AIDS is caused by a lentivirus, HIV-1. In addition to antiretroviral drugs that are currently in use for HIV/AIDS therapy, a number of gene therapy strategies have been designed as alternative therapies. Most of these therapies target HIV RNA/proteins, which are subject to high rate of mutation, resulting in escape mutants. Viral entry is mediated by CCR5 co-receptor in most routes of transmission. To downregulate CCR5 as a gene therapy approach, we targeted seven unique sites within the CCR5 mRNA by a multimeric hammerhead ribozyme, Rz1-7. Hammerhead ribozyme is a small RNA that cleaves a target RNA upon binding to it. Expressing the Rz1-7 from HIV-1- and MSCV-based vectors in otherwise susceptible cells inhibited replication of a CCR5-tropic strain of HIV-1 by 99-100%. The Rz1-7 will be tested for inhibition of HIV-1 replication in the CD4+ T-lymphoid and myeloid progeny of transduced human CD34+ hematopoietic progenitor stem cells. It may be preferable to interfere HIV-1 life cycle at the DNA level since a one-time inactivation might suffice to confer a complete and permanent inhibition of virus replication in the gene modified cells and their progeny. This is what other strategies that target the HIV-1 RNA/protein can hardly offer. For this purpose, group II introns, which are able to splice out and get incorporated into a specific DNA sequence, can be designed/modified to gain novel DNA targeting specificities. As a novel approach, we have examined whether insertion of a modified intron into an infectious HIV-1 clone at two sites within the integrase domain of HIV-1 pol gene could inhibit virus replication. Intron insertion into the HIV-1 clone was induced and mammalian cells were transfected with intron-inserted HIV-1 clones. Although similar amounts of HIV-1 RNA, protein, and progeny virus were produced from the clones as from wild-type HIV-1 provirus DNA, in the absence of a functional integrase, the HIV-1 reverse-transcribed DNA failed to integrate and virus replication was aborted. These results demonstrate that modified group II introns can confer complete inhibition of virus replication at the level of second round of infection. We are now developing vectors to assess whether intron insertion can take place in mammalian cells.

AIDS is caused by a lentivirus, HIV-1. In addition to antiretroviral drugs that are currently in use for HIV/AIDS therapy, a number of gene therapy strategies have been designed as alternative therapies. Most of these therapies target HIV RNA/proteins, which are subject to high rate of mutation, resulting in escape mutants. Viral entry is mediated by CCR5 co-receptor in most routes of transmission. To downregulate CCR5 as a gene therapy approach, we targeted seven unique sites within the CCR5 mRNA by a multimeric hammerhead ribozyme, Rz1-7. Hammerhead ribozyme is a small RNA that cleaves a target RNA upon binding to it. Expressing the Rz1-7 from HIV-1- and MSCV-based vectors in otherwise susceptible cells inhibited replication of a CCR5-tropic strain of HIV-1 by 99-100%. The Rz1-7 will be tested for inhibition of HIV-1 replication in the CD4+ T-lymphoid and myeloid progeny of transduced human CD34+ hematopoietic progenitor stem cells. It may be preferable to interfere HIV-1 life cycle at the DNA level since a one-time inactivation might suffice to confer a complete and permanent inhibition of virus replication in the gene modified cells and their progeny. This is what other strategies that target the HIV-1 RNA/protein can hardly offer. For this purpose, group II introns, which are able to splice out and get incorporated into a specific DNA sequence, can be designed/modified to gain novel DNA targeting specificities. As a novel approach, we have examined whether insertion of a modified intron into an infectious HIV-1 clone at two sites within the integrase domain of HIV-1 pol gene could inhibit virus replication. Intron insertion into the HIV-1 clone was induced and mammalian cells were transfected with intron-inserted HIV-1 clones. Although similar amounts of HIV-1 RNA, protein, and progeny virus were produced from the clones as from wild-type HIV-1 provirus DNA, in the absence of a functional integrase, the HIV-1 reverse-transcribed DNA failed to integrate and virus replication was aborted. These results demonstrate that modified group II introns can confer complete inhibition of virus replication at the level of second round of infection. We are now developing vectors to assess whether intron insertion can take place in mammalian cells.

The continued spread of HIV underscores the need to interrupt transmission. One attractive strategy is a topical microbicide. Sexual transmission of herpes simplex virus type 2 (HSV-2) in mice can be inhibited by intravaginal small inhibitory RNA (siRNA) application. To overcome the challenges of using siRNAs to knock down gene expression in immune cells susceptible to HIV infection, we used chimeric RNAs composed of an aptamer fused to an siRNA for targeted gene knockdown in cells bearing an aptamer-binding receptor. Here, we showed that CD4 aptamer-siRNA chimeras (CD4-AsiCs) specifically suppress gene expression in CD4+ T cells and macrophages in vitro, in polarized cervicovaginal tissue explants, and in both the genital and rectal tracts of humanized mice. CD4-AsiCs do not activate lymphocytes or stimulate innate immunity, provide durable target gene silencing for up to three weeks in vitro, and maintain effectiveness in a hydroxyethyl cellulose (HEC) gel formulation. CD4-AsiCs that knock down HIV genes and/or CCR5 inhibited HIV infection in vitro and in cervicovaginal explants. When applied intravaginally to humanized mice, CD4-AsiCs provided durable protection against transmission of the virus. Thus, CD4-AsiCs could be used as the active ingredient of a microbicide to prevent the sexual transmission of HIV.

Signal termination processes of GPCRs are well established, unlike processes that regulate the assembly and intracellular trafficking of these signaling complexes. Bimolecular fluorescence complementation was used to study GPCR dimer formation in two projects. Firstly, the importance of Rab GTPases on the cell surface expression and signaling of two chemokine receptors expressed on prostate cancer cells was examined. Rab GTPases necessary for CXCR4 and CCR2 cell surface expression and signaling were different from those necessary for the CXCR4/CCR2 heterodimer. Therefore, this project emphasizes the importance of studying heterodimers as unique entities from their constituent receptors. Secondly, interactions between molecular chaperones and two coreceptors necessary for HIV infection – CCR5, a chemokine GPCR, and the main HIV receptor, CD4, a glycoprotein – were investigated. Further emphasizing the unique characteristics of GPCR dimers, this project found that molecular chaperones interact differently with CCR5 homodimers, when compared to CCR5/CD4 heterodimers.

Abstract Early in the evolution of the vertebrates it is thought that two genomic duplications occurred, providing a basis for the evolution in body plan and neural crest of very early vertebrates and substantive material for further evolution of various gene families such as those making up a number of components of the adaptive vertebrate immune system. While the bony fish possibly had another, genome duplications are not generally a feature of vertebrate evolution and indeed the appearance of an antigen-adaptive immune recognition system may have served to limit the size that various vertebrate genomes, including that of the human, can in fact achieve. This initial step in vertebrate immune evolution, the establishment of recognition of non-self against the unique set of 'self' epitopes for an individual, provided an immensely powerful weapon in immune function with the ability to tailor a defense against as-yet-unseen dangers at any time albeit with the pitfall of autoimmune disease. As the recognition sites of the antigen receptor molecules such as TcR are produced by clonal modification of the segments provided in the germline and are thus not in the genome itself, pathogens have not been able to hijack this one component of the immune system in the way so many other components have been put to use throughout evolution, nor do these components necessarily reveal themselves as associated with disease through genome screens. Importantly, overall immune function is determined not just by the potential repertoire of recognition receptors but also by the ability of immunocompetent cells to migrate in a tissue specific fashion through the use of various chemokines and their receptors. Typical of the hijacking of an immune system component by a pathogen is the use of a chemokine ligand gene in the viral ancestor to SIV and HIV, allowing for virus binding to immunocompetent cells as is seen in the use of the CCR5 chemokine receptor by macrophage-tropic HIV strains. This thesis describes the allele and genotype frequencies for several TcR beta-chain variable segment polymorphisms in a population of MS patients compared with controls before and after stratification for HLA-DR15, polymorphism in the Apo-1 / Fas promoter, the DRB1 Val86/Val86 genotype, CCR5-delta32 and the HLA-DRA promoter. The thesis continues with CCR5-delta32 genotyping in IDDM, MS and SLE cohorts and then examines the question of the population of origin of the delta-32 allele of the CCR5 receptor for chemokine. Here, a case / control comparison of 122 RR-MS patients with 96 normal individuals was made for allele and genotype frequencies and for haplotypes formed by pairs of TCRB markers. Further analysis was made after HLA-DR15 stratification. Linkage disequilibrium was found between pairs of alleles of bv8s1, bv10s1, bv15s1 and bv3s1 loci in both patients and controls. In the RR-MS cohort, an increase in the allele frequency of bv8s1*2 was seen (p = 0.03) and the haplotype bv8s1*2 / bv3s1*1 was increased (p = 0.006), and both were found to be statistically significant. In the DR15-positive group, association between MS and TCRB was seen with the bv8s1*2 allele (p = 0.05) and the bv8s1*2 / bv10s1 haplotypes (p = 0.048), while the haplotype associations seen among the DR15-negative patients included the bv3s1*1 allele (bv10s1*1 / bv3s1*1, p = 0.022; bv8s1*2 / bv3s1*1, p = 0.048). While no associations were found after stratification for SDF1-3'A, Apo-1 / Fas or DRB1 there were modest interactions between bv3s1, bv10s1 and bv15s1 and the HLA-DRA promoter. These results support the involvement of the TCRB region in MS susceptibility. The further study of autoimmune disease here includes genotype analysis of CCR5-delta32 in type 1 diabetes (IDDM) and SLE. CCR5 is the major co-receptor for viral entry used by macrophage-tropic HIV strains and protection from infection is seen in homozygotes for CCR5-delta32. In diabetes, infiltration of pancreatic tissue by autoreactive T-cells involves secretion of multiple cytokines and chemokine receptor expression. Variation in the chemokine receptor CCR5 may result in differences in inflammatory cell migration in response to relevant chemokines. Adolescents with type 1 diabetes were genotyped for CCR5-delta32 (n = 626). The allele frequency was compared with that of 253 non-diabetic adolescents and with that of 92 adults with SLE. A reduced allele frequency was seen in type 1 diabetes compared with controls (0.092 vs 0.123, p = 0.05). This difference was not seen for the cohort of patients with SLE (freq = 0.114). A reduction in the number of CCR5-delta32/delta32 homozygotes, who lack CCR5, in the type 1 diabetes cohort was also seen and while not statistically significant (2 observed compared to 5.25 expected; p = 0.12) is interesting. These results suggest a partial protection from type 1 diabetes for CCR5-delta32 homozygous individuals is possible and that CCR5 has a potential role in the pathogenesis of type 1 diabetes. Global surveys of the CCR5-delta32 allele have confirmed a single mutation event in a Northeastern European population as the source of this allele. Here, Australian Ashkenazi Jews (n = 807) were found to have a CCR5-delta32 allele frequency of 14.6% while Australian Sephardic Jews (n = 35) had a frequency of 5.7% and non-Jewish Australian controls (n = 311) had an allele frequency of 11.25%. Data on birthplace of grandparents showed a gradient with highest CCR5-delta32 frequencies from Eastern European Ashkenazim (~19.5% for those whose four grandparents come only from Russia, Poland, Hungary, Austria and Czechoslovakia; n = 197) which differs significantly from the frequency seen in Ashkenazi Jews from Western Europe (n = 101, p = 0.001). Homozygotes for CCR5-delta32 were genotyped with 3p21 region microsatellites. This has defined an ancestral haplotype on which the mutation first occurred and helped to date this event to between 40 and 50 generations ago or just over a thousand years ago. The population gradient, combined with the dating of the mutation by microsatellite allele frequencies, suggests an origin for the CCR5-delta32 allele in a population ancestral to the Ashkenazim. The distribution in non-Jewish populations in northern Europe has led others to postulate spread of the mutation by Vikings. It is hypothesised here that the link between the two populations could be the kingdom of Khazaria with subsequent admixture into both Swedish Vikings and Ashkenazi Jews. The basic driving force of evolution is through selection and the immune system has a role which, through the survival pressure exerted by viruses and other pathogens, has the potential to exert a great deal of selective force on the various components of this system. The effects of this pronounced selection on an immune system component can be seen for example in the increase of the CCR5-delta32 allele over the last thousand years to the current frequency. As mentioned, some immune system components are not affected by such straightforward selection. In the case of the TCRBV segments, effects on the immune repertoire can occur through MHC interaction at the point of thymic entry and in the effects of various superantigens, but the actual binding pockets that recognise antigen are themselves unable to be selected for (or against). The findings presented in this thesis provide support for the association of TCRBV gene segments with multiple sclerosis and also provide support for the further study of the role of the CCR5-delta32 allele in type 1 diabetes. Furthermore, data presented here suggests that the CCR5-delta32 allele had an origin in the Khazar Kingdom just over a thousand years ago, accounting for the allele frequencies in both the Ashkenazi Jews and in lands frequented by the Vikings. The definition of an extended ancestral haplotype for the CCR5-delta32 allele shows how the effect of selection of an allele of one gene can carry with it specific alleles of a large number of other genes as well.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

Submitted by Natalia de Souza Gonçalves (natalia.goncalves@ufpe.br) on 2016-09-23T14:12:48Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) TESE-Plinio correta.pdf: 2445148 bytes, checksum: af634e24f9f6564ebd9b8c37b5d9c2f8 (MD5) / Made available in DSpace on 2016-09-23T14:12:48Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) TESE-Plinio correta.pdf: 2445148 bytes, checksum: af634e24f9f6564ebd9b8c37b5d9c2f8 (MD5) Previous issue date: 2016-03-10 / Serra Talhada, no sertão Pernambucano, registra grandes surtos de dengue. O armazenamento inadequado de água devido a estiagem prolongada, contribui para a perpetuação dos mosquitos. Além disso, Aedes albopictus também pode gerar maiores problemas para o município. Em nossas análises, o número de casos de dengue no período de 2012 a 2013, possui correlação com o número de ovos coletados no mês anterior. Apesar dos surtos intermitentes, o baixo número de casos de Dengue Hemorrágica, levanta questões a respeito de genes de suscetibilidade/resistência. Por isso, analisamos as frequências alélicas e genotípicas do marcador CCR5 e dos SNPs IL-4, IL-10 e Ly-6. Em nossos resultados, a mutação CCR5Δ32, apresentou baixa frequência. Os SNPs IL-4 e IL-10 encontraram-se em desequilíbrio de Hardy-Weinberg. Em relação às frequências alélicas, quando comparadas à população global, Ibérica, Peruana e Africana, observamos diferenças significativas para IL-4, mas não para IL-10. Já Ly-6 apresentou frequência alélica significativa em relação a Ibéricos, Peruanos e Africanos. A frequência genotípica dos genótipos CT e TT de IL-4, apresentaram diferenças significativas em relação a todas as populações. Enquanto que o genótipo TT de IL-10 foi significativamente diferente em relação aos Ibéricos. Para Ly-6, o genótipo GG foi significativamente diferente em relação à população africana, enquanto AG e AA diferiram das populações Ibérica, Peruana e Africana. Porém, só após o acréssimo de um grupo controle ou de outra categoria comparativa, teremos resultados conclusivos. / Serra Talhada, in semiarid of Pernambuco State, register every two years, dengue outbreaks. Inadequate water storage due prolonged drought, perpetuate the mosquito. Moreover, Aedes albopictus, may cause major problems for the municipality. Our analysis detected that the number of dengue cases in the period 2012-2013, has correlation with the number of eggs collected in the previous month. Despite facing intermittent outbreaks, the low number of cases of Hemorhagic Dengue, raises questions about susceptibility / resistance genes. Therefore, we analysed allelic and genotypic frequencies of the marker CCR5 and SNPs IL-4, IL-10 and Ly-6. About our results, the CCR5Δ32 mutation, showed a low frequency. About SNPs IL-4 and IL-10, Hardy-Weinberg disequilibrium were found. Regarding to allele frequency significant differences were observed for IL-4 but not IL-10 when compared to the overall population, Iberian, Peruvian and African. Ly-6 showed significant allele frequency compared to Iberian, African and Peruvian. Concerning to genotypic frequency, significant differences in relation to all populations were found to CT and TT genotype of IL-4. While the TT genotype of IL-10 was significantly different in relation to the Iberian. For Ly-6, the GG genotype was significantly different in relation to the African population, while AG and AA differ from the Iberian populations, Peruvian and African. However, to conclusive results regarding these markers, it becomes necessary to add a control group or another comparative category

Submitted by Caroline Falcao (caroline.rfalcao@ufpe.br) on 2017-04-06T18:31:16Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) 2010-Tese-AdrianaGomes.pdf: 1609955 bytes, checksum: bf29da9ef3c1a47f8b5309c4c3ba837e (MD5) / Made available in DSpace on 2017-04-06T18:31:16Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) 2010-Tese-AdrianaGomes.pdf: 1609955 bytes, checksum: bf29da9ef3c1a47f8b5309c4c3ba837e (MD5) Previous issue date: 2010-12-15 / O Brasil tornou-se objeto de interesse de estudos genético-populacionais devido às diferenças, fenotípica e social, verificadas entre as populações das cinco regiões geográficas do país. O estudo realizado nas populações atuais fazendo inferências históricas leva em consideração o polimorfismo genético de marcadores moleculares. Em vista disso, analisamos a população do Nordeste brasileiro, relacionando historicamente as populações atuais com as possíveis populações parentais, utilizando como marcadores moleculares o polimorfismo de 18locos de microssatélites e a frequência do alelo ccr5∆32 do gene ccr5, considerado excelente marcador de origem européia. A amostra foi constituída de 3.079indivíduos não aparentados dos nove Estados do Nordeste. A extração do DNA foi realizada através do método deMini-Salting Oute os amplificados separados por PAGE. Osalelos mais freqüentes dos locos de STRs foram os mesmos em todas as populações; o alelo ∆32apresentou menor freqüência no Maranhão-MA (0.0214) e maior freqüência no Rio Grande do Norte-RN (0.0626), não tendo sido encontrados homozigotos nos Estados do MA, CE, PB, AL e BA. Os dados permitem concluir que o loco ccr5 é bem mais eficiente do que as STRs para a análise histórica pretendida, mostrando que a contribuição das populações caucasianas européias na formação das populações brasileiras em geral e, em particular na das populações atuais do Nordeste brasileiro, é maior do que aquela prevista com base apenas nos dados históricos. / Brazil became the object of interest in population genetic studies because of differences, phenotypic and social observed among populations from five geographical regions of the country. The current study conducted in populations making historical inferences takes into account the genetic polymorphism of molecular markers. In view of this, we analyzed the population of Northeast Brazil, historically relation thepopulations present with the possible parental populations, using molecularmarkers polymorphism of 18microsatellite loci and the frequency of the alleleccr5Δ32, considered an excellent marker of European origin. The sample consisted of 3079 unrelated individuals from nine Northeastern States. DNA extraction was performed using the method ofMini Salting Out amplified and separated by PAGE. The most frequent alleles of STRs loci were the same in all populations, the Δ32 allele showed lower frequency in Maranhão-MA (0.0214) and more frequently in Rio Grande do Norte-RN (0.0626) and has not been homozygote found in the states of MA, CE, CP, AL and BA. The data shows that the ccr5 locusis much more efficient than STRs intended for historical analysis, showing that the contribution of European Caucasian populations in the formation of the Brazilian population in general and particularly in the current population of Northeastern Brazil, is larger than that predicted based only on historical data.

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第12957号 / 論医博第2099号 / 新制||医||1011(附属図書館) / 32356 / 京都大学大学院医学研究科医学専攻 / (主査)教授 生田 宏一, 教授 岩井 一宏, 教授 椛島 健治 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM