A imensa diversidade de micro-organismos no solo leva a uma inevitável riqueza de interações entre espécies. Neste intuito, esse projeto é inovador na identificação dos cogumelos da Mata Atlântica, e na descrição da comunidade bacteriana associada às suas micosferas. Usando corpos de frutificação dos fungos (cogumelos) como indicadores para sistemas ricos em nutrientes, as amostras foram coletadas para investigar as interações entre os fungos (maioria do domínio Basidiomycota) e as bactérias presentes no solo em volta das micélios fúngicos (ambiente micosférico). As análises foram feitas com técnicas independentes de cultivo (análise PCR-DGGE, sequenciamento Sanger de fragmentos de ITS/18S e pirosequenciamento de tags da região V4 de 16S DNAr). As famílias fúngicas Marasmiaceae e Lepiotaceae, do domínio Basidiomycota foram as mais abundantes entre os cogumelos amostrados (13 e 5 cogumelos, respectivamente), e estavam presentes entre todas as três parcelas de estudo. As demais amostras foram alocadas dentro das famílias Marasmiaceae, Lepiotaceae, Inocybaceae, Lachnocladiaceae, Bolbitiaceae, Entolomataceae, Hygrophoraceae, Hymenogastraceae, Mycenaceae e Strophariaceae, além de dois do domínio Ascomycota. Baseado na análise de DGGE, é bastante claro que existe uma grande diferença na comunidade bacteriana (de toda a comunidade bacteriana, de ?- proteobacteria e de ?-proteobacteria) entre os solos associados ou não com os corpos de frutificação. Os dados de pirosequenciamento indicaram que dentro dos tratamentos as amostras se agruparam baseado nas famílias fúngicas ou no substrato onde os cocumeglos ocorrem (solo ou serrapilheira), sendo clara em algumas micosferas as alterações na ocorrência de grupos microbianos. O grupo de UTOs mais induzidas na região da micosfera foi composto dos grupos Burkholderia, Acidobacteria Gp1, Comamonadaceae, Sphingobacteriaceae, Burkholderiaceae, Chitinophagaceae), Schlesneria, Acidobacteria Gp3 e Spartobacteria gênero incertae sedis. Isto indica que existe um processo de seleção para bactérias específicas dependendo das diversas variáveis e fatores ambientais presentes no microhabitat micosfera. / The immense microbial diversity in the soil leads to inevitable richness in inter-species interactions. For this reason, this is a novel project that focuses on identifying mushrooms and the associated bacterial community with their mycospheres in the Brazilian Atlantic Rainforest. Using fungal fruiting bodies (mushrooms) as indicators of nutrient-rich systems, samples were taken to investigate the interactions between fungi (mostly of the domain Basidiomycota) and bacteria present in the soil surrounding fungal mycelia (mycosphere environment). The analyses were conducted using culture-independent techniques, where isolating DNA of bacteria and/or mushrooms attempts to provide information on microbial functionality. These culture-independent analyses (PCR-DGGE, Sanger sequencing of ITS/18S fragments, and pyrosequencing of tags from the V4 region of 16S rDNA) generated extensive data on the bacterial diversity selected for in the presence of fungal structures in the soil. The fungal families Maramiaceae and Lepiotaceae, of the domain Basidiomycota were the most abundant among the mushrooms sampled (13 and 5 mushrooms, respectively) and were present in all of the three sampling sites. The rest of the mushrooms were found to be within the families Marasmiaceae, Lepiotaceae, Inocybaceae, Lachnocladiaceae, Bolbitiaceae, Entolomataceae, Hygrophoraceae, Hymenogastraceae, Mycenaceae, and Strophariaceae, in addition to two of the domain Ascomycota. Based on DGGE analysis, it is clear that there is a great difference in the bacterial community (entire bacterial community, of ?- proteobacteria and of ?-proteobacteria) between all fungal fruiting body-associated and non-associated soils. The pyrosequencing data indicated that within each treatment group, the samples did not separate according to fungal families or substrate where the mushrooms were found (in the soil or amoung the leaf litter). However, some mycospheres exhibited clearly altered bacterial communities. The group of OTUs most induced in the mycosphere region consisted of Burkholderia, Acidobacteria Gp1, Comamonadaceae, Sphingobacteriaceae, Burkholderiaceae, Chitinophagaceae), Schlesneria, Acidobacteria Gp3, and Spartobacteria genus incertae sedis. This indicates that there is a selection process for specific bacteria depending on a wide range of variable and environmental factors acting in the mycosphere microhabitat.

Neste estudo avaliou-se a remoção e degradação de surfactante aniônico linear alquilbenzeno sulfonado (LAS) e compostos orgânicos xenobióticos em água residuária de lavanderia comercial em reator anaeróbio de leito fluidificado (RALF) preenchido com areia como material suporte, em escala de bancada (1,2 L), bem como a comunidade microbiana do biofilme e biomassa do separador de fases ao final da operação. O reator foi inoculado com lodo proveniente de reator UASB utilizado no tratamento de dejetos de suinocultura e alimentado com substrato sintético acrescido de água residuária de lavanderia comercial. Caracterização da água residuária, análises de monitoramento da concentração de LAS e matéria orgânica, além de outros parâmetros físico-químicos foram realizadas durante as etapas de operação do sistema. Essa operação foi dividida em cinco etapas: I adaptação da biomassa (575±28mg.L-1 de DQO), II (9,5±3 mg.L-1 de LAS e 637±80mg.L-1 de DQO), III (23,3±8mg.L-1 de LAS e 686±92 mg.L-1 de DQO), IV (21,7±10mg.L-1 de LAS e 691±103 mg.L-1 de DQO), V (27,9±9,6mg.L-1 de LAS e 666±161mg.L-1 de DQO). Aplicação das técnicas de PCR/DGGE e pirosequenciamento da região do rRNA 16S foi realizada para constatar a diversidade microbiana nas etapas IV (com sacarose) e V (sem sacarose). Por meio da caracterização da água residuária de lavanderia comercial foi evidenciado grande variação na concentração de diversos parâmetros, principalmente matéria orgânica (704 mg.L-1 a 4.830 mg.L-1) e LAS (12,2 mg.L-1 a 11.949 mg.L-1). A eficiência média de remoção de matéria orgânica e LAS foi 88% e 60%, respectivamente, durante toda operação do reator. As populações dos Domínios Archaea e Bacteria foram 54% e 45%, similares, respectivamente, para a biomassa da Etapa IV e Etapa V. Por meio da análise de pirosequenciamento das amostras das Etapas IV e V da areia e separador de fases do reator foram identificados 92 gêneros dos quais 24 foram relacionados com a degradação de LAS (Bdellovibrio, Ferruginibacter, Gemmatimonas, etc.). / In this study the removal and degradation of anionic surfactant linear alkylbenzene sulfonate (LAS) and xenobiotic organic compounds in a commercial laundry wastewater was evaluated in anaerobic fluidized bed reactor (AFBR) filled with sand as support material, in a bench scale (1, 2 L), as well as the microbial community of the biofime and phase separator biomass at the end of the operation. The reactor was inoculated with sludge from a UASB reactor used in the swine manure treatment and fed with synthetic substrate plus commercial laundry wastewater. Wastewater characterisation, monitoring analyzes of LAS, organic matter and other physico-chemical parameters were performed during the stages of system operation. This operation was divided into five stages: Stage I - biomass adaptation (575 ± 28mg L-1 of COD), Stage II (9.5 ± 3 mg L-1 of LAS and 637 ± 80 mg L-1 of COD ), Stage III (23.3 ± 8 mg L-1 of LAS and 686 ± 92 mg L-1 of COD), Stage IV (21.7 ± 10 mg L-1 of LAS and 691 ± 103 mg L-1 of COD), Stage V (27.9 ± 9.6 mg L-1 of LAS and 666 ± 161 mg L-1 of COD). Application of PCR/DGGE and pyrosequencing of the 16S rRNA region was performed to verify the microbial diversity in the operational phase IV (with sucrose) and V (without sucrose). Through the commercial laundry wastewater characterization a wide variation in several parameters concentration was shown, mainly organic matter (704mg L-1 to 4.830mg L-1) and LAS (12.2mg L-1 to 11.949mg L-1). The average removal efficiency of organic matter and LAS was 88% and 60%, respectively, throughout the reactor operation. The populations of the Archaea and Bacteria Domains were 54% and 45% similar, respectively, for Stages IV and V biomass. By pyrosequencing analysis of sand and phase separator samples from the Stages IV and V, 92 genera of which 24 were related to the degradation of LAS (Bdellovibrio, Ferruginibacter, Gemmatimonas, Holophaga, Magnetospirillum, Zoogloea, etc.) were identified.

Neste estudo avaliou-se a remoção e degradação de surfactante aniônico linear alquilbenzeno sulfonado (LAS) e compostos orgânicos xenobióticos em água residuária de lavanderia comercial em reator anaeróbio de leito fluidificado (RALF) preenchido com areia como material suporte, em escala de bancada (1,2 L), bem como a comunidade microbiana do biofilme e biomassa do separador de fases ao final da operação. O reator foi inoculado com lodo proveniente de reator UASB utilizado no tratamento de dejetos de suinocultura e alimentado com substrato sintético acrescido de água residuária de lavanderia comercial. Caracterização da água residuária, análises de monitoramento da concentração de LAS e matéria orgânica, além de outros parâmetros físico-químicos foram realizadas durante as etapas de operação do sistema. Essa operação foi dividida em cinco etapas: I adaptação da biomassa (575±28mg.L-1 de DQO), II (9,5±3 mg.L-1 de LAS e 637±80mg.L-1 de DQO), III (23,3±8mg.L-1 de LAS e 686±92 mg.L-1 de DQO), IV (21,7±10mg.L-1 de LAS e 691±103 mg.L-1 de DQO), V (27,9±9,6mg.L-1 de LAS e 666±161mg.L-1 de DQO). Aplicação das técnicas de PCR/DGGE e pirosequenciamento da região do rRNA 16S foi realizada para constatar a diversidade microbiana nas etapas IV (com sacarose) e V (sem sacarose). Por meio da caracterização da água residuária de lavanderia comercial foi evidenciado grande variação na concentração de diversos parâmetros, principalmente matéria orgânica (704 mg.L-1 a 4.830 mg.L-1) e LAS (12,2 mg.L-1 a 11.949 mg.L-1). A eficiência média de remoção de matéria orgânica e LAS foi 88% e 60%, respectivamente, durante toda operação do reator. As populações dos Domínios Archaea e Bacteria foram 54% e 45%, similares, respectivamente, para a biomassa da Etapa IV e Etapa V. Por meio da análise de pirosequenciamento das amostras das Etapas IV e V da areia e separador de fases do reator foram identificados 92 gêneros dos quais 24 foram relacionados com a degradação de LAS (Bdellovibrio, Ferruginibacter, Gemmatimonas, etc.). / In this study the removal and degradation of anionic surfactant linear alkylbenzene sulfonate (LAS) and xenobiotic organic compounds in a commercial laundry wastewater was evaluated in anaerobic fluidized bed reactor (AFBR) filled with sand as support material, in a bench scale (1, 2 L), as well as the microbial community of the biofime and phase separator biomass at the end of the operation. The reactor was inoculated with sludge from a UASB reactor used in the swine manure treatment and fed with synthetic substrate plus commercial laundry wastewater. Wastewater characterisation, monitoring analyzes of LAS, organic matter and other physico-chemical parameters were performed during the stages of system operation. This operation was divided into five stages: Stage I - biomass adaptation (575 ± 28mg L-1 of COD), Stage II (9.5 ± 3 mg L-1 of LAS and 637 ± 80 mg L-1 of COD ), Stage III (23.3 ± 8 mg L-1 of LAS and 686 ± 92 mg L-1 of COD), Stage IV (21.7 ± 10 mg L-1 of LAS and 691 ± 103 mg L-1 of COD), Stage V (27.9 ± 9.6 mg L-1 of LAS and 666 ± 161 mg L-1 of COD). Application of PCR/DGGE and pyrosequencing of the 16S rRNA region was performed to verify the microbial diversity in the operational phase IV (with sucrose) and V (without sucrose). Through the commercial laundry wastewater characterization a wide variation in several parameters concentration was shown, mainly organic matter (704mg L-1 to 4.830mg L-1) and LAS (12.2mg L-1 to 11.949mg L-1). The average removal efficiency of organic matter and LAS was 88% and 60%, respectively, throughout the reactor operation. The populations of the Archaea and Bacteria Domains were 54% and 45% similar, respectively, for Stages IV and V biomass. By pyrosequencing analysis of sand and phase separator samples from the Stages IV and V, 92 genera of which 24 were related to the degradation of LAS (Bdellovibrio, Ferruginibacter, Gemmatimonas, Holophaga, Magnetospirillum, Zoogloea, etc.) were identified.

La diversité des protistes marins planctoniques, après avoir été historiquement étudiée sur des critères morphologiques, est depuis récemment sujette à une intense recherche à l’aide d’approches moléculaires. Notamment, les études basées sur l’amplification directe de marqueurs moléculaires à partir d’ADN environnemental ont révélées une exceptionnelle diversité. L’objectif central de ce travail est d’améliorer notre compréhension sur le lien existant entre la connaissance classique des eucaryotes unicellulaires et leur diversité estimée à partir des données moléculaires, en particulier pour une meilleure interprétation des processus évolutifs et écologiques. Pour cela, nous avons utilisé comme système modèle l’ordre des ciliés tintinnidés (Tintinnida) qui constituent un groupe riche en espèces, aisément identifiables au microscope grâce à leur coquille externe (lorica) et communément rencontrés dans l’ensemble des eaux marines et lacustres du monde. Un suivi approfondi sur deux ans des tintinnidés de la Baie de Villefranche-sur-Mer (Mer Méditerrannée, France), couplant des analyses moléculaires de la diversité à partir de cellules individuelles et à partir d’ADN environnemental, a permis de caractériser la composition de ces communautés et leur dynamique temporelle aux échelles macro- et micro-évolutives. La première partie de ce travail a été destinée à la réalisation d’une phylogénie moléculaire de référence pour les tintinnidés en incorporant les séquences de 62 individus de morphologies diverses pour lesquelles des données moléculaires n'étaient pas disponibles. Nous avons amplifié et séquencé les gènes codant pour les ARN ribosomiques (ARNr 18S, 5.8S et 28S) et les espaces intergéniques correspondants (ITS1 et ITS2). La classification taxonomique a été réévaluée d’après les données moléculaires. Dans un deuxième temps, nous avons testé l’efficacité des approches moléculaires conventionnelles (amplification, clonage et séquençage Sanger du gène de l'ARNr 18S) et plus récentes (amplification et pyroséquençage de régions de l'ARNr 18S et de l’ITS), pour décrire la composition des communautés des tintinnidés dans des échantillons environnementaux en les comparant avec des estimations de la diversité par observation morphologique sur les mêmes échantillons. Si il existe de légères incongruences entre les approches et/ou les différents marqueurs employés, les approches cultureindépendantes s’avèrent efficaces pour décrire la diversité morphologique. En revanche, afin de ne pas surévaluer artificiellement le nombre d’espèces estimées à partir des données de pyroséquençage, il faut que des méthodes de débruitage et de regroupement en unités taxonomiques opérationnelles (UTOs) contraignantes soient appliquées. La troisième partie de ce travail a été dédiée au suivi temporel des communautés de tintinnidés à différentes profondeurs dans la baie de Villefranche, basé sur le clonage et le séquençage du gène de l'ARNr 18S et des régions ITS. Il apparaît des différences de distribution au cours de l’année à une même profondeur, en particulier en termes d’abondance de séquences pour une UTO donnée. Malgré un cadre phylogénétique solide et assez enrichi, l’approche moléculaire révèle des séquences éloignées des espèces déjà séquencées. La découverte de ces clades environnementaux souligne potentiellement l’importance écologique d’espèces encore mal connues. Enfin, le séquençage direct du gène de l'ARNr 18S et de l'ITS2 à partir des cellules individuelles de l'espèce <Undella claparedei> a offert l’opportunité d’une étude populationnelle sur une période de deux ans. La diversité intra-spécifique mesurée met à jour des phénomènes d’hybridation entre variantes génétiques. Une structuration génétique temporelle a également été observée pour le gène de l'ARNr 18S. Les implications de ces différentes recherches sont discutées dans le cadre de l’étude de la diversité et de l’écologie des tintinnidés, et plus largement, des protistes marins. / The marine protistan diversity has been historically studied based on morphological characterization but has recently been the object of intense research using molecular approaches. Studies based on the amplification of molecular markers from environmental DNA revealed an outstanding diversity, partly new and uncharacterized. However, the actual extent of this diversity remains poorly known and highly debated. The main goal of this work was to improve our knowledge on protistan diversity to bridge the gap between molecular environmental surveys and classical protistology to better understand the ecology and evolution of unicellular eukaryotes. For this purpose, we used as a model the species-rich order of the tintinnid ciliates (Tintinnida, Ciliophora), which are easily distinguishable because of their secreted shell, the lorica, and commonly found in marine waters all around the globe. A two-year monitoring of the tintinnid populations in the Bay of Villefranche-sur- Mer (Mediterranean Sea, France), combining molecular analyses of the diversity based on single-cells and environmental DNA, gave us the opportunity to describe the tintinnid community composition and its temporal dynamics. In the first part of this work, we constructed a reference molecular phylogeny for the tintinnids including new sequences from 62 specimens of diverse morphologies, for which we amplified and sequenced the ribosomal coding genes (18S, 5.8S and 28S rRNA) and the corresponding intergenic spacers (ITS1 and ITS2). The taxonomic classification of the Tintinnida has been revised based on these molecular data. In the second part, in order to assess the accuracy of molecular-based approaches to describe the natural species assemblages of tintinnids, we compared the morphology-based diversity estimates with those derived from classical (amplification, cloning and Sanger sequencing of the 18S rRNA gene) and more recent (direct pyrosequencing of amplified 18S rRNA genes and ITS regions) molecular approaches. Even if there are still some disagreements between the different methods and/or molecular markers, the culture-independent approaches were efficient to describe the morphological diversity. However, a careful and rigorous analysis of pyrosequencing datasets, including sequence denoising and stringent sequence clustering in Operational Taxonomic Units (OTUs) with well-adjusted parameters, is necessary to avoid overestimating the species number. The third part of the thesis is dedicated to the study of the genetic diversity of tintinnids over a one-year survey in the Bay of Villefranche at five different depths by combining community fingerprinting analysis using denaturing gradient gel electrophoresis (DGGE) with direct PCR amplification and sequencing of 18S, 5.8S, and 28S rRNA genes and ITS regions. These analyses revealed marked seasonal changes, in particular in the sequence abundances of certain OTUs. In addition, despite an enriched phylogenetic reference sequence dataset for the tintinnids, we retrieved two abundant phylotypes without any closely related known species, highlighting the possible ecological relevance of unidentified species. Finally, we studied the intra-specific diversity of populations of the species <Undella claparedei> based on 18S rDNA and ITS direct sequencing of single-cells collected over a period of two years. We detected signals of hybridization and sexual recombination among different genetic variants. We also found genetic structuring of the 18S rRNA gene data differentiating populations collected at different times. The implications of all these results are discussed in the framework of the diversity and ecology of tintinnid ciliates and, more generally, of marine protists

A região anterior do intestino médio de Lepidoptera apresenta uma secreção microapócrina de enzimas digestivas com vesículas migrando pelo interior das microvilosidades. Essas vesículas brotam das microvilosidades intestinais como vesículas de membrana dupla e são descarregadas dentro do lúmen. O objetivo desse trabalho foi identificar as proteínas secretadas e aquelas envolvidas na maquinaria secretória microapócrina em Spodoptera frugiperda. Para isso, vesículas microapócrinas foram preparadas e usadas para a produção de anticorpo policlonal. Esse anticorpo foi utilizado para varrer uma biblioteca de expressão de cDNA do intestino médio de S. frugiperda. Também obtivemos um transcriptoma por pirosequenciamento de uma biblioteca de cDNA proveniente dos transcritos do intestino médio do mesmo inseto. Os clones positivos da varredura foram sequenciados, montados e submetidos a um BLASTN contra as sequências obtidas pelo pirosequenciamento, o que resultou na extensão dessas sequências. Usamos ainda as sequências geradas pelo pirosequenciamento para reanalisar sequências de proteínas presentes nas membranas microvilares, que tinham sido obtidas anteriormente em nosso laboratório (Ferreira et al., 2007). A reanálise das sequências de proteínas microvilares gerou 66 proteínas preditas. Dessas, 18 foram consideradas contaminantes de outros compartimentos celulares e 48 associadas às membranas microvilares. A análise das sequências obtidas das vesículas microapócrinas gerou 50 proteínas preditas que podem ser secretadas por essa rota. As sequências encontradas tanto em membrana microvilar quanto em vesículas microapócrinas podem ser classificadas em 8 grupos, de acordo com sua função: (1) enzimas digestivas; (2) proteínas da membrana peritrófica; (3) envolvidas com proteção; (4) transportadores; (5) receptores; (6) proteínas da maquinaria secretória; (7) proteínas de citoesqueleto; (8) com função desconhecida nesse local. Em ambas as preparações existem uma predominância de sequências de enzimas digestivas. Nas membranas microvilares a maioria das sequências são aminopeptidases, enquanto nas vesículas microapócrinas a maioria são lipases. Os cDNAs correspondentes as proteínas que poderiam estar envolvidas na maquinaria secretória foram clonados e sequenciados. São elas: fimbrina, cofilina, gelsolina-1 e miosina I. RT-PCRs semi-quantitativas dessas proteínas em diferentes tecidos do inseto (intestino, túbulos de Malpighi, corpo gorduroso e carcaça) mostraram que somente gelsolina-1 está presente exclusivamente no intestino. Os domínios G1-G3 da gelsolina característica do intestino (gelsolina-1) foram expressos e usados para produzir anticorpos em coelhos. Esses anticorpos reconhecem a proteína recombinante e uma proteína presente no epitélio intestinal com massa molecular compatível com a massa predita para gelsolina-1. Foi possível diminuir a expressão de gelsolina-1 utilizando RNA interferente. / Lepidoptera anterior midgut presents a microapocrine secretion of digestive enzymes with secretory vesicles migrating inside the microvilli. These vesicles bud from the midgut microvilli as double membrane vesicles and are discharged into the lumen. The aim of this work was to identify the proteins secreted and those involved in the microapocrine secretory machinery in Spodoptera frugiperda larvae. For this, microapocrine vesicles were prepared and used for polyclonal antibody production. This antibody was used to screen a cDNA expression library of S. frugiperda midgut. We also obtained a transcriptome by pyrosequencing a cDNA library derived from transcripts of the midgut. Positive clones from the screening were sequenced, assembled and N-blasted against S. frugiperda sequences obtained by pyrosequencing. This procedure led to the extension of the sequences previously obtained. We also used the sequences generated by pyrosequencing to reanalyze the sequences of microvillar membrane proteins obtained previously by Ferreira et al. (2007). This reanalysis generated 66 predicted proteins that are present in the microvillar membranes. Eighteen were considered to be contaminants from other compartments and 48 associated with the microvillar membranes. Analysing the sequences from microapocrine vesicles we found 50 predicted proteins that should be secreted by microapocrine vesicles. The sequences found in both microvillar membrane and microapocrine vesicles may be classified into 8 groups, according to their function: (1) digestive enzymes; (2) peritrophic membrane proteins; (3) protection; (4) transporters; (5) receptors; (6) secretory machinery; (7) cytoskeleton; (8) with unknown function. In both preparations there is a predominance of sequences of digestive enzymes. In microvillar membranes, there is a remarkable amount of aminopeptidases, while in microapocrine vesicles this is true for lipases. cDNAs coding for proteins that could be involved in the microapocrine secretory machinery were cloned and sequenced. They are: fimbrin, cofilin, gelsolin-1 and myosin I. Using RT-PCR, we showed that mRNAs coding for gelsolin-1 and myosin I are present only in the intestinal tissue. The mRNAs coding for other proteins were found in all tissues. The domains G1-G3 from gelsolin specific from intestinal midgut (gelsolin 1) were expressed and used to raise antibodies in rabbit. These antibodies were able to recognize the recombinant protein and a protein from the midgut epithelium that has a molecular weight similar to the one predicted from gelsolin-1 sequence. We succeed in decreasing the expression of gelsolin-1 by using interfering RNA

<p>DNA samples from crime scenes or mass disasters are often limited and degraded which limits the possibility of successful traditional STR analysis. Moreover, there is a need to decrease the turnaround time in criminal investigations. These circumstances require a wider set of assays and technologies to be investigated for potential use in forensic DNA analysis, which has been explored in this thesis work. DNA analysis can also provide a useful tool in forensic pathology investigations. </p><p>In a search for mutations involved in The Sudden Infant death Syndrome (SIDS), the entire mitochondrial genome was sequenced in six SIDS infants and shorter mtDNA regions were analysed in paraffin-embedded tissues from an additional 14 SIDS cases. In this sample material no mutations associated with SIDS were found that could explain the death of these infants. </p><p>To reduce time, cost and effort related to sequencing of the mtDNA HVI/HVII regions in caseworks, a HVI/HVII mtDNA linear array assay was used as a pre-screening for exclusions of suspects or evidence samples. Using this assay, 56% of the samples involved in casework analysis could be excluded before sequencing was undertaken.</p><p>The possibility to use the new array technology was explored in a SNP assay targeting both mtDNA and nuclear SNPs. The system relies on minisequencing in solution prior to hybridisation to tag arrays. Using this system, we demonstrate a rapid, highly multiplexable and flexible array-format for SNP analysis.</p><p>The properties of the Pyrosequencing technology being a fast and user-friendly assay was utilised in a study to investigate the possibility to use this method for limited and degraded samples. Ten STR loci, overlapping with standardised kits, were genotyped in 114 Swedish individuals. We found additional variation and higher resolution of repeats at some of these loci that are not detected using standard fragment analysis.</p>

<p>The field of forensic genetics is growing fast and the development and optimisation of more sensitive, faster and more discriminating forensic DNA analysis methods is highly important. In this thesis, an evaluation of the use of novel DNA technologies and the development of specific applications for use in forensic casework investigations are presented.</p><p>In order to maximise the use of valuable limited DNA samples, a fast and user-friendly Real-time PCR quantification assay, of nuclear and mitochondrial DNA copies, was developed. The system is based on the 5’ exonuclease detection assay and was evaluated and successfully used for quantification of a number of different evidence material types commonly found on crime scenes. Furthermore, a system is described that allows both nuclear DNA quantification and sex determination in limited samples, based on intercalation of the SYBR Green dye to double stranded DNA. </p><p>To enable highly sensitive DNA analysis, Pyrosequencing of short stretches of mitochondrial DNA was developed. The system covers both control region and coding region variation, thus providing increased discrimination power for mitochondrial DNA analysis. Finally, due to the lack of optimal assays for quantification of mitochondrial DNA mixture, an alternative use of the Pyrosequencing system was developed. This assay allows precise ratio quantification of mitochondrial DNA in samples showing contribution from more than one individual.</p><p>In conclusion, the development of optimised forensic DNA analysis methods in this thesis provides several novel quantification assays and increased knowledge of typical DNA amounts in various forensic samples. The new, fast and sensitive mitochondrial DNA Pyrosequencing assay was developed and has the potential for increased discrimination power.</p>

With the full sequence of the human genome now available, anexciting era in biomedical research has started. The sequenceprovides information about all our genes and greatly increasesthe scope to compare genetic activities in different cells, toanalyze genetic variation between individuals and betweendifferent species and, most importantly, to investigatesystematically the whole genome in a gene-by-gene manner, andthus increase our understanding of gene function. This thesis describes studies in which developments weremade in several areas of functional genomics. Messenger RNAlevels were analyzed by the use of an amplification procedure,in which the 3´-ends of the transcripts were selected inorder to amplify the mRNA population in an unbiased fashion. Bysonicating cDNA originating from expressed mRNA, uniformlysized representatives of the transcripts,“signaturetags”, were obtained. The mRNA levels in the original mRNApopulation correlated well with the levels in the amplifiedmaterial, as verified by microarray analysis and realtimequantitative PCR. The expressed transcripts can be identifiedusing pyrosequencing, by comparing the obtained sequenceinformation from the signature tags to information contained invarious sequence databases. In one of the articles, the use ofpyrosequencing is illustrated by efforts to find genes involvedin the disease progression of atherosclerosis. More challenging than the study of mRNA levels is to analyzewhen, where and how proteins fulfill their wide-ranging rolesin all the various cellular processes. Proteins are morecomplex biomolecules than mRNA, each having unique properties.Current techniques for studying proteins need much improvement,and are often limited to investigations of a specific portionof the proteome. One approach for studying the whole proteomeis to systematically generate reagents with specific affinityfor the proteins encoded by the genome, one by one. Theaffinity reagents can be used as flags for their targets,providing a flag-specific detection system, so that the targetproteins can be sub-cellularly localized in the majority ofhuman tissues in an array format. One of the articles includedin the thesis presents a pilot project for large-scale affinityreagent production. The aim was to provide a sound basis forwhole proteome studies, but as a pilot study this investigationwas limited to the proteins encoded by human chromosome 21. Allputative genes on the chromosome were subjected to antibodygeneration in a systematic manner. Small, uniform, and easilyproduced representative portions of the full-length proteinswere expressed. These were denoted“Protein EpitopeSignature Tags”and were designed to be unique for theirfull-length counterparts. The antibodies were produced inrabbits and two of the articles in the thesis discuss differentapproaches for affinity purification of the antibodies toachieve the highest possible specificity towards the targets.The resulting“mono-specific”, but still“multi-epitope”, antibodies can be used for a widerange of additional biochemical studies, such as protein arrayand protein pull-out analyses. <b>Keywords:</b>functional genomics, 3´-end signaturetags, pyrosequencing, amplification, PrEST, chromosome 21,polyclonal antibodies, dual expression, affinitypurification.

DNA samples from crime scenes or mass disasters are often limited and degraded which limits the possibility of successful traditional STR analysis. Moreover, there is a need to decrease the turnaround time in criminal investigations. These circumstances require a wider set of assays and technologies to be investigated for potential use in forensic DNA analysis, which has been explored in this thesis work. DNA analysis can also provide a useful tool in forensic pathology investigations. In a search for mutations involved in The Sudden Infant death Syndrome (SIDS), the entire mitochondrial genome was sequenced in six SIDS infants and shorter mtDNA regions were analysed in paraffin-embedded tissues from an additional 14 SIDS cases. In this sample material no mutations associated with SIDS were found that could explain the death of these infants. To reduce time, cost and effort related to sequencing of the mtDNA HVI/HVII regions in caseworks, a HVI/HVII mtDNA linear array assay was used as a pre-screening for exclusions of suspects or evidence samples. Using this assay, 56% of the samples involved in casework analysis could be excluded before sequencing was undertaken. The possibility to use the new array technology was explored in a SNP assay targeting both mtDNA and nuclear SNPs. The system relies on minisequencing in solution prior to hybridisation to tag arrays. Using this system, we demonstrate a rapid, highly multiplexable and flexible array-format for SNP analysis. The properties of the Pyrosequencing technology being a fast and user-friendly assay was utilised in a study to investigate the possibility to use this method for limited and degraded samples. Ten STR loci, overlapping with standardised kits, were genotyped in 114 Swedish individuals. We found additional variation and higher resolution of repeats at some of these loci that are not detected using standard fragment analysis.

The field of forensic genetics is growing fast and the development and optimisation of more sensitive, faster and more discriminating forensic DNA analysis methods is highly important. In this thesis, an evaluation of the use of novel DNA technologies and the development of specific applications for use in forensic casework investigations are presented. In order to maximise the use of valuable limited DNA samples, a fast and user-friendly Real-time PCR quantification assay, of nuclear and mitochondrial DNA copies, was developed. The system is based on the 5’ exonuclease detection assay and was evaluated and successfully used for quantification of a number of different evidence material types commonly found on crime scenes. Furthermore, a system is described that allows both nuclear DNA quantification and sex determination in limited samples, based on intercalation of the SYBR Green dye to double stranded DNA. To enable highly sensitive DNA analysis, Pyrosequencing of short stretches of mitochondrial DNA was developed. The system covers both control region and coding region variation, thus providing increased discrimination power for mitochondrial DNA analysis. Finally, due to the lack of optimal assays for quantification of mitochondrial DNA mixture, an alternative use of the Pyrosequencing system was developed. This assay allows precise ratio quantification of mitochondrial DNA in samples showing contribution from more than one individual. In conclusion, the development of optimised forensic DNA analysis methods in this thesis provides several novel quantification assays and increased knowledge of typical DNA amounts in various forensic samples. The new, fast and sensitive mitochondrial DNA Pyrosequencing assay was developed and has the potential for increased discrimination power.