Introdução: Variações genéticas podem influenciar a relação entre ácidos graxos (AG) do plasma e concentração plasmática de biomarcadores inflamatórios. Objetivo: Verificar a associação entre polimorfismos de nucleotídeo único (SNP) presentes nos genes da proteína C reativa (PCR), fator de necrose tumoral (TNF) e interleucina (IL)-10 e AG do plasma e seus efeitos sobre a concentração plasmática de biomarcadores inflamatórios em um estudo de base populacional ISACapital. Métodos: Foram coletadas informações sociodemográficas, de estilo de vida, de atividade física (IPAQ longo), hábito de fumar e beber, bem como amostras de sangue de 281 indivíduos (20 a 59 anos), oriundos de um estudo de base populacional (ISA-Capital). A partir do plasma, foram determinadas as concentrações de IL1, IL6, IL8, IL10, TNF, IL12p70, adiponectina, PCR, proteína quimiotática para macrófagos solúvel (sMCP)1, molécula de adesão intercelular solúvel (sICAM)1 e molécula de adesão celular vascular solúvel (sVCAM)1 por meio da técnica multiplex de imunoensaio e o perfil de ácidos graxos por cromatografia gasosa. O DNA genômico foi extraído e realizada a genotipagem dos SNP presentes no gene da PCR (rs1205, rs1417938, rs2808630), TNF (rs1799964, rs1799724, rs1800629 e rs361525) e IL10 (rs1800871, rs1800896 e rs1800872) pela ténica TaqMan Open Array. Foi realizada análise multivariada de cluster com base nos 11 biomarcadores inflamatórios, permitindo agrupar os indivíduos em grupo inflamado e cluster não inflamado. Resultados: Os indivíduos que possuíam os genótipos GA+AA do SNP -238 G>A (rs361525) do gene do TNF- apresentaram concentrações plasmáticas de TNF-, IL-1, IL-6, IL-10 e IL-12 aumentadas quando comparados com indivíduos homozigotos dominantes. Além disso, homozigotos recessivos do SNP e/i boundary C>T (rs1554286) do gene da IL-10 apresentaram maior concentração plasmática de IL-1 e de TNF- e menor de MCP-1 em relação aos indivíduos homozigotos dominantes. O grupo inflamado apresentou idade, circunferência da cintura, pressão arterial significantemente maiores que o grupo não inflamado. Em relação aos AG do plasma, o grupo inflamado apresentou concentração plasmática de AG palmítico (C16:0), razões AG saturados (SFA)/AG ômega-6 (n-6) e SFA/ AG poli-insaturados (PUFA) e atividade estimada da enzima estearoil CoA desaturase (SCD) aumentadas e concentrações plasmática de PUFA, n- 6 e AG araquidônico (AA) e atividade estimada da enzima delta-5-dessaturase (D5D) reduzidas em comparação com o grupo não inflamado. Interações SNP-AG plasmáticos estatisticamente significante foram detectadas entre o SNP +1919 A>T (rs1417938) do gene da PCR e C16:1n-7, SFA/n-6 e SFA/PUFA; entre o SNP +3872 G>A (rs1205) do gene da PCR e C16:1n-7; entre o SNP e/i boundary C>T (rs1554286) do gene da IL-10 e atividade estimada da enzima D6D; e entre o SNP -1082 A>G (rs1800896) do gene da IL-10 e C18:0, C14:0 e atividade estimada da enzima D5D. Conclusão: Os SNP analisados possuem associações com biomarcadores inflamatórios e os ácidos graxos plasmáticos palmítico, SFA/n-6, SFA/PUFA, SCD-18 foram associados positivamente com um padrão inflamatório, enquanto PUFA, n-6, ácido araquidônico e D5D foram negativamente associados. Dentre as interações encontradas, o AG palmitoleico e a razão SFA/PUFA interagiram com +1919 A>T (rs1417938), e os AG esteárico e mirístico e D5D com -1082 A>G.. / Introduction: Genetics variation can influence the relation between fatty acids (FA) and inflammatory biomarkers levels. Objective: To verify the association between Single Nucleotide Polymorphisms (SNP) in adiponectin, C-Reactive Protein (CRP), Tumor Necrosis Factor (TNF)- and Interleukin (IL)-10 genes and plasma fatty acids and their effects to a systemic inflammatory pattern at a population-based study. Methods: Sociodemographics information, life style, physical activity (IPAQ long form), smoking and drinking habits, as well as blood samples of 281 individuals (20 years 59 years) participants of a population based study (ISA-Capital). Plasma IL1, IL6, IL8, IL10, TNF, IL12p70, adiponectin, CRP, soluble monocyte chemoattractant protein-1 (sMCP-1), soluble intercellular adhesion molecule-1 (sICAM-1) and soluble vascular cell adhesion molecule 1 (sVCAM-1) levels were measured using a multiplex immunoassay and the fatty acid profile was measured by gas chromatography. The DNA was extracted and genotyping of SNP in CPR gene (rs1205, rs1417938, rs2808630), TNF (rs1799964, rs1799724, rs1800629 e rs361525) and IL10 (rs1800871, rs1800896 e rs1800872) was analyzed by TaqMan Open Array. Multivariate cluster analysis was applied on 11 inflammatory biomarkers, allowing to group individuals in inflammatory (INF) or non-inflammatory (NINF) group. Results: Individuals with GA+AA genotypes of SNP -238 G>A (rs361525) of TNF- gene had higher levels of TNF-, IL-1, IL-6, IL-10 and IL-12 compared to GG genotype. Besides that, recessive homozygous of SNP e/i boundary C>T (rs1554286) of IL-10 gene presented higher level of IL-1 and TNF- and lower level of sMCP-1 in relation to dominant homozygous. The INF group had significantly higher age, waist circumference, blood pressure, and total cholesterol than NINF group. Concerning fatty acid profile, INF group had palmitic acid (C16:0), saturated fatty acid (SFA)/omega-6 (n-6) polyunsaturated fatty acid (PUFA) ratio, SFA/PUFA and estimated enzyme activity of stearoyl-CoA desaturase (SCD) levels higher and PUFA, n-6, arachidonic acid and estimated enzyme activity of delta-5 desaturase (D5D) levels lower than NINF group. Significantly interactions were found between of SNP +1919 A>T (rs1417938) of PCR and C16:1n-7, SFA/n-6 and SFA/PUFA; between SNP +3872 G>A (rs1205) of PCR gene and C16:1n-7; between SNP e/i boundary C>T (rs1554286) of IL-10 and estimated enzyme activity of delta-6 desaturase (D6D); and between SNP -1082 A>G (rs1800896) of IL-10 gene and C18:0, C14:0 and estimated D5D activity. Conclusion: The SNP analyzed have associations with inflammatory biomarkers, and plasma palmitic, SFA/n-6, SFA/PUFA, SCD- 18 were positively associated with inflammatory pattern, while PUFA, n-6, arachidonic acid and D5D were negatively associated. Interactions were founded with palmitoleic and SFA/PUFA with +1919.A>T (rs1417938), and stearic and myristic acids and D5D with 1082 A>G.

Orientador: Ana Paula de Souza Pardo / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-22T12:44:41Z (GMT). No. of bitstreams: 1 Guedes_RogerAntoniaci_M.pdf: 960151 bytes, checksum: c0a68bdbdf663d427c182550b1d9fa14 (MD5) Previous issue date: 2013 / Resumo: A periodontite é caracterizada pela inflamação do periodonto, o tecido de suporte dos dentes. Este processo inflamatório pode evoluir da fase aguda para a fase crônica, acarretando severa destruição dos tecidos periodontais como também grave perda de inserção dos dentes ao osso alveolar. É evidente que o acúmulo de patógenos periodontais sobre a superfície dos dentes desencadeia a doença, porém seu agravamento e severidade também são dependentes de fatores ambientais, socioeconômicos, tabagismo, condição de saúde sistêmica e carga genética dos indivíduos. Desta forma, vários pesquisadores têm se dedicado a estudar a influência dos polimorfismos genéticos sobre a suscetibilidade e/ou risco aumentado à doença periodontal, uma vez que estes podem exercer efeito sobre o prognóstico da periodontite crônica. Estudos têm relatado associação entre vários polimorfismos genéticos com a inflamação periodontal. Há ainda estudos em larga escala onde grande parte do genoma (GWA) foi investigado, relatando efeito da genética do hospedeiro sobre a resposta à doença. Desde modo, considerando a hipótese de associação entre periodontite e polimorfismos no gene SOCS1 que expressa uma proteína chave no controle da via intracelular JAK/STAT ativada por diversas citocinas pró-inflamatórias presentes na inflamação periodontal, nosso objetivo neste estudo foi estudar a frequência dos genótipos, alelos e haplótipos dos polimorfismos SOCS1-1478 (rs33989964) e SOCS1-820 (rs33977706) em grupo de indivíduos com saúde periodontal e indivíduos com periodontite crônica, na tentativa de observar associação entre variações no gene SOCS1 com a doença periodontal crônica. Para tal, DNA genômico foi purificado de células epiteliais bucais obtidas por meio de enxágue com dextrose a 3%. Após, os genótipos foram identificados com a utilização das técnicas de PCR/ RFLP/ eletroforese. Análises estatísticas possibilitaram a observação da associação do polimorfismo SOCS1 -820 (rs33977706) com os casos de periodontite crônica mais severa / Abstract: The periodontitis is characterized by the periodontium inflammation, the supporting tissue of the teeth. This inflammatory disorder might evolve from the acute to the chronic phase, causing severe periodontal tissue destruction as well as teeth insertion loss in the alveolar bones. It is evident that the accumulation of periodontal pathogens on the teeth surface origins the disease, yet its aggravation and severity also depend on socioeconomic and environmental factors, smoking, systemic health condition and the genetic background of the subjects. Thus, several researchers have focused their studies on the influence of the genetic polymorphisms in the susceptibility and/or increased risk to the periodontal disease, once they might play a role on the chronic periodontitis prognosis. Studies have shown association between several genetic polymorphisms and periodontal inflammation. Still, there are large-scale studies in which the majority of the genome (GWA) has been investigated, reporting genetic host roles as responses to the disease. Thus, considering the hypothesis of association between periodontitis and polymorphisms in the SOCS1 gene which expresses a key protein in the control of the intracellular JAK/STAT via activated by varied pro-inflammatory cytokines found in the periodontal inflammation, we aimed to study the frequency of genotypes, alleles and haplotypes of the polymorphisms SOCS1-1478 (rs33989964) and SOCS1-820 (rs33977706) in a healthy periodontal subjects group and in a chronic periodontal subjects group, attempting to observe association between variations in the SOCS1 gene and the chronic periodontal disease. To do so, genomic DNA was purified from mouth epithelial cells collected through 3% dextrose rinse. Afterwards, the genotypes were identified by the use of PCR/RFLP electrophoresis techniques. Statistical analysis allowed us to observe association of SOCS1-820 polymorphism (rs33977706) with more severe chronic periodontitis cases / Mestrado / Histologia e Embriologia / Mestre em Biologia Buco-Dental

Orientador: Lilian Maria de Castilho / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-21T01:20:23Z (GMT). No. of bitstreams: 1 Mota_MarizaAparecida_D.pdf: 4935868 bytes, checksum: e4f0e5bb4b8dde3d39725064ffee9de2 (MD5) Previous issue date: 2012 / Resumo: Alelos RHD que levam à redução da expressão do antígeno D na superfície dos eritrócitos podem levar à tipagem errônea como D- por técnica sorológica e podem causar imunização anti-D quando transfundidos em pacientes. A fim de determinar a ocorrência de tais alelos em doadores de sangue aparentemente D-, foi implementada técnica molecular de rotina utilizando um pool de DNAs de doadores de sangue. Um total de 2450 amostras previamente tipadas como D- foram testadas em pools de 10 amostras para o polimorfismo RHD específico, intron 4 e éxon 7. Nos pools que apresentaram resultado de PCR positivo, as amostras foram reavaliadas individualmente utilizando PCR exon-específico, métodos sorológicos e sequenciamento genético. Dentre as 2.450 amostras de doadores D- testadas, 101 (4,1%) carreavam o gene RHD. Foi identificada a presença de RHD não funcional (RHD'psi', RHD*CE(2-9)-D e RHD*CE(3-7)-D), diferentes alelos de D fraco tais como RHD*weak D type 1, RHD*weak D type 4.3, RHD*weak D type 5, RHD*weak D type 38 e RHD*DEL. Uma metodologia utilizando a técnica de PCR foi empregada como teste de rotina para o gene RHD utilizando pools de 10 amostras de DNA de doadores de sangue. Foi possível a integração da genotipagem RHD em programas de triagem de rotina utilizando pools de DNA. Como resultado, 19 (0,8%) dos doadores de sangue que carreavam fenótipos D fraco ou Del, com potencial de causar imunização anti-D em receptores, foram reclassificados como D+. Entretanto, seria necessário adaptar a estratégia de genotipagem RHD ao espectro de alelos prevalente em cada população / Abstract: RHD alleles leading to a reduced expression of D antigen of the red blood cell (RBC) surface may be erroneously typed as D negative by serology and may cause anti-D immunizations when transfused to recipients. We have therefore investigated the occurrence of such alleles among apparent D negative blood donors, molecular typing was implemented as a routine test using a pool of DNA. A total of 2,450 pretyped D negative samples was tested in pools of 10 for the RHD-specific polymorphism in intron 4 and exon 7. Samples in PCR positive pools were individually reevaluated by exon-specific PCRs, sequencing and serologic methods. Our results showed that among 2,450 serologically D negative blood donor samples tested, 101 (4.1%) carried the RHD gene. Nonfunctional RHD (RHD*'psi', RHD*CE(2-9)-D and RHD*CE(3- 7)-D), different weak D alleles such as RHD*weak D type 1, RHD*weak D type 4.3, RHD*weak D type 5, RHD*weak D type 38 and RHD*DEL were identified. We employed a PCR-based assay for RHD as a routine test using pools of 10 DNA blood donor samples. The integration of RHD genotyping into the routine screening program using pools of DNA samples was straightforward. As a consequence, 19 (0.8%) blood donors carrying a weak D and Del phenotypes with the potential of causing anti-D immunizations in recipients were reclassified as RhD positive. For each population, it would be necessary to adapt the RHD genotyping strategy to the spectrum of prevalent alleles / Doutorado / Clinica Medica / Doutora em Ciências

There is a continuous development of different crop varieties in the crop trade. The cultivated crops tend to be more and more alike which require an effective method for crop identification. Crop type and crop type purity has become a quality measure in crop trade both nationally and internationally. A number of well known quality attributes of interest in the crop trade can be correlated to the specific crop type and therefore it is of great importance to reliably be able to identify different crop varieties. It is well known from the literature that there exist genomic variations at the nucleotide level between different crop varieties and these variations might potentially be useful for automated variety identification. This project deals with the crop variety identification area where the possibilities of distinguishing between different wheat varieties are investigated. Experience from performing wheat variety identification at protein level has shown unsatisfactory results and therefore DNA-based techniques are proposed instead. DNA-based techniques are dependent upon the availability of sequence data from the wheat genome and some work has concerned examining the availability of sequence data from wheat. But the focus of the work has been on defining a method for computational detection of single nucleotide variations in ESTs from wheat and to experimentally test that method. Results from these experiments show that the method defined in this project detects polymorphic variations that can be correlated to variety variations

Missense single-nucleotide polymorphisms (mSNPs) in titin are emerging as a main causative factor of heart failure. However, distinguishing between benign and disease-causing mSNPs is a substantial challenge. Here, we research the question of whether a single mSNP in a generic domain of titin can affect heart function as a whole and, if so, how. For this, we studied the mSNP T2850I, seemingly linked to arrhythmogenic right ventricular cardiomyopathy (ARVC). We used structural biology, computational simulations and transgenic muscle in vivo methods to track the effect of the mutation from the molecular to the organismal level. The data show that the T2850I exchange is compatible with the domain three-dimensional fold, but that it strongly destabilizes it. Further, it induces a change in the conformational dynamics of the titin chain that alters its reactivity, causing the formation of aberrant interactions in the sarcomere. Echocardiography of knock-in mice indicated a mild diastolic dysfunction arising from increased myocardial stiffness. In conclusion, our data provide evidence that single mSNPs in titin's I-band can alter overall muscle behaviour. Our suggested mechanisms of disease are the development of non-native sarcomeric interactions and titin instability leading to a reduced I-band compliance. However, understanding the T2850I-induced ARVC pathology mechanistically remains a complex problem and will require a deeper understanding of the sarcomeric context of the titin region affected.

CD40 signals are required for productive immune responses but also play a role in autoimmune disease pathogenesis. The major goal of this research was to investigate the contribution of two receptors to the development of autoimmune disease: (1) LMP1, an oncogenic EBV-encoded mimic of CD40 and (2) a naturally-occurring polymorphism in CD40, P227A, which appears to confer LMP1-like properties to the CD40 receptor. Interestingly, hCD40-P227A is overrepresented in individuals of Mexican and South American descent. Although this allele is not directly associated with SLE incidence in Hispanic populations, patients of Hispanic ethnicity have a tendency toward increased severity of SLE symptoms, particularly lupus nephritis. This work reports the initial genetic description of CD40-P227A and characterizes its gain-of-function signaling properties in mouse and human B cells. In comparison to Wt-CD40 signaling, CD40-P227A signaling results in increased binding of TRAF3, TRAF5, and Act1, as well as enhanced secretion of IL-6, TNF-α, and Ig due to a selective hyperactivation of the JNK pathway. Whereas TRAF3 is normally a negative regulator of Wt-CD40 signaling, TRAF3 is a required positive regulator of CD40-P227A signaling as demonstrated in TRAF3-deficient B cells. LMP1 is an EBV-encoded CD40 mimic which signals in an amplified and sustained manner. Although EBV is latent in >90% of humans, EBV infection is associated with autoimmunity, particularly SLE. Upon flares of autoimmunity, EBV reactivates and LMP1 is expressed, yet the contribution of LMP1 to exacerbation of disease is unknown. LMP1 transgenic mice generated in our lab have an autoreactive phenotype but do not die early due to autoimmune disease. To test the hypothesis that LMP1 cooperates with other genes in the host to exacerbate autoimmunity, mCD40-LMP1 transgenic mice were bred to two lupus-prone strains of mice. LMP1 signaling was able to enhance the autoimmune phenotype of the B6.Sle1, but not the B6.Sle3 strain. These data suggest that LMP1 is redundant with genes within the Sle3 interval, but acts cooperatively with genes within the Sle1 interval to exacerbate autoimmunity. Together, the research foci of this dissertation examine how the CD40 pathways of B cell activation can be amplified and dysregulated, by either a viral mimic of CD40, a polymorphism in its signaling domain, or its cooperation with additional gene products. Differential usage of TRAF3 as a positive, rather than a negative, regulator of signaling appears to be one common mechanism by which this occurs. In conditions where enhanced CD40 signaling may be desirable such as during chronic infections, manipulation of TRAF3-CD40 signaling may serve to enhance immune responses. It is hoped that these studies can additionally reveal important information about the normal regulation of this powerful activation pathway.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23087号 / 医博第4714号 / 新制||医||1050(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 木村 剛, 教授 YOUSSEFIAN Shohab, 教授 大森 孝一 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Single nucleotide polymorphism (SNP) arrays are important tools widely used for genotyping and copy number estimation. This technology utilizes the specific affinity of fragmented DNA for binding to surface-attached oligonucleotide DNA probes. This thesis contemplates the variability of the probe signals of Affymetrix GeneChip SNP arrays as a function of the probe sequence to identify relevant sequence motifs which potentially cause systematic biases of genotyping and copy number estimates.

Indiana University-Purdue University Indianapolis (IUPUI) / Determining an individual’s genetic susceptibility in complex diseases like Alzheimer’s disease (AD) is challenging as multiple variants each contribute a small portion of the overall risk. Polygenic Risk Scores (PRS) are a mathematical construct or composite that aggregates the small effects of multiple variants into a single score. Potential applications of PRS include risk stratification, biomarker discovery and increased prognostic accuracy. A systematic review demonstrated that methodological refinement of PRS is an active research area, mostly focused on large case-control genome-wide association studies (GWAS). In AD, where there is considerable phenotypic and genetic heterogeneity, we hypothesized that PRS based on endophenotypes, and pathway-relevant genetic information would be particularly informative. In the first study, data from the NIA Alzheimer’s Disease Neuroimaging Initiative (ADNI) was used to develop endophenotype-based PRS based on amyloid (A), tau (T), neurodegeneration (N) and cerebrovascular (V) biomarkers, as well as an overall/combined endophenotype-PRS. Results indicated that combined phenotype-PRS predicted neurodegeneration biomarkers and overall AD risk. By contrast, amyloid and tau-PRSs were strongly linked to the corresponding biomarkers. Finally, extrinsic significance of the PRS approach was demonstrated by application of AD biological pathway-informed PRS to prediction of cognitive changes among older women with breast cancer (BC). Results from PRS analysis of the multicenter Thinking and Living with Cancer (TLC) study indicated that older BC patients with high AD genetic susceptibility within the immune-response and endocytosis pathways have worse cognition following chemotherapy±hormonal therapy rather than hormonal-only therapy. In conclusion, PRSs based on biomarker- or pathway- specific genetic information may provide mechanistic insights beyond disease susceptibility, supporting development of precision medicine with potential application to AD and other age-associated cognitive disorders.

Meningeome sind Tumore der Hirnhäute und stellen zirka 24-30% aller intrakraniellen Tumore dar. Obwohl sie in den meisten Fällen als solitär, langsam wachsend und benigne (WHO Grad 1) beschrieben werden, ist ihr ausgeprägtes Rezidivverhalten die größte Herausforderung in der Therapie. Bisherige Arbeiten verwendeten zur genetischen Analyse von Meningeomen meist Untersuchungstechniken mit eingeschränkter (molekular-)zytogenetischer Aussagekraft. Mit der Kombination der Methoden Giemsa-Bandendarstellung (GTG-Bänderung), Spektrale Karyotypisierung (SKY-Technik), Fluoreszenz in situ Hybridisierungstechniken (FISH-Analyse) und molekulare Karyotypisierung unter Verwendung von 100K beziehungsweise 6.0 SNP-Arrays (SNP-A Karyotypisierung) sollte es möglich sein, in effizienterer Form bislang unentdeckte chromosomale Aberrationen zu identifizieren und weiterführende tumormechanistische Hinweise zu erhalten. In der vorliegenden Arbeit wurde zunächst ein multipel aufgetretenes Meningeom mit zwei Tumoren unterschiedlicher Malignität (1 WHO Grad 1; 1 WHO Grad 2) analysiert, anschließend erfolgte die Untersuchung einer Gruppe von 10 Meningeomen (5 WHO Grad 1; 5 WHO Grad 2). Bisher nicht beschriebene Aberrationen wie ein dizentrisches Chromosomen 4, die parazentrische Inversion im chromosomalen Bereich 1p36 und die balancierte reziproke Translokation t(4;10)(q12;q26) wurden detektiert. Die genomweite SNP-A Karyotypisierung ermöglichte neben der genaueren Betrachtung der zytogenetischen Ergebnisse die simultane Analyse von Blut und Tumor-DNA der Patienten und lieferte Hinweise auf konstitutionelle Aberrationen. Es zeigte sich eine signifikante Anreicherung von rekurrenten Regionen kopienneutraler Verluste der Heterozygotie als Hinweis auf das Vorliegen potenzieller segmentaler Uniparentaler Disomie (UPD) jeweils in Blut und Tumor der Patienten. Außerdem wurden nur im Tumor befindliche potentielle rekurrente segmentale UPD Regionen detektiert. Die weitere Analyse der konstitutionellen sowie somatischen segmentalen UPD hinsichtlich ihrer Rolle im Rahmen der Tumorgenese ist eine wichtige Aufgabe für die Zukunft.