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.

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Greitumas – vienas iš svarbiausių judamųjų gebėjimų. Jis įvairiose sporto šakose pasireiškia skirtingomis formomis. Greitumo pratybos, tai tokios pratybos, kurios nemažina maksimalaus raumens susitraukimo ir atsipalaidavimo greičio, o jį padidina. Yra žinoma, kad jėgos pratybos yra kenksmingos greitumui. Mūsų nuomone, didžiausia problema sporto moksle - kaip padidinti jėgą nesumažinant raumens susitraukimo ir atsipalaidavimo greičio. Taip pat labai aktualu sužinoti, kaip skirtingų genų variantų žmonės geba didinti greitumą ir jo pasireiškimo formas. Šiuo metu pasirodė daug straipsnių apie ACTN3 R577X polimorfizmą. Yra žinoma, kad α-aktinino-3 baltymo nėra pas 16 % pasaulio žmonių. Įdomu tai, kad mutavusio (X) alelio ir ypač pilno α-aktinino-3 (alelis XX) deficito dažnis yra ženkliai mažesnis tarp sprinto ir galingumo atletus. Todėl mes savo tyrime analizavome greitumo pratybų įtaką didesnio jėgos indėlio reikalaujantiems pratimams ir gautus rezultatus lyginome tarp skirtingų RR ir XX genotipų. Tyrimo tikslas – ištirti maksimalaus greitumo padidėjimo įtaką didesnio jėgos indėlio reikalaujantiems judamiesiems gebėjimams priklausomai nuo RR (gaminasi α-aktininas-3) ir XX (nesigamina α-aktininas-3) genotipo. Tyrimo objektas – greitumo pokytis ir to pokyčio įtaka didesnės jėgos reikalaujantiems judamiesiems gebėjimams RR ir XX genotipo grupėse. Tyrimo uždaviniai: 1. Nustatyti ir įvertinti, kaip po 10 maksimalaus greitumo lavinimo pratybų kito RR ir XX grupių greitumo judamasis... [toliau žr. visą tekstą] / Running speed is one of the most important physical properties. It has various forms. Speed training does not decrease muscle contraction and relaxation speed. It is well known, that strength training decrease running speed results. That’s why we think that maybe the most important problem in sport science is how to increase strength without maximum muscle contraction and relaxation speed decrease. Also it is important to know how people with different genotypes can increase running speed. There are only few articles about ACTN3 R577X polymorphism. A common nonsense mutation (R577X) in the ACTN3, resulting in a premature stop codon and lack of detectable protein in homozygous individuals for the ACTN3 null allele (XX genotype), has been demonstrated in the general human population with 16 % prevalence in Caucasians. Sprint athletes have lower 577XX genotype frequency endurance athletes. That’s why we analyze running speed workouts influence to more strength required physical properties between RR and XX groups. The aim of the study - to investigate running speed increment influence to more strength required physical properties and compare results between RR (with α-actinin-3) and XX (without α-actinin-3) groups. The object of the study - running speed change and this change influence to more strength required physical properties between RR (with α-actinin-3) and XX (without α-actinin-3) groups. Study tasks: 1. To investigate how after maximum speed workouts vary running speed... [to full text]

Das Ziel dieser Arbeit war zunächst in der Methodenoptimierung eines bereits grundlegend etablierten Protokolls zur Isolierung und Kultur equiner endometrialer Epithel (EEZ) und Stromazellen (ESZ) (BUSCHATZ 2007) zu sehen. Zudem wurde die Entwicklung weiterer Möglichkeiten des Handlings angestrebt (Passagierung, Kryokonservierung). So sollten den Zellen optimierte Rahmenbedingungen in vitro geboten werden, welche den Verhältnissen im Organismus weitgehend nahe kommen. Im Anschluss daran wurden die Zellen in vitro hinsichtlich ihrer morphologisch-funktionellen Charakteristika untersucht und die Befunde vergleichend zu den Gegebenheiten in situ betrachtet. Besonderes Augenmerk galt dabei der Expression von Progesteron- (PR) und Östrogenrezeptor-α (ERα) und von Inhibin-α. Wären vergleichbare Konstellationen in vitro und in vivo anzutreffen, könnte ein solches Kultursystem als Modell zum Studium interzellulärer Wechselwirkungen oder pathogenetischer Abläufe am Endometrium dienen. Voraussetzung hierfür wäre jedoch eine fortgeschrittene zelluläre Differenzierung, wie sie beispielsweise durch die Expression von Inhibin-α und der Steroidhormonrezeptoren angezeigt wird. Es wurden transzervikale Uterusbioptate und vollständige Uteri euthanasierter Stuten für die Zellaufreinigung sowie die vergleichende histologische Untersuchung gewonnen. Einer mechanischen und enzymatischen Dissoziation des Gewebes folgte die Separation beider Zellarten durch Filtration, Dichtegradientenzentrifugation und Differenzialadhärenz. Die Kultur erfolgte in wasserdampfgesättigter Raumluft bei 37 °C in 5 % CO2-Atmosphäre. Als Kulturmedium diente DMEM/Ham´s F-12 unter Zusatz von 2,5 % fötalem Kälberserums und diverser Additive. Es wurde eine morphologische Charakterisierung der Zellen während der Kultur vorgenommen und zudem ERα, PR und Inhibin-α an allen Kulturen und Gewebeproben immunhistologisch bestimmt. Das Ablösen der Zellen zur Passagierung erfolgte mit Trypsin-EDTA (ESZ) bzw. Alfazyme® (EEZ). Entsprechend abgelöste Zellen wurden zudem in DMSO-haltigem Nährmedium kryokonserviert. Die Kultivierung von EEZ und ESZ gelang sowohl bei Verwendung transzervikaler Uterusbioptate als auch bei Uteri euthanasierter Pferde. Zudem konnten alle physiologischerweise bei der Stute auftretenden Zyklusstände (Anöstrus, Interöstrus, Östrus) sowie ein gravider Uterus kultiviert werden. Die Konfluenz wird von EEZ nach 4 bis 16 d und von ESZ innerhalb von 7 bis 18 d erreicht, wobei Zellen aus ursprünglich proliferativen endometrialen Funktionszuständen tendenziell eher konfluent sind als sekretorisch aktive. Während der Kultur kann eine eindeutige morphologische Unterscheidung beider Zellarten voneinander erfolgen. ESZ besitzen eine spindelige, teils sternförmige, insgesamt „fibroblastenartige“ Gestalt. EEZ sind in zwei morphologischen Subtypen anzutreffen. Der monomorphe Zelltyp „A“ stellt kleine, polygonale Zellen mit regulären und regelmäßigen Zellgrenzen dar. Zelltyp „B“ ist größer, pleomorph, ebenfalls polygonal, mehrkernig und besitzt unregelmäßige, schlecht erkennbare Zellgrenzen. Subkultivierungen waren bis zu 20 (ESZ) bzw. 24 mal (EEZ) möglich. Zudem konnten beide Zellarten in flüssigem Stickstoff gelagert (kryokonserviert) und danach erfolgreich kultiviert werden. Weiterhin gelang der Nachweis von Inhibin-α im Uterus des Pferdes. Hierbei wurde eine zyklische Dynamik in der Expression festgestellt (stärkere Expression während der sekretorischen Phase), welche als Hinweis für eine sekretorische Differenzierung der EEZ anzusehen ist. Ebenfalls konnte dieses Protein in kultivierten EEZ und in viel geringerem Maße auch in den ESZ gefunden werden. Darüber hinaus war erstmalig der Nachweis von PR und ERα in beiden Zellarten in vitro möglich. Insgesamt ist die Expression dieser drei für uterines Gewebe essentiellen Rezeptoren/Proteine in kultivierten EEZ und ESZ als Hinweis für eine fortgeschrittene Differenzierung anzusehen, welche mit der vorgestellten Methode der Isolierung und Kultur auch erreicht werden konnte. Im Hinblick auf die Wachstumsgeschwindigkeit in vitro fanden sich zudem Hinweise auf eine Beibehaltung der ursprünglich im Gewebeverband erlangten zellulären Differenzierung, welche sich bei der Expression von ERα, PR bzw. Inhibin-α allerdings nicht nachvollziehen ließ. Abschließend betrachtet, deuten die vorliegenden Ergebnisse somit auf eine partielle Beibehaltung in situ erlangter endometrialer Funktionen und Spezifika hin, welche als Grundlage für weitere Arbeiten an endometrialen Zellkulturen des Pferdes anzusehen sind.

This thesis deals with the development and application of new synthetic methodology in organic chemistry. The first part describes the development of a new protocol for the synthesis of 3-pyrrolines by means of a microwave-assisted ring-expansion reaction of 2-vinylaziridines. In addition, this methodology is implemented as a key-step in a formal total synthesis of the antibiotic (-)-anisomycin. In the second part, a new methodology for the synthesis of arylglycines from Weinreb amides is described. In this procedure, a Grignard reagent is added to the iminium ion formed from the Weinreb amide upon treatment with a base. When a chiral amide is used, the nucleophilic addition proceeds with high diastereoselectivity. Finally, an easy and straightforward synthesis of α-amino amides via a base-mediated rearrangement of modified Weinreb amides into N,O-acetals is presented. Subsequent arylation, alkylation, alkenylation or alkynylation of this intermediate affords the corresponding α-amino amides in excellent yields. Furthermore, a more generalized protocol for the α-arylation of Weinreb amides lacking an α-amino moiety is also discussed. / QC 20100719

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Glutaric acidemia type I (GA-I) is an inborn error of metabolism (EIM), characterized biochemically by major accumulation of glutaric acid (GA) and pathologically by a characteristic striatal degeneration. The clinical manifestations are mainly neurological and develop during childhood (up to 5 years old). Among these changes, there are the seizures and cognitive deficits, which may be precipitated by infectious processes. From this, the first hypothesis to be tested in this study was to investigate whether lipopolysaccharide E. coli 055 B5 serotype (LPS; 2 mg/Kg; i.p.), an inflammatory agent, could facilitate seizures induced by GA in young rats (21 days of life). For this, firstly it was determined the acute dose of intrastriatal GA (1.3 μmol/striatum) that cause behavioral and electroencephalographic (EEG) seizures in young rats. Moreover, it was shown that LPS administration 3 hours before GA intrastriatal injection did not change the seizures, but when LPS was administered 6 hours before the GA, it reduced the latency and increased the duration of behavioral and EEG seizures induced by GA in young rats. It also was observed that LPS injection caused an initial drop in rectal temperature of young rats (up to 2 hours), followed by a rise in temperature that started at 3 hours and remained high until 6 hours after LPS injection. Furthermore, it was shown that LPS injection 3 and 6 hours before intrastriatal injection of GA caused an increase in striatal levels of IL-1β in young rats, and this increase was statistically higher in 6 than in 3 hours. In addition, it was observed that the increase in IL-1β striatal levels, caused by LPS administration, positively correlated with total time of seizures. Finally, it was observed that previous use of IL-1β antibody prevented the latency reduction and the increased duration of seizures caused by LPS administration 6 h before intrastriatal injection of GA in young rats. Thus, these findings suggest that the signaling of IL-1β present in inflammation produced by LPS contributes significantly to neuronal hyperexcitability, and thus to reduce latency and increase the duration of seizures induced by GA. Therefore, pharmacological treatments that block the specific functions or overproduction of IL-1β in GA-I, may represent an unconventional strategy to treat this condition. However, clinical studies should be conducted to evaluate the effectiveness of treatment in glutaricoacidemic patients with convulsions. Since patients with GA-I have other important neurological changes addition to the seizures, as cognitive impairments, the second hypothesis to be tested in this study was to determine whether chronic treatment with GA (5 μmol/g; s.c.; twice per day; from the 5th to the 28th day of life) could cause spatial memory impairment in young rats, and verify whether the inflammation produced by LPS (2 mg/Kg; i.p.; one per day; from the 25th to the 28th day of life) could facilitate the cognitive deficit induced by GA. In addition, it also was evaluated the possible impact of these treatments on functional and structural changes in the hippocampus of these animals. Initially it was shown that chronic treatment with GA, as well as the treatments with LPS and GA-LPS, caused a deficit in spatial learning of young rats. However, it was demonstrated that the treatment with GA-LPS produced a greater impairment in spatial memory compared to other treatments. In addition, it was observed that none of the treatments affected weight or locomotor activity/exploratory of animals. It also was shown that chronic treatment with GA, as well as treatments with LPS and GA-LPS, increased the hippocampal levels of IL-1β and TNF-α in young rats. Furthermore, it was demonstrated that treatments with GA, LPS and GA-LPS caused a reduction in total hippocampal volume of young rats. Finally it was observed that treatments with GA, LPS and GA-LPS caused a reduction of α1 subunit activity of Na+,K+-ATPase enzyme. On the other hand, it was shown that treatments with GA and LPS caused an increase in activity of α2/3 subunits of the enzyme. Thus, only treatment with GA-LPS showed a reduction in total activity of Na+,K+-ATPase in the hippocampus of young rats. These data indicate that the impairment in spatial learning observed in rats treated with GA, LPS and GA-LPS was due to increased levels of inflammatory cytokines, the reduction in hippocampal volume and the inhibition of α1 subunit activity of Na+,K+-ATPase enzyme. However, the worsening in spatial memory observed in rats treated with GA-LPS was due to inhibition of total activity of Na+,K+-ATPase, which was specific α2/3 isoforms, since only this group showed no compensatory response the activity of these subunits. Therefore, this second part of the study showed that chronic treatment with GA caused a deficit in spatial learning in young rats, and that the presence of an inflammatory process increased the impairment in spatial memory induced by GA alone. Thus, understanding the mechanisms involved in seizures and cognitive deficits observed in patients with GA-I in the presence of an inflammatory process is important for the development of new therapies to treat this condition, as well as other diseases associated with the presence of inflammatory mediators. / A acidemia glutárica tipo I (GA-I) é um erro inato do metabolismo (EIM) caracterizada bioquimicamente pelo acúmulo principal de ácido glutárico (GA) e patologicamente por uma característica degeneração estriatal. As manifestações clínicas são predominantemente neurológicas, e desenvolvem-se principalmente na infância (até os 5 anos de idade). Entre estas alterações, destacam-se as convulsões e os déficits cognitivos, os quais podem ser precipitados por processos infecciosos. A partir disso, a primeira hipótese a ser testada neste estudo foi investigar se o lipopolissacarídeo sorotipo E. coli 055 B5 (LPS; 2 mg/Kg; i.p.), um agente inflamatório, facilitaria as convulsões induzidas pelo GA em ratos jovens. Para isso, primeiramente determinou-se a dose intraestriatal aguda de GA (1.3 μmol/estriado) que causa convulsões comportamentais e eletroencefalográficas (EEG) em ratos jovens (21 dias). Em seguida foi verificado que a administração de LPS 3 horas antes da injeção intraestriatal de GA não alterou as convulsões, mas quando o LPS foi administrado 6 horas antes do GA, ele reduziu a latência e aumentou a duração das convulsões comportamentais e EEG induzidas pelo GA em ratos jovens. Observou-se também que injeção de LPS causou uma queda inicial na temperatura retal dos ratos jovens (até 2 horas), seguida de uma elevação na temperatura que iniciou em 3 horas e permaneceu alta até 6 horas após a injeção de LPS. Além disso, foi verificado que injeção de LPS 3 e 6 horas antes da injeção intraestriatal de GA causou um aumento nos níveis estriatais de IL-1β nos ratos jovens, sendo esse aumento estatisticamente maior em 6 do que em 3 horas. Também foi observado que o aumento nos níveis estriatais de IL-1β, causado pela administração de LPS, correlacionou-se positivamente com o tempo total de convulsões. Por fim, verificou-se que uso prévio do anticorpo da IL-1β preveniu a redução da latência e o aumento da duração das convulsões causadas pela administração de LPS 6 horas antes da injeção intraestriatal de GA nos ratos jovens. Assim, estes achados sugerem que a sinalização da IL-1β presente no processo inflamatório produzido pelo LPS contribui decisivamente para a hiperexcitabilidade neuronal e, consequentemente, para a redução da latência e o aumento da duração das convulsões induzidas pelo GA. Dessa maneira, tratamentos farmacológicos específicos que bloqueiam a superprodução ou as funções da IL-1β na GA-I, podem representar uma estratégia não convencional para o tratamento dessa patologia. Entretanto, estudos clínicos devem ser realizados a fim de avaliar a eficácia desse tratamento nos pacientes glutaricoacidêmicos que apresentam convulsões. Desde que os pacientes com GA-I apresentam outras alterações neurológicas importantes além das convulsões, como prejuízos cognitivos, a segunda hipótese a ser testada neste estudo foi verificar se o tratamento crônico com GA (5 μmol/g; s.c.; duas vezes por dia; do 5° ao 28° dia de vida) causaria déficit de memória espacial em ratos jovens, bem como se a inflamação produzida pelo LPS (2 mg/Kg; i.p.; uma vez por dia; do 25° ao 28° dia de vida) facilitaria o déficit cognitivo induzido pelo GA. Além disso, também foi objetivo avaliar o impacto desses tratamentos sobre possíveis alterações funcionais e estruturais no hipocampo desses animais. Inicialmente verificou-se que o tratamento crônico com GA, assim como os tratamentos com LPS e GA-LPS, causaram um déficit no aprendizado espacial dos ratos jovens. No entanto, foi observado que o tratamento com GA-LPS produziu um maior prejuízo na memória espacial comparado com os outros tratamentos. Em seguida foi observado que nenhum dos tratamentos alterou o peso ou a atividade locomotora/exploratória dos animais. Verificou-se também que o tratamento crônico com GA, assim como os tratamentos com LPS e GA-LPS, aumentaram os níveis hipocampais de IL-1β e TNF-α nos ratos jovens. Além disso, foi observado que tratamentos com GA, LPS e GA-LPS causaram uma redução no volume hipocampal total dos ratos jovens. Finalmente verificou-se que os tratamentos com GA, LPS e GA-LPS causaram uma redução na atividade da subunidade α1 da enzima Na+,K+-ATPase. Por outro lado, foi observado que os tratamentos com GA e LPS causaram um aumento na atividade das subunidades α2/3 da enzima. Assim, somente o tratamento com GA-LPS apresentou uma redução na atividade total da enzima Na+,K+-ATPase no hipocampo dos ratos jovens. Estes dados indicam que o prejuízo no aprendizado espacial observado nos ratos tratados com GA, LPS e GA-LPS parece estar relacionado a um aumento nos níveis de citocinas inflamatórias, a uma redução no volume hipocampal e a uma inibição na atividade da subunidade α1 da enzima Na+,K+-ATPase. No entanto, o maior prejuízo na memória espacial observado nos ratos tratados com GA-LPS ocorreu devido a inibição na atividade total da enzima Na+,K+-ATPase, que foi específica das isoformas α2/3, já que somente este grupo não apresentou resposta compensatória na atividade destas subunidades. Portanto, esta segunda parte do estudo demonstrou que o tratamento crônico com GA causou um déficit no aprendizado espacial de ratos jovens, e que a presença de um processo inflamatório potencializou o prejuízo na memória espacial induzida pelo GA sozinho. Assim, o entendimento dos mecanismos envolvidos nas convulsões e no déficit cognitivo observados nos paciente com GA-I frente a um processo inflamatório é importante para o desenvolvimento de novas terapias para o tratamento dessa patologia, bem como de outras doenças associadas à presença de mediadores inflamatórios.

Orientador: Ney Lemke / Resumo: Os sistemas de importação nuclear são responsáveis pelo intercâmbio entre o citoplasma e o núcleo da célula, permitindo que proteínas com função nuclear migrem através da membrana que separa essas duas regiões. A via de importação mais estudada é a via clássica de importação nuclear mediada pela Importina-α (Impα). A Impα é uma proteína solenóide, composta por repetições em tandem do motivo Armadillo (ARM) que formam uma estrutura longa e contorcida, com pequenos arcabouços ao longo do eixo da proteína. As sequências de localização nuclear clássicas (cNLSs) presentes nas proteínas-alvo de importação são compostas por resíduos carregados positivamente e estabelecem pontes salinas, ligações de hidrogênio e contatos hidrofóbicos com esses arcabouços da Impα. Esse reconhecimento pode ocorrer em um ou em dois sítios da Impα, caracterizando a cNLS como monopartida ou bipartida, respectivamente. A maioria das informações estruturais do complexo cNLS-Impα provém de dados de cristalografia e pouco se sabe sobre a dinâmica conformacional deste sistema. Uma abordagem para tratar da dinâmica de um sistema é o uso de técnicas de simulação de biomoléculas, tais como dinâmica molecular e análise de modos normais. Com base nessas técnicas de simulação, o presente estudo teve como objetivo compreender os mecanismos de interação e dinâmica conformacional envolvidos no reconhecimento de cNLSs pela Impα. Particularmente, este trabalho focou nas cNLSs das proteínas Nucleoplasmina e Ku70 complexa... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Nuclear import systems are responsible for the exchange between the cytoplasm and the nucleus of a cell, allowing nuclear proteins to migrate through the membrane that separates these two regions. The most studied import pathway is the classical nuclear import mediated by Importin-α (Impα). Impα is a solenoid protein consisting of tandem repeats of the Armadillo (ARM) motif, forming an extended and twisted structure with small grooves along the protein axis. The classical nuclear localization sequences (cNLSs) of cargo proteins are composed of positively charged residues and establish salt bridges, hydrogen bonds and hydrophobic contacts with the grooves of Impα. Such recognition can occur at one or two sites of Impα, thus characterizing the cNLS as monopartite or bipartite, respectively. Most structural information of the cNLS-Impα complex is from crystallographic data and little is known about the conformational dynamics of this system. One approach to address the dynamics of a system is the use of biomolecular simulation techniques such as molecular dynamics and normal modes analysis. Based on these techniques, this study aimed to understand the mechanisms of interaction and conformational dynamics involved in the recognition of cNLSs by Impα. In particular, this work focused on the cNLSs of Nucleoplasmin and Ku70 proteins complexed with Impα. The study of Nucleoplasmin determined two main motions of Impα that may be associated to the cNLS recognition: bending and twisting... (Complete abstract click electronic access below) / Doutor