SA. Materials and methods: For genotyping of APO E polymorphism was used PCR-RFLP. The detection of mutations in genes FV and FII was performed using by HRM. Laboratory markers of thyroid (TSH, a-TPO and fT4) were determined by an automated analyzer using chemiluminescent immunoassay. Results: APOE genotypes of investigated group of 410 samples abortioned embryonic/ fetal tissues were not significantly different from 2 606 adult controls (P = 0.653). In observed infertile group of 75 women with isolated SA was FVL detected in heterozygous constitution with a prevalence of 12 %. The prevalence of FVL in a group of women with early insulated SA was significantly higher than 76 controls (12 % vs. 2.6 %, P = 0.031). The difference of PTG20210A prevalence between women with isolated SA and controls was not significant (4 % vs. 5.3 %, P = 1). The prevalence of elevated TSH levels (higher than 2.5...