Diploma thesis deals with genetic variability and evolutionary relationships among selected species of the group Chenopodium album and is especially focused on the origin of hexaploid species C. album in the Czech Republic. The main aim of the study was detection of putative recent emergence of hexaploid C. album s. str. in mixed populations of diploid (C. ficifolium and C. suecicum) and tetraploid (C. strictum and C. striatiforme) species. To assess phylogenetical relationships I performed an analysis of nuclear microsatellite loci, that are suitable for detection parental lines within polyploid species. As long as microsatellite primers have been developed for studied group, therefore they had to be cross-amplified from closely related species C. quinoa. Three PCR multiplexes were assembled by cross-amplification of microsatellite primers. These PCR multiplexes were tested on representative sample sets to estimate genetic variability of individual microsatellite loci. A total of 911 individuals were analyzed from five localities from the Czech Republic. The results of analyses revealed (1) high interspecific differentiation, (2) gene flow among species of the same ploidy levels, (3) presence of hybrids among diploid species of C. ficifolium and C. suecium as well as among tetraploid species C. strictum...

Apesar de causar um enorme impacto na história da evolução e prognóstico a partir da infecção pelo HIV, a terapia anti-retroviral altamente potente prolongada apresenta vários efeitos colaterais. Dentre esses, a síndrome da lipodistrofia (SL) caracterizada por alterações metabólicas e morfológicas. Embora tenha sido descrito que a adesão à terapia esteja associada, sua patogenia ainda permanece desconhecida. Um enfoque têm sido dado aos mediadores pró-inflamatórios, como o Fator de Necrose Tumoral (TNF), sugerindo que o aumento nos níveis dessa citocina esteja associado com o desenvolvimento da SL. Como os sítios polimórficos têm sido associados com a magnitude da produção de citocinas, no presente estudo avaliamos a freqüência de alguns sítios polimórficos na região do gene que codifica o TNF em portadores do HIV/aids apresentando ou não a SL. Para avaliar o polimorfismo genético dos microssatélites TNFa-e e da região promotora do TNF (TNF-308 e TNF-238) foram estudados 117 portadores do HIV-1 usando terapia anti-retroviral (67 com SL e 50 sem SL) e 131 controles saudáveis. Os microssatélites e região promotora do TNF foram tipificados usando DNA genômico hibridizado com iniciadores específicos. Os pacientes foram arrolados no Hospital das Clínicas da Universidade de São Paulo (HCFMRP-USP). A analise estatística foi realizada utilizando-se o teste exato de Fisher. Quando consideramos as comparações das freqüências dos alelos dos microssatélites da região do TNF podemos inferir que a presença do alelo TNFa5 pode conferir proteção aos indivíduos portadores do HIV/aids no desenvolvimento da SL. Já as comparações dos alelos da região promotora do TNF nos sugerem que a presença do alelo TNF-308G, assim como seu homozigoto TNF-308GG, podem conferir susceptibilidade para o desenvolvimento da SL. A presença do haplótipo TNFe3 d3 -238G -308A c1 a5 b7 sugere proteção para o desenvolvimento dessa síndrome. Esse é o primeiro estudo associando o polimorfismo dos microssatelites do TNF com a SL e aponta diversas associações entre alelos da região do gene que codifica o TNF com a SL. Embora os mecanismos relacionados com a participação do TNF no desenvolvimento da SL não estejam bem esclarecidos, este estudo sugere que fatores imunogenéticos associados com a magnitude de expressão do TNF e, provavelmente da expressão de outras citocinas pró-inflamatórias, estejam envolvidas no desenvolvimento da SL em portadores do HIV/aids. / Despite causing a significant impact in the history of evolution and prognosis after HIV infection, the highly potent antiretroviral therapy causes various side effects, which include lipodystrophy syndrome (LS), characterized by metabolic and morphologic changes. Although it has been reported that treatment compliance is associated, LS pathogenesis remains unknown. Special attention has been given to proinflammatory mediators, such as the Tumoral Necrosis Factor (TNF), suggesting that the increase in levels of this cytokine is associated with the development of LS. Since polymorphic sites have been associated with the magnitude of cytokine production, in the present study we evaluated the frequency of some polymorphic sites in the gene region that codes TNF in HIV/aids-infected patients presenting LS or not. In order to evaluate the genetic polymorphism of TNFa-e microsatellites and of the TNF promoter region (TNF-308 and TNF-238), 117 HIV-1 infected patients using antiretroviral therapy (67 with LS and 50 without LS) and 131 healthy controls were studied. Microsatellites and the TNF promoter region were typified using hybridized genomic DNA with specific initiators. Patients were selected at the Clinics Hospital of the University of São Paulo (HCFMRP-USP). Statistical analysis was performed using the Fisher\'s exact test. Comparisons of the frequencies of microsatellite alleles of the TNF region suggest that the presence of the TNFa5 allele could provide HIV/aids patients with protection against developing LS. In addition, comparisons of alleles of the TNF promoter region suggest that the presence of TNF-308G allele, as well as of its homozygote TNF-308GG could pose susceptibility to developing LS. The presence of the haplotype, TNFe3 d3 -238G -308A c1 a5 b7, suggests protection against developing that syndrome. The present study is the first to associate TNF microsatellite polymorphism with LS, indicating several associations among alleles of the gene region that codes TNF with LS. Although the mechanisms regarding the participation of TNF in the development of LS are not clear, this study suggests that immunogenetic factors associated with the TNF expression magnitude and probably the expression of other proinflammatory cytokines are involved in the development of LS in HIV/aids infected patients.

As abelhas da tribo Meliponini (abelhas sem ferrão) estão amplamente distribuídas pelas regiões tropicais do planeta, tendo um importante papel na polinização, sendo o gênero Melipona o que contém o maior número de espécies. A espécie Melipona marginata é uma das menores e mais ancestrais do grupo, e a exemplo de outras espécies nidifica em ocos de árvore. M. marginata, assim como outras espécies do gênero Melipona, vêm sofrendo com a destruição do seu ambiente natural, pelo desmatamento, sendo, aparentemente mais exigente que outras espécies quanto ao tamanho do fragmento florestal para se manter, devido a isso é encontrada apenas em fragmentos maiores, mais antigos e menos perturbados. Tendo em vista a perda de hábitat e o pouco conhecimento da biologia dessa espécie, este trabalho pretende analisar populações de M. marginata, através da técnica PCR+RFLP do DNA mitocondrial e marcadores microssatélites, visando contribuir para entendimento da estrutura populacional de M. marginata. Foram analisados 54 ninhos, provenientes dos estados de MG, SP, PR, SC e RS. Oito regiões do DNA mitocondrial foram amplificadas, e posteriormente digeridas com 12 enzimas de restrição. Foram detectados 14 haplótipos, sendo apenas um compartilhado. A população de SP apresentou o maior número de haplótipos. Os testes estatísticos demonstraram que as populações estão estruturadas e isoladas, não havendo fluxo gênico entre as populações. Já nas análises dos microssatélites foram analisados 4 locos, apresentando alta variabilidade genética, onde também foi verificado que as populações se encontram estruturadas. Os resultados obtidos podem ser explicados principalmente pela redução da área de floresta, mas, podem se dever a eventos antigos em conseqüência de mudanças climáticas ocorridas durante as últimas glaciações. / The tribe Meliponini (stingless bees) is present in all tropical regions of the world and has an important role in pollination. The genus Melipona has the highest number of species in the tribe. The specie Melipona marginata is considered the most ancestral within the genus, and like other species builds the nests in hollow of trees. Unfortunately several bee species have suffered with the devastation of their natural environment. M. marginata seems to be very dependent on the size of the forest fragment, being found only in the biggest, oldest and consequently more preserved ones. In view of the habitat loss and few biological knowledge about this specie, this research intended to analyse M. marginata populations molecularly, through mitochondrial DNA PCR-RFLP and microsatellite data., Fifty four colonies were analyzed, from MG, SP, PR, SC and RS states. Eight mitochondrial DNA regions were amplified and screened with 12 restriction enzymes. Fourteen haplotypes were verified and among them just one was shared. SP population showed the highest number of haplotypes. Statistic tests showed that the 5 populations were genetic structured and isolated, therefore not presenting gene flow. The 4 microsatellites loci analyzed showed high genetic variability. The statistics analysis indicated that the 5 populations are structure and isolated. These results can be explained mainly because the decrease of forest leading to population isolation, however we can not discard the hypothesis that this current scenario is a consequence of climatic changes occurred during the last glaciations.

Molecular ecology has already provided profound insights into behaviour, ecology and systematics of organisms improving our understanding of the relationship between genetic variation and biodiversity. The objectives of my PhD were to develop new genetic markers and use these markers to address fundamental issues in evolutionary biology using shorebirds as model organisms. Shorebirds are part of the ancient avian Charadriiformes order and are characterised by extraordinary ecological and behavioural diversity. However, due to the lack of appropriate genetic markers the molecular ecology of many shorebirds has not been investigated previously. Therefore, first, I developed polymorphic microsatellite markers from genomic libraries for a behaviourally diverse shorebird, the Kentish plover Charadrius alexandrinus (Chapter II). Second, using the genomic data-bases I expended this work to develop further markers that cannot only be used in the Kentish plover, but also a large number of other shorebird species (Chapter III). Third, I investigated population differentiation and genetic diversity of Eurasian and American Kentish plover populations using the newly developed microsatellite markers and further mitochondrial markers (Chapter IV). The genetic differences between Eurasian and American populations that are mirrored by phenotypic differences call for a reconsideration of the current taxonomic status of the species; Eurasian and American populations should be recognised as belonging to two separate species. Finally, I asked how genetic diversity influences the fitness of precocial Kentish plover young (Chapter V). I found that survival of chicks until fledging was associated with genetic diversity (measured as heterozygosity) at three of eleven marker loci. Genetic diversity at one marker locus had a positive effect on survival whilst it had negative effects at two loci. The results of my PhD have brought up many new questions and I propose promising lines that need to be explored in the future (Chapter VI).

"Local genetic adaptation and homing behavior in anadromous fish favors the formation of local populations across their geographic range of distribution. Spawning- and natal-site fidelity repeated over generations restricts gene flow and allows genetic differences to accumulate resulting in reproductive isolation. This leads to progressive genetic differentiation and population structuring among different river populations. Alewife, Alosa pseudoharengus, are anadromous fish which are estimated to have high rates of reproductive fidelity and hence might show population structuring among different breeding streams. Alewife are fish of economic importance since they have both commercial and recreational value. Alewife populations have been declining over the past decades and conservation measures to restore the populations have been implemented. Since maintaining genetic integrity of natural populations is one of the main concerns, identification of population structure can assist in designing appropriate restocking programs. In this study, I used microsatellite markers developed for shad to study population structuring in alewife. Samples were collected from two sites in Connecticut and one in Lake Michigan and genetic differentiation among these populations was estimated using five microsatellite loci. My studies indicate that microsatellite loci developed for shad can be used for alewife. Results from this preliminary study indicated subtle but significant genetic differentiation among populations. This suggests that care should be taken when restocking alewife from different sites in order to maintain genetic diversity among these populations."

O gênero Partamona compreende 33 espécies, distribuídas do sul do México ao sul do Brasil. O gênero tem sido amplamente estudado em diferentes níveis: citogenético, etológico e morfológico. O presente trabalho teve como objetivo contribuir com dados moleculares para o conhecimento do grupo, realizando estudos populacionais por meio da caracterização do DNA mitocondrial por PCR+RFLP e da análise de regiões de microssatélites do DNA genômico de duas espécies: P. mulata de distribuição restrita ao sul de Mato Grosso e norte do Mato Grosso do Sul, e P. helleri de distribuição mais ampla, do sul da Bahia até Santa Catarina. Foram detectados apenas dois haplótipos em P. mulata, os quais diferiram entre si por apenas um sítio de restrição. As análises estatísticas demonstraram não haver estruturação entre as populações sugerindo que esta espécie possa ter passado por recente afunilamento populacional. Em P. helleri foram observados dez haplótipos sendo alguns exclusivos e outros compartilhados. Análises estatísticas apontaram alta estruturação entre as populações e a distribuição filogeográfica observada sugere um possível isolamento por fragmentação da Mata Atlântica durante o Pleistoceno. A análise dos locos microssatélites mostrou baixa variabilidade genética em ambas espécies e discreta estruturação entre as populações, não relacionada com a distribuição geográfica das mesmas. Isto pode ser conseqüência de migração de machos entre populações visto que as rainhas são filopátricas ou, fragmentação dos habitats pela rápida degradação do cerrado e da Mata Atlântica, ou por alelos nulos causados pelo uso de primers heteroespecíficos. A análise de parentesco entre abelhas de um mesmo ninho apontou a existência de apenas uma patrilínea em P. mulata sugerindo monoandria para esta espécie. Foram encontradas duas patrilíneas em algumas colônias de P. helleri, o que pode ser resultante de fecundação por mais de um macho ou substituição recente da rainha. A caracterização parcial do DNAmt de duas espécies de Partamona poderá contribuir em estudos filogenéticos tanto do gênero quanto de outras espécies de Meliponini. A análise populacional mostrou o status da variabilidade genética das espécies, suas possíveis histórias evolutivas e a possível relação desta com degradação dos ambientes onde estas estão distribuídas. / The Partamona genus comprises 33 species distributed from south Mexico to south Brazil. This genus has been studied at different levels: cytogenetical, ethological and morphological. This work aimed at to contribute with molecular data for the knowledge about the group performing a population study employing the PCR+RFLP of mtDNA, and analysis of microsatellite loci from nuclear DNA of two species, P. mulata which is distributed in south Mato Grosso and north Mato Grosso do Sul, and P. helleri which geographic distribution is wider, from Santa Catarina to southern Bahia. It was detected two haplotypes in 58 colonies of P. mulata, each one differing by one single restriction site. The statistical analyses indicated no differentiation among populations suggesting that the species could have passed through a recent populational bottleneck. It was observed ten haplotypes in 47 colonies of P. helleri, some exclusive and others shared among populations. Statistical analysis pointed high population differentiation and the observed phylogeography distribution suggested a possible recent isolation probably by Atlantic Forest fragmentation during the Pleistocene. The microsatellite analysis showed low genetic variability in both species and discrete population structuring, not related to the geographic distribution. This might be consequence of migration of males, since the queens are highly phylopatric, or habitat fragmentation by degradation of savanna and Atlantic forest areas, or null alleles caused by the use of heterospecific primers. The relatedness investigation revealed only one patriline in nest mates of P. mulata that suggests monoandry for this species. It was found two patrilines in P. helleri that can be resulted from more than one mating or recent queen replacement. The partial characterization of the mtDNA of two Partamona species can contribute to further phylogenetic studies among bees of this genus or among other Meliponini species. The populational analysis showed the genetic variability status of the species, their putative evolutionary histories and the possible relation between the results and the environmental degradation in their distribution areas.

Apesar de causar um enorme impacto na história da evolução e prognóstico a partir da infecção pelo HIV, a terapia anti-retroviral altamente potente prolongada apresenta vários efeitos colaterais. Dentre esses, a síndrome da lipodistrofia (SL) caracterizada por alterações metabólicas e morfológicas. Embora tenha sido descrito que a adesão à terapia esteja associada, sua patogenia ainda permanece desconhecida. Um enfoque têm sido dado aos mediadores pró-inflamatórios, como o Fator de Necrose Tumoral (TNF), sugerindo que o aumento nos níveis dessa citocina esteja associado com o desenvolvimento da SL. Como os sítios polimórficos têm sido associados com a magnitude da produção de citocinas, no presente estudo avaliamos a freqüência de alguns sítios polimórficos na região do gene que codifica o TNF em portadores do HIV/aids apresentando ou não a SL. Para avaliar o polimorfismo genético dos microssatélites TNFa-e e da região promotora do TNF (TNF-308 e TNF-238) foram estudados 117 portadores do HIV-1 usando terapia anti-retroviral (67 com SL e 50 sem SL) e 131 controles saudáveis. Os microssatélites e região promotora do TNF foram tipificados usando DNA genômico hibridizado com iniciadores específicos. Os pacientes foram arrolados no Hospital das Clínicas da Universidade de São Paulo (HCFMRP-USP). A analise estatística foi realizada utilizando-se o teste exato de Fisher. Quando consideramos as comparações das freqüências dos alelos dos microssatélites da região do TNF podemos inferir que a presença do alelo TNFa5 pode conferir proteção aos indivíduos portadores do HIV/aids no desenvolvimento da SL. Já as comparações dos alelos da região promotora do TNF nos sugerem que a presença do alelo TNF-308G, assim como seu homozigoto TNF-308GG, podem conferir susceptibilidade para o desenvolvimento da SL. A presença do haplótipo TNFe3 d3 -238G -308A c1 a5 b7 sugere proteção para o desenvolvimento dessa síndrome. Esse é o primeiro estudo associando o polimorfismo dos microssatelites do TNF com a SL e aponta diversas associações entre alelos da região do gene que codifica o TNF com a SL. Embora os mecanismos relacionados com a participação do TNF no desenvolvimento da SL não estejam bem esclarecidos, este estudo sugere que fatores imunogenéticos associados com a magnitude de expressão do TNF e, provavelmente da expressão de outras citocinas pró-inflamatórias, estejam envolvidas no desenvolvimento da SL em portadores do HIV/aids. / Despite causing a significant impact in the history of evolution and prognosis after HIV infection, the highly potent antiretroviral therapy causes various side effects, which include lipodystrophy syndrome (LS), characterized by metabolic and morphologic changes. Although it has been reported that treatment compliance is associated, LS pathogenesis remains unknown. Special attention has been given to proinflammatory mediators, such as the Tumoral Necrosis Factor (TNF), suggesting that the increase in levels of this cytokine is associated with the development of LS. Since polymorphic sites have been associated with the magnitude of cytokine production, in the present study we evaluated the frequency of some polymorphic sites in the gene region that codes TNF in HIV/aids-infected patients presenting LS or not. In order to evaluate the genetic polymorphism of TNFa-e microsatellites and of the TNF promoter region (TNF-308 and TNF-238), 117 HIV-1 infected patients using antiretroviral therapy (67 with LS and 50 without LS) and 131 healthy controls were studied. Microsatellites and the TNF promoter region were typified using hybridized genomic DNA with specific initiators. Patients were selected at the Clinics Hospital of the University of São Paulo (HCFMRP-USP). Statistical analysis was performed using the Fisher\'s exact test. Comparisons of the frequencies of microsatellite alleles of the TNF region suggest that the presence of the TNFa5 allele could provide HIV/aids patients with protection against developing LS. In addition, comparisons of alleles of the TNF promoter region suggest that the presence of TNF-308G allele, as well as of its homozygote TNF-308GG could pose susceptibility to developing LS. The presence of the haplotype, TNFe3 d3 -238G -308A c1 a5 b7, suggests protection against developing that syndrome. The present study is the first to associate TNF microsatellite polymorphism with LS, indicating several associations among alleles of the gene region that codes TNF with LS. Although the mechanisms regarding the participation of TNF in the development of LS are not clear, this study suggests that immunogenetic factors associated with the TNF expression magnitude and probably the expression of other proinflammatory cytokines are involved in the development of LS in HIV/aids infected patients.

The grain amaranths (Amaranthus hypochondriacus L., A. cruentus L., and A. caudatus L.) are important pseudocereals native to the Americas that have received increased attention for their nutritional content, specifically their balance of amino acids. The objective of this project was to produce and characterize a set of highly informative, reproducible microsatellite markers for the grain amaranths. A total of 1457 clones were sequenced from three genomic libraries enriched for the microsatellite motifs AAC, AAT and AC. Of these, 353 (24%) contained unique microsatellites. An additional 29 microsatellite loci were identified among 728 BAC-end sequences of a newly developed amaranth BAC library. Flanking primers were designed for 319 of the microsatellite loci and all were screened on a panel of diverse amaranths, including grain and weedy Amaranthus species. A total of 179 (56%) microsatellites were polymorphic across accessions from the three grain amaranths. Among these polymorphic microsatellite loci, a total of 731 alleles were identified with average of four alleles per locus. Heterozygosity values ranged from 0.14 to 0.83 with a mean value of 0.62. Thirty-seven (21%) of the markers were polymorphic between the parents of a segregating population and were shown to be inherited in a normal Mendelian fashion based on chi-squared analysis, demonstrating the utility of these markers for linkage mapping of the amaranth genome. Phylogenetic analysis using the marker data showed A. hybridus accessions in two of the three major grain amaranth clades, suggesting the polyphyletic evolution of the three cultivated species from different A. hybridus ancestors. The microsatellite markers reported here will be useful for further evaluating the relationships among the grain amaranths and their relatives and are an ideal resource for use in marker-assisted breeding programs, germplasm analysis and varietal identification. The transferability of these markers to A. hybridus, A. powellii, and A. retroflexus as reported here suggests that the markers may be useful to other species with the genus Amaranthus, including economically important weeds, vegetable amaranths, and ornamentals.

Deleterious gene interactions due to accumulation of individual genetic variations between different lineages are a cause of population diversification by creating reproductive barriers that ultimately lead to differentiation of species. One type of deleterious interactions is called “hybrid necrosis”, in which epistatic interactions between some plant immunity genes (usually very variable) cause autoimmunities that produce a necrotic and dwarf phenotype. Hybrid necrosis has been widely studied in several plant species, such as Arabidopsis thaliana and many gene interactions were found for that plant. This study tests the applicability of these results on a close relative, A. lyrata, by crossing individuals from different populations and genotyping F2 progeny with polymorphic markers close to homologous sequences to those involved in hybrid necrosis in A. thaliana. Results suggest the possibility of a homologous gene to DM8 or DM9 in chromosome 7 to be involved in formation of hybrid necrosis.

The genetics of colonization is understudied in salamanders but has large conservation implications as new habitats are formed or restored to their previous condition. The 1980 eruption of Mount St. Helens provided a natural experiment to study the genetic effects of a large infrequent environmental disturbance on two species of salamander, Taricha granulosa (Rough-skinned newt) and Ambystoma gracile (Northwestern salamander). Both these species breed in ponds, and are thought to exhibit high breeding site fidelity and low vagility. I designated three treatments based on the effects of the eruption: new ponds (created by the eruption, immigrants only), recovery lakes (in blast zone, survivors plus immigrants), and reference lakes (unaffected by eruption, assumed to represent pre-eruption genetic diversity measures). Salamanders took at least nine years to colonize the new ponds. I studied the population genetics of colonization and recovery using microsatellites and AFLPs (amplified fragment length polymorphisms) to measure genetic diversity, gene flow, and population substructure at Mount St. Helens National Volcanic Monument. Based on population genetics theory and the life history characteristics of these pond-breeding amphibians, I predicted that genetic diversity would be lower in newly colonized ponds compared to recovery or reference sites. I also expected significant levels of population substructuring. Finally, I predicted that because of their lower vagility and large number of neotenes, that A. gracile would have less gene flow and a greater degree of population substructuring than T. granulosa. My predictions were not supported by my data. There was no loss of genetic diversity in new or recovery populations in either species. There was no strong evidence for population substructure by either AMOVA, isolation by distance or principal components analysis. Gene flow (Fst) was high in both species. Taricha granulosa and A. gracile were found to be resistant to a large infrequent environmental disturbance. Loss of genetic variability in new populations cannot automatically be assumed. Predicting dispersal and colonization ability based on the broad category of pond-breeding amphibian is not always reliable.