Taxonomic changes are made based on checkered beetle (Coleoptera: Cleridae) types of the Natural History Museum, London (BMNH).Lectotypes are designated (and holotypes and paralectotypes recognized) for 44 species of Hydnocerinae, including the type species for Isolemidia, Parmius, Paupris, Allelidea, Blaesiopthalmus and Lemidia, four species of Enoclerus (Clerinae), and 14 species of Cymatodera (Tillinae). Annotations include comments on additional type material, new type locality, previous (type series) locality, and questionable or missing types. Phyllobaenus pallipes(Gorham) and P. rufithorax (Gorham) are synonymized with P. flavifemoratus(Gorham), P. chapini (Wolcott) is synonymized under P. lateralis (Gorham), and P. villosus (Schenkling) is synonymized under P. longus (LeConte), new synonymies. The first molecular phylogeny of the clerid lineage (Coleoptera: Cleridae, Thanerocleridae) is presented and compared with the two most recent phylogenetic hypotheses of the group. Phylogenetic relationships of checkered beetles wareere inferred from approximately 5,000 nucleotides amplified from four loci (28S, 16S, 12S, COI). A worldwide sample of ~70 genera is included and phylogenies are reconstructed using Bayesian Inference and Maximum Likelihood. The results are not entirely congruent with either of the current classification systems. Three major lineages are recognized. Tillinae are supported as the sister group to all other subfamilies, whereas Thaneroclerinae, Korynetinae sensu latu and a new subfamily formally described here, Epiclininae, new subfamily, form a sister group to Clerinae + Hydnocerinae. To assess the phylogeny and evolution of Hemiptera, a comprehensive mitogenomic analysis integrating mitogenome-based molecular phylogenetics, fossil-calibrated divergence dating (using BEAST), and ancestral state reconstructions are presented. The 81 sampled mitogenomes represent the most extensive mitogenomic analyses of Hemiptera to date. The putatively primitive “Homoptera” was previously rendered paraphyletic by Heteroptera, whereas the presented results support each group as monophyletic. The results from both diet and habitat ancestral state reconstructions support that 1) Heteroptera (and Homoptera) evolved from a phytophagous ancestor, contrary to the popular hypothesis that the ancestor was predaceous; and 2) family-level radiation of Heteroptera is coincident with the apically-produced labium and the novel hemelytron. It is here proposed these morphological innovations facilitated multiple independent shifts from phytophagy to predation and multiple independent colonizations of aquatic habitats.

The mitochondrial small-subunit ribosomal RNA (mt. SSU rRNA = rns) gene appears to be a reservoir for a number of group I and II introns along with the intron- encoded proteins (IEPs) such as homing endonucleases (HEases) and reverse transcriptases. The key objective for this thesis was to examine the rns gene among different groups of ophiostomatoid fungi for the presence of introns and IEPs. Overall the distribution of the introns does not appear to follow evolutionary lineages suggesting the possibility of rare horizontal gains and frequent loses. Some of the novel findings of this work were the discovery of a twintron complex inserted at position S1247 within the rns gene, here a group IIA1 intron invaded the ORF embedded within a group IC2 intron. Another new element was discovered within strains of Ophiostoma minus where a group II introns has inserted at the rns position S379; the mS379 intron represents the first mitochondrial group II intron that has an RT-ORF encoded outside Domain IV and it is the first intron reported to at position S379. The rns gene of O. minus WIN(M)371 was found to be interrupted with a group IC2 intron at position mS569 and a group IIB1 intron at position mS952 and they both encode double motif LAGLIDADG HEases referred as I-OmiI and I-OmiII respectively. These IEPs were examined in more detail to evaluate if these proteins represent functional HEases. To express I-OmiI and I-OmiII in Escherichia. coli, a codon-optimized versions of I-OmiI and I-OmiII sequences were synthesized based on differences between the fungal mitochondrial and bacterial genetic code. The optimized I-OmiI and I-OmiII sequences were cloned in the pET200/D TOPO expression vector system and transformed into E. coli BL21 (DE3). These two proteins were biochemically characterized and the results showed that: both I-OmiI and I-OmiII are functional HEases. Detailed data for I-OmiII showed that this endonuclease cleaves the target site two nucleotides upstream of the intron insertion site generating 4 nucleotide 3’overhangs.

In spite of significant progress in our knowledge of mechanisms that control blood pressure, our understanding of the pathogenesis of hypertension, its genetics, and population efforts to control blood pressure, hypertension remains the leading risk factor for mortality worldwide. It’s estimated that 1 out of every 3 adults has hypertension. Hypertension is a major risk factor for cardiovascular disease and stroke, and is considered a primary or contributing cause of death to more than 2.4 million US deaths each year. Although spontaneous hypertension has been the subject of substantial research, many critical questions remain unanswered. To investigate mechanisms underlying spontaneous hypertension, a unique rodent breeding approach was used to isolate nuclear and mitochondrial genes contributing to the disease. By diluting the nuclear genome of the Spontaneously Hypertensive Rat on a normotensive Brown Norway background while maintaining the SHR mitochondrial genome, I investigated both intrinsic and extrinsic mechanisms of the kidney and its relationship to hypertension. Chapter 2 documents the dominance of the hypertensive phenotype in our rodent colony, despite the dilution of the nuclear genome of the SHR. Chapter 3 presents data indicating that the renin-angiotensin system, particularly the location and abundance of the AT1 receptor may play an important role in the manifestation of spontaneous hypertension. Chapter 4 presents that rats in our rodent colony exhibited normal pressure-natriuresis and kidney function; however, hypertensive rats had a reduced ability to sense orally ingested sodium chloride, thus necessitating chronic elevations of arterial pressure in order to maintain sodium balance. This chronic pressure-natriuresis relationship shifts the renal function curve to the right, thus sustaining elevated blood pressure. Chapter 5 presents data that genes important for oxidative phosphorylation may play a critical role in the development of hypertension. Both nuclear and mitochondrial oxidative phosphorylation genes were downregulated in hypertensive rats compared with normotensive rats. Data presented in every chapter highlights the importance of the kidney in the pathogenesis of hypertension. Humoral, genetic and genomic mechanisms of the kidney appear to play a dominant role in the development and maintenance of the disease.

The mitochondrial small-subunit ribosomal RNA (mt. SSU rRNA = rns) gene appears to be a reservoir for a number of group I and II introns along with the intron- encoded proteins (IEPs) such as homing endonucleases (HEases) and reverse transcriptases. The key objective for this thesis was to examine the rns gene among different groups of ophiostomatoid fungi for the presence of introns and IEPs. Overall the distribution of the introns does not appear to follow evolutionary lineages suggesting the possibility of rare horizontal gains and frequent loses. Some of the novel findings of this work were the discovery of a twintron complex inserted at position S1247 within the rns gene, here a group IIA1 intron invaded the ORF embedded within a group IC2 intron. Another new element was discovered within strains of Ophiostoma minus where a group II introns has inserted at the rns position S379; the mS379 intron represents the first mitochondrial group II intron that has an RT-ORF encoded outside Domain IV and it is the first intron reported to at position S379. The rns gene of O. minus WIN(M)371 was found to be interrupted with a group IC2 intron at position mS569 and a group IIB1 intron at position mS952 and they both encode double motif LAGLIDADG HEases referred as I-OmiI and I-OmiII respectively. These IEPs were examined in more detail to evaluate if these proteins represent functional HEases. To express I-OmiI and I-OmiII in Escherichia. coli, a codon-optimized versions of I-OmiI and I-OmiII sequences were synthesized based on differences between the fungal mitochondrial and bacterial genetic code. The optimized I-OmiI and I-OmiII sequences were cloned in the pET200/D TOPO expression vector system and transformed into E. coli BL21 (DE3). These two proteins were biochemically characterized and the results showed that: both I-OmiI and I-OmiII are functional HEases. Detailed data for I-OmiII showed that this endonuclease cleaves the target site two nucleotides upstream of the intron insertion site generating 4 nucleotide 3’overhangs.

A fundamental problem in biology is the reconstruction of the relatedness of all (extant) species. Traditionally, systematists employ visually recognizable characters of organisms for classification and evolutionary analysis. Recent developments in molecular and computational biology, however, lead to a whole different perspective on how to address the problem of inferring relatedness. The discovery of molecules, carrying genetic information, and the comparison of their primary structure has, in a rather short period of time, revolutionized our understanding of the phylogenetic relationship of many organisms. These novel approaches, however, turned out to bear similar problems as previous techniques. Moreover, they created new ones. Hence, taxonomists came to realize that even with this new type of data not all problematic relationships could be unambiguously resolved. The search for complementary approaches has led to the utilization of rare genomic changes and other characters which are largely independent from the primary structure of the underlying sequence(s). These “higher order” characters are thought to be evolutionary conserved in certain lineages and largely unaffected by primary sequence data-based problems, allowing for a better resolution of the Tree of Life. The central aim of this thesis is the utilization of molecular characters of higher order in connection with their consistent and comparable extraction from a given data set. Two novel methods are presented that allow such an inference. This is complemented with the search for and analysis of known and novel molecular characteristics to study the relationships among Metazoa, both intra- as well as interspecific. The first method tackles a common problem in phylogenetic analyses: the inference of reliable data set. As part of this thesis a pipeline was created for the automated annotation of metazoan mitochondrial genomes. Data thus obtained constitutes a reliable and standardized starting point for all downstream analyses, e.g. genome rearrangement studies. The second method utilizes a subclass of gaps, namely those which define an approximate split of a given data set. The definition and inference of such split-inducing indels (splids) is based on two basic principles. First, indels at the same position, i.e. sharing the same end points in two sequences, are likely homologous. Second, independent single-residue insertions and deletions tend to occur more frequently than multi-residue indels. It is shown that trees based on splids recover most of the undisputed monophyletic groups while influence of the underlying alignment algorithm is relatively small. Mitochondrial markers are a valuable tool for the understanding of small and large scale population structure. The non-coding control region of mitochondrial DNA (mtDNA) often contains a higher amount of variability compared to genes encoding proteins and non-coding RNAs. A case study on a small scale population structure investigates the control region of the European Fire-bellied Toad in order to find highly variable parts which are of potential importance to develop informative genetic markers. A particular focus is placed on the investigation of the evolutionary dynamics of the repetitive region at an inter- and intraspecific level. This includes understanding mechanisms underlying its evolution, i.e. by exploring the impact of secondary structure on slipped strand mispairing during mtDNA replication. The 7SK RNA is a key player in the regulation of polymerase II (Pol-II) transcription, interacting with at least three known proteins: It mediates the inhibition of the Positive Transcription Elongation Factor b (P-TEFb) by the HEXIM1/2 proteins, thereby repressing transcript elongation by Pol-II. A highly specific interaction with LARP7 (La-Related Protein 7), on the other hand, regulates its stability. 7SK RNA is capped at its 5’ end by a highly specific methyltransferase MePCE (Methylphosphate Capping Enzyme). Employing sequence and structure similarity it is shown that the 7SK RNA as well as its protein binding partners have a much earlier evolutionary origin than previously expected. Furthermore, this study presents a good illustration of the pitfalls of using markers of higher order for phylogenetic inference.

A mitocôndria é a organela citoplasmática responsável pelo maior sistema produtor de energia, a fosforilação oxidativa (OXPHOS). Foi proposto que em células tumorais a hiper-regulação da glicólise em condições normais de oxigênio (Efeito Warburg), está associada a defeitos na OXPHOS e pode regular o fenótipo tumoral, por exemplo, o potencial metastático da célula por meio da indução de vias pseudohipóxicas durante a normóxia. Estudos recentes mostraram que vários tipos de tumores possuem mutações somáticas em seu genoma mitocondrial, o que pode alterar as funções da OXPHOS levando a troca de metabolismo energético nas células tumorais e induzindo a tumorigênese. Diante disto, o presente trabalho avaliou a instabilidade do genoma mitocondrial em etapas bem definidas da progressão do câncer colorretal. O DNA genômico foi extraído de amostras de adenoma, adenocarcinoma, tecido adjacente e sangue periférico de nove pacientes diagnosticados com Câncer colorretal. O genoma mitocondrial foi amplificado e sequenciado para que fossem feitas as buscas por mutações nas amostras de sangue periférico, adenomas e adenocarcinoma. Foi também medido o número de cópias relativas do mtDNA. Foram encontradas um total de 233 mutações, das quais 162 foram em comum entre os três tecidos avaliados. As amostras de adenocarcinoma foram as que apresentaram uma maior média de mutações por amostra (44,6), seguidas dos adenoma (40,2) e do sangue periférico (34). As amostras de adenocarcinoma apresentaram uma maior instabilidade do mtDNA refletidas a partir de um maior número de mutações somáticas (tanto do tipo InDel como mutações de uma única base), mutações não sinônimas com maior patogenicidade, maior número de mutações em heteroplasmia e com taxa de heteroplasmia elevada. Já as amostras de adenoma apresentaram instabilidade dos seus mtDNA intermediários entre o tecido não tumoral e tumoral, refletindo bem a etapa de modificação celular no qual esses tecidos se encontram. Na análise do número de cópias relativas, as amostras de adenocarcinoma tiveram diminuição no número de cópias relativas quando comparadas com tecido adjacente (p= 0,01) e com adenomas (p= 0,04). Em síntese, o presente trabalho sugere que a instabilidade do genoma mitocondrial parece ter um papel importante no desenvolvimento de tumores colorretais. / The mitochondrion is a cytoplasmic organelle responsible for the major energy producing system, which is the oxidative phosphorylation enzyme pathway (OXPHOS). It was proposed that glycolysis up-regulation during normal oxygen conditions (Warburg effect) may induce defects in the mitochondrial respiration and regulate tumoral phenotypes, for example, metastatic potential through the induction of pseudohipoxic pathways during normoxia. Recent studies have shown that many kinds of tumors have mtDNA somatic mutations, which could alter the OXPHOS functions, leading to changes in glucose metabolismo and improvind tumorigenesis. This study analyzed the mitochondrial genome instability of well defined stages of colorectal cancer. Genomic DNA was extracted from adenoma, adenocarcinoma, adjacente tissue and peripheral blood of patients diagnosed with Colorectal cancer. The mitochondrial genome was amplified and sequenced for mutations screening in adenoma, adenocarcinoma e blood samples. It was also analyzed the relative mtDNA copy number. It was find a total of 233 mutations, which 162 were in common among the three analyzed tissues. The adenocarcinoma samples presented a greater mutation mean per sample (44.6) followed by adenomas samples (40.2) and blood samples (34). The adenocarcinoma samples also shown a greater mitochondrial genome instability refleted by increased of somatic mutations (InDels and single nucleotide variation), non sinonimous mutations with higher patogenicity, increased number of heteroplasmatic mutations and higher heteroplasmatic levels. The adenoma samples showed intermadiate instability of its mtDNA, which well reflects the intermediate stage of cellular modifications of this tissue. The mtDN copy number analysis shown that the adenocarcinoma samples presented decreased number of mtDNA content when compared with adjacente tissue (p= 0.01) and adenoma samples (p= 0.04). In summary the presente study suggests that the mitochondrial genomic instability seems to play an importante role in colorectal tumorigenesis.

Estudos recentes relataram oncogenes induzindo a reprogramação metabólica no câncer. Essa reprogramação é fundamental para que as células cancerosas tenham nutrientes e biomoléculas suficiente para manter sua alta taxa proliferativa. A mitocôndria tem um papel central no metabolismo energético da célula e alterações no seu genoma, tanto em relação a mutações como em número de cópias, já foram bastante observados em vários tipos tumorais. Além disso, deficiência no fator de transcrição mitocondrial A (TFAM), fundamental para a transcrição e estabilidade do mtDNA, já foi associada com o crescimento tumoral. Diante disso, nosso estudo teve como objetivo avaliar o papel da instabilidade do genoma mitocondrial no metabolismo energético e crescimento do melanoma. Para isso, nós medimos a instabilidade do mtDNA utilizando como parâmetros: o acúmulo de mutações no mtDNA, alterações no mtDNAcn e a expressão do TFAM. O impacto da instabilidade do mtDNA foi avaliado em três modelos diferentes de melanoma: um modelo in vitro de linhagens celulares, dados de expressão gênica de tumores de melanoma metastático proveniente do TCGA e um modelo murino induzível de melanoma (BrafV600E/Ptennull), adicionado a um background alternativo de deficiência para o TFAM/mtDNAcn. Esse modelo murino também nos permitiu avaliar a deficiência do TFAM limitada a células tumorais (Tfamflox) e tanto em células tumorais, como no seu microambiente (Tfam+/-). Nas análises in vitro, nós observamos correlações positivas entre o mtDNAcn e a expressão do TFAM com a taxa de consumo de glicose e produção de ATP, indicando um impacto desses parâmetros na bioenergética celular. Análises de expressão gênica, utilizando tanto as linhagens de melanoma como tumores de melanoma metastático, nos sugeriram que o TFAM regula genes indutores de angiogênese, a resposta imunológica humoral e vias metabólicas de aminoácidos. Nas análises in vivo, nós observamos um aumento dos tumores em camundongos Tfam+/-, indicando que a deficiência de TFAM/mtDNAcn em células tumorais e no seu microambiente induz a tumorigênese, o que confirma os dados de expressão gênica encontrados com linhagens e tecido de melanoma. Além disso, análises de metabolômica e transcriptômica combinadas nos sugeriram que as células de melanoma com deficiência no TFAM/mtDNAcn são mais dependentes do metabolismo de glutamina. Diante disso, nós concluímos que a deficiência do TFAM/mtDNAcn tem um papel importante no crescimento do melanoma, induzindo a expressão de genes pro-tumorigênicos e aumentando o consumo da glutamina para suprir as necessidades proliferativas das células cancerosas. Esses dados são relevantes e podem nos ajudar a entender melhor o papel da mitocondrial na progressão do melanoma. / Recent studies have shown many oncogenes triggering metabolic reprogramming in cancer. The metabolic switch in cancer cells is necessary to supply the high demand for nutrients and biomolecules for proliferative cells. In this context, mitochondria play a central role in the energetic metabolism of the cell and changes in its genome, such as an increased load of mutations and alterations in mtDNA content, have been reported in several cancers. In addition, deficiency in the Mitochondrial Transcription Factor A (TFAM), responsible for transcription and maintenance of mtDNA stability, was previously associated with tumor growth. Based on that, our goal was to evaluate the impact of the mitochondrial genome instability in the energetic metabolism and melanoma growth. mtDNA instability was inferred measuring mtDNA mutations load and content, as well as TFAM expression. Its impact was evaluated in three different melanoma models: an in vitro model using melanoma cell lines, gene expression data from metastatic melanoma tumors, publicly available at TCGA, and an inducible murine model of melanoma (BRAFV600E/PTENnull), crossed onto different TFAMdeficient backgrounds. The murine model also provides us a tractable model to examine the consequences of mtDNA instability limited to cancer cells (Tfamflox) and in both cancer cells and tumor microenvironment (Tfam+/-). In vitro analysis showed us a positive correlation between mtDNA copy number (mtDNAcn) and TFAM expression with glucose consumption and ATP production, pointing an impact of these parameters in cellular bioenergetics. Further gene expression analysis, using both cell lines and metastatic melanoma data, suggested that TFAM could regulate the expression of angiogenesis genes, humoral immunity and amino acid metabolism. In vivo analysis confirmed the gene expression data, and revealed a higher melanoma growth in Tfam+/-. Also, combined metabolomics and transcriptomics data suggested that TFAM/mtDNAcn deficient melanoma cells rely mostly on glutamine metabolism to supply their energetic requirements. In conclusion, these data indicate that TFAM/mtDNAcn influences melanoma growth by triggering pro-tumorigenic signals and inducing metabolic reprogramming towards glutamine metabolism. These results are relevant and might help us understand how mitochondria affect melanoma progression.

The basking shark, Cetorhinus maximus, has historically been a target of international fisheries, leading to well-documented declines in parts of its global distribution. Currently, the basking shark is listed as globally ‘Vulnerable’ and regionally ‘Endangered’ (North Pacific and Northeast Atlantic) on the IUCN Red List of Threatened Species, rendering the species an international conservation priority. Here, we assessed the global matrilineal genetic population structure and evolutionary history of the basking shark by completing the first whole mitochondrial genome sequence level survey of animals sampled from three globally widespread geographic regions: the western North Atlantic (n = 11), the eastern North Atlantic (n = 11), and within New Zealand territorial waters (n = 12). Despite the relatively large amount of sequence data assessed (~16,669 bp per individual), whole mitogenome analyses showed no evidence of population differentiation (ΦST = -0.047, P > 0.05) and very low nucleotide diversity (π = 0.0014 ± 0.000) across a global seascape. The absence of population structure across large distances and even between ocean basins is indicative of long-dispersal by this species, including an ability to cross known biogeographic barriers known to differentiate populations of other highly vagile pelagic fishes. Notably, evolutionary analyses of the mitogenome sequences revealed two globally sympatric but evolutionary divergent lineages, with a Bayesian framework estimated coalescence time of ~2.46 million years ago. Coalescent-based Bayesian skyline analysis uncovered subtle evidence of Pleistocene demographic flux for this species, including a potential decline in female effective population size. Thus, historical population changes may be responsible for the occurrence of the two highly divergent, yet sympatric lineages, as population declines may have resulted in the loss of intermediate haplotypes and resulted in an overall loss of genetic diversity. This work supports the recognition of basking sharks as a single matrilineal global population, and as such requires the application of a cooperative multiagency and international approach to fisheries management to conserve this highly vulnerable and ecologically unique species.

The aim of this work is a literature review on the topic of the mitochondrial genome and DNA barcoding, building a dataset of mitochondrial sequences from GenBank database and creatione of a software function for extraction of individual genes that are present in the mitochondrial genome. This function was developed in Matlab. DNA barcoding is a method that uses short DNA sequence of mitochondrial genome for identification of species. There is no comprehensive work examining the appropriateness of different mitochondrial genes. This aim investigates the potential of other mitochondrial genes and evaluate their effectiveness for DNA barcoding and calculation of intra-and interspecific variability.

The phylogenetic position of Xenoturbella bocki has been a matter of controversy since its description in 1949. We sequenced a second complete mitochondrial genome of this species and performed phylogenetic analyses based on the amino acid sequences of all 13 mitochondrial protein-coding genes and on its gene order. Our results confirm the deuterostome relationship of Xenoturbella. However, in contrast to a recently published study (Bourlat et al. in Nature 444:85–88, 2006), our data analysis suggests a more basal branching of Xenoturbella within the deuterostomes, rather than a sister-group relationship to the Ambulacraria (Hemichordata and Echinodermata).