A genética forense tem grande importância na geração de provas em casos de violência sexual, paternidade criminal, identificação de cadáveres e investigação de evidências de locais de crime. A análise de STRs apresenta grande poder de discriminação, mas é uma metodologia multi-etapas, trabalhosa, cara e em muitos casos a análise genética é prejudicada pela baixa quantidade e qualidade das evidências coletadas. Este estudo teve como objetivo desenvolver e caracterizar uma metodologia de triagem de amostras forenses através da análise de perfis de dissociação em alta resolução (HRM) de regiões do DNA mitocondrial, o qual está presente em maior número de cópias e mais resistente a degradação. Para tanto, foram extraídos DNAs de 68 doadores. Estas amostras foram sequenciadas e analisadas por HRM para sete alvos no DNA mitocondrial. Também foram realizados ensaios para determinar a influência do método de extração, da concentração e nível de degradação do DNA no perfil de HRM obtido para uma amostra. Os resultados demonstraram a capacidade da técnica de excluir indivíduos com sequências diferentes da referência comparativa em cinco regiões amplificadas. Podem ser analisadas em conjunto, amostras de DNA com variação de concentração de até a ordem de 100 vezes e extraídas por diferentes metodologias. Condições de degradação de material genético não prejudicaram a obtenção de perfis de dissociação em alta resolução. A sensibilidade da técnica foi aprimorada com a análise de produtos de amplificação de tamanho reduzido. A fim de otimizar o ensaio foi testada a análise de HRM em reações de PCR duplex. Um dos pares de amplificação forneceu perfis de HRM compatíveis com resultados obtidos de reações com amplificação de apenas um dos alvos. Através da análise conjunta das cinco regiões, esta metodologia visa a identificação de indivíduos não relacionados com as referências comparativas, diminuindo o número de amostras a serem analisadas por STRs, reduzindo gastos e aumentando a eficiência da rotina de laboratórios de genética forense. / The forensic genetics has an important role in the generation of evidence in cases of sexual assault, criminal paternity, identification of corpses and crime scenes investigation. The analysis of STRs has great power of discrimination, but it is a multi-stage methodology, complex, expensive and in many cases the genetic analysis is hampered by the low quantity and quality of evidence collected. This study aimed to develop and characterize a forensic samples screening methodology to examine high resolution melting profiles (HRM) of regions of the mitochondrial DNA, which is present in more copies and more resistant to degradation. Thus, we extracted DNA from 68 donors. These samples were sequenced and analyzed by HRM to seven mitochondrial DNA targets. Tests were also conducted to determine the influence of extraction method, concentration and DNA degradation level of HRM profile obtained for a sample. The results demonstrated the technical ability to exclude individuals with different sequences of comparative reference amplified in five regions. Can be analyzed together samples with varying concentration to the order of 100 times and extracted by different methods. Genetic material degradation conditions did not prevent obtaining high resolution melting profiles. The sensitivity of the technique was improved with the analysis of reduced size amplification products. In order to optimize the assay HRM analysis was tested in duplex PCR reactions. A pair of amplification provided HRM profiles consistent with results from amplification in reactions with only one of the targets. Through the joint analysis of the five regions, this approach aims to identify individuals not related to comparative references, reducing the number of samples to be analyzed by STRs, reducing costs and increasing the efficiency of the routine of forensic genetics laboratories.

INTRODUÇÃO: A forma clássica de encefalomiopatia mitocondrial associada à mutação do DNA mitocondrial m.3243A>G é a Mitochondrial Encephalomyopathy with Lactic Acidosis and Stroke-like episodes (síndrome de MELAS). Entretanto, o espectro de manifestações clínicas dos indivíduos que apresentam essa mutação é bastante amplo. OBJETIVO: Descrever o espectro clínico, laboratorial e de imagem de pacientes com doença mitocondrial decorrente da mutação m.3243A>G. MÉTODOS: Estudo descritivo retrospectivo de uma série de casos de pacientes com a mutação m.3243A>G em seguimento no ANEM/HCFMRP-USP. Os dados clínicos e informações sobre exames complementares foram coletados através de revisão sistemática dos prontuários médicos dos pacientes selecionados. Os exames de neuroimagem foram revisados juntamente com neurorradiologista experiente para descrição das lesões encontradas. RESULTADOS: No período compreendido entre maio de 2000 e maio de 2015, a mutação m.3243A>G foi pesquisada em um total de 817 pacientes, em DNA extraído de células do sangue periférico (n= 441), de fragmentos de biópsia de músculo esquelético (n= 293), de ambos (n= 82) e mais raramente de células do sedimento urinário (n=1). Dentre esses, 16 indivíduos de 12 famílias apresentaram a referida mutação, resultando em uma taxa de detecção da mutação de 1,96% nessa população. Foram incluídos no estudo 12 indivíduos de 9 famílias que estavam em seguimento no nosso serviço. Os achados mais comuns nesta série foram em ordem de frequência: sinais de miopatia, transtornos neurocomportamentais, epilepsia, endocrinopatias, ataxia cerebelar, migrânea, episódios semelhantes a AVC, vômitos recorrentes, distúrbios de condução cardíaca, neuropatia periférica e sinais de disautonomia, mioclonias, surdez neurossensorial, cegueira cortical, comprometimento ocular, e proteinúria. Em nossa série, identificamos que cinco pacientes foram classificados com a forma clássica de MELAS, dois apresentaram CPEO associada a outros sintomas como transtornos psiquiátricos e diabetes mellitus. Os demais pacientes apresentavam características clínicas que não configuravam uma síndrome clinica definida. Além das lesões semelhantes a AVC, as lesões reveladas por neuroimagem mais frequentes nessa população foram alteração de sinal dos núcleos da base, atrofia encefálica e alteração de sinal da substância branca, sendo igualmente prevalentes entre os pacientes com a síndrome clássica de MELAS e os pacientes que não apresentaram lesões semelhantes a AVC. Dos pacientes com MELAS, 100% apresentaram pico anômalo de lactado e 60% redução do NAA à espectroscopia de prótons; enquanto que entre os pacientes sem lesões semelhantes a AVC essas alterações foram encontradas em dois e em um paciente Caracterização clínica, laboratorial e de neuroimagem de pacientes com doença mitocondrial associada à mutação m.3243A>G 8 respectivamente. Nós identificamos o achado inédito de azoospermia associada à mutação m.3243A>G. Essa é a maior série de casos de pacientes brasileiros com a mutação m.3243A>G até o momento. CONCLUSÃO: O amplo espectro de apresentação clínica e de neuroimagem é uma característica notável entre os pacientes com a mutação m.3243A>G do DNAmt. Essa desordem deve ser considerada em pacientes com evidência de sinais e sintomas que sugiram acometimento multissistêmico. / INTRODUCTION: The classic form of mitochondrial encephalomyopathy associated with m.3243A>G mutation is the Mitochondrial Encephalomyopathy with Lactic Acidosis and Stroke-like episodes (MELAS syndrome). However, the spectrum of clinical manifestations of patients with this mutation is quite wide. OBJECTIVE: To describe the clinical, laboratory and imaging spectrum of patients with mitochondrial disease due to m.3243A>G mutation METHODS: A retrospective descriptive study of a series of cases of patients with the m.3243A>G mutation in follow-up in ANEM/HCFMRP-USP. Clinical data and information about additional tests were collected through systematic review of medical records of selected patients. Neuroimaging studies were reviewed with supervision of experienced neuroradiologist and the lesions were described. RESULTS: Between May 2000 and May 2015, the mutation m.3243A>G was evaluated in a total of 817 patients, on DNA extracted from peripheral blood (n = 441), skeletal muscle biopsy samples (n = 293), both (n = 82) and more rarely urinary sediment cells (n = 1). We founded 16 individuals 12 families with the mutation, resulting in a mutation detection rate of 1.96 % that population. 12 individuals from nine families, who were following at our service, were included in this study. The most common findings in this series were in order of frequency: myopathy signs, neurobehavioral disorders, epilepsy, endocrine disorders, cerebellar ataxia, migraine, stroke-like episodes, recurrent vomiting, cardiac conduction disorders, peripheral neuropathy and signs of dysautonomia, myoclonus, sensorineural deafness, cortical blindness, uveitis, and proteinuria. In our series, we found that five of the patients were classified with the classical MELAS syndrome, two patients had CPEO associated with other symptoms such as psychiatric disorders and diabetes mellitus. The remaining patients had other features of mitochondrial disease not consistent with another recognised syndrome. In addition to stroke-like lesions, the more frequent lesions revealed in neuroimaging studies were deep gray matter changes, brain atrophy and white matter changes. These changes had similar prevalences between the patients with the classic syndrome of MELAS and patients who did not have stroke-like lesions. All patients with classical MELAS have lactate peak and 60% of them have reduction of NAA at spectroscpopy; while these changes were found in two and one patient respectively, in the group of patients without stroke-like lesions. We identified azoospermia in one paciente with classic MELAS, a finding not previously associated with m.3243A>G. At moment, this is the largest Brazilian case series of patients with the m.3243A>G mutation. Clinical, laboratory and neuroimaging features of patients with mitochondrial disease associated with mutation m.3243A > G 10 CONCLUSION: The wide spectrum of clinical presentation and neuroimaging is a notable feature among patients with the mutation m.3243A> G mtDNA. This disorder should be considered in patients with evidence of signs and symptoms suggestive of multisystem involvement.

As abelhas da tribo Euglossini, possuem elevada representatividade nas florestas tropicais (25% da comunidade apícola existente) distribuindo-se do sul dos EUA ao centro da Argentina, sendo restritas à região Neotropical. A espécie Euglossa pleosticta, com ocorrência relatada exclusivamente no território brasileiro, possui hábito de vida solitário e costuma estar presente em levantamentos realizados em remanescentes da Mata Atlântica. Pouco se sabe sobre sua estrutura populacional e os efeitos da fragmentação florestal sobre as mesmas. Sendo assim, a realização de um estudo populacional para esta espécie é algo relevante, uma vez que colabora para a compreensão dos efeitos da fragmentação florestal na variabilidade genética do grupo, sendo objetivo deste trabalho, caracterizar a variabilidade genética e morfológica desta espécie, utilizando ferramentas morfológicas (Morfometria Geométrica de asas e Assimetria Flutuante), e moleculares (DNA microssatélite e DNA mitocondriais). Sendo assim, trabalhamos com 293 amostras de E. pleosticta coletadas em três fragmentos de mata no Estado de São Paulo. Para as análises morfológicas, foram marcados 18 marcos anatômicos nas intersecções das nervuras das asas anterior direita de cada abelha. Nas análises de microssatélite, trabalhamos com oito loci heterólogos e para realizarmos as análises de DNA mitocondrial, analisamos 20 abelhas por localidade, amplificando segmentos do Citocromo B. Nas análises de Morfometria Geométrica e Microssatélites de DNA, as localidades estudadas formaram dois grupos, os quais corroboraram quanto às características de fitofisionomia, clima, altitude e distância geográfica compartilhadas entre as localidades. Nossos dados sugerem a presença de ecotipos localmente adaptados, o que pode ser explicado em função da plasticidade fenotípica, a qual é muito comum em insetos que vivem em ambientes variados. Na análise de Assimetria Flutuante, observamos valores significativos de assimetria flutuante e assimetria direcional para as três localidades estudadas. Já na análise de DNA mitocondrial, encontramos nove haplótipos, com diversidade nucleotídica (? = 0,01636) e diversidade haplotípica (Hd = 0,777), sendo apenas um destes haplótipos compartilhado (H2). O teste de Fst par a par indicou estruturação genética populacional, e na AMOVA a maior taxa de variação observada foi quando consideramos a estruturação de um único grupo com variação interpopulacional (61,9%). Os testes de Mantel realizados tanto para distância genética quanto para distância morfológica, não apresentaram correlação quando relacionadas com distância geográfica. A falta de concordância entre os marcadores moleculares, é algo que já foi relatado em outros trabalhos e pode ser explicado em função da diferente taxa de evolução dos mesmos, uma vez que o marcador mitocondrial é mais conservado quando comparado com o de microssatélite. Em conjunto, estes marcadores apontam para a formação de subpopulações localmente adaptadas (ecotipos) para a espécie de E. pleosticta, provavelmente em decorrência dos processos recentes de fragmentação de habitat. / The bees from Euglossini tribe have high representativeness in tropical forests (25% of existing bee community) and are distributed from the south of USA to the center of Argentina, being restricted to Neotropical region. The Euglossa pleosticta species, with occurrence reported exclusively in the Brazilian territory, has solitary behaviour and, according to some researches, use to be present in Atlantic forest remnants. Little is known about its population structure and the forest fragments effects over them. Therefore, the realization of a population study of this species is relevant, since it contributes to the comprehension of the forest fragmentation effects in the genetic variability of this group. The ultimate goal of this research was to characterize the genetic and morphological variability of this species, usinging morphological tools (Geometric Morphometrics of forewings and Fluctuation Asymmetry) and molecular tools (Microsatellite DNA and Mitocondrial DNA). Thus, we have worked with 293 samples of E. pleosticta, which were collected from three forest fragments in São Paulo state. For the morphological analyses, 18 landmarks were plotted in the vein intersections of the right forewing of each bee. On the Microsatellite DNA analysis, we have worked with eight heterologous loci and on the Mitocondrial DNA analysis, we amplified the Cytochrome B segments to 20 bees per locality. In the Geometric Morphometry and DNA Microsatellites analyzes, two groups were formed from the studied localities, which corroborated to the characteristics of phytophysiognomy, climate, altitude and geographic distance shared between the localities. Our data suggest the presence of locally adapted ecotypes, which can be explained by phenotypic plasticity, which is very common in insects living in different environments. In the Fluctuation Asymmetry analysis, we observed significant values of Fluctuation and Directional Asymmetry for all studied localities. In the mitochondrial DNA analysis, we found nine haplotypes with nucleotide diversity (? = 0.01636) and haplotypic diversity (Hd = 0.777), where only one was shared (H2). The Fst pairwise test indicated population genetic structuring and, in AMOVA, the highest rate of variation was observed when we considered the structuring of a single group with interpopulation variation (61.9%). The Mantel test performed for both genetic and morphological distances, did not present correlation when related to geographic distance. The disagreement between the molecular markers has already been reported in other studies and can be explained by the different rate of evolution of these markers, since the mitochondrial marker is more conserved when compared to the microsatellite. Together, these markers indicate the formation of locally adapted subpopulations (ecotypes) for the species E. pleosticta, probably due to the recent cases of habitat fragmentation.

As abelhas da tribo Euglossini, possuem elevada representatividade nas florestas tropicais (25% da comunidade apícola existente) distribuindo-se do sul dos EUA ao centro da Argentina, sendo restritas à região Neotropical. A espécie Euglossa pleosticta, com ocorrência relatada exclusivamente no território brasileiro, possui hábito de vida solitário e costuma estar presente em levantamentos realizados em remanescentes da Mata Atlântica. Pouco se sabe sobre sua estrutura populacional e os efeitos da fragmentação florestal sobre as mesmas. Sendo assim, a realização de um estudo populacional para esta espécie é algo relevante, uma vez que colabora para a compreensão dos efeitos da fragmentação florestal na variabilidade genética do grupo, sendo objetivo deste trabalho, caracterizar a variabilidade genética e morfológica desta espécie, utilizando ferramentas morfológicas (Morfometria Geométrica de asas e Assimetria Flutuante), e moleculares (DNA microssatélite e DNA mitocondriais). Sendo assim, trabalhamos com 293 amostras de E. pleosticta coletadas em três fragmentos de mata no Estado de São Paulo. Para as análises morfológicas, foram marcados 18 marcos anatômicos nas intersecções das nervuras das asas anterior direita de cada abelha. Nas análises de microssatélite, trabalhamos com oito loci heterólogos e para realizarmos as análises de DNA mitocondrial, analisamos 20 abelhas por localidade, amplificando segmentos do Citocromo B. Nas análises de Morfometria Geométrica e Microssatélites de DNA, as localidades estudadas formaram dois grupos, os quais corroboraram quanto às características de fitofisionomia, clima, altitude e distância geográfica compartilhadas entre as localidades. Nossos dados sugerem a presença de ecotipos localmente adaptados, o que pode ser explicado em função da plasticidade fenotípica, a qual é muito comum em insetos que vivem em ambientes variados. Na análise de Assimetria Flutuante, observamos valores significativos de assimetria flutuante e assimetria direcional para as três localidades estudadas. Já na análise de DNA mitocondrial, encontramos nove haplótipos, com diversidade nucleotídica (? = 0,01636) e diversidade haplotípica (Hd = 0,777), sendo apenas um destes haplótipos compartilhado (H2). O teste de Fst par a par indicou estruturação genética populacional, e na AMOVA a maior taxa de variação observada foi quando consideramos a estruturação de um único grupo com variação interpopulacional (61,9%). Os testes de Mantel realizados tanto para distância genética quanto para distância morfológica, não apresentaram correlação quando relacionadas com distância geográfica. A falta de concordância entre os marcadores moleculares, é algo que já foi relatado em outros trabalhos e pode ser explicado em função da diferente taxa de evolução dos mesmos, uma vez que o marcador mitocondrial é mais conservado quando comparado com o de microssatélite. Em conjunto, estes marcadores apontam para a formação de subpopulações localmente adaptadas (ecotipos) para a espécie de E. pleosticta, provavelmente em decorrência dos processos recentes de fragmentação de habitat. / The bees from Euglossini tribe have high representativeness in tropical forests (25% of existing bee community) and are distributed from the south of USA to the center of Argentina, being restricted to Neotropical region. The Euglossa pleosticta species, with occurrence reported exclusively in the Brazilian territory, has solitary behaviour and, according to some researches, use to be present in Atlantic forest remnants. Little is known about its population structure and the forest fragments effects over them. Therefore, the realization of a population study of this species is relevant, since it contributes to the comprehension of the forest fragmentation effects in the genetic variability of this group. The ultimate goal of this research was to characterize the genetic and morphological variability of this species, usinging morphological tools (Geometric Morphometrics of forewings and Fluctuation Asymmetry) and molecular tools (Microsatellite DNA and Mitocondrial DNA). Thus, we have worked with 293 samples of E. pleosticta, which were collected from three forest fragments in São Paulo state. For the morphological analyses, 18 landmarks were plotted in the vein intersections of the right forewing of each bee. On the Microsatellite DNA analysis, we have worked with eight heterologous loci and on the Mitocondrial DNA analysis, we amplified the Cytochrome B segments to 20 bees per locality. In the Geometric Morphometry and DNA Microsatellites analyzes, two groups were formed from the studied localities, which corroborated to the characteristics of phytophysiognomy, climate, altitude and geographic distance shared between the localities. Our data suggest the presence of locally adapted ecotypes, which can be explained by phenotypic plasticity, which is very common in insects living in different environments. In the Fluctuation Asymmetry analysis, we observed significant values of Fluctuation and Directional Asymmetry for all studied localities. In the mitochondrial DNA analysis, we found nine haplotypes with nucleotide diversity (? = 0.01636) and haplotypic diversity (Hd = 0.777), where only one was shared (H2). The Fst pairwise test indicated population genetic structuring and, in AMOVA, the highest rate of variation was observed when we considered the structuring of a single group with interpopulation variation (61.9%). The Mantel test performed for both genetic and morphological distances, did not present correlation when related to geographic distance. The disagreement between the molecular markers has already been reported in other studies and can be explained by the different rate of evolution of these markers, since the mitochondrial marker is more conserved when compared to the microsatellite. Together, these markers indicate the formation of locally adapted subpopulations (ecotypes) for the species E. pleosticta, probably due to the recent cases of habitat fragmentation.

The White-bellied Sea-Eagle Haliaeetus leucogaster is widespread in Australia, but has been the subject of conservation concern due to suggested localised declines and extinctions. Regionalised monitoring programmes have addressed some aspects of local concern, however a broader approach is needed to gain an understanding of large-scale processes affecting long-term persistence at scales equivalent to the species Australian range. Ultimately, the ability to predict change in population size over time accurately depends on the scale of analysis. By necessity, ecological studies using direct sampling techniques are often made across spatial scales smaller than a species geographic range and across relatively short time frames. This seems counter-intuitive considering that long-term species persistence is often dependent on large-scale processes. The principal aim of this thesis was to identify historical and contemporary forces responsible for the current pattern of population structure in H. leucogaster. This required a multi-scale approach, and the resulting research uses genetic, distributional and morphometric data. Haliaeetus leucogaster is a large territorial raptor that historically has been associated with coastal regions, lakes and perennial river systems. It has an extensive worldwide distribution from the western coast of India throughout the Indomalaysian region, Papua New Guinea and Australia. By virtue of the species' large-scale distribution, in Australia it is fairly cosmopolitan in its use of habitat and prey types. Haliaeetus leucogaster is monomorphic for adult plumage colouration, but in body size displays reversed sexual dimorphism with female birds significantly larger. A discriminant function based on 10 morphometric characters was 100% effective in discriminating between 19 males and 18 females that had been sexed using molecular genetic methods. Re-classification using a jackknife procedure correctly identified 92% of individuals. The discriminant function should be a viable alternative to genetic sexing or laparoscopy for a large proportion of individuals within the Australo-Papuan range of this species; and can also be used to identify a small proportion of "ambiguous" individuals for which reliable sexing will require those other techniques. I used mitochondrial (mtDNA) control region sequence data to investigate the current distribution of genetic variation in this species at the continental level and within and between specified regional units. I was specifically interested in identifying breaks in genetic connectivity between the west and east of the continent and between Tasmania and the Australian mainland. Overall, genetic diversity was low and there was no significant level of genetic subdivision between regions. The observed genetic distribution suggests that the population expanded from a bottleneck approximately 160 000 years ago during the late Pleistocene, and spread throughout the continent through a contiguous range expansion. There is insufficient evidence to suggest division of the population into different units for conservation management purposes based on the theoretical definition of the 'evolutionary significant unit'. It is clear from the analysis that there are signatures of both historical and contemporary processes affecting the current distribution. Given the suggestion that population expansion has been relatively recent, additional sampling and confirmation of the perceived pattern of population structure using a nuclear marker is recommended to validate conservation monitoring and management at a continental scale. To determine the existence of perceived population declines across ecological time scales, I analysed the Australian Bird Atlas Data to identify the extent and pattern of change in range and density of the species between three Atlas Periods (1901-1976, 1977-1981 and 1998-2001) using a new standardised frequency measure, the Occupancy Index (OI) for 1° blocks (approx. 100km2) across the continent. At the continental scale, there was no significant difference in the spatial extent of occupancy between Atlas Periods. However, there were considerable changes in frequency and range extent between defined regions, and there were distinct differences in the pattern of change in OI between coastal and inland blocks over time. Coastal blocks showed much more change than inland blocks, with a clear increase in the use of coastal blocks, accompanied by a decrease in inland blocks, during the 1977-1981 Atlas Period, relative to both other Atlas Periods. The over-riding factor associated with distributional shifts and frequency changes was apparently climatic fluctuation (the 1977-1981 period showing the influence of El Nino associated drought). The impression of abundance was strongly dependent on both the temporal and spatial scale of analysis. To test for correspondence between geographic variation in morphology and geographic variation in mtDNA I analysed morphometric data from 95 individuals from Australia and Papua New Guinea. First, the degree of morphometric variation between specified regions was determined. This was then compared with the pattern of genetic differentiation. There was a strong latitudinal cline in body dimensions. However, there was no relationship between morphometric variation and patterns of genetic variation at least for mtDNA. Females showed a pattern of isolation by distance based on morphometric characters whereas males did not. Three hypotheses to explain the pattern of morphometric variation were considered: phenotypic plasticity, natural selection and secondary contact between previously isolated populations. I conclude that the pattern of morphometric variation is best explained by the suggestion that there is sufficient local recruitment for natural selection to maintain the observed pattern of morphometric variation. This implies that gene flow may not be as widespread as the mtDNA analysis suggested. In this instance either the relatively recent colonisation history of the species or the inability of the mtDNA marker to detect high mutation rates among traits responsible for maintaining morphometric variation may be overestimating the levels of mixing among regions. As might be expected given the physical scale over which this study was conducted, the pattern of genetic, morphometric and physical distribution varied dependent on the scale of analysis. Regional patterns of genetic variation, trends in occupancy and density and morphometric variation did not reflect continental patterns, reinforcing the contention that extrapolation of data from local or regional levels is often inappropriate. The combined indirect methodologies applied in this study circumvent the restrictions imposed by direct ecological sampling, because they allow survey across large geographic and temporal scales effectively covering the entire Australian range of H. leucogaster. They also allow exploration of the evolutionary factors underpinning the species' current distribution.

<p>I use morphological, vocal, molecular, behavioural, ecological and distributional data to re-evaluate the systematics of three passerine bird groups, the <i>Mirafraassamica </i>complex (bush-larks), the genus <i>Seicercus</i> ("spectacled-warblers"; with emphasis on the the <i>S. burkii</i> complex) and the genus <i>Motacilla</i> (wagtails). Two new species are described: <i>Seicercus soror</i> and <i>Motacilla samveasnae</i>. I propose that the polytypic species <i>M. assamica</i> should be treated as four separate species: <i>M. assamica</i>, <i>M. affinis</i>, <i>M. microptera</i> and <i>M. marionae</i> (it is also remarked that the proper name of the latter is <i>M. erythrocephala</i>). That is primarily supported by vocalisations and mitochondrial DNA. The latter data set also suggests that <i>M. assamica</i> sensu lato is paraphyletic, since <i>M. erythroptera</i>, which is always treated as a separate species, is nested within the <i>M. assamica</i> complex. I propose that the polytypic species <i>S. burkii</i> comprises six sibling species. Some of these are found to breed sympatrically, although mainly or entirely segregated altitudinally. Mitochondrial DNA suggests that the <i>S. burkii</i> complex is non-monophyletic, and also that the divergence of the different taxa is much older than indicated by morphological and vocal data. According to the molecular phylogeny, both the genera <i>Seicercus</i> and its assumed sister genus <i>Phylloscopus</i> are paraphyletic. That is corroborated by independent data. The phylogenetic study of the genus <i>Motacilla</i> reveals incongruence between mitochondrial DNA, nuclear DNA and non-molecular data. I conclude that the nuclear gene tree reflects the organismal phylogeny more faithfully than the mitochondrial gene tree. The latter is likely to have been affected by introgressive hybridisation, possibly also stochastic lineage sorting. The most remarkable result that is strongly supported by both mitochondrial and nuclear DNA is that <i>M. flava</i> is non-monophyletic.</p>

<p>Genetic profiling is commonly performed on the autosomes using multiple DNA markers. Although routine forensic DNA analysis is robust and based on reliable technologies, samples with degraded or limited amounts of DNA often fail. In these cases, the analysis of mitochondrial DNA (mtDNA) can be very valuable due to the high copy number per cell. This thesis describes evaluation and modifications of existing technologies that are useful in forensic DNA typing, mainly focusing on mtDNA.</p><p>DNA quantities isolated from common evidence materials such as hairs, fingerprints and accessories were estimated using a real-time quantification assay. Knowledge of quantitative differences between materials can guide forensic scientists to perform the best analysis (Paper I).</p><p>The current mtDNA analysis is based on hypervariable region (HVI/HVII) sequencing, which is the most rigorous and time-consuming forensic DNA analysis. Therefore, we evaluated the possibility to exclude individuals by screening for non-matching samples using the rapid and easy mtDNA Linear Array Assay (Paper II). </p><p>The major disadvantage using mtDNA is the lower discrimination power compared to multiple nuclear DNA markers. In contrast to the nuclear genome, due to the uniparental (maternal) mode of inheritance, no individual has unique mtDNA. We investigated the possibility of increasing the discrimination power by using pyrosequencing technology to analyse parts of the coding region in addition to HVI/HVII (Paper III). Furthermore, the addition of coding mtDNA information was evaluated by comparing several recently published mtDNA coding region assays (Paper IV). </p><p>Mixtures of DNA are common in forensic genetics due to contribution of DNA from several individuals, contamination or heteroplasmy. To resolve mixtures we have developed a pyrosequencing-based assay for the accurate quantification of the mtDNA mixture components (Paper V).</p><p>In conclusion, this thesis describes several assays that are valuable in forensic genetics for DNA quantification, improved mtDNA analysis, and mtDNA mixture interpretation.</p>

I use morphological, vocal, molecular, behavioural, ecological and distributional data to re-evaluate the systematics of three passerine bird groups, the Mirafraassamica complex (bush-larks), the genus Seicercus ("spectacled-warblers"; with emphasis on the the S. burkii complex) and the genus Motacilla (wagtails). Two new species are described: Seicercus soror and Motacilla samveasnae. I propose that the polytypic species M. assamica should be treated as four separate species: M. assamica, M. affinis, M. microptera and M. marionae (it is also remarked that the proper name of the latter is M. erythrocephala). That is primarily supported by vocalisations and mitochondrial DNA. The latter data set also suggests that M. assamica sensu lato is paraphyletic, since M. erythroptera, which is always treated as a separate species, is nested within the M. assamica complex. I propose that the polytypic species S. burkii comprises six sibling species. Some of these are found to breed sympatrically, although mainly or entirely segregated altitudinally. Mitochondrial DNA suggests that the S. burkii complex is non-monophyletic, and also that the divergence of the different taxa is much older than indicated by morphological and vocal data. According to the molecular phylogeny, both the genera Seicercus and its assumed sister genus Phylloscopus are paraphyletic. That is corroborated by independent data. The phylogenetic study of the genus Motacilla reveals incongruence between mitochondrial DNA, nuclear DNA and non-molecular data. I conclude that the nuclear gene tree reflects the organismal phylogeny more faithfully than the mitochondrial gene tree. The latter is likely to have been affected by introgressive hybridisation, possibly also stochastic lineage sorting. The most remarkable result that is strongly supported by both mitochondrial and nuclear DNA is that M. flava is non-monophyletic.

Genetic profiling is commonly performed on the autosomes using multiple DNA markers. Although routine forensic DNA analysis is robust and based on reliable technologies, samples with degraded or limited amounts of DNA often fail. In these cases, the analysis of mitochondrial DNA (mtDNA) can be very valuable due to the high copy number per cell. This thesis describes evaluation and modifications of existing technologies that are useful in forensic DNA typing, mainly focusing on mtDNA. DNA quantities isolated from common evidence materials such as hairs, fingerprints and accessories were estimated using a real-time quantification assay. Knowledge of quantitative differences between materials can guide forensic scientists to perform the best analysis (Paper I). The current mtDNA analysis is based on hypervariable region (HVI/HVII) sequencing, which is the most rigorous and time-consuming forensic DNA analysis. Therefore, we evaluated the possibility to exclude individuals by screening for non-matching samples using the rapid and easy mtDNA Linear Array Assay (Paper II). The major disadvantage using mtDNA is the lower discrimination power compared to multiple nuclear DNA markers. In contrast to the nuclear genome, due to the uniparental (maternal) mode of inheritance, no individual has unique mtDNA. We investigated the possibility of increasing the discrimination power by using pyrosequencing technology to analyse parts of the coding region in addition to HVI/HVII (Paper III). Furthermore, the addition of coding mtDNA information was evaluated by comparing several recently published mtDNA coding region assays (Paper IV). Mixtures of DNA are common in forensic genetics due to contribution of DNA from several individuals, contamination or heteroplasmy. To resolve mixtures we have developed a pyrosequencing-based assay for the accurate quantification of the mtDNA mixture components (Paper V). In conclusion, this thesis describes several assays that are valuable in forensic genetics for DNA quantification, improved mtDNA analysis, and mtDNA mixture interpretation.

La transferencia nuclear de células somáticas (SCNT) en la especie porcina se ha convertido en una herramienta muy útil para para la elaboración de modelos genéticos de enfermedades humanas y para el uso en xenotransplantes. Aunque el número de cerdos clonados aumenta cada año, la eficiencia total de esta tecnología es todavía muy baja. Uno de los pasos más difíciles de la SCNT en porcino es la enucleación del ovocito, principalmente debido a que su citoplasma contiene numerosas gotas lipídicas. El principal objetivo de la tesis fue evaluar el efecto de diversas técnicas para visualizar la placa metafásica y el corpúsculo polar sobre la capacidad de desarrollo de ovocitos porcinos madurados in vitro. / Somatic cell nuclear transfer (SCNT) technology in porcine has become a very useful tool for the elaboration of genetic models for human diseases and the use in xenotransplantation. The efficiency of SCNT is still very low, although the number of cloned pigs increases each year. One of the hardest steps of porcine SCNT is the enucleation of the oocyte because its cytoplasm contains many lipid droplets. The main objective of this thesis was to assess the effect of several approaches to visualize the metaphase II plate and the first polar body on the developmental ability of in vitro mature porcine oocytes.