Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser + 1 appendix.

Black wattle (Acacia mearnsii), which belongs to the genus Acacia, is one of the many species of trees or hardwoods grown commercially in South Africa. Black wattle is a species indigenous to Australia and was introduced into South Africa by the van der Plank brothers in 1864. These trees are grown in South Africa because of its tannin-rich bark, the extract of which is used by the leather tanning industry. Black wattle is also grown for its timber, timber products and pulp. The introduction and cultivation history of black wattle suggests that the South African plantations contain limited genetic variation with relatedness amongst groups estimated to be high, thus implying a narrow genetic base in the South African black wattle population. In this investigation, Random Amplified Polymorphic DNA (RAPD) was used to estimate the genetic variation between seven different black wattle groups. A total number of 34 individuals obtained from different areas in South Africa were examined; Piet Retief (group 47 and 50: half-sibs), Kumbula (group 85: unrelated individuals), Howick (group 400: unrelated individuals) and an unknown area (groups 88, 89, 91: full-sibs). As this investigation was the first of its kind, a DNA isolation method as well as a PCR-RAPD protocol had to be modified. Total genomic DNA was successfully extracted using the CTAB DNA extraction method. This method removed large amounts of tannin present in the cells of the black wattle leaves and extracted high quality DNA to conduct between 50-100 RAPD reactions. The DNA purities ranged from 0.1 to 1.8, with an average of 1.46. A total of fourteen 10-mer RAPD primer sequences were randomly selected from the Operon Technologies primer list A, and tested in this investigation. Of the 14 primers used, only nine primers produced clear, single and repeatable bands. Therefore nine primers were selected for subsequent analyses. Ninety one loci that generated bands ranging from 300-3050 base pairs were produced. Seven to 13 loci per primer were generated. A total of 95.6 % of the loci were polymorphic. The overall expected mean heterozygosity (H = 0.3) obtained in this study was high in comparison to other studies conducted on acacias. The high levels of genetic variation were attributed to mating systems, dissortative mating and geographic distribution. The statistical packages POPGENE and ARLEQUIN were used to analyse the RAPD fingerprints. The genetic measures, Nei's diversity and Shannon's Information Index, showed that there was greater diversity exhibited (Nei's gene diversity = 32.09 % and Shannon's = 48.31 %), in the whole population than in each of the groups (with average of Nei's gene diversity = 20.33 % and Shannon's = 34.64 %). With regards to individual group analyses, low levels of genetic variation was obtained in group 400 (unrelated), from the Howick region, and group 85 (unrelated), from the Kumbula region, (mean 0.14 and 0.17 respectively). The low genetic values were attributed to limited gene exchange occurring in these two areas, bottlenecks and selection pressures. Groups 88, 89 and 91, from the unknown region (full-sib groups), were the most variable in comparison to the other groups, with means of (0.27,0.24 and 0.18 respectively). These high genetic variation values could be due to the fact that gene migration could have occurred between these groups and others in the area. It is thought that most acacias are insect-pollinated and this could have lead to gene migration between groups or populations, thereby explaining the high mean values. The gene flow obtained for the seven groups (FST = 0.174) indicated that great genetic differentiation existed in this population of black wattle studied. This value is higher in comparison to other woody species; however it is similar to other acacia species. UPGMA cluster analysis using Nei's unbiased genetic distance, revealed four distinct clusters of groups corresponding to the distribution areas represented in this study. The Howick (group 400: unrelated) and Kumbula (group 85: unrelated) were more closely related to each other than to the other groups, since both these groups are from Natal. The Piet Retief groups (groups 47 and 50: half-sibs), branched-off together, indicating that they are distinct from the other groups. The pairwise analysis of identity showed that the relationship between the group from Howick (group 400: unrelated) and all the other groups from the other regions was the lowest, ranging from 64 % to 79 %. The relationship between all the groups beside the group from Howick (group 400: unrelated) was reasonably high, ranging from 78 % to 90 %. This distance displayed by group 400 (unrelated) from Howick in relation to the groups, is attributed to the fact that it is frost resistant and the other groups not. Genetic variation was also detected and partitioned, between and within groups, by Analysis of Molecular Variance (AMQVA). Majority of the variation existed within groups (82.65 %) but significant differentiation was recorded between groups (17.44 %). This high level of within group differentiation may be explained by many aspects, such as the species breeding system, genetic drift or genetic isolation of groups or populations. The application of RAPD fingerprinting in black wattle has provided a more in depth understanding of the genetic variation residing in the South African population. The results achieved implementing this technique has shown that significant genetic variation exists within the black wattle population in South Africa. The results obtained in this study are also important since it is contrary to the expectation that the black wattle population in South Africa has low genetic variation. This knowledge is of great value to genetically discriminate between individuals or groups, to improve the selection of superior genotypes and allowing improved quality control in breeding programmes and seed orchard management. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2005.

The generation of memory T cells following primary exposure to a pathogen is a critical feature of the vertebrate immune system which has evolved as a protective mechanism in order to defend the host against repeated assaults by the patnogen. Memory T cells are long-lived, undergo rapid proliferation upon re-activation, mediate a robust secondary response and clear the pathogen much more efficiently. These aspects have made the generation of memory T cells an attractive goal for the production of both prophylactic and therapeutic vaccines. However, the degeneracy of the T cell receptor, whereby a given T cell recognizes more than one epitope, allows the T cell to be modulated by epitope variants which could be self-ligands, ligands related to the original epitope but altered in sequence, or completely unrelated epitopes. Experiments in both mice and humans show that such cross-reactive stimulation of memory T cells results in complete, partial, or no activation of T cells, and in some cases, even alters the functional identity of the T cell (for example, T helper 1 cells start secreting IL-4, IL-5 and become part of a T helper 2 response). In the context of secondary infection of immune organisms with pathogens containing mutated or related T cell epitopes, such alterations at the cellular level translate into drastic changes in the overall clinical outcome of the infection. Thus, the presence of cross-reactive T cells in the memory population implies that the protective or pathologic nature of the secondary immune response is a consequence of the host's infection history. Although several murine models of heterologous infection resulting in altered pathological outcome have been studied, the exact immune correlates of protection versus immunopathology are still unclear. This thesis addresses this issue in dengue virus infections in humans. Dengue fever (DF) and Dengue Hemorrhagic Fever (DHF) are two disease manifestations caused by infections of humans by the dengue viruses. These are a group of 4 serologically distinct flaviviruses (D1-4) which often co-circulate among endemic populations. While primary infection with any of the four serotypes can result in the more severe clinical disease characterized by DHF, epidemiological data from several outbreaks show that 80% - 90% of DHF cases occur among individuals with secondary infection. This implies that prior immunity to dengue is actually a risk factor for developing severe disease. In these DHF cases, there are increased numbers of CD69+ CD8+ T cells in circulation, with increases observed in the frequency of epitope-specific T cells, and the serum levels of several T cell produced cytokines, chemokines, and immune receptors are highly elevated. Since the four serotypes share 65% - 75% amino acid sequence homology, the possibility that unconserved T cell epitope sequences stimulated cross-reactive responses was borne out in in vitroexaminations. In these studies, peripheral blood mononuclear cells (PBMC) and cloned T cells from both vaccinated and infected donors contained large populations of memory T cells that were cross-reactive for heterologous viral serotypes in proliferation and CTL assays. These data suggest that the severity of disease seen in DHF patients can be attributed to an immunopathologic secondary response during heterologous infection, and highlight a role for serotype cross-reactive T cells in this process. This thesis addresses the hypothesis that the recognition of the natural variants of dengue virus T cell epitopes by serotype cross-reactive CD8+ T cells of a dengue-immune donor results in an altered secondary response profile, with the changes reflected in both the quantitative and qualitative nature of the response. In order to compare the functional profile of the secondary response of dengue-immune PBMC re-activated with heterologous serotypes, we focused on a panel of 4 donors who were vaccinated with live attenuated monovalent vaccines corresponding to D1, D2, or D4 serotypes. We screened a panel of peptides predicted to bind to HLA-A*0201 for cytokine responses and identified 4 novel epitopes that were highly immunogenic in all four donors. Direct ex vivo stimulation of donor PBMC with the heterologous sequences of these epitopes also showed sizeable serotype cross-reactive T cell populations. CFSE- and intracellular staining for cytokines and chemokines showed that these cross-reactive T cells not only expanded but also produced IFNγ, TNFα, and MIP-1β. Multi-parameter staining revealed functionally diverse populations comprised of single cytokine (IFNγ+, TNFα+, MIP-1β+, double cytokine (IFNγ+TNFα+, IFNγ+MIP-1β+, TNFα+MIP-1β+, and triple cytokine (IFNγ+TNFα+MIP-1β+ secreting sub-sets. Stimulation with the epitope variants altered the magnitude of the overall response as well as the relative sizes of these sub-sets. The patterns of responses revealed the effects of epitope immunogenicity, infection history and donor-specific variability. All 4 donors showed the highest cytokine response to a -single epitope (NS4b 2353). The same two peptide variants (D2 NS4a 2148 and D3 NS4b 2343) induced the highest response in all 4 donors regardless of the serotype of primary dengue infection. Interestingly, the epitope variants which showed the highest immunogenecity in our donors corresponded to the D2 and D3 serotypes which have been documented as being more virulent as well as a viral risk factor for DHF. In one donor, the response to all peptide variants was dominated by the same cytokine sub-sets. These data suggested that the dengue-immune memory T cell repertoire was functionally diverse and underwent alterations in size after secondary stimulation. Therefore, we also investigated the effect of epitope variants on dengue-specific CD8+T cell clones isolated from vaccinated and infected donors in order to determine if epitope variants induced altered functional outcomes at the clonal level. The epitope variants functioned either as strong agonists (particularly the D2 and D3 sequences), partial agonists, or null ligands. Some variants were able to induce cytolysis but not other effector functions at low concentrations. The variant ligands also influenced the hierarchy of cytokine responses within each clone. The third part of this thesis focused on the characterization of the frequency and phenotypic profile of epitope-specific CD8+ T cells in patients with DHF and DF at different times in the disease course in order to better understand the kinetics of the response and delineate any differences between the immune profile of severe vs. moderate disease. Tetramer staining for a previously identified HLA-B*07 restricted epitope was combined with staining for activation markers (CD69, CD38, HLA-DR), homing receptors (CCR7, CD62L), and programmed death receptor 1 (PD-1). The DHF subjects had early T cell activation with higher frequencies of tetramer+CD69+ cells as compared to DF subjects, in whom T cell frequencies peaked around the time of defervescence. While each subject had a unique phenotypic profile of tetramer+ cells, there was a difference between DF and DHF subjects in terms of CCR 7 expression; all subjects expressed low levels of CCR7 during acute illness but only the DHF subjects did not show upregulation of CCR7 on tetramer+ cells during convalescence. These data suggest that there is a sustained alteration in memory phenotype in those who recovered from severe dengue disease. A majority of the tetramer+cells also expressed PD-1 during acute illness but not during convalescence. Double-staining with variant tetramers allowed us to directly visualize serotype cross-reactivity of the epitope-specific population, and showed that secondary stimulation did induce the expansion of cells with low avidity for that secondary serotype and higher avidity to the variant. Furthermore, the ratios of these sub-sets changed during the course of the response. Taken together, these studies suggest that the immune response to heterologous secondary dengue infection is mediated by a heterogeneous population of serotype-cross reactive T cells that have different functional avidities to epitope variants and is influenced by the serotype of the secondary infection as well as the prior infection history of the individual. The preferential expansion of clones which secrete IFNγ but not inflammatory MIP-1β or TNFα or a repertoire characterized by a higher ratio of cytolytic to cytokine producing clones could limit immune mediated damage while efficiently clearing the virus. This information will be useful in the design of vaccine strategies aimed at inducing protective cross-reactive responses against all 4 dengue serotypes while preventing immunopathological outcomes following secondary infection.

Made available in DSpace on 2016-01-26T12:51:55Z (GMT). No. of bitstreams: 1 vaniapiatto_tese.pdf: 1479841 bytes, checksum: 96e04c84851d635167398b1a4196d2fe (MD5) Previous issue date: 2003-11-28 / Mutações no gene que codifica a proteína conexina 26 têm contribuído para a maioria das deficiências auditivas pré-lingual, sensorioneural não-sindrômicas recessivas. Uma mutação específica, a 35delG, é a mais freqüente das mutações detectadas no gene GJB2 nos vários grupos étnicos estudados. O objetivo foi determinar a prevalência de mutações no gene GJB2 em pacientes com deficiência auditiva sensorioneural não sindrômica, do Serviço de Otorrinolaringologia da FAMERP, em São José do Rio Preto, SP. Trinta e três casos-índice foram avaliados por exames fisico e complementares para excluir formas sindrômicas e causas ambientais da deficiência auditiva e, pela reação em cadeia da polimerase alelo-específico (AS-PCR) para a detecção da mutação 35delG. Os pacientes heterozigotos para a mutação 35delG e aqueles que não tiveram a mutação detectada foram submetidos, posteriormente, ao PCR para detecção da mutação A(GJB6-D 1381830) e ao sequenciamento automático direto para análise da região codificante do gene GJB2. A mutação 35delG foi detectada em 27,3% dos casos-índice (9/3 3) ou em 2 1,2% dos alelos (14/66). A mutação A(GJB6-DI3SI 830) foi encontrada em um (3,0%) caso-índice heterozigoto 35delG. O seqüenciamento direto nos casos-índice heterozigotos identificou um paciente (3,0%) com as mutações 35delG/V3 71. As mutações no gene GJB2 são responsáveis por mais de um quarto das deficiências auditivas sensorioneural não-sindrômica na população do estudo e, o teste PCR alelo-específico (AS-PCR) é um método fácil para rastreamento da mutação 35delG e os resultados positivos podem estabelecer o diagnóstico etiológico e aconselhamento genético nos pacientes afetados.

Thesis (Ph. D.)--Uniformed Services University of the Health Sciences, 2005. / Typescript (photocopy).