Thesis (Ph. D.)--University of Adelaide, Dept. of Plant Science, 1994. / Includes bibliographical references (leaves 93-114).

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2008. / Härtill 5 uppsatser.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 69-76).

Orientador: Raquel Mantuaneli Scarel Caminaga / Banca: Silvana Regina Perez Orrico / Banca: José Eduardo Tanus dos Santos / Resumo: Objetivo: O presente estudo foi realizado para investigar a associação entre polimorfismos +785(C/T), +1208(T/C) e +1440(G/A) no gene do receptor 2 da interleucina 8 (CXCR2), bem como de seus haplótipos, e a suscetibilidade à Periodontite em indivíduos brasileiros. Material e Método: Foram selecionados 500 indivíduos de ambos os gêneros (Grupo Controle: n = 224, idade média 35,3 anos; Grupo Periodontite [GP] n = 276, idade média 43,4 anos) que procuraram atendimento na Faculdade de Odontologia de Araraquara. A partir de células da mucosa bucal, o DNA foi extraído por uma solução de solventes orgânicos (fenol:clorofórmio:álcool isoamílico; - 25:24:1). Os polimorfismos +785 e +1208 foram investigados pela técnica SSP-PCR (Sequence Specific Primer - PCR) enquanto que o polimorfismo +1440 foi analisado por PCR-RFLP (Polymerase Chain Reaction - Restriction Fragment Length Polymorphism). Os fragmentos obtidos por PCR-RFLP e os produtos obtidos por SSP-PCR foram submetidos à eletroforese vertical em gel de poliacrilamida a 10% (locus +785 e +1208) e 14% (locus +1440), sendo depois corados com nitrato de prata. Resultados: Considerando a frequência de alelos e genótipos de cada polimorfismo isoladamente, não foi encontrada diferença estatisticamente significante entre os grupos. Quando analisados em haplótipos, a freqüência do haplótipo TCG foi significativamente maior no Grupo Periodontite (p=0,005; odds ratio [OR] = 3,54) do que no Controle. O haplótipo heterozigoto TCG/CCA foi relacionado com um aumento de suscetibilidade à periodontite na população total (p=0,015; OR=3,22). Conclusão: Os resultados deste estudo sugerem que haplótipos no gene CXCR2 estão associados com suscetibilidade à periodontite em indivíduos brasileiros. / Abstract: Objective: This study investigated whether the +785(C/T), +1208(T/C) and +1440(G/A) single nucleotide polymorphisms (SNPs) in the CXCR2 gene, as well as their haplotypes, would be associated with susceptibility to periodontitis in Brazilian individuals. Methods: Five hundred individuals in both genders (Control Group [CG] = 224, mean age 35.3 years; Periodontitis Group [PG] = 276, mean age 43.4 years) were recruited for this study from the patient pool of the School of Dentistry of Araraquara - FOAr/UNESP. DNA was extracted from buccal epithelial cells with a solution of organic solvents (phenol/chlorophorm/isoamilic alcohol, - 25:24:1). The +785 and +1208 SNPs were investigated using the Sequence Specific Primers Polymerase Chain Reaction method (SSP-PCR). The +1440 SNP was genotyped by the PCR-RFLP method (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism). The SSP-PCR products and the RFLP fragments were submitted to polyacrylamide gel electrophoresis in a concentration of 10% and 14%, respectively, which were stained by silver staining method. Results: Considering each polymorphism allele and genotype frequency isolated, no differences were found between groups. The frequency of TCG haplotype was significantly higher in the Periodontitis group (p=0.005, odds ratio [OR] =3.54) than in the Controls. The heterozygous haplotype TCG/CCA was related to an increased susceptibility to Periodontitis in the total casuistic (p=0.015, OR=3.22). Conclusion: These data indicate that haplotypes in the CXCR2 gene were associated with susceptibility to periodontitis in brazilian individuals. / Mestre

Orientador: Raquel Mantuaneli Scarel Caminaga / Banca: Sérgio Roberto Line / Banca: Joni Augusto Cirelli / Resumo: O estudo investigou se há associação individual dos polimorfismos -353(A/T), -738(T/A) e -845(T/C) na região promotora do gene interleucina 8 (IL8), bem como de seus haplótipos, com suscetibilidade à periodontite em indivíduos Brasileiros. Foram selecionados 500 indivíduos de ambos os gêneros (Grupo Controle n=224 e Grupo Periodontite n=276) que procuraram atendimento na Faculdade de Odontologia de Araraquara. A partir do DNA obtido de células da mucosa oral, os polimorfismos -738(T/A) e -845(T/C) foram analisados por PCR-RFLP (Polymerase Chain Reaction - Restriction Fragment Length Polymorphism), e o -353(A/T) por SSP-PCR (Sequence Specific Primer - PCR). Os fragmentos obtidos foram submetidos à eletroforese vertical em gel de poliacrilamida a 10%. Para analisar a freqüência dos genótipos e alelos foi utilizado o teste χ2, ou o programa CLUMP para os polimorfismos que mostraram alelos raros. Os haplótipos foram estimados pelo programa ARLEQUIN e a diferença na distribuição deles entre os grupos foi investigada por meio do programa CLUMP. Os resultados da análise individual dos polimorfismos não evidenciaram associação com a periodontite. Em relação aos haplótipos, as freqüências dos mesmos como alelos e/ou genótipos entre Grupo Controle e Grupo Periodontite apresentaram diferenças estatisticamente significantes considerando a população total e também a não fumante. Os indivíduos com o haplótipo CTA apresentaram-se 2,14 vezes mais susceptíveis à doença periodontal (p=0,0012, OR=2,14; 95% CI =1,6-3,38). Concluiu-se que houve associação dos haplótipos formados pelos polimorfismos -845(T/C), -738(T/A) e -353(A/T) no gene IL8 com a suscetibilidade a periodontite na população brasileira estudada. / Abstract: The purpose of this study was to investigate whether there is an individual association of the -845(T/C), -738(T/A) and -353(A/T) single nucleotide polymorphisms (SNPs) in the IL8 gene, as well as their haplotypes with susceptibility to periodontitis in Brazilian population. DNA was extracted from buccal epithelial cells from 500 Brazilian individuals (Control group n = 224, Periodontitis group n = 276). The -845 and -738 SNPs were genotyped by PCR-RFLP method (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism). The -353 SNP was investigated using the Sequence Specific Primers Polymerase Chain Reaction method (SSP-PCR). Differences of the allelic and genotypic frequencies were assessed by Chi-squared test or by using the CLUMP program for rare alleles. Haplotypes were estimated using the ARLEQUIN program. Differences in the haplotype frequencies between the studied groups were assessed by the CLUMP program. No differences were observed in the allelic and genotypic distribution of all the investigated SNPs between control and periodontitis groups when they were analysed individually. The haplotypes distribution in the periodontitis and controls groups was significantly different both considering the total casuistic or the non-smokers subgroup only. Individuals with CTA haplotype seemed to be over twice more likely to develop periodontitis than individuals with other haplotypes (OR=2.14, 95% CI =1.36-3.38). The analysis of haplotypes constructed from -845(T/C), - 738(T/A) and -353 (A/T) polymorphisms in the IL8 gene demonstrated association with susceptibility to periodontitis in Brazilian individuals. / Mestre

Orientador: Raquel Mantuanelli Scarel Caminaga / Resumo: Estudos realizados por nosso grupo investigaram polimorfismos no gene Interleucina 8 (IL8) em 500 indivíduos com e sem periodontite e identificaram que o haplótipo ATC/TTC conferiu 2 vezes maior suscetibilidade à doença periodontal crônica (DP) em comparação com o haplótipo ATT/TTC. Estudos clínicos revelaram que indivíduos portadores do haplótipo no gene IL8 associado à DP, mesmo com baixo desafio microbiano, desenvolviam periodontite de maneira mais exacerbada quando comparados aos indivíduos portadores do haplótipo não associado à DP. No entanto, a funcionalidade biológica desses haplótipos no gene IL8 ainda não foi investigada por meio de controlados ensaios in vitro. A proposta deste estudo é avaliar a funcionalidade dos haplótipos no gene IL8 em células imunes, incluindo a investigação por meio da regulação da expressão do gene repórter presente em plasmídeos recombinantes construídos artificialmente (constructos). Foi coletado sangue periférico de pacientes que carregam cada haplótipo e estimulados com mediadores inflamatórios e bactérias periodontopatogênicas para se avaliar a expressão gênica (mRNA, RT-qPCR), proteínas secretadas (Multiplex), perfil fenotípico celular (Citometria de fluxo), fagocitose e produção de espécies reativas de oxigênio (ROS) por macrófagos (Citometria de fluxo) e potencial de migração de linfócitos e neutrófilos. Constructos contendo cada haplótipo foram transfectados em linfócitos T humanos (células JM) e a expressão do gene repórter GFP fo... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Studies by this group investigated polymorphisms in the Interleukin 8 (IL8) gene in 500 individuals with and without periodontitis and identified that the ATC/TTC haplotype conferred 2-fold increased susceptibility to periodontal disease (PD) compared to the ATT/TTC haplotype. Clinical studies showed that individuals with IL8 haplotype associated with PD, even with low microbial challenge, responded in a more exacerbated manner when compared to individuals with not susceptible haplotype. However, the biological functionality of these haplotypes in the IL8 gene was not investigated by in vitro controlled assays. . The purpose of this study is to evaluate the functionality of haplotypes in the IL8 gene in immune cells including the investigantion of gene reporter expression present in the recombinant plasmids constructed artificially (constructs). Peripheral blood was collected from patients from each haplotype and stimulated with inflammatory mediators and periodontopathogenic bacteria to evaluate the gene expression (mRNA, RT-qPCR), secreted proteins (Multiplex), cellular phenotype profile (flow cytometry), phagocytosis and reactive oxygen species (ROS) by macrophages (flow cytometry) and migration potential of lymphocytes and neutrophils. Constructs containing each haplotype were transfected into Human lymphocytes (JM cells) and expression of the GFP reporter gene was evaluated using flow cytometry. The individuals with the susceptible haplotype (ATC / TTC) had higher levels... (Complete abstract click electronic access below) / Doutor

Les récepteurs Fc jouent un rôle important en permettant aux cellules de l’immunité d’interagir avec lesanticorps, notamment thérapeutiques. Dans cette thèse, nous montrons que des mécanismes de recombinaisonhomologue, de knock-in par insertion rétrovirale et de duplication segmentale ont permis l’acquisition chez lesPrimates (FCGR2A) puis chez les Hominidés (FCGR2C et FCGR3B) de gènes codant des récepteurs Fc ayantde nouvelles propriétés, tout en rendant instable ce cluster (variation de nombre de copies), et très complexeson analyse chez l’Homme. Grâce à une approche originale de pyroséquencage, nous sommes parvenus àétudier simultanément le polymorphisme allélique ORF/STOP du FCGR2C, ainsi que son nombre de copies.Nous avons ainsi révélé de nouveaux déséquilibres de liaison, s’ajoutant au déséquilibre FCGR3A-FCGR2Adont nous avons montré l’importance d’une prise en compte adaptée dans les études d’association avec laréponse aux anticorps thérapeutiques. Ces résultats devraient contribuer à améliorer le développement préclinique(pertinence des modèles animaux) et clinique (variabilité des effets) des anticorps thérapeutiques. / Fc receptors play an important allowing connexion between immune cells and antibody notably therapeutic. Inthis thesis, we have shown that homologous recombination events, knock-in by retroviral insertion andsegmental duplication led to the acquisition in primates (FCGR2A) then in Hominids (FCGR2C and FCGR3B)of genes coding for Fc receptors with new properties, led to genomic instability of the cluster (copy numbervariation) and to complex analysis in human. Through a original pyrosequencing approach, we have studiedsimultaneously ORF/STOP polymorphism and copy number variation of FCGR2C. We have also revealed newlinkage disequilibrium, additionnaly to FCGR3A-FCGR2A disequilibrium which we have shown theimportance of a suitable methdology in association studies with responses to therapeutic antibodies. Theseresults contribute to improve pre-clinical (of animal models) and clinical (variability effects) development oftherapeutic antibodies.

The common human GNB3 825C > T variant, which is present in 50% of the world’s chromosomes, has previously been shown to predispose individuals to hypertension, cardiac and neural disorders. This variant causes the production of a stable and gain of function protein Gβ_3S- This thesis describes the discovery of a novel D153del mutation that produces an unstable, loss of function, protein Gβ_3D in the recessively inherited, retinopathy globe enlarged (rge) chickens. This thesis also demonstrates that the normal Gβ₃ downstream phosphorylation signalling pathways are significantly altered in a tissue specific manner in rge chicken organs and in a human GNB3 825TT lymphoblast cell line. In rge tissues expressing Gβ_3D protein, the cAMP induced GRK2 phosphorylation activity is significantly altered. Moreover MAPK1 (ERK2) phosphorylation is significantly decreased compared to normal tissues. In contrast human 825TT cell lines expressing the Gβ_3S protein, showed enhanced cAMP induced GRK2 and MAPK (ERK1 and ERK2) phosphorylation activity. These results confirm previous findings of 825C > T Gβ₃ studies, that Gβ_3S is indeed a hyper-activating structural variant, in contrast to the D153del Gp3D is a classical recessively inherited non-functional mutation.

Introdução: Existem fortes evidências de um fator genético estar envolvido na etiologia do transtorno bipolar (TB), contudo a interação entre polimorfismos genéticos e alterações bioquímicas permanecem desconhecidas. O fator neurotrófico derivado do cérebro (BDNF) parece exercer um papel importante na patofisiologia do TB. Objetivos: O presente estudo tem por objetivo avaliar a associação do polimorfismo localizado no gene do fator neurotrófico derivado do cérebro (BDNF) e os níveis séricos desta substância em pacientes com transtorno bipolar. Material e Métodos: Foram selecionados 114 pacientes com TB tipo I de acordo com critério do DSMIV e 137 controles pareados por sexo, idade e anos de estudo para a análise do polimorfismo val66met do BDNF e do BDNF sérico. Suas associações foram medidas através da análise de variância (ANOVA).Resultados: Não houve diferenças significativas na freqüência dos genótipos do polimorfismo val66met do BDNF entre pacientes e controles (p>0.05; teste Qui-quadrado). Não foi encontrada associação entre o polimorfismo do gene do BDNF e o diagnósticode transtorno bipolar(eutímicos) nos níveis séricos de BDNF (p=0.34; ANOVA Fatorial) Conclusão: O polimorfismo do BDNF val66met parece não interferir no nível sérico de BDNF em pacientes bipolares em tratamento e controles sem TB, sugerindo que a variante BDNFMet não diminui a secreção constitutiva; possivelmente este polimorfismo do BDNF exerça alguma influencia nos níveis séricos do BDNF durante os episódios agudos da doença. / Introduction: There is strong evidence demonstrating that genetic inheritance is associated with higher susceptibility to bipolar disorder (BD) but the interaction between gene polymorphisms and biochemical changes remains largely unknown. The brainderived neurotrophic factor (BDNF) may play a role in the pathophysiology of BD. Objectives: The aim of the present study was to evaluate the association between BDNF polymorphism val66met and its serum levels in bipolar patients. Methods: One hundred and seven Caucasian type-I bipolar patients were recruited from the Bipolar Disorders Program and underwent Structured Clinical Interview for DSMIV- Axis I for diagnosis. The subjects were matched by age, gender and education with137 controls without BD. The association between BDNF serum levels and polymorphism was analysed by ANOVA. Results: No significant differences were found in the frequency of the BDNF val66met genotype or allele distribution between patients and controls (p>0.05; Chi-square test). We have found no significant interaction between BDNF polymorphism anddiagnostic status (bipolar disorder and controls) on serum BDNF levels (p=0.34; Factorial ANOVA) Conclusion: BDNF val66met polymorphism does not affect serum BDNF levels in a sample of mostly euthymic BD subjects currently on medication. Considering that the BDNFMet variant decreases only the activity-dependent but not the constitutive BDNF secretion, it is conceivable that BDNF polymorphism may exert some influence on serum BDNF levels during acute mood episodes.

The aim of this thesis was to analyze the polymorphism in position -371bp (related to ATG start codone) of MSTN gene, SNP in position 1984bp of MYF5 gene and influence of these polymorphisms on tenderness, water holding capacity, pH and color of meat. Samples (241) of bull meat of Czech pied cattle were used for analysis. PCR-RFLP method was applied to genotype MSTN and MYF5 genes. For polymorphisms detection was used restriction endonucleases DraI (for MSTN gene) and TaqI (for MYF5 gene). From established genotypes were computed their frequencies and alleles frequencies. The frequencies of genotypes in MSTN gene were AA=0,729, AB=0,258, BB=0,013 and alleles frequencies were A=0,858, B=0,142. The frequencies of genotypes in MYF5 gene were AA=0,181, AB=0,542, BB=0,278 and alleles frequencies were A=0,452 a B=0,548. Using statistical analysis, the influence of genotypes of MYF5 gene on the water holding capacity, pH and the color of meat was determined.