Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 5 uppsatser.

Diss. (sammanfattning) Uppsala : Univ., 2004. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 5 uppsatser.

Thesis (Ph. D.)--Case Western Reserve University, 2006. / [School of Medicine] Department of Epidemiology and Biostatistics. Includes bibliographical references. Available online via OhioLINK's ETD Center.

Persistent infection of the cervix with high risk (HR) types of Human Papilloma Virus (HPV) (HR-HPV) can result in precancerous lesions and cancers. However, most HPV infections can be cleared naturally by the immune response without causing disease. Although genetic variations have long been considered as the main explanation for individual heterogeneity in cancer susceptibility, the underlying mechanisms remain unclear. In this project, a panel of routinely taken clinical samples was assessed for 32 rationally selected SNPs with allele frequency related to disease outcome using the Taqman® OpenArray® system. The panel incorporated 475 HR-HPV negative, cytologically-normal cervical samples, 413 HR-HPV positive cervical high grade squamous intraepithelial lesion (HSIL) cases and 62 HR-HPV positive cervical cancers. Two SNPs, rs2234671 and rs2623047, were found with significant differences between HR-HPV negative, cytologically-normal samples and HR-HPV positive cervical HSIL cases. In the validation step, these two SNPs were further genotyped in the same set of samples using TaqMan® SNP genotyping assay and/or LightSNiP assay and in additional samples including 83 HR-HPV positive, cytologically-normal cervical samples, 21 HR-HPV positive cervical cancer cases, 129 HR-HPV positive vulval intraepithelial neoplasia cases and 23 HR-HPV positive vulval cancer cases. Statistical analysis was then performed based on pooled and re-grouped genotyping data of the above-mentioned samples under different genetic models so as to evaluate the associations with different stages in the disease process. After validation, SULF1 rs2623047 revealed a strong significant association with the susceptibility to HR-HPV infection but not with the development of high-grade squamous intraepithelial lesion and the progression to cervical cancer. CXCR1 rs2234671, by contrast, was associated with the progression of HR-HPV-related cancers and the minor allele CXCR1 827C was significantly enriched in HPV16 positive cancers. CXCR1 is a receptor for the chemokine CXCL8/IL-8 and CXCR1 rs2234671 leads to a serine to threonine change in an extracellular loop of the receptor. Functionally, the CXCR1 827C allele was shown to enhance cell motility in response to IL-8 stimulation in a chemotaxis assay with transiently transfected fibroblasts (HEK293 cells) and also in a wound healing assay with stably transduced cervical cancer (CaSki) cells. In addition, significantly increased cell proliferation upon IL-8 treatment was observed in two cervical cancer derived cell lines, CaSki and SiHa, transduced with CXCR1-827C allele, but not in their CXCR1 827G transduced counterparts. These findings suggest that SULF1 rs2623047 and CXCR1 rs2234671 may be genetic risk factors for HR-HPV-related cervical disease and CXCR1 rs2234671 might affect HR-HPV-related cancer susceptibility by functionally altering IL-8-CXCR1 signalling. This information has potential for use in the risk stratification of HR-HPV infected women and may also suggest new therapeutic targets to be exploited for treatment of cervical cancer patients.

The majority of human proteins are post-translationally modified by covalent addition of one or more complex oligosaccharides (glycans). Alterations in glycosylation processing are associated with numerous diseases and glycans are attracting increasing attention both as disease biomarkers and as targets for novel therapeutic approaches. Using a recently developed high performance liquid chromatography (HPLC) method for high-throughput glycan analysis, genome-wide association studies (GWAS) of 33 directly measured and 13 derived N-glycan features were performed in 3533 individuals from four European isolated populations. Polymorphisms at six loci were found to show genome-wide significant association with plasma concentrations of N-glycans. Several of these gene products have well characterised roles in glycosylation, however, SLC9A9 and HNF1A were two of the novel findings. Subsequent work performed by collaborators found HNF1A to be a “master regulator” of genes involved in the fucosylation of plasma N-glycans. Additionally, this work led to the discovery that N-glycans could act as biomarkers to discriminate HNF1A-MODY from type 1 and type 2 diabetes mellitus (T1D, T2D) patients. After the success of the total plasma N-glycan GWAS, it was thought that stronger and more biologically interpretable associations may be found from the investigation of N-glycans isolated from a single protein. Glycosylation of immunoglobulin G (IgG) influences IgG effector function by modulating binding to Fc receptors. To identify genetic networks that govern IgG glycosylation, N-linked IgG glycans were quantitated using ultra performance liquid chromatography (UPLC) in 2247 individuals from the same four European populations from the previous study. GWAS of the 77 N-glycan measures identified 15 loci with a p-value < 5x10-08. Four loci contained genes encoding glycosyltransferases, while the remaining loci contained genes that have not previously been implicated in protein glycosylation. However, most have been associated with autoimmune and inflammatory conditions and/or hematological cancers. Several high-throughput methods for the analysis of N-glycans have been developed in the past few years but thorough validation and standardization of these methods is required before significant resources are invested in large-scale studies. To this end, four of these methods were compared, UPLC, multiplexed capillary gel electrophoresis (xCGE), and two mass spectrometric (MS) methods, for quantitative profiling of N-glycosylation of plasma IgG in a subset of 1201 individuals recruited from two of the cohorts used in the previous GWAS studies. A “minimal” dataset was compiled of N-glycan structures able to be measured by all four methods. To evaluate their accuracy, correlations were calculated for each structure in the minimal dataset. Additionally, GWAS was performed to test if the same associations would be observed across methodologies. Chromatographic methods with either fluorescent or MS-detection yielded slightly stronger associations than MS-only and xCGE, but at the expense of lower levels of throughput. Advantages and disadvantages of each method were identified, which should aid in the selection of the most appropriate method for future studies. This work shows that it is possible to identify new loci that control glycosylation of plasma proteins using GWAS and the potential of N-glycans for biomarker development. It also provides some guidelines for methodology selection for future studies of N-glycans.

Orientadores: Eliana Cotta de Faria, Helena Coutinho Franco de Oliveira, Daniel Zanetti Scherrer / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-26T20:49:38Z (GMT). No. of bitstreams: 1 Zago_VanessaHelenadeSouza_D.pdf: 5059555 bytes, checksum: 854d9d1d1674a14d2ebcf5798acc31b3 (MD5) Previous issue date: 2015 / Resumo: Dado o importante papel desempenhado pelos transportadores ATP binding cassete A1 (ABCA1), G1 (ABCG1), G5 (ABCG5), G8 (ABCG8) e pelo scavenger receptor class B type I (SR-BI) para a homeostase corpórea de colesterol e desenvolvimento da aterosclerose, este trabalho se propôs a: (i) investigar a relação dos polimorfismos rs2275543 (ABCA1), rs1893590 (ABCG1), rs6720173 (ABCG5), rs6544718 (ABCG8) e rs5888 (SCARB1) com gênero, idade e índice de massa corpórea (IMC) e suas interações sobre variáveis clínicas e bioquímicas (n=654); (ii) determinar a repercussão destes polimorfismos sobre os parâmetros estudados na população total e de forma gênero-específica (n=590) e (iii) avaliar se os miRNAs hsa-miR-33a e hsa-miR-128a são diferencialmente expressos em um subgrupo da população (n=51) e averiguar sua associação com as concentrações plasmáticas do colesterol da lipoproteína de alta densidade (HDL-C), aterosclerose subclínica e expressão de ABCA1, ABCG1 e SCARB1. Para tanto, foram selecionados voluntários normolipidêmicos e assintomáticos, de ambos os gêneros, com idade entre 20 e 75 anos. Dados clínicos e antropométricos foram obtidos, assim como sangue venoso periférico para as determinações bioquímicas e extração de DNA e RNA. O subgrupo de 51 voluntários foi classificado de acordo com HDL-C (mg/dL) em hipoalfalipoproteinêmicos (hipo, HDL-C?39), hiperalfalipoproteinêmicos (hiper, HDL-C?68) e controles (CTL, HDL-C?40<68) e determinadas a espessura íntimo-medial das artérias carótidas e proteínas relacionadas ao metabolismo de HDL. Determinamos que o rs1893590 interage com a idade e o IMC, modulando as concentrações de HDL-C, bem como o tamanho e volume da partícula, sugerindo que este pode modificar seu metabolismo e composição. Nas análises comparativas o rs2275543 apresentou efeitos diferentes, porém benéficos para ambos os gêneros; adicionalmente, o rs6720173 determinou um fenótipo lipoproteico proaterogênico no gênero masculino, enquanto as variantes rs5888 e rs6544718 repercutiram sobre marcadores de adiposidade no gênero feminino. A análise dos cinco polimorfismos nesta população fornece evidências de que estes atuam em diferentes vias do metabolismo lipoproteico, e tem na maioria dos casos características gênero-específicas. Adicionalmente, a avaliação da expressão de hsa-miR-33a, hsa-miR-128a, ABCA1, ABCG1 e SCARB1 revelou que os indivíduos hiper apresentam um aumento da expressão de ABCA1 e ABCG1 em relação ao grupo CTL, somado a uma redução de 72% na expressão do hsa-miR-33a; em conjunto, estes resultados indicam um potencial papel regulatório deste miRNA em indivíduos assintomáticos, possivelmente contribuindo para o aumento do efluxo e do transporte reverso de colesterol / Abstract: Given the important role played by ATP binding cassete transporters A1 (ABCA1), G1 (ABCG1), G5 (ABCG5), G8 (ABCG8) and by scavenger receptor class B type I (SR-BI) on body cholesterol homeostasis and atherosclerosis development, this study proposes to: (i) investigate the relationship of polymorphisms rs2275543 (ABCA1), rs1893590 (ABCG1), rs6720173 (ABCG5), rs6544718 (ABCG8) e rs5888 (SCARB1) with gender, age and body mass index (BMI) and its interactions with clinical and biochemical variables (n=654); (ii) determine the effects of these polymorphisms on the studied parameters in the total population and in a gender-specific manner (n=590) and (iii) evaluate if miRNAs hsa-miR-33a e hsa-miR-128a are differentially expressed in a subgroup of the population (n=51) and verify its association with plasma levels of high-density lipoprotein cholesterol (HDL-C), subclinical atherosclerosis plus ABCA1, ABCG1 and SCARB1 expression. Thus, normolipidemic and asymptomatic volunteers from both genders, with ages ranging from 20 to 75 years were selected. Clinical and anthropometric data were obtained, as well as peripheral venous blood for biochemical determinations plus DNA and RNA extraction. The subgroup of 51 individuals was classified according HDL-C (mg/dL) in hypoalphalipoproteinemics (hypo, HDL-C?39), hyperalphalipoproteinemics (hyper, HDL-C?68) and controls (CTL, HDL-C?40<68); then, were determinated the carotid intima-media thickness and proteins related to HDL metabolism. The polymorphism rs1893590 interacts with age and BMI, modulating HDL-C levels as well as the particle size and volume, suggesting its role on HDL metabolism and composition. Comparative analysis demonstrated that rs2275543 has different, but beneficial repercussions in both genders; furthermore, rs6720173 determines a pro-atherogenic lipoprotein profile in males, while the variants rs5888 and rs6544718 affect positively adiposity markers in females. The analyses of the five studied polymorphism in this population provide evidences of its role in several pathways of lipoproteins metabolism, in most cases in a gender-specific manner. Moreover, the ABCA1, ABCG1, SCARB1, hsa-miR-33a and hsa-miR-128a expression analysis revealed that hyper group presents a significant increase of ABCA1 and ABCG1 expression in relation to the control group; additionally, hsa-miR-33a decreased by 72%. Together, these results indicate a potential regulatory role of this miRNA in asymptomatic individuals, probably contributing to increased cholesterol efflux and reverse cholesterol transport / Doutorado / Ciencias Biomedicas / Doutora em Ciências Médicas

Ziel der vorliegenden Arbeit war die Charakterisierung von Punktmutationen in ifng und ncam1 in Hinblick auf eine veränderte Funktionalität von NK-Zellen bei der Interaktion mit A. fumigatus. Die gewonnenen Erkenntnisse sollen langfristig zur Verbesserung der Diagnostik, Prophylaxe und Therapie einer Invasiven Aspergillose, die zum Beispiel im Rahmen einer Stammzelltransplantation auftreten könnte, beitragen. In dieser Arbeit wurde die DNA von zwanzig gesunden Spendern auf einen ifng-SNP (rs2069705) und einen ncam1-SNP (rs10502171) untersucht. Von je drei ausgewählten Spendern mit SNP und sechs Kontrollspendern wurden NK-Zellen isoliert. Diese wurden unstimuliert belassen, mit Interleukin 2/15 oder A. fumigatus stimuliert. Bei der Versuchsreihe zum ifng-SNP wurde eine qPCR zur Ermittlung der relativen Expression von ifng und ccl4, bei den Versuchen zum ncam1-SNP eine durchflusszytometrische Analyse zur Messung der Expression verschiedener Oberflächenmarker durchgeführt. Bei beiden wurde mittels ELISA die Freisetzung von IFN-gamma bzw. CCL4/MIP-1ß bestimmt. Die in dieser Arbeit gewonnenen Ergebnisse zum ifng-SNP lassen vermuten, dass das Vorliegen dieses ifng-SNP keine durch NK-Zellen vermittelten Auswirkungen auf das Risiko der Patienten, an einer Invasiven Aspergillose zu erkranken, hat. In Bezug auf den ncam1-SNP konnte die Hypothese bestätigt werden, dass der SNP die Interaktion zwischen der NK-Zelle und A. fumigatus verändert. Der SNP korreliert zwar mit einer erhöhten Grundaktivierung von NK-Zellen, jedoch auch mit einem schwächeren Aktivierungspotential bei Stimulation mit dem Pilz. / The aim of this thesis was the characterization of point mutations in ifng and ncam1 with respect to an altered functionality of NK cells during interaction with A. fumigatus. In the long term, the findings should contribute to the improvement of the diagnosis, prophylaxis and therapy of invasive aspergillosis, which can occur, for example, after stem cell transplantation. In this work, the DNA of twenty healthy donors was examined for one ifng-SNP (rs2069705) and one ncam1-SNP (rs10502171). NK cells were isolated from three donors for each SNP and six control donors. These were left unstimulated, stimulated with interleukin 2/15 or A. fumigatus. In the ifng-SNP series qPCR was performed to determine the relative expression of ifng and ccl4, in the ncam1-SNP series flow cytometric analysis was performed to measure the expression of different surface markers. In both cases the release of IFN-gamma and CCL4/MIP-1ß was determined by ELISA. The results obtained in this study on ifng-SNP suggest that the presence of this ifng-SNP has no NK cell mediated effects on the risk of patients suffering from invasive aspergillosis. With regard to the ncam1-SNP, the hypothesis that the SNP alters the interaction between the NK cell and A. fumigatus was confirmed. The SNP correlates with an increased basic activation of NK cells, but also with a weaker activation potential when stimulated with the fungus.

Altered intracellular calcium homeostasis and oxidative stress are involved in the pathophysiology of bipolar disorder (BD)-I. To explore the genes contributing to these abnormalities, we examined the association with BD of the iPLA2β (PLA2G6), a signaling enzyme that mobilizes the arachidonic acid signaling cascade and activates oxidative stress, and assessed whether it interacts genetically with type 2 transient receptor potential channel gene (TRPM2), an oxidative stress-responsive calcium channel implicated both functionally and genetically in BD-I. Two tag single nucleotide polymorphisms rs4375 and rs3788533 in iPLA2β were genotyped in 446 White case-control individuals and 296 BD families using a 5′-nuclease TaqMan assay. The results were analyzed using χ-test and transmission disequilibrium tests, and Haploview. In a secondary analysis, we tested gene-gene interactions between TRPM2 and iPLA2β on BD vulnerability by logistic regression using a case-only design in PLINK. iPLA2β-rs3788533 showed a borderline association with BD-I in patients with a history of psychosis in both case-control and family designs. Association with BD as a whole was observed in the family study (significant over transmissions of rs3788533-allele C, P=0.015, PBonferroni=0.03, TDTPHASE). A borderline interaction was found between rs749909 within TRPM2 and rs4375 within iPLA2β (Puncorrected=0.009), on the basis of the case-only design analyzed with PLINK. A significant association of iPLA2β variants with BD-I and a trend gene-gene interaction between iPLA2β and TRPM2 provides additional support for the notion that genetic variation in these two functionally implicated candidates contributes toward the risk and pathophysiology of this illness.