Submitted by admin tede (tede@pucgoias.edu.br) on 2016-10-07T13:26:52Z No. of bitstreams: 1 SIMONE SOUZA FAGUNDES.pdf: 1864838 bytes, checksum: 28db816101558781ede4b2fb42be7695 (MD5) / Made available in DSpace on 2016-10-07T13:26:52Z (GMT). No. of bitstreams: 1 SIMONE SOUZA FAGUNDES.pdf: 1864838 bytes, checksum: 28db816101558781ede4b2fb42be7695 (MD5) Previous issue date: 2016-06-27 / Breast cancer is the most frequent in the world and Brazilian women, except for cases of skin cancer nonmelanoma. It is a complex disease that has no single cause, results from the interaction of multiple risk factors (environmental, hormonal, lifestyle) with an individual genome (Pruthi et al., 2007). Polymorphism of codon 72 of TP53 gene is well studied because of the impact on the coding sequence of the gene is also associated with increased risk for the development of some cancers. The TP53 polymorphism in exon 4 of the 72 codon results in the substitution of arginine (Arg) or proline (Pro), which may encode an arginine (p53Arg) or a proline (p53Pro). This is a job that made the update meta-analysis on the association between the R72P polymorphism of the TP53 gene in patients with breast cancer considering a broad sample obtained from studies with conflicting data, making a statistical method that combines results of relevant studies to answer a question, search available evidence and points out the areas where there is need for more research. After search of the articles surveyed in multiple databases were selected for performing the metaanalysis a total of 47 articles totaling 27,068 cases and 28,065 controls. The organization of the articles was according to the year of publication, covering studies from 2002 to 2015. The results found worldwide about codon 72 polymorphism in p53 are quite conflicting, suggesting that it is due to ethnic and geographic factors that impact each continent evident. Although several studies show the wide variety can occur between genotypes and codon 72 alleles, identifying significant results for Arg / Arg genotype and allele arginine suggesting that this meta-analysis corroborates for therapies, diagnostics and more studies in this area. / O câncer de mama é o mais incidente na população feminina mundial e brasileira, excetuando-se os casos de câncer de pele não melanoma. É uma doença complexa que não tem causa única, resulta da interação de múltiplos fatores de risco (ambientais, hormonais, estilo de vida) com um genoma individual (Pruthi et al., 2007). O polimorfismo do códon 72 do gene TP53 é bastante estudado devido ao impacto na sequência codificadora do gene, além de estar associado ao maior risco para o desenvolvimento de alguns tipos de câncer. O polimorfismo do gene TP53 no éxon 4 do códon 72, resulta na substituição de Arginina (Arg) ou Prolina (Pro), que pode codificar um aminoácido arginina (p53Arg) ou uma prolina (p53Pro). Este é um trabalho que fez a atualização de metanálise sobre associação entre o polimorfismo R72P do gene TP53 em pacientes com câncer de mama considerando uma ampla amostragem obtidos de estudos com dados conflitantes, fazendo uma abordagem estatística que combina resultados de estudos relevantes para responder a uma questão, busca evidências disponíveis e aponta as áreas onde há necessidade de mais pesquisas. Após busca dos artigos pesquisados em várias bases de dados foram selecionados para a realização da metanálise um total de 47 artigos somando 27.068 casos e 28.065 controles. A organização dos artigos foi de acordo com o ano de publicação, contemplando estudos de 2002 a 2015. Os resultados encontrados mundialmente a respeito do polimorfismo do códon 72 na p53 são bastantes conflitantes, sugerindo que seja devido a fatores étnicos e geográficos que impactam em cada continente de forma evidente. Apesar de vários estudos mostrarem a grande variedade pode ocorrer entre os genótipos e alelos do códon 72, identificando resultados significativos para o genótipo Arg/Arg e o alelo arginina sugerindo que esta metanálise corrobora para terapias, diagnósticos e mais estudos nesta área.

Testes de paternidade geralmente são feitos analisando amostras de DNA do suposto pai, mãe e criança. Para realizar esse exame antes de a criança nascer era preciso recorrer à métodos invasivos, tais como amniocentese e biópsia de vilo corial. Com a descoberta de DNA fetal livre (fcfDNA) no soro e plasma materno, hoje é possível utilizar técnicas que usem esse fcfDNA diminuindo assim os riscos à saúde do feto e da mãe. Testes de pa- ternidade que analisam Short Tandem Repeats (STRs) do fcfDNA, embora possíveis, não são confiáveis, pois muitas vezes há degradação do DNA. Por sua vez, Single Nucleotide Polymorphisms (SNPs) têm sido demonstrados como bons candidatos para identificação humana e podem ser obtidos de fragmentos pequenos de DNA (ou seja, mesmo com o DNA degradado). No entanto, SNPs possuem um número limitado de alelos diferentes (entre dois e quatro). Micro-haplótipos são segmentos cromossomais menores do que 200 pb (pares de bases), contendo dois ou mais SNPs que formam pelo menos três haplótipos distintos. Ao utilizá-los como marcadores genéticos, aumentamos o número de possíveis alelos formados a partir dos SNPs. Como o fcfDNA possui um tamanho de aproximada- mente 145 pb, isso é suficiente para conter micro-haplótipos que podem ser sequenciados usando tecnologia de Sequenciamento de Nova Geração (NGS). O objetivo desse projeto é determinar a probabilidade de paternidade usando SNPs dentro de micro-haplótipos. Os micro-haplótipos foram escolhidos com base em literatura prévia e as frequências relativas destes foram calculadas com base nos grupos étnicos dos dados do 1000 Genomes. Dados brutos de sequenciamento de três amostras de DNA são analisados: o suposto pai, a mãe e o plasma materno (mistura de DNA livre da mãe e do feto). Em seguida, desenvolvemos scripts para obter e analisar os genótipos do suposto pai e da mãe, para cada um dos micro-haplótipos escolhidos. Combinando informação genotípica, frequências populacio- nais e frações fetais (plasma), desenvolvemos um método para calcular a probabilidade de paternidade em casos de não exclusão da mesma. / Paternity tests are usually done by analyzing DNA samples from the alleged father, the mother, and the child. To perform this exam before the birth, invasive methods such as am- niocentesis and chorionic villus sampling are usually necessary. Fortunately, the discovery of fetal cell-free DNA (fcfDNA) in maternal plasma and serum, and the development of te- chniques to analyze this fcfDNA have allowed researchers to reduce the health risk for both fetus and mother. Although paternity tests that analyze Short Tandem Repeats (STRs) from fcfDNA are possible, they are not reliable because DNA degradation often occurs. Single Nucleotide Polymorphisms (SNPs) have been demonstrated as good candidates for human identification and they can be obtained from small DNA fragments (even from de- graded DNA). However, SNPs have a limited number of different alleles (between two and four). Microhaplotypes are chromosomal segments smaller than 200 bp (base pairs) con- taining two or more SNPs that form at least three distinct haplotypes. By using them as genetic markers, we increased the number of possible alleles formed from the SNPs. Since fcfDNA has approximately 145 bp, this is sufficient to contain microhaplotypes that can be sequenced using Next Generation Sequencing (NGS) technology. The aim of this project is to determine the probability of paternity using SNPs within microhaplotypes. Microha- plotypes were chosen based on previous literature review. The haplotype frequencies were calculated based on the ethnic groups from 1000 Genomes database. Raw DNA sequence data from three DNA samples were analyzed: the alleged father, the mother, and the maternal plasma (mixture of mother and fcfDNA). Then, we developed scripts to analyse and obtain the genotypes of the alleged father and mother, for each microhaplotype. By combining genotypic information, population frequencies, and fetal fractions (plasma), we developed a method to calculate the probability of paternity in cases of non-exclusion.

O Transtorno Bipolar (TB) tipo I é uma doença caracterizada por episódios de mania e depressão recorrentes com importante prejuízo do funcionamento global e comprometimento das funções cognitivas. Além disso, sabe-se que o número de episódios de humor patológico ao longo da vida pode também influenciar o funcionamento cognitivo destes sujeitos. Neste cenário, ocorreu a necessidade de se investigar marcadores genéticos para disfunção cognitiva no TB com o objetivo de estudar este fenômeno. Dentre os potenciais genes responsáveis por influenciar a cognição destacam-se os polimorfismos funcionais do fator neurotrófico derivado do cérebro (BDNF), da catecol-O-metiltransferase (COMT), da apolipoproteína-E (APOE) e do canal de cálcio de baixa voltagem subunidade 1-C (CACNA1C). Sabe-se, também, que no TB os marcadores de estresse oxidativo estão aumentados durante todas as fases da doença, entretanto, não é claro qual impacto destes na disfunção cognitiva de indivíduos com TB. O objetivo dessa tese foi avaliar o desempenho cognitivo de pacientes jovens com bipolaridade tipo I e sua associação com o genótipo de BDNF, COMT, APOE e CACNA1C e também com os níveis plasmáticos de oxidação da guanosina (8-OHdG) e citosina (5-Mec) durante os episódios de humor, eutimia e em controles. Para investigar essa associação foram incluídos 116 pacientes (79 em episódio de humor patológico e 37 eutímicos) com diagnóstico de TB tipo I (DSMIV-TR); 97 controles saudáveis foram submetidos à avaliação neuropsicológica e coleta de sangue para extração de DNA visando genotipagem para BDNF (rs6265), COMT (rs4680; rs165599), APOE (rs429358 e rs7412), CACNA1C (rs1006737), 8-OhdG e 5-Mec. A análise dos dados obtidos revelou que pacientes portadores do genótipo Met/Met rs4680/rs165599 do COMT apresentam comprometimento cognitivo mais grave (função executiva, fluência verbal, memória e inteligência) comparado ao genótipo Val/Met ou Val/Val durante episódios maníacos ou mistos. Na mesma direção destes resultados, verificou-se que pacientes portadores do alelo Met rs4680 do COMT apresentam comprometimento do reconhecimento de emoções faciais em episódios de mania e depressão. Nenhum efeito do COMT foi observado em controles. O alelo de risco Met do CACNA1C se associou a um pior comprometimento executivo independente dos sintomas maníacos ou depressivos no TB, porém nenhum efeito se observou nos controles. O alelo Met do BDNF rs6265 ou a presença do alelo 4 da APOE não representa um fator que identifique um grupo com desempenho cognitivo diferenciado durante as fases do TB ou em controles. Sujeitos com TB apresentaram níveis mais elevados de 8-OHdG e tais níveis eram diretamente proporcionais ao número de episódios maníacos ao longo da vida, sugerindo um papel dos episódios hiperdopaminérgicos na oxidação das bases de DNA. Concluiu-se que a genotipagem para COMT e CACNA1C em pacientes com TB pode identificar um grupo de pacientes associados a pior disfunção cognitiva durante as fases maníacas e mistas do TB. Tal dado pode ser um indicador do envolvimento do sistema dopaminérgico e dos canais de cálcio de baixa voltagem na fisiopatologia da disfunção cognitiva no TB e deve ser explorado em outros estudos / Bipolar I disorder (BD) is a disease whose main features include severe mood swings that cause severe impairment in global functioning and cognitive domains. Moreover, the number of mood episodes throughout patients life is also associated with deterioration in cognitive functions. In this context, it is important to study genetic markers for the cognitive dysfunction observed in BD to elucidate the physiopathology of this phenomenon. The main candidates for genetic modulation of cognition are the genes brain derived neurotrophic factor (BDNF), catechol-o-methyltransferase (COMT), apolipoprotein E (APOE) and 1-C subunit of the L-type voltage-gated calcium channel (CACNA1C). Furthermore, elevated levels of oxidative stress have been reported in BD for all types of mood episodes but no data is available on their impact on cognitive functioning of BD patients. The aim of this thesis was to investigate whether cognitive functioning of BD patients is influenced by BDNF, COMT, APOE, CACNA1C genotypes or by levels of oxidative damage to the DNA base guanosine (8-OHdG) and cytosine (5-Mec). One hundred sixteen patients (79 during mood episode and 37 euthymic) with BD type I (mania, depression or euthymia) and 97 healthy controls were submitted to neuropsychological evaluation and blood collection for DNA analysis. All subjects were genotyped for BDNF (rs6265), COMT (rs4680; rs165599), APOE (rs429358 and rs7412), CACNA1C (rs1006737), DNA levels of 8-OHdG and 5-Mec were also measured. Our results revealed that BD subjects that carried the rs4680/rs165599 Met/Met genotype had more severe cognitive dysfunction (executive function, verbal fluency, memory and intelligence) than carriers of other genotypes during manic or mixed episodes. Moreover, patients carrying the COMT rs4680 Met allele had worse performance on facial emotion recognition tests during manic and depressive episodes. BD carriers of the Met allele of CACNA1C had more severe executive dysfunction than non-carriers, regardless of manic or depressive symptoms. No effect of CACNA1C or COMT genotypes was observed in controls. The genotypes of BDNF or APOE were not associated with cognitive dysfunction in BD patients or controls. The BD group exhibited higher levels of 8-OHdG than the control group and these levels were influenced by the lifetime number of manic episodes, suggesting that hyperdopaminergic episodes may influence the oxidation of DNA bases. In summary, the genotype of COMT and CACNA1C may represent a useful tool for identifying BD subjects at risk of developing more severe cognitive dysfunction in all mood states of the disease. This evidence associating dopamine catabolism and calcium channels to degree of cognitive dysfunction in BD should be further explored by future research

Introdução. A paracoccidioidomicose (PCM) é uma micose sistêmica crônica, endêmica na América Latina, principalmente Brasil, sendo a oitava causa de morte entre as doenças infecciosas crônicas recorrentes. A PCM infecção é caracterizada por uma resposta Th1, a forma aguda por um perfil misto da resposta Th2/Th9, enquanto na forma crônica caracteriza-se pelo perfil Th17/Th22. A ocorrência e gravidade da PCM humana podem também estar associadas a fatores genéticos como os polimorfismos dos genes de citocinas. Objetivos. 1. Descrever a frequência dos polimorfismos de (SNPs) IFNG +874 T/A, IL12B 3\' UTR +1188 A/C e IL12RB1 11014 A/G no éxon 7 em pacientes e controles; 2. Investigar a associação entre esses polimorfismos e as diferentes formas clínicas da micose; 3. Verificar se há associação entre esses polimorfismos e a secreção das citocinas IFN-y, IL-12p40 e IL-12p70. Materiais e Métodos. 143 pacientes com PCM foram incluídos (40 com a forma aguda, 100 com a forma crônica multifocal e 17 unifocal). Critérios de inclusão: ter doença ativa (DA) comprovada por exame micológico ou histopatológico positivo ou presença de anticorpos anti-Paracoccidioides brasiliensis (>= 1/32 por contraimunoeletroforese) ou ter doença curada/tratada (CT) quando comprovada anteriormente pelos critérios de DA e atualmente com títulos de anticorpos estáveis e <= 4 em dois períodos com intervalo >= 6 meses. Analisaram-se os SNPs IFNG pela técnica de PCR-ARMS (\"Polymerase Chain Reaction - Amplification Refractory Mutational System\"), IL12B e IL12RB1 por RFLP (\"PCR-Restriction Fragment Lenght Polymorphism\"). Para a dosagem de citocinas foram utilizadas as técnicas de ELISA (n=29) e CBA (\"Cytometric Bead Array\"; n= 18), sendo considerados estatisticamente significantes, os valores de p < 0,05 para os testes de x2 e o teste de Kruskal-Wallis, com pós-teste de Dunn. Resultados. O genótipo AA do SNP IL12RB1 foi mais frequente na forma crônica multifocal e o genótipo AG, na forma unifocal masculina (p= 0,048). À análise desta forma clínica entre ambos os sexos, o genótipo AG foi também mais frequente no sexo masculino (p= 0,009). Segundo a etnia, foi demonstrada diferença estatisticamente significante nas frequências dos genótipos e alelos dos SNPs IFNG e IL12RB1 (p < 0,05). Em relação às formas clínicas da PCM, houve similaridade nas frequências dos genótipos e alelos dos SNPs estudados. Quanto aos níveis das citocinas, para os SNPs IFNG, IL12B e IL12RB1, maiores níveis de secreção de citocinas, frente a PHA, foram registrados nos grupos CT e CO em relação ao DA, sugerindo relação com a evolução da doença e com a imunossupressão já descrita na doença ativa. Conclusão. Não houve associação entre os SNPs IFNG, IL12B e IL12RB1 e as diferentes formas da doença quando todos os pacientes foram analisados; no sexo masculino, sugere-se que o genótipo AA esteja associado à doença crônica mais disseminada (IL12RB1). Houve diferença significante entre as etnias nos SNPs IFNG e IL12RB1, sugerindo-se a ampliação do número de pacientes em determinadas etnias e na forma clínica unifocal para melhor compreensão dessas associações / Introduction. Paracoccidioidomycosis (PCM) is a systemic chronic mycosis, endemic in Latin America, mainly in Brazil where it is the eighth cause of death among chronic recurrent infectious diseases. PCM infection is characterized by the Th1 immune response, the acute form, by a mixed Th2/Th9 profile, while the chronic form is characterized by Th17/Th22 profile. The occurrence and severity of human PCM can also be associated with genetic factors such as polymorphisms on genes of cytokines. Objectives. 1. To describe the frequencies of the single nucleotide polymorphisms (SNPs) IFNG +874 T/A, IL12B 3\'UTR +1188 A/C and IL12RB1 11014 A/G on exon 7, on patients with PCM and non-PCM controls; 2. To investigate the association between those SNPs and the different clinical forms of PCM. 3. To verify the possible association between those SNPs and the secretion of the cytokines IFN-?, IL-12p40 and IL12p70. Materials and Methods. 143 patients with PCM were included (40 with acute form, 100 with multifocal chronic form and 17 unifocal). Inclusion criteria: active disease (DA) proved by fungal identification on direct microscopy/histopathology or culture, or presence of antibodies antiParacoccidioides brasiliensis ( >= 1/32 by counterimmunoelectrophoresis) or cured/treated disease (CT) when previously proved by criteria of DA and present stable antibodies titles =6 months in between. The SNP IFNG was analyzed by PCR-ARMS (Polymerase Chain Reaction - Amplification Refractory Mutational System) and the SNPs IL12B and IL12RB1 by PCR-RFLP (PCR-Restriction Fragment Length Polymorphism). The levels of cytokines were detected by ELISA (n= 29) and CBA (Cytometric Bead Array; n= 18) and values of p < 0.05 for ?2 test and Kruskal-Wallis\' test, with Dunn\'s post-test were considered statistically significant. Results. The AA genotype of SNP IL12RB1 was the most frequent in the multifocal chronic form while the AG was more frequent in men with the unifocal chronic form of PCM (p = 0.048). On this clinical form in the comparison between genres, the AG genotype was also more frequent in men (p= 0.009). On ethnicity, it was demonstrated statistical difference between the frequencies of genotypes and alleles of SNPs IFNG and IL12RB1 (p < 0.05). In the comparison between the clinical forms of PCM, the frequencies of genotypes and alleles of the evaluated SNPs were similar. On the levels of cytokines, for SNPs IFNG, IL12B and IL12RB1, increased levels of cytokines were observed with PHA on the CT and CO groups compared with DA, suggesting a connection with the evolution of the disease and the previously described immunosuppression during active disease. Conclusion. There was no association between the SNPs IFNG, IL12B and IL12RB1 and the different forms of PCM when all patients were analyzed; among men, it is suggested that the AA genotype of IL12RB1 is associated with a more disseminated chronic disease. There was a significant difference between the ethnicities on SNPs IFNG and IL12RB1, being the latter also associated with the chronic form in men. The increase in the number of patients in certain ethnic groups and in the unifocal clinical form of PCM might help the better understanding of these associations

Duzentas amostras de DNA obtidas de voluntários brasileiros não aparentados foram triadas para SNPs em uma região de 4079 pares de bases do gene PLAC4 através de reação em cadeia da polimerase (PCR) - utilizando o kit Taq Platinum DNA polymerase (Invitrogen, USA) ciclada em termociclador Eppendorf Mastercycle Gradient (Eppendorf, Germany); e posterior sequenciamento - utilizando o kit Big Dye Terminator Cycle Sequencing v3.1 (Applied Biosystems, USA) corrida em sequenciador ABI 3100 DNA Sequencer (Applied Biosystems, USA). Sete fragmentos foram amplificados utilizando pares de iniciadores desenhados com o auxílio do programa Primer 3 baseado em uma sequência do gene PLAC4 obtida do GenBank. Dez SNPs com taxa de heterozigozidade superior a 25% foram identificados, localizados em seis dos sete fragmentos estudados, que fazem a cobertura de 93% da população brasileira. Um painel combinando estes 10 SNPs apresenta potencial utilidade clínica em um teste pré-natal não invasivo da síndrome de Down fetal baseado na abordagem SNP/mRNA / Two hundred DNA samples obtained from unrelated Brazilian individuals were screened for SNPs in a region of 4079 bp of the exon of PLAC4 gene by polymerase chain reaction (PCR) - using Taq Platinum DNA polymerase kit (Invitrogen, USA) cycled on Eppendorf Mastercycle Gradient thermocycle (Eppendorf, Germany); and subsequent sequencing using Big Dye Terminator Cycle Sequencing v3.1 kit (Applied Biosystems, USA) on ABI 3100 DNA Sequencer (Applied Biosystems, USA). Seven fragments were amplified using primer pairs designed by primer 3 software based on PLAC4 sequence obtained from GenBank. Ten SNPs with heterozigosity rate above 25% were identified, located in six of the seven fragments studied, that covers up to 93% of Brazilian population. A panel combining this 10 SNPs show potential utility in clinical setting for a noninvasive prenatal diagnostic test for Down syndrome based in the SNP/mRNA approach

Sickle Cell Disease (SCD) is a hereditary monogenic disorder that affects millions of people worldwide and is associated with symptoms such as stroke, lethargy, chronic anemia, and increased mortality. SCD can be quickly detected and diagnosed using a simple blood test as an infant, but as of now, there is currently limited treatment to cure an individual of sickle cell disease. Recently, there have been several promising developments in CRISPR-Cas-associated gene-editing therapeutics; however, there have been limitations in gene-editing efficiency monitoring, which if improved, could be beneficial to advancing CRISPR-based therapy, especially in SCD. The CRISPR-Chip, a three-terminal graphene-based field effect transistor (gFET), was used to detect genomic samples of individuals with SCD, with and without amplification. With the dRNP-HTY3’ complex, CRISPR-Chip was able to specifically detect its target sequence with and without pre-amplification. With the dRNP-MUT3’ complex, CRISPR-Chip was only able to specifically detect one of its two target sequences. Facile detection, analysis, and editing of sickle cell disease using CRISPR-based editing and monitoring would be beneficial for simple diagnostic and gene-editing therapeutic treatment of other single nucleotide polymorphisms as well, such as beta-thalassemia and cystic fibrosis.

<p>In Molecular Medicine a wide range of methods are applied to analyze the genome to find genetic predictors of human disease. Apart from predisposing disease, genetic variations may also serve as genetic markers in the search for factors underlying complex diseases. Additionally, they provide a means to distinguish between species, analyze evolutionary relationships and subdivide species into strains. </p><p>The development and improvement of laboratory techniques and computational methods was a spin-off effect of the Human Genome Project. The same techniques for analyzing genomic sequence variations may be used independent of organism or source of DNA or RNA. In this thesis, methods for high-throughput analysis of sequence variations were developed, evaluated and applied. </p><p>The performance of several genotyping assays were investigated prior to genotyping 4000 samples in a co-operative genetic epidemiological study. Sequence variations in the estrogen receptor alpha gene were found to be associated with an increased risk of breast and endometrial cancer in Swedish women.</p><p>Whole genome amplification (WGA) enables large scale genetic analysis of sparse amounts of biobanked DNA samples. The performance of two WGA methods was evaluated using four-color minisequencing on tag-arrays. Our in-house developed assay and “array of arrays” format allow up to 80 samples to be analyzed in parallel on a single microscope slide. Multiple displacement amplification by the Φ29 DNA polymerase gave essentially identical genotyping results as genomic DNA. To facilitate accurate method comparisons, a cluster quality assessment approach was established and applied to assess the performance of four commercially available DNA polymerases in the tag-array minisequencing assay. </p><p>A microarray method for genotyping human group A rotavirus (HRV) was developed and applied to an epidemiological survey of infectious HRV strains in Nicaragua. The method combines specific capture of amplified viral sequences on microarrays with genotype-specific DNA-polymerase mediated extension of capture oligonucleotides with fluorescent dNTPs.</p>

<p>In this thesis, the four-color fluorescence tag-microarray minisequencing system pioneered by our group was further developed and applied for analysing genetic variation in human DNA and RNA. A SNP marker panel representing different chromosomal regions was established and used for identification of informative SNP markers for monitoring chimerism after stem cell transplantation (SCT). The success of SCT was monitored by measuring the allelic ratios of informative SNPs in follow-up samples from nine patients with leukaemia. The results agreed with data obtained using microsatellite markers. Further the same SNP marker panel was used for evaluation of two whole genome amplification methods, primer extension preamplification (PEP) and multiple displacement amplification (MDA) in comparison with genomic DNA with respect to SNP genotyping success and accuracy in tag-array minisequencing. Identical results were obtained from MDA products and genomic DNA.</p><p>The tag-microarray minisequencing system was also established for multiplexed quantification of imbalanced expression of SNP alleles. Two endothelial cell lines and a panel of ten coding SNPs in five genes were used as model system. Six heterozygous SNPs were genotyped in RNA (cDNA) from the cell lines. Comparison of the relative amounts of the SNPs alleles in cDNA to heterozygote SNPs in genomic DNA displayed four SNPs with significant imbalanced expression between the SNP alleles. Finally, the tag-array minisequencing system was modified for detection of splice variants in mRNA from five leukaemia cell lines. A panel of 20 cancer-related genes with 74 alternatively splice variants was screened. Over half of the splice variants were detected in the cell lines, and similar alternative splicing patterns were observed in each cell line. The results were verified by size analysis of the PCR product subjected to the minisequencing primer extension reaction. The data from both methods agreed well, evidencing for a high sensitivity of our system.</p>

<p>Autoimmune mechanisms and genetic susceptibility contribute to the pathogenesis of primary Sjögren’s syndrome and SLE. These chronic systemic autoimmune diseases have many serological and clinical features in common and have an impact on daily life. The studies in this thesis aim to elucidate their autoimmune mechanisms, define susceptibility genes and evaluate effects of androgen supplement on health-related quality of life.</p><p>Autoantibodies against α-fodrin, a widely distributed cytoskeletal protein, were detected at similar frequencies in sera from patients with primary and secondary Sjögren’s syndrome and SLE. Consequently, testing for antibodies against α-fodrin would not add diagnostic value compared to conventional serological analysis and does not discriminate between these diseases.</p><p>The type I interferon (IFN) system was found to be activated in primary Sjögren’s syndrome. IFN-α containing cells were detected in minor salivary gland biopsies, while sera from patients with primary Sjögren’s syndrome induced IFN-α production in the presence of apoptotic and necrotic cell material. This ability of sera correlated with the presence of antibodies against RNA-binding proteins and IFN-α production was dependent on RNA in immune complexes. The natural interferon producing cells/plasmacytoid dendritic cells (NIPC/PDC) were the IFN-α producers and blocking of FcγRIIa inhibited the production. Single nucleotide polymorphisms (SNPs) in two genes in the type I IFN signalling pathway, those for tyrosine kinase 2 and interferon regulatory factor 5, were strongly associated with SLE in a Swedish, Finnish and Icelandic population. The minor allele frequencies were lower in SLE patients than in healthy controls. These SNPs may decrease the function of the type I IFN system, thereby conferring protection against SLE. </p><p>Supplementation with dehydroepiandrosterone (DHEA) in glucocorticoid treated women with SLE led to mild improvements in health-related quality of life in respect of mental well-being and sexuality, whereas physical well-being was unaffected.</p>

Autoimmune mechanisms and genetic susceptibility contribute to the pathogenesis of primary Sjögren’s syndrome and SLE. These chronic systemic autoimmune diseases have many serological and clinical features in common and have an impact on daily life. The studies in this thesis aim to elucidate their autoimmune mechanisms, define susceptibility genes and evaluate effects of androgen supplement on health-related quality of life. Autoantibodies against α-fodrin, a widely distributed cytoskeletal protein, were detected at similar frequencies in sera from patients with primary and secondary Sjögren’s syndrome and SLE. Consequently, testing for antibodies against α-fodrin would not add diagnostic value compared to conventional serological analysis and does not discriminate between these diseases. The type I interferon (IFN) system was found to be activated in primary Sjögren’s syndrome. IFN-α containing cells were detected in minor salivary gland biopsies, while sera from patients with primary Sjögren’s syndrome induced IFN-α production in the presence of apoptotic and necrotic cell material. This ability of sera correlated with the presence of antibodies against RNA-binding proteins and IFN-α production was dependent on RNA in immune complexes. The natural interferon producing cells/plasmacytoid dendritic cells (NIPC/PDC) were the IFN-α producers and blocking of FcγRIIa inhibited the production. Single nucleotide polymorphisms (SNPs) in two genes in the type I IFN signalling pathway, those for tyrosine kinase 2 and interferon regulatory factor 5, were strongly associated with SLE in a Swedish, Finnish and Icelandic population. The minor allele frequencies were lower in SLE patients than in healthy controls. These SNPs may decrease the function of the type I IFN system, thereby conferring protection against SLE. Supplementation with dehydroepiandrosterone (DHEA) in glucocorticoid treated women with SLE led to mild improvements in health-related quality of life in respect of mental well-being and sexuality, whereas physical well-being was unaffected.