As abelhas da tribo Meliponini apresentam ampla distribuição, ocupando principalmente as regiões neotropicais do planeta. São encontradas mais de 400 espécies pertencentes a 50 gêneros. Tetragonisca angustula é uma espécie que se distribui por praticamente toda a América Latina, sendo em geral bastante abundante.No presente projeto foram empregadas técnicas moleculares (sequenciamento de DNA mitocondrial e análise de loci de microssatélites) no intuito de se verificar a variabilidade em duas pequenas populações de Tetragonisca angustula distribuídas nos campi da USP de Ribeirão Preto e São Paulo. Para a obtenção de um panorama da variabilidade da espécie, foram coletados e analisados indivíduos de áreas externas aos campi. Os resultados de sequenciamento mostraram que a variabilidade do genoma mitocondrial das amostras externas é muito maior que a variabilidade nas amostras dos campi. Além disso, ficou claro que os campi contêm populações monofiléticas bastante isoladas entre si, e que cada uma possui maior similaridade com as amostras externas do que com as amostras do outro campus. Em contrapartida, os resultados de microssatélites mostraram que a variabilidade dessas populações para os loci estudados é extremamente similar. Isso sugere que a filopatria da espécie é muito grande, e que o vôo dos machos é responsável por, de certa forma, homogeneizar a variabilidade genética das populações, impedindo que haja muita estruturação. Dessa maneira, em uma população de T. angustula, existem dois números efetivos populacionais: um para as fêmeas (estimado pela variabilidade do DNA mitocondrial) e outro maior para machos (estimado pela variabilidade dos microssatélites). Os resultados indicam que a colonização dos campi se deu por um número pequeno de rainhas que posteriormente aumentaram a população por conta da grande quantidade de recursos que os campi possuem / The bees of the Meliponini tribe have a broad distribution, mainly throughout the neotropical regions of the planet. There are over 400 species distributed among 50 genera. Tetragonisca angustula is a species which occupies almost all Latin America, generally in large individual numbers. In this study we used molecular techniques (mitochondrial DNA sequencing and analysis of microsatellite loci) to obtain measures of the genetic variability in two small populations of T. angustula located in both the campi of USP, Ribeirão Preto and São Paulo. In order to get a better idea of the variability of the species as a whole, we also analyzed individuals from populations from outside the campi. The results of the DNA sequencing showed that the variability of these external samples is far bigger than the variability of the other two populations. Also, it became clear that both campi have monophyletic populations that are very isolated from each other, and that each one of these campus populations is phylogenetically closer to the external samples than they are to each other. On the other hand, the results obtained with microsatellites showed that the variability of the populations from both campi are very similar to the variability of the external samples. This suggests that there is a high degree of filopatry in the species, and that the male flight is responsible, in a way, for mixing the genetic variability of the populations, this way keeping them from getting structured. Thus, in a given population of T. angustula, there are two different effective population sizes: of for the females (measured by the variability in the mitochondrial DNA) and another for the males (measured by the variability in the microsatellites). Our results indicate that the process of colonization in both campi was carried out by a few queens, which lately were responsible for increasing the population sizes due to the big amount of resources found in the campi

Trypanosoma brucei is the causative agent of Human African Trypanosomiasis (HAT), also known as African sleeping sickness. T. brucei is unique in several ways that distinguish this organism from other eukaryotes. One of the unique features of T. brucei is the organism’s mitochondrial DNA, which is organized in a complex structure called kinetoplast DNA (kDNA). Since kDNA is unique to the kinetoplastids, kDNA may serve as a good drug target against T. brucei. Previews studies have shown that kDNA has 4 different family A mitochondrial DNA polymerases. Three of these mitochondrial DNA polymerases (POLIB, POLIC, and POLID) are essential components of kDNA synthesis and replication. POLID and POLIC dynamically localize throughout the cell cycle. POLID is found dispersed in the matrix before the kDNA has undergone replication and is re-localized at the antipodal sites when the kDNA is dividing. POLIC is found in the kinetoflagellar zone (KFZ) at low concentrations when the kDNA is not replicating and relocalizes to the antipodal sites when dividing. Based on the dynamic localization of these two DNA polymerases, we hypothesize that POLIB undergoes dynamic localization at some point during the cell cycle stage. Here, a POLIB/PTP single expressor cell line was analyzed by immunofluorescence microscopy in an unsynchronized population. We characterized the localization pattern of POLIB-PTP at different cell cycle stages and found different localization patterns throughout cell cycle. Cells at 1N1K (the majority of cell in an unsynchronized population) have single foci, but at 1N1Kdiv two different patterns are mainly observed, diffuse and segregated. When the kDNAs are separated POLIB-PTP is again seen as a distinct foci in each kDNA. By doing TdT labeling and a quantitative analysis, we found that at early stages of minicircles replication POLIB-PTP start relocalizing to the kDNA disk with a diffuse pattern being the main. By the time the minicircles are being reattached in the disk (late TdT), POLIB is seen in the disk as a bilobe shape.

In most tropical river systems there has been a lack of integrated ecological research to investigate the dynamics and impacts of invasive species on recipient river systems. This is in sharp contrast to temperate river systems. This thesis investigated the nature, extent, and impact of Nile tilapia, Oreochromis niloticus (Linnaeus, 1758), on indigenous congenerics within the Limpopo River basin in northern South Africa. An integrated approach was adopted to gain a better understanding of factors that allow Nile tilapia to be a successful invader and also to gain an insight into its invasion rate and conservation implications within South Africa. Morphometric and genetic variation between Nile tilapia, indigenous congenerics and their associated hybrids were determined. Intermediate meristic characters obscured the identification of hybrid specimens from pure morpho-specimens and species identity was only confirmed through mtDNA analysis. Preliminary evidence points to unidirectional hybridization among Oreochromis congeners in the Limpopo River system. The hypothesis that bigger Nile tilapia males may have a competitive advantage over spawning grounds and in female mate choice is proposed. The trophic ecology of Nile tilapia was investigated using both stomach contents and stable isotope analysis. A high similarity in stomach contents was observed but interspecific differences were revealed in the isotopic composition of diets that suggest fine scale patterns of resource partitioning that could be achieved by the ability of fish to selectively feed on what is immediately available and the ability to perceive the dynamics that determine food resource availability. Ecological niche models were used to determine the potential invasive range of Nile tilapia and revealed broad invasive potential over most river systems in southern Africa that overlapped the natural range of endemic congenerics. It was noted that model performance and the degree of niche conservatism varied significantly with variable selection and spatial extent of study area. This implied that the spatial distribution of suitable and unsuitable environmental variables varied between the native and introduced ranges of Nile tilapia and also indicated the ability of Nile tilapia to survive in conditions incongruent with its native range. The extreme hardiness and adaptive life history characteristics of Nile tilapia have probably predisposed it to be a successful invader in novel systems within southern Africa. Lastly, a qualitative risk assessment method was developed as a potential application to determine the risk of establishment and spread of the invasive Nile tilapia. Results showed that in the absence of quantitative data on ecosystem structure and functioning, habitat suitability analysis in terms of known physiological tolerance limits to minimum water temperature, presence or absence of dams, seasonality of river flows and the presence of indigenous fish species of concern could be adequate for identifying vulnerable river systems. The model developed also provides an objective method that is easy to implement, modify and improve on as new data become available. Furthermore, the model can be applied to highlight areas of uncertainty where future research should be directed. / Thesis (PhD)--University of Pretoria, 2012. / Zoology and Entomology / Unrestricted

Despite recent advances in the understanding of the molecular bases of hematological malignancies, the specific mechanisms on how they originate and why some subtypes are more prevalent than others still remain to be elucidated. These two important aspects have been even more difficult to analyze when dealing with individuals under immune suppression because other factors must be considered. Questions still remain as to why individuals with AIDS tend to develop lymphoproliferative disorders differently from those observed in individuals under iatrogenic immunosuppressive therapy. Most of lymphomas occurring in transplant recipients are B-cell neoplasias typically associated with Epstein-Barr virus (EBV) infection. In contrast, only about 50% of lymphomas of patients with AIDS are associated with lymphotrophic herpesviruses such as EBV and Kaposi's sarcoma-associated herpesvirus (KSHV). No known infectious agent has been detected in the remaining 50% of AIDS-associated lymphomas, suggesting the involvement of novel viruses or unique molecular mechanisms. Since most oncogenic viruses persist as episomal circular viral genomes in the nuclei of tumor cells, we developed a method to visualize and identify covalently closed circular DNA (cccDNA) in lymphoma samples. Although this study revealed no novel viruses, we identified concatemers of the mitochondrial genome in all lymphoma samples tested. We further studied in detail one AIDS-associated lymphoma (denominated EL) whose mitochondrial DNA primarily consisted of tandem head-to-tail genome duplications. Insertion of cytosine residues was noted in the EL mitochondrial genome sequence near the origin of replication. EL cells responded weakly to Fas-apoptotic stimulus, displayed reduced mitochondrial activity and mass, and produced higher levels of reactive oxygen species (ROS) than control cells. Screening of several other AIDS-associated lymphomas and established lymphoma cell lines revealed a different kind of mitochondrial genome concatemers consisting of interlinks of DNA monomer molecules. Concatemers were not detected in normal T-lymphocytes suggesting an association with neoplastic transformation. This dissertation describes the two distinct types of mitochondrial genome concatemers identified in transformed lymphoid cells and presents a detailed analysis of their structure and implications in cellular homeostasis.

I developed and tested a protocol for determining the sex of individual pinnipeds using the sex-chromosome specific genes ZFX and ZFY. I screened a total of 368 seals (168 crabeater, Lobodon carcinophagus; 159 Weddell, Leptonychotes weddellii; and 41 Ross, Ommatophoca rossii) of known or unknown sex and compared the molecular sex to the sex assigned at the time of collection in the Ross and Amundsen seas, Antarctica. Discrepancies ranged from 0.0% - 6.7% among species. It is unclear, however, if mis-assignment of sex occurred in situ or in the laboratory. It also is possible, however, that the assigned morphological and molecular sex both are correct, owing perhaps to developmental effects of environmental pollution. I sequenced a portion (ca 475 bp) of the mitochondrial control region of Weddell seals (N = 181); crabeater seals (N = 143); and Ross seals (N = 41). I resolved 251 haplotypes with a haplotype diversity of 0.98 to 0.99. Bayesian estimates of Θ from the program LAMARC ranged from 0.075 for Weddell seals to 0.576 for crabeater seals. I used the values of theta to estimate female effective population sizes (NEF), which were 40,700 to 63,000 for Weddell seals, 44,400 to 97,800 for Ross seals, and 358,500 to 531,900 for crabeater seals. Weddell seals and crabeater seals had significant, unimodal mean pairwise difference mismatch distributions (p = 0.56 and 0.36, respectively), suggesting that their populations expanded suddenly around 731,000 years ago (Weddell seals) and around 1.6 million years ago (crabeater seals). Both of these expansions occurred during times of intensified glaciations and may have been fostered by expanding pack ice habitat. Autosomal microsatellite based NEs were 147,850 for L. Weddellii, 344,950 for O. rossii, and 939,600 for L. carcinophagus. I screened one X-linked microsatellite (Lw18), which yielded a larger NE estimate for O. rossii than the other two species. Microsatellite NE estimates are compared with previously published mitochondrial NE estimates and this comparison indicates that the Ross seal may have a serially monogamous system of mating. I find no sign of a recent, sustained genetic bottleneck in any of the three species.

Siberia is an ideal region for exploring population histories from a molecular anthropological perspective given the diverse human populations, in terms of linguistic affiliation and lifestyle, currently inhabiting this geographically large region. As such, this thesis explores new methodologies for the investigation of the genetic histories of Siberian populations. While previous genetic work in this area of the world was able to provide detailed insights into paternal histories based on Y chromosomal data, it was not as successful on the maternal side. There existed difficulties in exploring the complex maternal demographic histories due to high levels of sequence identity between individuals in different populations when using only a very small region of the mitochondrial DNA (mtDNA), known as the hypervariable region I (HV1). This realization led to the initial focus of this dissertation which was to identify and test improved methods of sequencing entire mtDNA genomes. This was necessary because the mtDNA genomes that were published for human Siberian populations and across the globe prior to the work described here were chosen based on specific sub-sample selection criteria that introduced an ascertainment bias rendering them unusable for population-wide analyses. After testing multiple next generation DNA sequencing methods, I helped develop a sequencing library preparation method based on multiplexing and hybridization enrichment of mtDNAs for sequencing by synthesis that has since become widely used in labs across the globe. Comparing the same samples sequenced by both the traditional and new methods for five ethnolinguistic populations showed that these new methods were robust and could lead to different inferences about population histories while avoiding a sampling bias. Based on the results of this thesis it is now recommended for researchers to sequence complete mtDNA genomes for all relevant samples within a collection. By applying these methods to additional Siberian populations it was possible to better describe maternal population contact and identify demographic changes over time. This additional information allowed for the identification of putative drops in the maternal effective population sizes in the Siberian populations examined here. When examining the potential migrations and population contact between Turkic-speaking Yakuts and the Tungusic-speaking Even and Evenks, there exists a differential sharing of haplotypes suggesting that the Tungusic speaking populations herein were already in the northern region and split prior to the expansion of the Yakuts into their territory. The putative origin of the Yakuts as being around Lake Baikal was given additional support from the analyses included in this study and the origins of the Dolgans were shown to predominately include the admixture of Yakuts and Evenks.

The main goal of this study is the comparison of sequences of the HVRII region of the mitochondrial genome in the cord blood sample and the saliva sample of the same individual, taken at average ten years from his/her birth. It is known that during ontogenesis the human genome changes. All the more the mitochondrial genome which shows a higher mutation rate, and moreover it is not taken care of it by repair mechanisms. In older individuals, there was found a distinctive amount of mitochondrial variations cumulated in different tissues in the process of the ontogenesis. This study is focused on the detection of these changes already in younger individuals. The tissue-specific variability which is created during ontogenesis might have an adverse influence on all sorts of the mtDNA based studies. The samples were taken in two regions (Teplice / Prachatice) that differ in the pollution of environment. With regard to that, the samples with discovered changes were compared from the standpoint of the region, which they had come from, with the aim to prove the influence of environment on the mutagenesis of the mitochondrial DNA. Samples were also compared from the point of view of sex. Furthermore the variability of the collection of Czech population was evaluated and the estimation of the genetic...

During the climate oscillations of the Pleistocene an ice sheet formed covering Fennoscandia. Traditionally, Scots pine and many other species were believed to have persisted in glacial refugia in the south, only returning to northern latitudes as the ice retreated. Recent studies have demonstrated the existence of glacial refugia in higher latitudes, in fact, cryptic refugia as far north as Lofoten have been suggested. I investigated the potential existence of such a refugium for Scots pine using mtDNA markers Nad 1-B/C, Nad 7-1 and NODE_new_663. Results show an east/west distribution of the mtDNA polymorphisms at Nad 1 and Nad 7 across Fennoscandia, where multi-locus mitotype bb is more common in the east and ba is more common in the west, in accordance with the hypothesis of a glacial refugium in northeast Europe. Further, the bb mitotype was discovered to be more widespread in Fennoscandia than previously documented. No unique mitotype was discovered in northwestern Fennoscandian populations to support the hypothesis of a glacial refugium along the northwest coast of Norway. Genetic diversity was high and even across Fennoscandia and differences in diversity were not significantly correlated with distance between populations, possibly due to high mutation-rates for the NODE_new_663 minisatellite locus or as a result from admixture between multiple glacial refugia in the area. No conclusive evidence regarding the existence of a glacial refugium in Fennoscandia was discovered and needs to be studied further.

Once so abundant as to be called the ‘common’ angelshark, Squatina squatina has been extirpated from nearly the entirety of its historical range, from the eastern North Atlantic, to the Mediterranean Sea [International Union for the Conservation of Nature (IUCN) Red List: Critically Endangered]. The angelshark now only occurs in any abundance in the waters surrounding the Canary Islands. I present the first genetic assessment of the angelshark’s population dynamics and diversity from three locations within the Canary Islands archipelago: Gran Canaria, Tenerife, and Lanzarote. Using a suite of individual mitochondrial genome regions [Control region (CR), NADH dehydrogenase subunit 2 (ND2), NADH dehydrogenase subunit 4 (ND4), and Cytochrome c oxidase subunit 1 (COI)], the complete mitogenome, and nuclear markers [microsatellites and Internal Transcribed Spacer 2 (ITS2)] this work aimed to i) assess the genetic diversity of the angelshark in comparison to other endangered or historically overfished elasmobranchs, ii) examine the relative nucleotide variability across different marker sets, and iii) assess fine-scale multi-locus population structure within the Canary Islands, as well as broad-scale population genetic structure of angelsharks throughout its historic Eastern Atlantic and Mediterranean range. Results revealed exceptionally low genetic diversity across all individual mitochondrial regions sequenced (CR, π % = 0.0046 ± 0.016; ND2, π % = 0; ND4, π % = 0; COI, π % = 0), yielding some of the lowest values reported to date in any elasmobranch. Mitogenome analysis followed this low diversity trend with only 11 single nucleotide polymorphisms seen across all Canary Island individuals in a genome of 16,689 bp (π % = 0.0257 ± 0.0166). Furthermore, screening of 35 microsatellite markers across 34 individuals revealed all but two loci to be monomorphic and nuclear ITS2 showed negligible diversity. Lanzarote showed significant population differentiation from both Gran Canaria (ΦST = 0.073, p = 0.004, FST = 0.113, p = 0.00) and Tenerife (ΦST = 0.029, p = 0.001, FST = 0.065, p = 0.001) at the CR. Haplotype analysis of whole mitogenomes also demonstrated Lanzarote individuals forming a separate lineage from angelsharks at the other two islands. Broad- scale structure across the angelshark’s historical range was detected between the Canary Islands and Mediterranean (ΦST = 0.792, p = 0.000, FST = 0.785, p = 0.000), indicating a regional break between populations. The exceptionally low levels of genetic diversity in angelsharks in the Canary Islands, as well as indications of population isolation from the rest of the angelshark’s historical range, suggest an immediate need for strong conservation measures to ensure the protection and continued persistence of this highly vulnerable and ecologically unique species.

Genetic mutation is the ultimate source of new phenotypic variation in populations. The importance of mutation cannot be understated, and constitutes a significant evolutionary force. Although single mutations may have little to no impact on organismal performance or fitness, when multiplied across the total number of potential sites within the genome, mutation can have a large impact. Accurate measurement of the rates, molecular mechanisms, and distributions of effects of mutations are critical for many applications of evolutionary theory. Despite the importance of both deleterious and beneficial mutations, their genome-wide patterns and phenotypic consequences are poorly understood when considering the mitochondrial genome. Mitochondria are organelles that are essential for eukaryotic life. They contain their own genome and generate bioenergy (ATP) necessary to sustain life via the electron transport chain (ETC). Because of their role in eukaryotic physiology, understanding how mitochondrial genetic and phenotypic variation can impact populations and evolutionary outcomes is essential. Past studies have implicated DNA-damaging oxidative stress as a source of mutations within somatic tissue, but there is a gap in knowledge regarding its role in heritable damage within the germ line. In this thesis, I aimed to test this possibility by characterizing the phenotypic and mutational consequences of high intracellular ROS levels caused by mitochondrial ETC genetic damage. I performed experiments using Caenorhabditis elegans ETC mutant, gas-1, and mutation-accumulation (MA) lines generated from this ancestral genotype. I quantified organismal fitness (fecundity and longevity), reactive oxygen species (ROS) levels, mitochondrial membrane potential (delta psi m), and ATP levels in these lines, and compared the results to those from a set of wildtype control lines. I begin with a general introduction to the hypothesis and the C. elegans system in Chapter I. In Chapter II, I report the findings from this work. In short, I found that while gas-1 MA lines began the experiment with low lifetime fecundity in comparison to the wildtype strain, their fecundity showed no further decline as expected, and even exhibited higher fecundity levels on days 3-5 of reproduction relative to the gas-1 progenitor. The gas-1 progenitor exhibited higher rates of ROS compared to wildtype, whereas the MA lines reverted back to wildtype levels; a similar pattern was observed for delta psi m, while ATP levels were low in the gas-1 progenitor and remained low in the MA lines. I interpret these findings in light of high-throughput sequencing results from these lines showing that, while nuclear and mitochondrial DNA mutation rates were equal to wildtype in these lines, the genomic pattern of mutation was highly nonrandom and indicative of selection for beneficial or compensatory sequence changes. Because ROS levels declined to wildtype in the evolved (MA), this study was unable to address whether ROS is a major contributor to heritable mutation in this system. I hypothesize that, in addition to their putatively compensatory genetic changes, gas-1 lineages experienced physiological compensation allowing them to survive, and that this was associated with a "slow living" phenotype. In Chapter III, I summarize general conclusions and implications of this study and end by providing suggestions for further study.