Rozprawy doktorskie na temat „GEWE”

Tumor cells can be modified with cytokine genes such as the Interleukin-2 (IL-2) gene. The levels of IL-2 expressed are critical for successful treatment. We have tried to achieve higher levels of IL-2 than those currently available by conventional plasmids. Use of a transcriptional activator, e.g; the tat gene along with the HIV promoter driving the IL-2 gene, greatly increased IL-2 levels compared to widely used cytomegalovirus (CMV) driven plasmids. Control of the tat gene with an inducible promoter, i.e; the human HSP70B promoter, permitted control of gene expression. The inducibility of the HSP70B promoter by heat, γ-radiation and geldanamycin (a chemotherapeutic drug) allowed for a combinatorial approach to cancer treatment with hyperthermia, radiation therapy and chemotherapy. Also a brief heat treatment of 10 min at 42°C of target cells increased plasmid uptake, and higher levels of gene expression could be achieved. Another arm of immunotherapy is adoptive therapy with Tumor Infiltrating Lymphocytes (TILs). Insufficient numbers of tumor-specific T-cells limit the success of TIL therapy. An alternative approach to overcome this limitation is to transfer tumor-specific T cell receptor (TCR) into peripheral T-cells, redirecting their specificity to the tumor cell. To prove the feasibility of this technique, T-cell receptors were identified and cloned from hybridomas specific for the tumor cell line, MO5. A three domain single chain T-cell receptor was also constructed from the tumor-specific TCR genes to investigate the ability of a single chain T-cell receptor to activate T-cells. The CD3ζ chain was linked to the single chain to allow signal transduction upon antigen recognition by the TCR. The full length and the single chain TCR were cloned into a retroviral vector and transfected into mouse and human T cell lines. Cell surface expression of the chains were detected by flow cytometry. Functionality of the transfected TCR chains was assessed by IL-2 secretion on co-culture of the tumor cell line MO5 and the transfected T-cells. The two different approaches described here, i.e; higher levels of IL-2 for IL-2 gene therapy and specific redirection of T-cells can potentially greatly enhance the success rate of cancer treatment.

Rheumatoid arthritis (RA) is a systematic autoimmune disorder characterized by a persistent joint inflammation. A subset of HLA-DRB1 alleles known as shared epitope (SE) are the strongest genetic risk factors to develop anti-citrullinated protein antibody positive (ACPA-positive) RA. A strong enrichment of interactions exists between ACPA-positive RA-associated genetic variants and HLA-DRB1 SE alleles in disease development. Pathway analysis was performed to investigate how the interactions between risk variants (SNPs) with HLA-DRB1 from a previous study related to ACPA-positive RA. Gene-gene interactions analysis was performed between non-HLA risk variants and HLA-DRB1 SE alleles in SRQ biobank (SRQb) case-control cohort. We also evaluated whether the reported gene-gene interactions from a previous study relate to methotrexate (MTX) response for RA patients, at three and six months of follow-up in EIRA study. Interaction analysis based on an additive model was performed to understand the combined effect of two risk factors in the disease and treatment response. Two out of three genes from pathway analysis that were RXRA and NR3C1, pointed to ACPA-positive RA related important pathways including vitamin D receptor (VDR) pathway and adipocytokine signaling pathway. The replication analysis in SRQ-case-control study showed 2.627% of the evaluated SNPs insignificant additive interaction with HLA-DRB1 SE alleles. No interactions were significant in relation to the response to MTX monotherapy after 3 and 6 months follow-up. This project provides new insights into the gene-gene interactions in the study of ACPA-positive RA and suggests candidate genes for future functional studies.

The DNA microarray biotechnology simultaneously monitors the expression of thousands of genes and aims to identify genes that are differently expressed under different conditions. From the statistical point of view, it can be restated as identify genes strongly associated with the response or covariant of interest. The Gene Set Enrichment Analysis (GSEA) method is one method which focuses the analysis at the functional related gene sets level instead of single genes. It helps biologists to interpret the DNA microarray data by their previous biological knowledge of the genes in a gene set. GSEA has been shown to efficiently identify gene sets containing known disease-related genes in the real experiments. Here we want to evaluate the statistical power of this method by simulation studies. The results show that the the power of GSEA is good enough to identify the gene sets highly associated with the response or covariant of interest.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior
A bactÃria Helicobacter pylori, à um agente etiolÃgico bem estabelecido para o desenvolvimento de lesÃes gÃstricas como gastrite, Ãlcera pÃptica, metaplasia e doenÃas malignas, com alta incidÃncia de infecÃÃo em todo mundo, porÃm cerca de 80% dos indivÃduos infectados permanecem assintomÃticos e apenas uma minoria desenvolve doenÃas a ela relacionadas. Estudos tÃm sido realizados na tentativa de identificar a relaÃÃo de genes determinantes da patogenicidade de H. pylori, entretanto, atà o momento apenas os genes cagA e o alelo de vacA s1m1 sÃo considerados marcadores de virulÃncia para o desenvolvimento de lesÃes gÃstricas mais graves. A bactÃria H. pylori possui uma alta variabilidade genÃtica, sendo que um dos mecanismos propostos para o desenvolvimento de lesÃo seria atravÃs da inflamaÃÃo. DiferenÃas na intensidade de respostas inflamatÃrias poderiam ser decorrentes do perfil genotÃpico da cepa infectante. Recentes trabalhos apontam a importÃncia dos genes cagE, virB11 de H. pylori, em cÃncer gÃstrico. Entretanto, estudos em lesÃes gÃstricas sÃo restritos. Assim, o objetivo deste trabalho foi determinar o perfil genotÃpico das cepas de H. pylori quanto a presenÃa dos genes de virulÃncia cagA, cagE, virB11, vacA e flaA, circulantes no estado do Cearà em 201 casos de lesÃes gÃstricas de diferentes gravidades, coletadas de pacientes dispÃpticos atendidos em trÃs hospitais de Fortaleza-CE. A detecÃÃo de H. pylori foi feita atravÃs da amplificaÃÃo do gene ureC, os genes estudados por amplificaÃÃo de fragmentos especÃficos, usando a tÃcnica de PCR, foram observados em gel de agarose 1% e poliacrilamida a 6% e 8%. Neste estudo houve um predomÃnio do sexo feminino 59% (119/201), principalmente na faixa etÃria (15-44) 30% (61/201), alta taxa de infecÃÃo por H. pylori 97,5% (196/201). A gastrite crÃnica ativa (GCA) foi a lesÃo gÃstrica mais frequente (55,7%;112/201), associada a pacientes na faixa etÃria de 15-44 (36,6% : 41/112) opondo-se à metaplasia intestinal em que, a frequÃncia dos casos aumentou com a idade sendo maior em pacientes mais velhos 46% (16/35), o que concorda com a literatura. O Ãnico caso de displasia ocorreu numa paciente > 65 anos. As lesÃes gÃstricas foram predominantemente localizadas no antro. O gene cagA foi mais frequente na gastrite crÃnica ativa (GCA), estando tambÃm em alta frequÃncia na gastrite atrÃfica (GA) e metaplasia intestinal (MI). Na GCA foi observado tambÃm alta frequÃncia dos genes cagE, virB11, vacAm1 e flaA, com diferenÃa estatÃstica, quando comparada com a gastrite crÃnica inativa (GCI) (cagA - p=0,007 cagE- p=0,000, virB11 - p=0,005, vacAm1- p=0,047 e flaA - p=0,001), sendo que os genes virB11 e flaA foram mais frequentes na GCA do que na Ãlcera. Os alelos s1 e m1 de vacA , bem como a combinaÃÃo s1m1 foram os mais frequentes nas lesÃes gÃstricas. Os casos H. pylori positivos foram agrupados de acordo com a presenÃa dos genes estudados, levando em consideraÃÃo a presenÃa do alelo s1 e os genes da ilha. Nessas anÃlises foi observada maior frequÃncia de cepas contendo os genes vacA s1 e [Ia (cagA+, cagE+ e virB11+ ) + Ib (cagA+ e virB11+; cagE+ e virB11+) ; 47,7% ] na gastrite crÃnica ativa em relaÃÃo a gastrite crÃnica inativa (GCI), esta com maior frequÃncia de cepas dos grupos [Ic (cagA+ ou cagE+ ou virB11+ ou cagA+ e cagE+) +Id (ureC+); 62%]. Adicionalmente, maior frequÃncia de cepas pertencentes aos grupos Ia e Ib foi observada na metaplasia intestinal incompleta, (59%), enquanto que na metaplasia intestinal completa uma maior frequencia de cepas pertencentes aos grupos Ic + Id, (78%) (p=0,027). Todos os casos de gastrite atrÃfica e Ãlcera eram do grupo I, e um Ãnico caso de displasia pertencia ao grupo Ia. Esses dados evidenciam uma associaÃÃo de cepas com genÃtipos mais virulentos em lesÃes com potencialidade de malignizaÃÃo.
A bactÃria Helicobacter pylori à um agente etiolÃgico bem estabelecido para o desenvolvimento de lesÃes gÃstricas como gastrite, Ãlcera pÃptica, metaplasia e doenÃas malignas, com alta incidÃncia de infecÃÃo todo mundo, porÃm cerca de 80% dos indivÃduos infectados permanecem assintomÃticos e apenas uma minoria desenvolve doenÃas a ela relacionadas. Estudos tem sido realizados na tentativa de identificar a relaÃÃo de genes determinantes da patogenicidade de H. pylori, entretanto, atà o momento apenas os genes cagA e o alelo de vacA s1m1 sÃo considerados marcadores de virulÃncia para o desenvolvimento de lesÃes gÃstricas mais graves. A bactÃria H. pylori possui uma alta variabilidade genÃtica sendo que um dos mecanismos propostos para o desenvolvimento de lesÃo seria atravÃs da inflamaÃÃo. DiferenÃas na intensidade de respostas inflamatÃrias poderiam ser decorrentes do perfil genotÃpico da cepa infectante. Recentes estudos apontam a importÃncia dos genes cagE, virB11 de H. pylori, em cÃncer gÃstrico. Entretanto, estudos em lesÃes gÃstricas sÃo restritos. Assim, o objetivo deste trabalho foi determinar o perfil genotÃpico das cepas de H. pylori quanto a presenÃa dos genes de virulÃncia cagA, cagE, virB11, vacA e flaA, circulantes no estado do Cearà em 201 casos de lesÃes gÃstricas de diferentes gravidades, coletadas de pacientes dispÃpticos atendidos em trÃs hospitais de Fortaleza-CE. A detecÃÃo de H. pylori foi feita atravÃs da amplificaÃÃo do gene ureC, e os genes estudados por amplificaÃÃo de fragmentos especÃficos, usando a tÃcnica de PCR, e foram observados em gel de agarose 1% e poliacrilamida a 6% e 8%. Nesse estudo houve um predomÃnio do sexo feminino 59% (119/201), principalmente na faixa etÃria (15-44) 30% (61/201), e alta taxa de infecÃÃo por H. pylori 97,5% (196/201). A gastrite crÃnica ativa (GCA), foi a lesÃo gÃstrica mais frequente (55,7%;112/201), associada a pacientes na faixa etÃria de 15-44 (36,6% : 41/112) opondo-se à metaplasia intestinal onde a frequÃncia dos casos aumentou com a idade sendo maior em pacientes mais velhos 46% (16/35), o que concorda com a literatura. O Ãnico caso de displasia ocorreu numa paciente > 65 anos. As lesÃes gÃstricas foram predominantemente localizadas no antro. O gene cagA foi mais frequente na gastrite crÃnica ativa (GCA), estando tambÃm em alta frequÃncia na gastrite atrÃfica (GA) e metaplasia intestinal (MI). Na GCA foi observado tambÃm alta freqÃÃncias dos genes cagE, virB11, vacAm1 e flaA com diferenÃa estatÃstica quando comparada com a gastrite crÃnica inativa (GCI) (cagA - p=0,007 cagE- p=0,000, virB11 - p=0,005, vacAm1- p=0,047 e flaA - p=0,001), sendo que os genes virB11 e flaA foram mais frequentes na GCA que na Ãlcera. Os alelos s1 e m1 de vacA , bem como a combinaÃÃo s1m1 foram os mais frequentes nas lesÃes gÃstricas. Os casos H. pylori positivos foram agrupados de acordo com a presenÃa dos genes estudados, levando em consideraÃÃo a presenÃa do alelo s1 e os genes da ilha. Nestas anÃlises foi observada maior frequÃncia de cepas contendo os genes vacA s1 e [Ia (cagA+, cagE+ e virB11+ ) + Ib (cagA+ e virB11+; cagE+ e virB11+) ; 47,7% ] na gastrite crÃnica ativa em relaÃÃo a gastrite crÃnica inativa (GCI), esta Ãltima com maior frequÃncia de cepas dos grupos [Ic (cagA+ ou cagE+ ou virB11+ ou cagA+ e cagE+) +Id (ureC+); 62%]. Adicionalmente, maior frequÃncia de cepas pertencentes aos grupos Ia e Ib foi observada na metaplasia intestinal incompleta, (59%), enquanto que na metaplasia intestinal completa uma maior frequencia de cepas pertencentes aos grupos Ic + Id, (78%) (p=0,027). Todos os casos de gastrite atrÃfica e Ãlcera eram do grupo I, e o Ãnico caso de displasia pertencia ao grupo Ia. Esses dados evidenciam uma associaÃÃo de cepas com genÃtipos mais virulentos em lesÃes com potencialidade de malignizaÃÃo.
The bacterium Helicobacter pylori is a well-established etiological factor in the development of gastric lesions such as gastritis, peptic ulcers, metaplasia and malignancy. The incidence of infection by this pathogen is high worldwide, but about 80% of infected individuals remain asymptomatic, and only a minority develops related diseases. Many studies have been conducted in an attempt to identify the involvement of genes in determining the pathogenicity of H. pylori, but so far, only the genes cagA and vacA allele s1m1 are considered virulence markers for the development of the more severe gastric lesions. H. pylori bacteria have a high genetic variability and one of the proposed mechanisms for lesion development is through inflammation. Differences in the intensity of inflammatory responses could be due to the genotypic profile of the infecting strain. Recent studies indicate the importance of the genes cagE and virB11, but mostly involving gastric cancer, while their role in gastric lesions is limited. The objective of this study was to determine the genetic subtypes of H. pylori strains and the presence of the virulence genes cagA, cagE, virB11 and flaA genes and vacA alleles, circulating in Ceara state, Brazil. Samples were collected from 201 cases of gastric lesions of varying severity, in dyspeptic patients treated at three hospitals in Fortaleza, Ceara State. The detection of H. pylori was performed using ureC gene amplification by PCR, and the detection of the genes studied was carried out by amplification of gene-specific fragments separated in 1% agarose gels. The sample was predominantly female (59%, 119/201) and mainly in the age group 15-44 years old (30%, 61/201), and had a high rate of H. pylori infection (97.5%, 196/201). Active chronic gastritis (ACG) was the most common gastric lesion (55.7%, 112/201), associated with patients aged 15-44 (36.6%, 41/112), unlike intestinal metaplasia, in which the frequency of cases increased with age, being higher in older patients (46%, 16/35), which agrees with the literature. A single case of dysplasia occurred in a patient > 65 years. The lesions were predominantly located in the gastric antrum and 30% of the cases had lesions located in the body and antrum simultaneously. The cagA gene was more frequent in ACG, showing a statistically significant correlation (r = 0.220, p = 0.007); it also showed a high frequency in atrophic gastritis and in intestinal metaplasia. In ACG, a high frequency of the genes cagE, virB11, flaA and vacAm1 was also statistically associated when compared to chronic inactive gastritis (ICG) (cagA - p = 0.007, cagE - p = 0.000, virB11 - p = 0.005, vacAm1 p = 0.047 and flaA - p = 0.001), and the genes virB11 and flaA were more frequent in ACG than in ulcer. The vacA alleles s1 and m1, and the combination s1m1, were the most frequent ones in gastric lesions. Considering the genotypes of H. pylori grouped by the presence of the genes studied, we observed a higher frequency of the most virulent strains in the ACG group (Ia + Ib, 47.7%) when compared to ICG, the latter showing a higher frequency of less virulent strains (Ic + Id, 62%). Additionally, a higher frequency of more virulent strains, belonging to groups Ia and Ib, was observed in incomplete intestinal metaplasia (59%), while in complete intestinal metaplasia an increased frequency of less virulent strains, belonging to the groups Ic + Id (78%) (p = 0.027), was found. All cases of atrophic gastritis and ulcer were in group I, and the single case of dysplasia belonged to group Ia (high virulence). These data indicate the important role of more virulent strains in potential malignant lesions.

Genome sequencing projects from diverse bacteria and the release of genetically engineered microorganisms make the topic of horizontal gene transfer (HGT) fashionable. Sequence analysis indicates the high frequency and the importance of horizontal gene transfer to the microbial evolution. Much knowledge about horizontal transfer has been derived from studies using soil microcosms, fluorescent-marked donors and recipients and from study of the increase of the antibiotic resistance as a result of use and often abuse of antibiotics. However, little is known about the transfer of chromosomally-located antibiotic biosynthesis genes in natural populations. The streptomycin pathway-specific regulator, strR, has been found in a set of diverse streptomycetes both phenotypically and genetically that previously have already been described to carry the resistance (strA) and one biosynthetic (strBl) gene from the same cluster. Phylogenetic analysis of both 16S rRNA and trpB gene fragments showed that two isolates were closely related to S. coelicolor which are known not to produce streptomycin or to contain any biosynthesis or resistance genes. The remaining pair did appear to be closely related to each other, particularly from the trpB analysis. trpB gene proved to be useful for resolving phylogenetic relationships between strains with highly conserved gamma region of the 16S rRNA gene. However, one of these strains is the only one from the S. griseus distant isolates that produced streptomycin and possessed many other genes from the cluster. The phylogenetic incongruency between `species' tree and `gene' tree can be attributed to horizontal gene transfer. The sequence identity of the detected genes is extremely high (99%) indicating a recent transfer event. Besides the physical proximity of strRABl genes, there is also a well-characterised functional correlation of these genes in the streptomycin producer, S. griseus. However, strRA genes are silent in coelicolor-like isolates ASSF15 and ASB37. Therefore, this transfer may have other ecological role than a simple resistance phenotype such as the evolution of antibiotic cluster. It was not possible to prove the integration site of strRABl genes within ASB37 genome but a possible site was identified. Pathway-specific regulator StrR activates the transcription of strBl and other genes from the streptomycin cluster by binding to the upstream promoter regions. This protein binds as a tetramer. N-terminal deleted StrR derivatives were still able to bind as pseudodimers (two monomers). Two putative domains in the N'-terminus of the protein responsible for the protein dimerisation during the binding process have been identified.

Genome wide association studies (GWAS) have revolutionized our approach to mapping genetic determinants of complex human diseases. However, even with success from recent studies, we have typically been able to explain only a fraction of the trait heritability. GWAS are typically analysed by testing for the marginal effects of single variants. Consequently, it has been suggested that gene-gene interactions might contribute to the missing heritability of complex diseases. GWAS incorporating interaction effects have not been routinely applied because of statistical and computational challenges relating to the number of tests performed, genome-wide. To overcome this issue, I have developed novel methodology to allow rapid testing of pairwise interactions in GWAS of complex traits, implemented in the IntRapid software. Simulations demonstrated that the power of this approach was equivalent to computationally demanding exhaustive searches of the genome, but required only a fraction of the computing time. Application of IntRapid to GWAS of a range of complex human traits undertaken by the Wellcome Trust Case Control Consortium (WTCCC) identified several interaction effects at nominal significance, which warrant further investigation in independent studies. In an attempt to fine-map the identified interacting loci, I undertook imputation of the WTCCC genotype data up to the 1000 Genomes Project reference panel (Phase 1 integrated release, March 2012) in the neighbourhood of the lead SNPs. I modified the IntRapid software to take account of imputed genotypes, and identified stronger signals of interaction after imputation at the majority of loci, where the lead SNP often had moved by hundreds of kilobases. The X-chromosome is often overlooked in GWAS of complex human traits, primarily because of the difference in the distribution of genotypes in males and females. I have extended IntRapid to allow for interactions with the X chromosome by considering males and females separately, and combining effect estimates across the sexes in a fixed-effects meta-analysis. Application to genotype data from the WTCCC failed to identify any strong signals of association with the X-chromosome, despite known epidemiological differences between the sexes for the traits considered. The novel methods developed as part of this doctoral work enable a user friendly, computationally efficient and powerful way of implementing genome-wide gene-gene interaction studies. Further work would be required to allow for more complex interaction modelling and deal with the associated computational burden, particularly when using next-generation sequencing (NGS) data which includes a much larger set of SNPs. However, IntRapid is demonstrably efficient in exhaustively searching for pairwise interactions in GWAS of complex traits, potentially leading to novel insights into the genetic architecture and biology of human disease.

Mestrado em Engenharia Biomédica - Biomateriais
O quitosano é um policatião de origem natural que tem vindo a ser investigado como sistema não viral de vectorização de genes devido à sua biocompatibilidade e baixa toxicidade. No entanto, a sua baixa eficiência de transfecção tem dificultado o seu uso generalizado. Num estudo anterior mostrámos que a conjugação de resíduos de imidazol a cadeias de quitosano resulta numa melhoria da eficiência de transfecção do polímero. O principal objectivo deste estudo foi avaliar a aplicação de quitosano modificado com imidazol (CHimi) como vector para entrega de genes em medicina regenerativa, bem como encontrar novas vias para melhorar a eficiência. A expressão genética mediada por CHimi com dois graus de substituição - 13% e 22% das aminas primárias do quitosano - foi avaliada em células 293T (células embrionárias humanas do epitélio do rim) por um período de 8 dias, usando o gene da β-Galactosidase (β-gal) como gene repórter. Os complexos de CHimi-DNA foram preparados numa razão molar de 18 entre aminas primárias e grupos fosfato. As células transfectadas com estes complexos apresentam um pico de actividade da β-gal às 72 horas pós-transfecção, verificando-se a expressão sustentada da proteína repórter durante todo o período de avaliação. Nestas condições a viabilidade celular não é comprometida. Quando se efectua um segundo tratamento das células com complexos à base de CHimi, a actividade de transfecção volta a aumentar, sem haver alterações na viabilidade celular. Verificou-se também que células transfectadas com estes vectores sobrevivem a um ciclo de congelação/descongelação, mantendo uma actividade de transfecção sustentada no tempo. Uma polietilenimina comercial (Escort V) foi usada como referência neste estudo. Apesar de os níveis de transfecção mediados por este vector serem duas ordens de grandeza mais elevados, a viabilidade celular decresce até aos 50% após cada tratamento. De forma a investigar o processo de transfecção mediado por polímeros de CHimi, o tráfego intracelular destes complexos foi estudado por microscopia confocal de varrimento laser. Complexos de CHimi e DNA marcados com fluoróforos foram encontrados no citoplasma celular 2 horas depois da transfecção, sendo detectados até 48 horas pós-transfecção. Estes resultados podem em parte explicar a expressão sustentada de β-gal ao longo do tempo. Os complexos foram detectados no interior do núcleo 4 horas pós-transfecção. O DNA marcado com fluorescência não foi observado na forma livre em nenhum dos momentos analisados, enquanto que CHimi foi detectado num evento único no citoplasma. Num ensaio “cell-free” de transcrição/tradução in vitro não foi detectada a síntese de proteína quando o DNA estava complexado com CHimi, apesar de este ser expresso na ausência do polímero. Este conjunto de resultados sugere que, apesar dos complexos poderem ser encontrados no interior do núcleo rapidamente após a transfecção, a expressão genética parece depender da desintegração do complexo. O CHimi é um potencial candidato a vector para transporte de genes num cenário de regeneração. Este material medeia uma expressão proteica sustentada sem afectar a viabilidade celular. Com este sistema, as células toleram uma segunda adição de complexos, pelo que a administração repetida poderá ser potencialmente usada como estratégia para prolongar o efeito terapêutico de uma proteína de interesse. Em relação aos resultados de tráfego intracelular dos complexos, e considerando o perfil de expressão genética obtido, pode pôr-se a hipótese de que a expressão sustentada do gene resulta de um processo de libertação dependente do tempo. Neste sentido, ajustar a velocidade de degradação dos polímeros de CHimi pode ser usado como estratégia para melhorar o processo de expressão do gene tendo em vista o fim terapêutico pretendido. ABSTRACT: Chitosan is a polycation of natural origin, emerging in the non-viral gene delivery vectors scene due to its biocompability and low cytotoxicity. However, its low transfection efficiency has hampered its wide application so far. We have previously shown that grafting imidazole moieties into the chitosan backbone results in improved transfection efficiency of this polymer. The main goal of this study was to assess the application of imidazole-grafted chitosan (CHimi) as gene delivery vector in a regenerative medicine scenario and to find avenues to further improve its efficiency. Gene expression mediated by CHimi with two degrees of substitution - 13% and 22% of chitosan primary amines - was assessed in 293T cells for periods up to 8 days, using the β-Galactosidase (β-gal) gene as reporter gene. CHimi- DNA complexes were prepared at a primary amine to phosphate groups molar ratio of 18. Cells transfected with the CHimi-based complexes have a peak of β-gal activity 72 hours post-transfection and show a sustained β-gal production for 8 days. During this time period cell viability is not impaired. When a second treatment with CHimi-based complexes is performed, transfection activity increases, without changes on cell viability. Additionally, cells transfected with CHimi-based vectors are able to withstand a freeze/thawing cycle, maintaining a sustained transfection activity. A commercially available polyethylenimine (Escort V) was used as a reference. Though transfection levels are two orders of magnitude higher, cell viability decreases up to 50% after each treatment. In order to investigate the transfection process mediated by CHimi-based vectors a study of the intracellular pathway of the complexes has been performed by confocal laser scanning microscopy. Complexes formed by fluorescently labeled CHimi and DNA were found inside the cell cytoplasm 2 hours after transfection and were detected up to 48 hours post-transfection. These results could explain in part the sustained gene expression over time. Complexes were detected inside cell nucleus since 4 hours post-transfection. Fluorescently labeled DNA in the free form was not observed at any of the time points analyzed. Free CHimi was detected in the cytoplasm in an atypical event. In a cell-free in vitro transcription/translation assay no protein production was detected when DNA was complexed with CHimi, though expressed when using plasmid DNA in the absence of CHimi. Taken together these results suggest that, though CHimi-based complexes can be detected inside cell nucleus promptly after transfection, gene expression is dependent on the complex disassembling. CHimi is a potential candidate vector for gene delivery in a regenerative scenario. This material is able to mediate a sustained protein expression without impairing cell viability. In our system, cells can sustain another addition of the complexes suggesting that repeated administration could be used as a strategy to prolong the therapeutic effect. In view of the trafficking results and considering the gene expression profile, one can hypothesize that the observed sustained transgene expression is a time dependent release process. Thus, tuning the degradation rate of CHimi-based polymers could be a strategy to further improve the overall transgene expression process to fulfill the therapeutic end.

Introduction – Animal studies suggested that NFKB1, SOCS3 and IKBKB genes could be involved in the association between overnutrition and obesity. This study aims to investigate interactions involving these genes and nutrition affecting obesity-related phenotypes. Methods – We used multifactor dimensionality reduction (MDR) and penalized logistic regression (PLR) to better detect gene/environment interactions in data from the Toronto Nutrigenomics and Health Study (n=1639) using dichotomized body mass index (BMI) and waist circumference (WC) as obesity-related phenotypes. Exposure variables included genotypes on 54 single nucleotide polymorphisms, dietary factors and ethnicity. Results – MDR identified interactions between SOCS3 rs6501199 and rs4969172, and IKBKB rs3747811 affecting BMI in whites; SOCS3 rs6501199 and NFKB1 rs1609798 affecting WC in whites; and SOCS3 rs4436839 and IKBKB rs3747811 affecting WC in South Asians. PLR found a main effect of SOCS3 rs12944581 on BMI among South Asians. Conclusion – MDR and PLR gave different results, but support some results from previous studies.

A coloração dos animais é uma característica que apresenta uma grande diversidade de fenótipos nas diferentes espécies. Diferentes abordagens podem ser utilizadas para o entendimento da diversidade na coloração existente nas espécies animais. Através da análise de genes candidatos as mutações responsáveis pela variação na coloração têm sido descritas em diferentes espécies, demonstrando o envolvimento de mecanismos moleculares variados na sua regulação. Este trabalho tem por objetivo a utilização de duas abordagens genéticas para o estudo da variação na coloração, a análise de genes candidatos e a predição computacional do efeito de polimorfismos não sinônimos. Em ovinos a coloração da lã é uma característica com importância na produção e para a identificação das raças. Polimorfismos em diferentes genes foram associados com a coloração da lã, entretanto, estes não foram estudados em muitas raças que apresentam variação fenotípica. A ovelha crioula é uma raça local existente no sul do Brasil que apresenta uma ampla diversidade de cores na lã, incluindo branco, preto e diversos tons intermediários. O gene receptor de melanocortina 1 (MC1R) foi previamente associado com a coloração na raça crioula, entretanto, outros genes também devem estar envolvidos na regulação da coloração na raça. Este trabalho avaliou a influência dos genes MC1R, ASIP (proteína sinalizadora agouti), TYRP1 (proteína relacionada à tirosinase 1) e KIT (homólogo do oncogene de sarcoma felino viral v-kit Hardy-Zuckerman 4) na coloração da lã na raça ovina crioula. Amostras de 410 animais de diferentes cores foram analisadas, sendo a variação na coloração da lã determinada por colorimetria. O padrão de herança dos fenótipos foi avaliado através de cruzamentos dirigidos entre indivíduos de diferentes cores. Os polimorfismos nos genes foram avaliados através da realização do sequenciamento e da análise de fragmentos e a quantificação da expressão do gene ASIP foi realizada por PCR em Tempo Real. Foi observada a associação significativa entre polimorfismos nos genes MC1R e ASIP e a cor da lã na raça crioula. O alelo dominante do gene MC1R, provocado pelas mutações p.M73K e p.D121N, foi encontrado apenas em indivíduos pigmentados. Este alelo resulta na ativação constitutiva do receptor, e consequentemente na produção constante de eumelanina, sendo epistático sobre o gene ASIP. Nos animais homozigotos para o alelo selvagem do MC1R a manifestação do fenótipo branco ocorreu somente nos portadores de um alelo contendo a duplicação do gene ASIP. Os portadores da duplicação do ASIP apresentaram níveis elevados de expressão do gene enquanto os homozigotos para a cópia simples do ASIP não expressaram o gene e apresentaram fenótipos pigmentados. Os resultados obtidos permitiram identificar a influência da interação epistática dos genes MC1R e ASIP na coloração da lã nos ovinos crioulos. O estudo de genes candidatos envolvidos na rota da pigmentação mostrou-se uma abordagem adequada para a análise da variação na coloração nestes animais. Espera-se que o conhecimento adquirido neste trabalho auxilie na criação e na preservação da raça através da manutenção da diversidade fenotípica existente. A avaliação computacional dos polimorfismos não sinônimos vem sendo utilizada recentemente a fim de determinar os SNPs que potencialmente afetam o funcionamento dos genes e identificar os mecanismos responsáveis por doenças complexas e pela variação nos fenótipos. A predição do efeito de polimorfismos nos genes utilizando ferramentas computacionais apresenta-se como uma abordagem alternativa para o estudo da genética da coloração. O gene MC1R humano apresenta uma grande quantidade de polimorfismos alguns dos quais foram associados com a variação na pigmentação e com suscetibilidade a tumores de pele. Entretanto, muitas das variações existentes no gene não foram avaliadas quanto às suas consequências funcionais e o seu papel na variação da pigmentação. Foi realizada a predição computacional dos polimorfismos não sinônimos no gene MC1R humano com o objetivo de identificar os nsSNPs mais provavelmente danosos, e estabelecer aqueles com potencial efeito na função do MC1R. Foram utilizadas 11 ferramentas de predição individuais (SIFT, MutPred, Polyphen-2, PROVEAN, I-Mutant 3.0, PANTHER, SNPs3D, Mutation Assessor, PhD-SNP, SNPs&GO e SNAP) e dois programas consenso (PON-P e PredictSNP 1.0) para a análise de 92 nsSNPs localizados no gene. Os programas utilizados baseiam-se em métodos evolutivos, estruturais e computacionais, resultando na identificação dos 14 nsSNPs mais danosos (L48P, R67W, H70Y, P72L, S83P, R151H, S172I, L206P, T242I, G255R, P256S, C273Y, C289R e R306H). Apesar das diferenças nos resultados de cada programa a combinação dos diferentes métodos permitiu diferenciar os polimorfismos neutros dos danosos, mostrando concordância com os programas consenso. A predição computacional demonstrou ser uma abordagem eficiente para a identificação dos alelos danosos no gene MC1R e para a priorização de mutações para posteriores estudos funcionais e populacionais.
Animal color is a characteristic that presents a large diversity of phenotypes. Different approaches can be used to understand the color diversity existing among and within species. Through analysis of candidate genes the mutations responsible for the color variation have been described in different species, showing the involvement of various molecular mechanisms of regulation. The objective of this work is the use of two genetic approaches to the study of color variation, the analysis of candidate genes and the computational prediction of non-synonym polymorphism effects (nsSNPs). In sheep the wool color is a feature with commercial importance and in identifying breeds. Polymorphisms in different genes have been associated with wool color, but they have not been studied in many breeds that show phenotypic variation regarding such a charactere. The Creole is a local breed from southern most Brazil that presents a wide range of wool color, varying from white to black, and including several intermediate hues. The melanocortin 1 receptor (MC1R) was previously associated with the wool color in the Creole, however, other genes might also be involved in the regulation of color in the breed. This study evaluated the influence of the genes MC1R, ASIP (agouti signaling protein), TYRP1 (tyrosinase related protein 1) and KIT (v-kit Hardy- Zuckerman 4 feline sarcoma viral oncogene homolog) in the Creole breed wool color. Samples from 410 specimens of different colors were analyzed. The variation in the color of the wool was performed by colorimetry. The inheritance pattern of the phenotypes was assessed by crossbreeding individuals of different colors. Polymorphisms in the genes were evaluated by performing sequencing and fragment analysis, and the quantification of the ASIP gene expression was performed by Real Time-PCR. It was observed a significant association between polymorphisms in MC1R and ASIP gene and the wool color in Creole breed. The dominant allele of the MC1R gene, caused by p.M73K and p.D121N mutations was found only in pigmented individuals. This allele leads to the constitutive activation of the receptor and therefore in constant production of eumelanin and is epistatic on the ASIP gene. In the homozygous to the wild-type allele of MC1R the manifestation of white phenotype occurred only in individuals with one allele containing a duplication of the ASIP gene. The carriers of the duplicated copy of ASIP showed high levels of gene expression while homozygous for the simple copy of the ASIP did not expressed the gene, and showed pigmented phenotypes. The results allowed the identification of the influence of epistatic interaction of MC1R and ASIP gene in the wool color in Creole breed. The study of candidate genes involved in the pigmentation pathway proved to be a suitable approach for the analysis of variation in pigmentation in these animals. It is expected that the knowledge acquired in this work will assist on stablishment of commercial breeding and preservation policies of this sheep breed. The computational evaluation of non-synonymous polymorphism has been used to determine SNPs that potentially affect the function of the genes and identify the mechanisms responsible for complex diseases and by the variation in phenotypes. The prediction of the effect of polymorphisms in genes using computational tools presents an alternative approach to the study of the genetic of coloration. The human MC1R gene has a large number of know polymorphisms, some of which were associated with changes in pigmentation and susceptibility to skin tumors. However, many existing variations in the gene have not been evaluated regarding the functional consequences and its role in the variation of pigmentation. Computational prediction of nonsynonymous polymorphisms was performed in the human MC1R gene in order to identify the most likely harmful nsSNPs and to establish those with potential effect on the function of MC1R. Eleven individual tools (SIFT, MutPred, Polyphen-2, PROVEAN, I-Mutant 3.0, PANTHER, SNPs3D, Mutation Assessor, PhD-SNP, SNPs&GO and SNAP) and two consensus programs (PON-P and PredictSNP 1.0) were used to the analysis of 92 nsSNPs located in the gene. The programs used are based in evolutionary, structural and computational methods, resulting in the identification of the 14 most damaging nsSNPs (L48P, R67W, H70Y, P72L, S83P, R151H, S172I, L206P, T242I, G255R, P256S, C273Y, C289R and R306H). Despite the differences in the results of the each program the combination of different methods allowed the differentiation of the neutral polymorphisms from the most damaging, showing agreement with the consensus programs. The computational prediction has proved to be an efficient approach for the identification of harmful alleles in the MC1R gene and for the prioritization of mutations for further functional and population studies.

Microarray experiments generate vast amounts of data that evidently reflect many aspects of the underlying biological processes. A major challenge in computational biology is to extract, from such data, significant information and knowledge about the complex interplay between genes/proteins. An analytical approach that has recently gained much interest is reverse engineering of genetic networks. This is a very challenging approach, primarily due to the dimensionality of the gene expression data (many genes, few time points) and the potentially low information content of the data. Bayesian networks (BNs) and its extension, dynamic Bayesian networks (DBNs) are statistical machine learning approaches that have become popular for reverse engineering. In the present study, a DBN learning algorithm was applied to gene expression data produced from experiments that aimed to study the etiology of necrotizing enterocolitis (NEC), a gastrointestinal inflammatory (GI) disease that is the most common GI emergency in neonates. The data sets were particularly challenging for the DBN learning algorithm in that they contain gene expression measurements for relatively few time points, between which the sampling intervals are long. The aim of this study was, therefore, to evaluate the applicability of DBNs when learning genetic networks for the NEC disease, i.e. from the above-mentioned data sets, and use biological knowledge to assess the hypothesized gene interactions. From the results, it was concluded that the NEC gene expression data sets were not informative enough for effective derivation of genetic networks for the NEC disease with DBNs and Bayesian learning.

We used the GENECONV program, the Hsu et al. (2010) method and phylogenetic analyses to analyze the gene conversions which occurred in the growth hormone, folate receptor and trypsin gene families of six primate species. Significant positive correlations were found between sequence similarity and conversion length in all but the trypsin gene family. Converted regions, when compared to non-converted ones, also displayed a significantly higher GC-content in the growth hormone and folate receptor gene families. Finally, all detected gene conversions were found to be less frequent in conserved gene regions and towards functionally important genes. This suggests that purifying selection is eliminating all gene conversions having a negative functional impact.

This thesis presents the use of Virus Induced Gene Silencing (VIGS) for the discovery of enzymes and transporters involved in monoterpene indole alkaloid (MIA) metabolism in the medicinal plant Catharanthus roseus. C. roseus is the source of a number of MIAs that are used as chemotherapeutic agents in the treatment of a variety of cancers, however the complete biosynthetic pathway for these metabolites remains to be elucidated. Additionally, this metabolic pathway is subcellulary compartmented with the key branch point enzyme, strictosidine synthase, localised to the plant vacuole. There is therefore a need for the import of the substrates for strictosidine biosynthesis; secologanin and tryptamine, across the vacuolar membrane, and export of the product, strictosidine, for synthesis of the downstream alkaloids. This thesis presents the identification of two proteins that act as trans-tonoplastic transporters in MIA metabolism. The multidrug and toxic compound extrusion (MATE) protein, CrMATE1952, was localised to the vacuolar membrane and silencing its expression in planta resulted in the accumulation of a secologanin derivative. This implicates CrMATE1952 in the transport of secologanin into the vacuole and highlights the importance of the spatial organisation of the pathway in preventing secologanin derivatisation. Secondly silencing the expression of a tonoplast localised nitrate/peptide (NPF) transporter, CrNPF2.9, resulted in the 20-fold accumulation of strictosidine, suggesting this transporter is the exporter of strictosidine from the vacuole. Furthermore, VIGS also allowed the identification of a reticuline oxidase like protein, CrRO, which resulted in the accumulation of two new MIAs in leaf tissue upon silencing. This thesis highlights a reverse genetics strategy for gene identification in metabolic pathways and is the first time the MATE and NPF transporters, and the reticuline oxidase like enzymes, have been shown to be involved in MIA metabolism in C. roseus.

The regulation of gene expression in differentiated neurons is poorly understood. By studying the transcriptional regulation of particular "model genes", whose expression is neuron specific, we hope to gain insight into the mechanisms governing neuron specific gene expression as a whole. The rat hippocalcin gene encodes a small calcium binding protein. Northern blot analysis and an in-situ hybridization study demonstrated that rat hippocalcin gene expression is brain specific, with different neuronal populations transcribing the gene to varying extents. Particularly high hippocalcin mRNA levels are found in the hippocampus, caudate putamen and olfactory tubercle of the rat brain. RT-PCR analysis detected hippocalcin gene expression in neuronal x glial hybridoma (NG108) and rat pheochromocytoma (PC12) cell lines; however not in 3T3 fibroblast (3T3) or human embryonic kidney (HEK) cell lines. To investigate the regulation of this cell-specific expression, the rat hippocalcin gene was cloned and it's structure determined. Genomic regions were then tested for transcriptional activity in transgenic mice. 3.2 kb of the hippocalcin gene upstream region directs neuron-specific expression of a lacZ reporter gene, in a pattern partially corresponding with endogenous hippocalcin gene expression. Reporter gene expression was invariably observed in the hippocampus, suggesting the presence of a hippocampal-specific-cis-regulatory element. Transcriptional activity of the -3.2 to +1 kb hippocalcin upstream region was investigated in more detail using transient transfection of reporter constructs into neuronal (NG108 and PC12) and non-neuronal (3T3 and HEK) cell lines. This analysis identified three, cell-specific, positively activating, elements between -3.2 to 1.4 kb. One of these elements is a small 300 bp fragment, that independently directs high levels of neuronal-cell-line-specific reporter gene expression.

The main aims of this work to set up two new mouse models, by conventional and conditional gene targeting, to investigate the functions of ERCC1, which has not, thus far, been associated with any known human disorders. To facilitate gene targeting studies, a series of overlapping ERCC1 genomic clones were retrieved from a bacteriophage library. Analysis of the mouse ERCC1 5'-flanking region revealed a novel transcription initiation site. Furthermore, several potential liver- and brain-specific transcription factor binding sites were present. A novel ERCC1-deficient mouse line, designated ET#209, was generated by the targeted deletion of the ERCC1 exon 3-5 region. In addition to the aberrant nuclei in the KT#209 ERCC1-/- livers, as reported in previous ERCC1 knockouts, we also found microvesicular steatosis and mitochondrial abnormalities in livers of both our ERCC1-deficient lines. These findings suggested that oxidative stress may damage nuclear and/or mitochondrial DNA, leading to the abnormal lipid deposits. We also found that other repair proteins (MSH2, PMS2 and PCNA) and an immediate early gene product, c-fos, were elevated in both ERCC1-deficient liver extracts. A delay in cerebellar development was found in both our ERCC1-deficient lines and the possibility of a cerebellar disorder was investigated. Also, we have demonstrated that some thymocyte markers were changes in flow cytometric profiles, suggesting a possible role for ECC1 in V(D)J recombination. To overcome the limitations of premature death in the ERCC1-deficient mice, we used a conditional gene targeting strategy. We reconstructed a functional ERCC1 allele with loxP sites flanking exons 3 and 5. In vitro analysis demonstrated that the ERCC1 exon 3-5 region between the loxP sites was deleted after transient expression of Cre recombinase. We have generated mice with this floxed ERCC1 allele, which will be used for Cre/loxP-mediated recombination in a tissue- and/or developmentally-specific manner.

Recent studies suggest that horizontal gene transfer (HGT) plays an important role in niche adaptation in some eukaryotes and may be a major evolutionary force in unicellular lineages. One subcategory of HGT is endosymbiotic gene transfer (EGT), which is characterized by a large influx of genes from endosymbiont to host nuclear genome and is a critical step in the establishment of permanent organelles, such as plastids. The dinoflagellates are a diverse group of mostly marine eukaryotes that have a propensity for both HGT and plastid endosymbiosis. Many dinoflagellates are predators and can acquire both genes and plastids from prey, blurring the distinction between HGT and EGT. Here, I measure genome mosaicism in dinoflagellates to investigate how HGT has impacted gene innovation and plastid endosymbiosis in this group. Because analysis of HGT depends on accurate phylogenetic trees, I first assessed the sensitivity of automated phylogenomic methods to variation in taxon sampling due to homolog selection parameters. Using methods based on this analysis, I showed that a large amount of HGT has occurred in dinoflagellates, particularly from bacterial donors. Further, I demonstrated that the dinoflagellate Alexandrium tamarense has the largest number of genes gained relative to related eukaryotes using ancestral gene content reconstruction. Additionally, dinoflagellates have lost several ubiquitous eukaryotic metabolic genes, but missing genes have been functionally replaced by xenologs from many evolutionarysources. Other transferred genes are involved in diverse functions. These results suggest that dinoflagellate genomes are heavily impacted by HGT. Also, I investigated the timing and consequences of HGT in plastid endosymbiosis. Using the dinflagellate Dinophysis acuminata, a mixotrophic species that sequesters and maintains prey plastids, I identified plastid-targeted proteins that function in photosystem stabilization and metabolite transport. Dinophysis acuminate may be able to extend the useful life of the stolen plastid by protecting the photosystem and replacing damaged transporters. Phylogenetic analyses showed that genes are derived from multiple sources indicating a complex evolutionary history involving HGT. Dinophysis acuminate can acquire both genes and plastids from prey, which suggests that HGT could play an important role in plastid acquisition during the earliest stages of this transition.

Die epitaktische Wachstum von GeTe Dünnschichten und Sb2Te3/GeTe Übergittern durch Molekularstrahlepitaxie wird auf drei verschiedenen Silizium Oberflächen gezeigt: Si(111)−(7×7), Si(111)−(√3×√3)R30°−Sb, und Si(111)−(1×1)−H. Mit Röntgenstrukturanalyse wird bewiesen, dass die epitaktische Beziehung der GeTe Schicht von der Oberflächepassievierung abhängig ist; auf einer passivierten Fläche können verdrehte Domänen unterdrückt sein. Dieses Verhalten ähnelt dem, welches bei 2D Materialien zu erwarten wäre, und wird auf die Schwäche der Resonanten ungebundenen Zustände zurückgeführt, die durch Peierls Verzerrung noch schwächer werden.
The growth by molecular beam epitaxy of GeTe and Sb2Te3/GeTe superlattices on three differently reconstructed Si(111) surfaces is demonstrated. Namely, these are the Si(111)−(7×7), Si(111)−(√3×√3)R30°−Sb, and Si(111)−(1×1)−H reconstructions. Through X-ray diffraction, the epitaxial relationship of GeTe is shown to depend on the passivation of the surface; in-plane twisted and twinned domains could be suppressed on a passivated surface. This behavior which resembles what would be expected from lamellar materials, is attributed to the relative weakness of resonant dangling bonds, that are further weakened by Peierls distortion.

Genes envolvidos com a interação hospedeiro-patógeno são fortemente afetados pela seleção natural positiva. O gene codificante do antígeno sangüíneo Duffy, também conhecido como DARC (Duffy Antigen Receptor for Chemokines), tem um importante papel na invasão dos eritrócitos pelos parasitas causadores da malária, Plasmodium vivax em humanos e Plasmodium knowlesi em outros primatas. A estrutura do gene DARC já é conhecida, estando este presente na região 1q22q23 do cromossomo 1, e sendo composto por dois éxons separados por um grande íntron. Em uma população africana a deleção de um nucleotídeo no domínio GATA-1 da região promotora do gene é responsável pela não expressão de DARC nos eritrócitos e pela resistência à invasão pelo P. Vivax. Além disso, o antígeno DARC age como um receptor promíscuo de quimiocinas, sendo expresso nos eritrócitos, células endoteliais de vênulas e outros tecidos. Devido a esse papel dual, no presente estudo seqüenciou-se regiões homólogas do gene DARC em macacos do Novo e Velho Mundo e utilizando métodos estatísticos procurou-se indícios da seleção natural positiva na sua história evolutiva. Nenhuma nova mutação foi encontrada no promotor ou na região codificante. As árvores filogenéticas pelos métodos de máxima parcimônia, máxima verossimilhança e neighbor-join apresentaram topologias semelhantes com três grande clados monofiléticos reconhecíveis e com a espécie Macaca fascicularis apresentando um perfil polifilético. O teste de seleção positiva pelos métodos de Nei-Gojobori, máxima verossimilhança por ramos e máxima verossimilhança por sítios não demonstraram, estatisticamente, à ação da seleção positiva sobre o gene DARC. Porém, o teste de máxima verossimilhança por sítio em domínios demonstrou que existem regiões do gene DARC sujeitas à diferentes pressões seletivas, mas também falhou em detectar a assinatura da seleção positiva. Os resultados indicam a presença da seleção darwiniana sobre a região de ligação do P. vivax, porém os testes de máxima verossimilhança utilizados, aparentemente, não possuem poder suficiente para detectar a sua assinatura. Além disso, os resultados sugerem que a região de ligação do P. vivax está sob influência de duas pressões seletivas antagônicas (seleção positiva exercida pelo parasita e seleção purificadora exercida pelas quimiocinas) o que pode, também, explicar a não detecção da seleção positiva.
Genes involved in pathogen-host interactions are strongly affected by positive natural selection. The gene of blood Duffy antigen, also known as DARC (Duffy Antigen Receptor for Chemokines), has an important role in the invasion of red blood cells by the parasites that cause malaria, Plasmodium vivax in humans and Plasmodium knowlesi in other primates. The structure of the DARC gene is known, it was mapped in 1q22-q23 region of chromosome 1, and is composed by two exons separated by a large intron. In an African population a nucleic acid deletion in GATA-1 of the gene promoter is responsible for the non-expression of DARC on red blood cells and the resistance to invasion by P. vivax. Moreover, the DARC antigen acts as a promiscuous receptor for chemokines and is expressed in red blood cells, endothelial venules cells and other tissues. Because of this dual role, in this study we sequenced homologous regions of the DARC gene in monkeys of the New and Old World and using statistical methods we tried to detect positive natural selection in their evolutionary history. New mutations were not found at promoter or in coding region. The phylogenetic trees by the methods of maximum parsimony, maximum likelihood and neighbor-join showed similar topologies with three large monophyletic clades recognizable and with the Macaca fascicularis showing a poliphyletic profile. The test of positive selection by the methods of Nei-Gojobori, maximum likelihood by branchs and maximum likelihood by sites not shown, statistically, the action of positive selection on the DARC gene. But the maximum likelihood test using sites divided in domains showed that some regions of the DARC gene are subject to different selective pressures, but also failed to detect the signature of positive selection. The results indicate the presence of darwinian selection on P. vivax binding region, but the maximum likelihood tests used, apparently, do not have enough power to detect its signature. Moreover, the results suggest that P. vivax binding region is under the influence of two opposing selective pressures (positive selection exerted by the parasite and purifying selection exerced by chemokines) that can also explain the non-detection of positive selection.

DNA was directly extracted from a number of water and sediment samples taken from the soda lakes of the Kenyan-Tanzanian Rift Valley. A limited number of microbial specific enrichments of these were also extracted in the same way. 16S rDNA genes were amplified by the polymerase chain reaction (PCR) and microbial diversity studied using denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (t-RFLP). DGGE had relatively poor resolution, as shown by the limited number of bands on gels as compared to the minimal number of amplicons represented by t-RFLP profiles. Cloning and sequencing of some of the DGGE bands indicated that they usually contained mixed amplicons. Amplicon sequencing enabled the identification of possible members of the soda lake community. Several sequences had high identity to the 16S rDNA genes of organisms previously isolated from soda lakes, others were only distantly related. Amplicons recovered from enrichment cultures were similar to those recovered from environmental DNA. In general, enrichment cultures showed comparable diversity to environmental samples in terms of DGGE and t-RFLP analysis. Genomic DNA libraries were also made from DNA recovered from an environmental soda lake sample, and three different enrichment cultures. DNA was extracted from the samples and digested with Sau 3AI. Fragments of between 2 and 10kbp were gel purified and ligated into ZAP express vector (Stratagene) for packaging into the XL1-Blue MRF' Escherichia coli host cells. pBK-CMV phagemids were excised from the libraries and screened by direct enzyme assays for enzyme activities. Two cellulase and three esterase/lipase activity clones were found. Sequence data indicated that one cellulase activity clone had highest amino acid identity to an endo-beta-1,4-glucanase gene from the glycoside hydrolase family 5. The second active cellulase clone was not fully sequenced.

Thesis (Ph. D.)--Massachusetts Institute of Technology, School of Architecture and Planning, Program in Media Arts and Sciences, 2008.
"June 2008."
Includes bibliographical references.
The ability to synthesize custom de novo DNA constructs rapidly, accurately, and inexpensively is highly desired by researchers, as synthetic genes and longer DNA constructs are enabling to numerous powerful applications in both traditional molecular biology and the emerging field of synthetic biology, from the synthesis of large sets of novel proteins to the complete re-writing of bacterial genomes. However, the current cost of de novo synthesis--driven largely by reagent and handling costs-is a significant barrier to the widespread availability of such technology. The use of microfluidic technology greatly reduces reaction volumes and corresponding reagent and handling costs. Additionally, microfluidic technology enables large numbers of complex reactions to be performed in parallel, while facilitating the automation and integration of multiple processes in a single device. While microfluidic devices have been used to miniaturize a variety of chemical and biological processes, the benefits of such devices have yet to be realized in the area of de novo DNA synthesis. This thesis reports the first demonstration of gene synthesis in a microfluidic environment. A variety of DNA constructs with sizes as large as 1 kb were fabricated in parallel in a multi-chamber microfluidic device at volumes one to two orders of magnitude lower than those utilized in conventional bench top techniques. This thesis also reports on progress toward the direct synthesis of genes from hybrid microfluidic-DNA microarray devices, the integration of microfluidic gene synthesis with on-chip protein synthesis, and the microfluidic hierarchical synthesis of long DNA molecules.
by David Sun Kong.
Ph.D.

Zusätzlich zu der eubakteriellen RNA-Polymerase (RNAP) der Plastiden sind im Zellkern von Arabidopsis thaliana drei weitere, phagentypische RNAP kodiert, die jeweils aus nur einer Einheit aufgebaut sind. Die Enzyme RpoTp und RpoTm werden in die Plastiden, bzw. die Mitochondrien transportiert, während RpoTmp in beiden Organellen zu finden ist. Um die Lichtabhängigkeit der RpoT-Gene zu untersuchen, wurde die lichtinduzierte Akkumulation ihrer Transkripte in 7-Tage alten Keimlingen, sowie 3- bzw. 9-Wochen alten Rosettenblättern mittels quantitativer real-time PCR ermittelt. Die entwicklungsabhängige Regulation der RpoT-Transkript-Akkumulation wurde außerdem während der Blattentwicklung analysiert. Zusätzlich wurde der Einfluss des circadianen Rhythmus untersucht. Es stellte sich heraus, dass die Transkriptakkumulation aller drei RpoT-Gene stark lichtinduziert war und nur marginalen circadianen Schwankungen unterlag. In weiteren Versuchen mit verschiedenen Lichtrezeptor-Mutanten und unterschiedlichen Lichtqualitäten wurde der Einfluss multipler Rezeptoren auf den Prozess der Lichtinduktion gezeigt. In den Zellen höherer Pflanzen finden sich drei Genome. Die Biogenese von Chloroplasten und Mitochondrien, sowie lebenswichtige Prozesse, wie Atmung und Photosynthese setzen oftmals die Aktivität von Genen auf mindestens zwei dieser Genome voraus. Eine intrazelluläre Kommunikation zwischen den verschiedenen Genomen ist daher unumgänglich für einen funktionierenden Stoffwechsel der Pflanze. In dieser Arbeit wurde herausgestellt, dass die Zahl mitochondrialer Genkopien in photosynthetisch inaktiven Arabidopsis-Keimlingen drastisch erhöht ist. Bei der Untersuchung des DNA-Gehaltes in Proben, die Altersstufen von 2-Tage alten Keimblättern bis hin zu 37-Tage alten, seneszenten Rosettenblättern umfassten, fand sich ein deutlicher Anstieg der Kopienzahlen in älteren Rosettenblättern. Außerdem unterschieden sich die Kopienzahlen der untersuchten Gene zum Teil erheblich voneinander.
In addition to eubacterial-like multi-subunit RNA polymerases (RNAP) localized in plastids and the nucleus, Arabidopsis thaliana contains three phage-like single-unit, nuclear-encoded, organellar RNAPs. The enzymes RpoTp and RpoTm are imported into plastids and mitochondria, respectively, whereas RpoTmp shows dual targeting properties into both organelles. To investigate if expression of the RpoT genes is light-dependent, light-induced transcript accumulation of RpoTm, RpoTp and RpoTmp was analyzed using quantitative real-time-PCR in 7-day-old seedlings as well as in 3- and 9-week-old rosette leaves. To address the question whether RpoT transcript accumulation is regulated differentially during plant development transcript abundance was measured during leaf development. Additionally, effects of the plants circadian rhythm on RpoT transcript accumulation were analyzed. Transcripts of all three RpoT genes were found to be strongly light-induced even in senescent leaves and only marginally influenced by the circadian clock. Further analyses employing different photoreceptor mutants and light qualities revealed the involvement of multiple receptors in the light-induction process. The biogenesis of mitochondria and chloroplasts as well as processes like respiration and photosynthesis require the activity of genes residing in at least two distinct genomes. There have to be ways of intracellular communication between different genomes to control gene activities in response to developmental and metabolic needs of the plant. In this study, it was shown that gene copy numbers drastically increased in photosynthetically inactive Arabidopsis seedlings. Mitochondrial DNA contents in cotyledons and leaves ranging in age from 2-day-old cotyledons to 37-day-old senescent rosette leaves were examined. A common increase in senescing rosette leaves and drastic differences between individual genes were found, revealing the importance of an integrative chondriome in higher plant cells.

Orientador: Maricilda Palandi de Mello
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: Para um correto desenvolvimento sexual masculino em humanos, é necessária a presença, entre outros, de dois hormônios esteróides: a testosterona (T) e a diidrotestosterona (DHT). A T é o hormônio responsável pelo desenvolvimento da genitália interna masculina, já a DHT é o hormônio chave da virilização da genitália externa masculina e responsável pelo estabelecimento dos caracteres sexuais secundários durante a puberdade. Duas enzimas são responsáveis pela produção destes hormônios: a enzima 17?-hidroxiesteróide desidrogenase tipo 3 (gene HSD17B3), a qual é responsável pela conversão do hormônio androstenediona em T, reação realizada na última etapa da biossíntese da T e a enzima 5?-redutase tipo 2 (gene SRD5A2), que é responsável por catalisar a conversão da T em DHT. Mutações nos genes HSD17B3 ou SRD5A2 causam alterações que levam à não produção ou à síntese defeituosa das enzimas 17?-hidroxiesteróide desidrogenase tipo 3 e 5?-redutase tipo 2, promovendo deficiência na virilização de indivíduos 46,XY. Indivíduos estes cujas gônadas são representadas por testículos, apresentam pseudo-hermafroditismo masculino (PHM), agora denominado distúrbio da diferenciação do sexo em indivíduos 46,XY (DDS-XY). Estes podem apresentar genitália ambígua, sendo o sexo de criação predominantemente feminino, com virilização na puberdade. O diagnóstico pode ser confirmado com a identificação de mutações nos genes específicos HSD17B3 e SRD5A2. Assim, o objetivo desse estudo foi investigar alterações moleculares nos genes HSD17B3 e SRD5A2, em pacientes com ambiguidade genital com cariótipo 46,XY, e contribuir para a confirmação do diagnóstico clínico e laboratorial de DDS em indivíduos 46,XY por deficiências nas enzimas 17?-hidroxiesteróide desidrogenase tipo 3 e 5?-redutase tipo 2. A metodologia empregada neste estudo teve por base a amplificação dos 11 exons do gene HSD17B3 e dos 5 exons do gene SRD5A2 pela reação em cadeia da polimerase (PCR), seguida por rastreamento das mutações através do sequenciamento direto dos produtos de amplificação. Das 2 famílias estudadas para diagnóstico de alterações no gene HSD17B, foi encontrada uma alteração, p.R80Q, em homozigose em um paciente. E para o para o gene SRD5A2, foram estudadas 45 famílias, e foi verificada a presença de três mutações: a c.418delT em homozigose em um paciente, a c.278delG em heterozigose em um paciente e a p.Q126R em homozigose em duas irmãs. Vários polimorfismos freqüentes foram observados e, também, algumas riações nucleotídicas novas ou raras foram identificadas. Fez parte do estudo a avaliação in vitro da mutação g.49529G>A, já descrita previamente como uma mutação missense (p.G183S), quanto ao efeito deletério no processo de splicing, uma vez que esta alteração encontra-se no último nucleotídeo do exon 3 do gene SRD5A2. Foi verificado que esta alteração promove a excisão do exon 3, mostrando que o efeito primário desta mutação não é a troca de aminoácidos e sim uma alteração no processo de splicing deste gene.
Abstract: The male sexual development requires the production of normal amounts of two steroid hormones: testosterone (T) and dihydrotestosterone (DHT). The T is responsible for development of male internal genitalia, whereas DHT is the key for the virilization of the male external genitalia and responsible for the establishment of secondary sexual characteristics during puberty. Two enzymes are responsible for the production of these hormones: 17?-hydroxysteroid dehydrogenase type 3 enzyme (HSD17B3 gene), which is responsible for converting androstenedione to T, the last step of the biosynthesis of T; and 5?-reductase type 2 enzyme (SRD5A2 gene), which is responsible for catalyzing the conversion of T into DHT. Individuals with 46,XY karyotype and mutations in either HSD17B3 gene or SRD5A2 gene, present a deficiency of enzyme activity that leads to phenotypes ranging from male with ambiguous genitalia to normal female. Such individuals may be assigned at birth and raised as females. The gonads of those individuals are represented by testes. This condition defines the male-pseudohermaphroditism (MPH), recently renamed as disorder of sexual development in 46,XY karyotype (DSD-XY). The diagnosis of these deficiencies can be confirmed with the identification of mutations in either HSD17B3 or SRD5A2 genes. The aim of this investigation was to identify nucleotide alterations in the HSD17B3 gene or in the SRD5A2 gene in patients with clinical and hormonal characteristics suggestive of 17?-hydroxysteroid dehydrogenase type 3 deficiency, or 5?-redutase type 2 deficiency. Molecular analysis was performed by amplification of the eleven exons of HSD17B3 gene, and the five exons of SRD5A2 gene followed by sequencing. In two families studied for the HSD17B3 gene, one alteration was detect, p.R80Q, in a homozygous patient. Forty-five families were studied for SRD5A2 gene and the presence of three mutations was verified: the c.418delT in one homozygous patient, the heterozygosis for c.278delG in one patient and the homozygosis for p.Q126R in two sisters. Several frequent polymorphisms in the SRD5A2 gene were observed and some novel or rare variations were identified as well. The study in vitro with the g.49529G>A mutation was also performed due to the possibility of deleterious effect on the splicing process, although his mutation had been previously described as a missense mutation (p.G183S). It was verified exon 3 skipping in the mRNA as a result of the mutation, showing that the primary effect is not the change of amino acids but the anomalous splicing process of the SRD5A2 gene.
Mestrado
Genetica Animal e Evolução
Mestre em Genética e Biologia Molecular

TCC(graduação) - Universidade Federal de Santa Catarina. Centro de Ciências Biológicas. Biologia.
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O biocombustível tornou-se uma alternativa ao uso do petróleo. Nesse cenário o Brasil apostou no etanol. Industrialmente, sua produção acontece através do processo de fermentação alcóolica pela levedura Saccharomyces cerevisiae em matéria vegetal, como a cana-de-açucar (Saccharum spp.). Porém, a levedura não apresenta o metabolismo necessário para fermentar pentoses, como a xilose, um dos carboidratos mais abundantes em matéria lignocelulósica. Investigando leveduras que fermentam xilose, encontramos um processo de redução e oxidação mediado pelas enzimas Xilose Redutase (XR) e Xilitol Desidrogenase (XDH). Portanto, uma maneira de aumentar a produção de etanol no país, sem aumentar a área plantada de cana-de-açucar, é a criação de linhagens de S. cerevisiea transformantes com as enzimas supracitadas. Para isso, buscamos as enzimas com maior atividade entre as espécides fermentadoras de xilose, chegando as seguintes duas espécies: Spathaspora arborarie e Spathaspora passalidarum. Sendo que a enzima XR de S. arborarie, além de apresentar alta atividade, apresenta atividade com dois cosubstratos, NADPH e NADH. Já a enzima XDH de S. passalidarum apresentou a maior atividade entre as enzimas XDH já descritas, e uma dependência do cosubstrato NAD+. O uso conjunto das enzimas é interessante pela reciclagem dos cosubstratos. Portanto, nesse trabalho, buscamos criar um plasmídeo com um forte promotor constitutivo PGK para XR e TEF para XDH com a intenção de transformar o organismo S. cerevisiae para futura produção de etanol a partir de xilose. Além disso, estudos de engenharia genética demonstraram um efeito positivo da deleção do gene PHO13 de S. cerevisiae, já que essa mudança parece aumentar a expressão de enzimas da Via Glicolítica e da Via das Pentoses Fosfato. Por isso, utilizamos linhagens recombinantes pho13∆ com expressão das enzimas heterólogas supracitadas e comparamos seu perfil fermentativo com linhagens com a mesma expressão de enzimas, porém não pho13∆. Como resultado, percebemos que a expressão das enzimas permite o consumo de xilose, porém percebemos pouca produção de etanol. A linhagem pho13∆ se torna vantajosa em fermentação com alta densidade de xilose, 10%, produzindo o dobro de etanol da linhagem não pho13∆.

A cell is a the basic structural and functional unit of every living thing, it is protein-based an that regulates itself. The cell eats to stay alive, it grows and develops; reacting to the environment, while subjected to evolution. It also makes copies of itself. These processes are governed by chain of chemical reactions, creating a complex system. The scientific community has proposed to model the whole process with Gene Regulatory Networks (GRN). The understanding of these networks allows gaining a systems-level acknowledgment of biological organisms and also to genetically related diseases. This thesis focused on network inference from gene expression data, will contribute to this field of knowledge by studying different techniques that allows a better reconstruction of GRN. Gene expression datasets, are characterised by having thousands of noisy variables measured only with tens of samples. Moreover, these variables presents non-linear dependencies between them. Therefore, recovering a model that is capable of capturing the relationships contained in this data, constitutes a major challenge. The main contribution of this thesis is a set of fair and sound studies of different GRN inference methods and post-processing algorithms. First, we present a novel approach for inferring gene networks and we compare it with other methods. It is inspired by the concept of "variable importance" in feature selection. However, many algorithms can be proposed to infer GRNs, so there is a need to assess the quality of these algorithms. Secondly, and motivated by the fact that the previous comparison was not informative enough, we introduce a new framework for in silico performance assessment of GRN inference methods. This work has led to an open source R/Bioconductor package called NetBenchmark. Finally, and thanks to this tool we have corroborated that inferring gene regulatory networks from expression data is a tough problem. The different algorithms have some particular biases and strengths, and none of them is the best across all types of data and datasets. Therefore, we present a framework for evaluating and standardising network consensus methods to aggregate various network inferences
Una célula es es la unidad estructural y funcional básica de todo ser viviente capaz de autoregularse mediante proteínas. La célula come para mantenerse viva, crece y se desarrolla; Reaccionando al medio ambiente y está sometida a la evolución. También hace copias de sí misma. Estos procesos se rigen por una cadena de reacciones químicas, creando un sistema complejo. La comunidad científica ha propuesto modelar todo el proceso con las redes reguladoras de genes (GRN). La comprensión de estas redes permite entender los sistemas de los organismos biológicos y también las enfermedades genéticas. Esta tesis se centra en la inferencia de GRN a partir de datos de expresión génica, contribuye a este campo de conocimiento mediante el estudio de diferentes técnicas que permiten una mejor reconstrucción de GRN. Los conjuntos de datos de expresión génica se caracterizan por tener miles de variables ruidosas de las que sólo se disponen decenas de muestras. Además, estas variables presentan dependencias no lineales entre ellas. Por lo tanto, recuperar un modelo capaz de capturar las relaciones contenidas en estos datos, constituye un reto importante. La principal contribución de esta tesis es un conjunto de estudios de los diferentes métodos de inferencia de GRN y algoritmos de posprocesamiento. En primer lugar, presentamos un nuevo enfoque para inferir redes de genes y lo comparamos con otros métodos del estado del arte. Se inspira en el concepto de "importancia de variable" propio de la selección de características (feature selection). Sin embargo, muchos algoritmos pueden ser propuestos para inferir GRNs, por lo que hay una necesidad de evaluar la calidad de estos algoritmos. En segundo lugar, y motivado por el hecho de que la comparación anterior no era lo suficientemente informativa, introducimos un nuevo marco para la evaluación en bases de datos sintéticas de los métodos de inferencia GRN. Este trabajo ha llevado a un paquete de código abierto de R / Bioconductor llamado NetBenchmark. Finalmente, y gracias a esta herramienta hemos corroborado que inferir las redes reguladoras de genes a partir de los datos de expresión es un problema difícil. Los diferentes algoritmos tienen algunos sesgos y fortalezas particulares, y ninguno de ellos es el mejor en todos los tipos de datos y conjuntos de datos. Por lo tanto, presentamos un marco para evaluar y estandarizar los métodos de consenso de redes para agregar varias inferencias de red.

Systems biology is an interdisciplinary field that combines engineering and molecular biology to better understand the 'design principles' of biological systems. One of the main goals in systems biology is to understand and map complex biological networks. In order to achieve this goal, tools able to process the non-linearity and high dimensionality of biological systems are urgently needed. To develop, test and benchmark tools for investigation of biological processes, it is important to utilize a model system that is able to provide a glimpse of complexity found in higher organisms, and be simple enough such that detailed studies can be performed rapidly, accurately, and reproducibly. Here, we have developed a system-identification framework for the extraction of quantitative and mechanistic information about causal relationship among genes using the canonical galactose utilization pathway in Saccharomyces cerevisiae. This framework, referred to as s&barbelow;ystematic g&barbelow;ene p&barbelow;erturbation a&barbelow;nalysis (SGPA), is based on the effects of systematic pair-wise gene deletions. In essence, the method establishes dynamical models of the regulatory network from single-cell measurements of steady-state input-output relationships, in systematically perturbed networks. SGPA framework is successful in identifying the network structure for the GAL system. This strategy leads the way to a better application of available resources and provides a scalable framework for system-identification and reverse-engineering of biological networks based on in vivo systematic data generation.

The SA gene encodes a 578 amino acid protein of unknown function. SA was first identified as a candidate gene for hypertension and blood pressure regulation due to its increased expression in the kidneys of genetically hypertensive compared with normotensive rats. The aim of the work undertaken in this thesis was to investigate the function of the SA gene by gene targeting in the mouse and to assess any possible involvement of the SA protein in BP homeostasis. We utilised ES cell technology to generate a mouse model carrying a null mutation of the SA gene. Mice lacking the protein product of the SA gene are viable, reproductively normal and have no overt phenotype. Body weight and kidney, liver and heart weights are not affected by the absence of the SA protein. Comparison of basal blood pressures (BP) revealed no differences between SA-null and wildtype littermate controls in either male or female mice. Exposure of male mice to a high salt diet caused an increase in BP in wildtype mice. However in SA-null mice no effect of salt intake on BP was observed. It therefore appears that absence of the SA protein may offer some protection against a sodium induced rise in BP. The mechanism for this remains to be elucidated but may include an involvement of the SA protein in sodium retention. Administration of dihydrotestosterone (DHT) to female wildtype mice caused no increase in BP. However in SA-null mice BP was increased in response to DHT administration implying a protective effect of SA against a DHT induced rise in BP. These findings provide for the first time direct evidence of the involvement of the SA protein in BP regulation under certain conditions.

It has been proposed that CD4+ T cells and cell-mediated immunity play a central role in corneal allograft rejection. Two subsets of CD4+ T cells, Th1 and Th2 cells, are known to cross-regulate each other through their cytokine pattern and the immune response might be directed predominantly in one or the other direction. As such, it has been hypothesized here that predominant Th1 type immune response could lead to corneal allograft rejection, and that previous inflamed corneal beds (high-risk eyes) might augment the Th1 response and thus accelerate the graft rejection.
Reverse transcription of mRNA followed by polymerase chain reaction amplification was used to determine the relative gene expression in ocular tissues (cornea and iris/ciliary body) obtained from syngeneic grafts, low- and high-risk allografts. Compared with the syngeneic grafts, mRNA analysis of the low- and high-risk allografts showed a significantly decreasing expression pattern for the Th3 cytokine TGF-beta2, an early peak followed by a decline in the Th2 cytokines IL-4 and IL-10 expression, and a progressively increasing expression of the Th1 cytokines IL-2 and IFN-gamma and the proinflammatory cytokines IL-1beta and TNF-alpha, which paralleled the course of graft rejection. Prevascularization of the recipient eye (high-risk) significantly accelerated the rejection of corneal allografts and the mRNA levels of the Th1 cytokines IL-2 and IFN-gamma and proinflammatory cytokines IL-1beta and TNF-alpha in high-risk allografts were significantly higher and peaked faster than that in low-risk allografts.
In vivo gene transfer using plasmid DNA encoding cytokines is an attractive alternative to modulate the Th1 inflammatory reaction and immune response. This has led to the hypothesis that transferring the gene encoding Th2 cytokine IL-10 into the recipient could prevent or reduce the subsequent corneal allograft rejection through the suppression of Th1-mediated alloimmune response.
Intramuscular injection with in vivo electroporation of IL-10 plasmid DNA was administered at one week before and at one week after corneal transplantation. Corneal allograft survival was significantly prolonged and the rejection rate was significantly reduced after gene therapy with IL-10 plasmid DNA, compared with that in control groups treated with the empty plasmid vector. In IL-10 treated rats, the mRNA expression for the Th1 cytokines IL-2 and IFN-gamma was depressed, and the IL-10 mRNA expression was significantly increased. However, graft survival was not permanent. (Abstract shortened by UMI.)