Urophycis Gill, 1863 é um gênero de peixes marinhos demersais de médio porte, popularmente conhecidos como abróteas, distribuídos ao longo da costa do Atlântico Ocidental, do Canadá à Argentina. Atualmente são reconhecidas sete espécies válidas dentro deste gênero. Apesar de sua importância comercial, até então poucos estudos taxonômicos e biogeográficos haviam sido realizados com estas espécies. Três espécies foram descritas do Atlântico Sul ocidental: U. brasiliensis (Kaup, 1858), descrita de Motevideo, Uruguai, U. latus Miranda Ribeiro, 1903 e Urophycis mystacea Miranda Ribeiro, 1903, ambas descritas do Rio de Janeiro, Brasil. Urophycis mystacea é morfologicamente semelhante à U. cirrata (Goode & Bean, 1896), descrita do Golfo do México nos Estados Unidos, sendo frequentemente confundidas. Os objetivos desta contribuição foram (1) redescrever U. brasiliensis, e alocar U. latus como um sinônimo júnior ; (2) redescrever U. cirrata e alocar U. mystacea como um sinônimo júnior; (3) estabelecer diagnoses e mapas de distribuição atualizados para ambas espécies. Tecidos e exemplares foram obtidos com pescadores e em coleções científicas. Foram utilizados métodos tradicionais de morfologia para comparação dos exemplares, além de técnicas de identificação molecular utilizando o gene mitocondrial citocromo oxidase I (COI). Testes para a amplificação da região controle do DNA mitocondrial de U. brasiliensis foram realizados e discutidos. / Urophycis Gill, 1863 is a genus of marine, demersal, mid-sized fish, popularly known as codlings or hakes, and distributed along the the Western Atlantic coast, from Canada to Argentina. Seven species are currently recognized as valid within this genus. Despite its economical importance, a few taxomomic an biogeographic studies had been carried out with those species. Three species were described from western South Atlantic: U. brasiliensis (Kaup, 1858), described from Montevideo, Uruguay, U. latus Miranda Ribeiro, 1903 and Urophycis mystacea Miranda Ribeiro, 1903, both described from Rio de Janeiro, Brazil. Urophycis mystacea is morfologically similar to U. cirrata (Goode & Bean, 1896), described from Gulf of Mexico, USA, and these species are repeteadly confused. The objectives of this contributions were (1) redescribe U. brasilienis, placing U. latus as a junior synonym; (2) redescribe U. cirrata, placing U. mystacea as a junior synonym; (3) provide updated diagnosis and maps of distributions for both species. Tissues and specimens were obtained from fisherman and scientific collections. Traditional morphological methods were used to compare the specimens, besides molecular identification techniques using the mithocondrial gene citochrome c oxidadse I (COI). Testst for amplification of the DNA mitochondrial control region in U. brasiliensis were performed and discussed.

Submitted by Sandra Pereira (srpereira@ufrrj.br) on 2017-01-11T12:09:07Z No. of bitstreams: 1 2016 - Joao Bosco Varela.pdf: 1890246 bytes, checksum: 359c15e2f52f2ea873d394cbbfa5479c (MD5) / Made available in DSpace on 2017-01-11T12:09:07Z (GMT). No. of bitstreams: 1 2016 - Joao Bosco Varela.pdf: 1890246 bytes, checksum: 359c15e2f52f2ea873d394cbbfa5479c (MD5) Previous issue date: 2016-02-24 / The study of ticks and tick-borne disease is increasingly dependent upon the use of molecular biological techniques that are employed in pathogen detection and for the accurate identification of ticks, particularly immature stages. The successful application of molecular methods, principally the polymerase chain reaction (PCR), can only be achieved if the DNA present in the tick was effectively preserved and could be extracted efficiently. The current study compared three fixatives (RNAlater, zinc salts (ZN) and isopropanol) for the ability to preserve the nuclear and mitochondrial (mt) DNA of larvae and nymphs of Amblyomma parvum and larvae of Amblyomma scultptum. DNA was extracted from ticks at times between 72h to 12 months post fixation using a phenol-chloroform procedure and examined using PCR assays for nuclear (internal transcribed spacer 2; ITS2) and mitochondrial (12S rDNA, subunit 1 of cytochrome c oxidase (COI) and D-loop) sequences. The efficiency of amplification was analyzed quantitatively (number of samples which produced amplicon) and qualitatively (relative intensity of bands observed on agarose gels). It was observed that the ITS2 sequence could be amplified in the majority (93,39%, n= 283/303) of the samples, in each of the three fixatives, although qualitative differences were observed, particularly with A. sculptum preserved in ZN. In contrast, fixation in isopropanol effectively abolished the ability to amplify the mitochondrial marker sequences of A. parvum and also resulted in inferior amplification (qualitative), of the D-loop target with A. sculptum. Those effects were observed in samples fixed for as little as 72h. The detrimental effects of isopropanol were also observed in samples extracted using an alkaline lysis method (Hotshot). Samples of A. parvum larvae preserved in RNAlater for 30 months showed an amplification efficacy of 100% in the ITS2 and COI assays, irrespective of the extraction method. Mitochondrial sequences are a central component of the majority of molecular studies of ticks. The findings of this study indicate that isopropanol should be avoided as a fixative for immature stages of ticks. Instead, the use of RNAlater is recommended in order to permit the consistent recovery of amplifiable mtDNA / O estudo de carrapatos e doen?as transmitidas por eles ? cada vez mais dependente da utiliza??o de t?cnicas de biologia molecular que s?o empregadas na detec??o de pat?genos, e a acurada identifica??o desses artr?podes, em particular os est?gios imaturos. A aplica??o bem-sucedida dos m?todos moleculares, principalmente, a rea??o em cadeia da polimerase (PCR), s? pode ser alcan?ada se o DNA presente no carrapato foi eficazmente preservado e extra?do de forma eficiente. O estudo comparou tr?s fixadores (RNAlater, sais de zinco (ZN) e isopropanol) avaliando a sua capacidade de preservar DNA mitocondrial (mtDNA) e nuclear de larvas e ninfas de Amblyomma parvum e larvas de Amblyomma scultptum. O DNA foi extra?do dos carrapatos, em tempos entre 72h e 12 meses ap?s a fixa??o por meio da t?cnica de fenol-clorof?rmio e lise alcalina (?Hot Shot?) e examinadas usando ensaios de PCR para sequ?ncias nucleares (espa?ador interno transcrito 2; ITS2) e mitocondriais (12S rDNA, subunidade 1 do citocromo c oxidase (COI) e D-loop). A efici?ncia de amplifica??o foi analisada quantitativamente (n?mero de amostras que produziram ?amplicon?) e qualitativamente (intensidade relativa das bandas observadas em g?is de agarose). Foi observado que a sequ?ncia ITS2 foi amplificada na maioria (93,39%, n= 283/303) das amostras em cada um dos tr?s fixadores, embora tenham sido observadas diferen?as qualitativas, particularmente com A. sculptum preservado em ZN. Em contraste, a fixa??o em isopropanol afetou negativamente a capacidade de amplificar as sequ?ncias dos marcadores mitocondriais de A. parvum e tamb?m resultou na amplifica??o inferior (qualitativa), do alvo D-loop com A. sculptum. Esses efeitos foram observados em amostras fixadas por apenas 72 horas. Os efeitos prejudiciais de isopropanol tamb?m foram observados igualmente em amostras extra?das usando um m?todo de lise alcalina (?HotShot?). As amostras de larvas de A. parvum preservadas em RNAlater durante 30 meses, mostrou uma efic?cia de amplifica??o de 100% nos ensaios para ITS2 e COI, independentemente do m?todo de extra??o usado. Sequ?ncias mitocondriais s?o um componente central da maioria das pesquisas moleculares com carrapatos. Os resultados deste estudo indicam que isopropanol deve ser evitado como um fixador para fases imaturas de carrapatos. Em vez disso, o uso de RNAlater ? recomendado, a fim de permitir a recupera??o consistente de mtDNA amplific?ve

As mitocôndrias desempenham um papel fundamental na sobrevivência e morte celular. Alterações do DNA mitocondrial (DNAmt) - como, por exemplo, amplificação, mutação homoplásmica, deleção e depleção -, bem como suas implicações clínico-patológicas, tem sido analisadas em inúmeras neoplasias humanas. No intuito de se pesquisar alterações mitocondriais associadas à tumorigênese, o presente trabalho teve como objetivos analisar a expressão de genes implicados no metabolismo energético e envolvidos na replicação e transcrição mitocondriais, quantificar o número de organelas mitocondriais e de cópias de DNAmt e analisar a expressão dos genes em astrocitomas de diferentes graus de malignidade (23 OMS grau I, 26 grau II, 18 grau III e 84 grau IV ou GBM) em relação ao tecido cerebral não tumoral (22 amostras). As expressões relativas dos genes selecionados, bem como as quantificações relativa e absoluta do DNA mitocondrial, foram realizadas por PCR em tempo real. O aumento de expressão relativa de genes-chave da via glicolítica, alterações nos níveis de expressão dos genes do ciclo dos ácidos tricarboxílicos e hipoexpressão de genes da fosforilação oxidativa detectados corroboraram o efeito Warburg. Foi demonstrado que a redução do número de cópias do DNAmt está associada com o grau de malignidade dos astrocitomas difusamente infiltrativos, sendo GBM o mais depletado e independente do número de organelas. As médias observadas para tecido não tumoral, astrocitoma grau I, grau II, grau III e GBM foram, respectivamente, 1,28, 0,26, 0,45, 0,42 e 0,17. Níveis aumentados de expressão relativa dos genes dos fatores de transcrição mitocondriais A (TFAM), B1 (TFB1M), B2 (TFB2M) e da subunidade catalítica da polimerase mitocondrial (POLG) foram detectados em todos os graus de astrocitomas, exceto TFB2M em astrocitoma grau II. Embora exista forte correlação entre os fatores de transcrição mitocondriais, somente os níveis de expressão de POLG se correlacionaram inversamente com o número de cópias de DNAmt. A expressão elevada de TFAM está associada a uma maior sobrevida no grupo de pacientes com GBM, interpretada como compensatório. As hiperexpressões de TFAM e POLG estão relacionadas a um melhor prognóstico em pacientes com GBM. Embora nossos achados da disfunção do metabolismo intermediário e depleção do DNAmt em astrocitomas corroborem a literatura, ainda não está bem esclarecida sua implicação na iniciação e manutenção da transformação maligna. Investigações futuras são necessárias para o esclarecimento destas questões. / Mitochondria has a key role in cell survival and death. Mitochondrial DNA (mtDNA) alterations, for example, amplification, homoplasmic mutation, deletion and depletion, and their clinical and pathological implications have been analyzed in human malignancies. In order to search for mitochondrial alterations associated to tumorigenesis, this study aimed to analyze the expression levels of genes involved in energetic metabolism, and in mitochondrial replication and transcription, to quantify the number of mitochondrial organelle and mtDNA copy number in astrocytomas of different grades of malignancy (23 WHO grade I, 26 grade II, 18 grade III and 84 grade IV or GBM) related to non-neoplastic brain tissue (22 samples). The relative expression level of the selected genes as well as the relative and absolute quantification of mtDNA were performed by real-time PCR. Relative expression increase of glycolytic pathway key genes, change of citric acid cycle genes and hipoexpression of oxidative phosphorylation genes were detected, and confirmed the presence of Warburg effect. The reduced mtDNA copy number was associated to the grade of malignancy of diffusely infiltrating astrocytoma, being GBM the most depleted, and not related to parallel decrease in the number of organelle. The mean mtDNA copy number for non neoplastic tissue, astrocytoma grade I, grade II, grade III and GBM were respectively 1.28, 0.26, 0.45, 0.42 and 0.17. The increased relative gene expression of mitochondrial transcription factor A (TFAM), B1 (TFB1M), B2 (TFB2M) and the catalytic subunit of mitochondrial polymerase (POLG) were observed in all grades of astrocytoma, except TFB2M in grade II astrocytoma. Although a strong correlation was observed among the mitochondrial transcription factors, only the expression level of POLG correlated inversely to the mtDNA copy number. The overexpression of TFAM was associated with long-term survival in the GBM patients and interpreted as compensatory. TFAM and POLG overexpressions were related to better prognosis in GBM patients. Although our findings concerning the impairment of intermediary metabolism and depletion of mtDNA in astrocytomas confirmed previous reports, their role in initiation or maintenance of malignant transformation were not fully understood. Further investigations are needed to clarify these issues.

The incidence of Squamous Cell Carcinoma is growing in certain populations to the extent that it is now the most common skin lesion in young men and women in high ultraviolet exposure regions such as Queensland. In terms of incidence up to 45% of the Australian population over 40 years of age is thought to possess the precancerous Solar Keratosis lesion and with a small but significant chance of progression into SCC, understanding the genetic events that play a role in this process is essential. The major aims of this study were to analyse whole blood derived samples for DNA aberrations in genes associated with tumour development and cellular maintenance, with the ultimate aim of identifying genes associated with non-melanoma skin cancer development. This study had an explicit emphasis on the mitochondrial genome and nuclear genes that encode for subunits in the mitochondrial regulated energy transducing oxidative phosphorylation pathways. More specifically the first aim of this project was to analyse the NDUFA8, PTCH, NDUFAS, SMOH, SDHD, MMPI2, NDUFV1, EMSI, COXVIIc, and RASAI genes via non-specific fluorophoric Real-Time PCR for genetic aberrations in an affected Solar Keratosis and control cohort. The second aim was to analyse two specific genes, SDHD and MMPI2, for copy number aberrations via Dual-Labelled Probe Real-Time PCR in the same affected Solar Keratosis and control cohort. The third aim was to analyse Mitochondrial DNA Depletion syndrome (MDS) in a chemically exposed RAAF personnel cohort via Dual-Labelled Probe Real-Time PCR. The significance of these studies is in their contribution to the knowledge of the genetic pathways that are malformed in the progression and development of the pre-cancerous skin lesion Solar Keratosis. Furthermore, it would determine whether the genes analysed in this study exist in greater prevalence in the affected Solar Keratosis population compared to the control cohort. With regard to the MDS component, identifying the presence of this disease in these individuals was initially undertaken as part of a study to provide evidence in compensation claims. The diagnosis may assist in their medical therapy, insofar as some of them were now suffering from liver malfunctions and atypical male breast cancer. Another application of this effective and low cost method of diagnosing MDS is in populations with high HTV incidences. This is due to the fact that the most common drug used to treat this disease can give rise to the expression of MDS, thus further complicating the health status of HIV infected individuals. The analysis of this research was accomplished via the Real-Time PCR technique, with a non-specific fluorophore component in addition to specific Dual-Labelled Probe components, to ascertain the general nature of any aberration identified in the sample cohort. This project also employed additional methods of analysis such as DHPLC and DNA sequencing to assist in determining the veracity of its aims, particularly in terms of the precise detection of genetic aberrations via Real-Time PCR. Patients exhibiting male breast cancer and liver malftinctions were also analysed via Dual-Labelled Probe RealTime PCR to ascertain the presence of Mitochondrial DNA Depletion syndrome, a disorder characterised by lactic acidosis, liver failure, seizures, and congestive heart failure. Determining the presence of this syndrome in these patients would assist in their medical treatment, and contribute to the analytical methods available to diagnose this syndrome, which is known to occur in HIV sufferers due to the nucleoside drugs used to combat the disease. Real-Time PCR can adequately gauge the integrity of a genetic area in terms of amplicon malformities (non-specific-fluorophoric) and DNA copy number aberrations (Dual-Labelled Probe) via fluorophore signal differentials compared to wild-type samples and housekeeper profiles. The results of the first component of this project, namely the analysis of five gene pairs by non-specific fluorophoric Real-Time PCR, highlighted that a significantly higher incidence of putative aberrants is evident in the affected population when compared to the control cohort. The genes analysed were NDUFA8, PTCH, NDUFA5, SMOH, SDHD, MMP 12, NDUFVI, EMS 1, COXVIIc, and RASA 1. These ten genes were subdivided into five pairs; one of the pair being a gene associated with the development of a non-melanotic skin cancer (NMSC), the other a gene encoding for a subunit of the Electron Transport Chain (ETC). Each of these pairs exists in close proximity to one another on a particular chromosomal locale. Differences were highlighted in the single gene triplicate run population. The ETC genes (NDUFA8, NDUFA5, SDHD, NIDUFVI, COXVIIc) exhibited 10 / 720 (1.37%) as being putative mutants in the control population, compared to 117 / 675 (17.3%) for the affected population (p value less than 0.0001). The NMSC gene analysis (PTCH, SMOH, MMPI2, EMSI, RASA1) produced a 16 / 720 (2.22%) ratio for the control population, with the affected population having an incidence of 97 / 675 (14.4 %) for putative mutants (p value less than 0.0001). The observance of putative aberrants in the NDUFVI (p less than 0.018), EMS1 (p less than 0.003), COXVTIc (p less than 0.001), and RASA I (p less than 0.009) genes in the affected Solar Keratosis (SK) population was significantly higher than that observed in the control population. The majority of aberrations detected via the non-specific fluorophoric Real-Time PCR technique were small nucleotide base insertions and deletions. The analysis of the SK affected and control cohort via Real-Time PCR proved a cost-effective and reliable method in identifying the presence of DNA aberrations such as non-instructional sites. The results of the second component extended the findings of the non-specific fluorophoric analysis. The SDHD and MMPI 2 genes were analysed for copy number aberrations via Dual-Labelled Probe Real-Time PCR for genetic aberrations the same affected and control Solar Keratosis cohort. It was found that 12 of 279 samples had identifiable copy-number aberrations in either the SDHD or MMPI2 gene (this means that a genetic section of either of these two genes is aberrantly amplified or deleted), with five of the samples exhibiting aberrations in both genes. The MMPI2 gene also had nine samples identified as possessing an intronic heterozygous base-pair substitution anomaly via DNA sequencing. The NDUFA8 gene had 12 samples identified as anomalous via the DHPLC technique, 11 of which were identified via non-specific fluorophoric Real-Time PCR, with the analysis performed to verify the accuracy of the Real-Time technique in identifying DNA aberrations. This study identified DNA aberrations in an affected Solar Keratosis and control cohort and ascertained several particular genomic abnomialities in the SDHD, MMPI2 and NDUFA8 genes, with an emphasis on copy-number aberrations and amplicon abnormalities. In the third component of this study, namely the analysis of Mitochondrial DNA Depletion syndrome (MDS) in a jet-fuel exposed RAAF personnel cohort via Dual-Labelled Probe Real-Time PCR, the results indicated that four of the seven patients were expressing MDS. Of the four patients who exhibited a reduction in mitochondrial copy-number the average decrease was of a four-fold level, or approximately a depletion of mitochondrial copies from 200 plus to ~ 54 (74 % reduction in MtDNA). The patients who contributed DNA for investigation into the presence of MDS were suffering from liver malfunction and atypical male breast cancer. The Dual-Labelled Probe technique proved a reliable and cost effective method in identifying the presence of MDS in these patients, with the DNA extracted from fresh white blood cells that had been isolated using the Ficoll-Hypaque method. The importance of this is that accurate levels of Mitochondrial DNA copy numbers can be ascertained in white blood cells as it removes the presence of platelets, which also contain mitochondria but no nucleus. The analysis of ETC and NMSC associated genes in addition to mitochondrial copy number integrity means that this study investigated two aspects of the carcinogenetic pathway i.e. abnormal energy regulation and the regulation of micromolecular and macromolecular cellular homeostatic mechanisms. The mechanism of programmed cell death or apoptosis is regulated by the mitochondria and the ability of a genetically damaged cell to evade the apoptotic process is directly linked to a cell becoming cancerous. It is only after the evasion of apoptosis and the replication of the damaged cells' DNA into daughter cells that neoplastic events can occur. Thus, this study contributed to the understanding of how neo-plastic lesions may develop and progress into invasive tumours. It additionally assisted in proving the effectiveness of the RealTime PCR technique in detecting DNA aberrations and mitochondrial copy number anomalies.

Les anàlisis realitzades en cent deu poblacions de truita comuna (Salmo trutta) que abarquen el seu rang natural de distribució indiquen que el patró filogenètic es relaciona amb les tres grans vessants on es troba distribuïda l'espècie: ponto-càspia, atlàntica i mediterrània. Aquesta diferenciació estaria associada a l'aïllament de les vessants durant el Quaternari. L'origen de l'espècie es relaciona amb la vessant ponto-càspia, d'acord amb els models biogeogràfics que postulen l'origen asiàtic de la ictiofauna europea. S'ha detectat també un segon nivell de divergència dins de cada vessant que dóna com a resultat l'existència de sis llinatges evolutius: Atlàntic i Duero a la vessant atlàntica, els llinatges Adriàtic, Mediterrani i Marmoratus als rius mediterranis, i el llinatge Danubi a la zona ponto-càspia.Les glaciacions del Pleistocè han modificat profundament el rang de distribució de la truita comuna, especialment a la vessant atlàntica, on s'han proposat quatre grans refugis glacials: a l'est de la capa de gel, a Europa central, a l'entorn del canal de la Mànega i a l'entorn del golf de Biscaia; tot i que només els tres primers haurien participat en la recolonització del nord d'Europa al final de l'última glaciació. El quart refugi, que inclou el sud de França i el Cantàbric hauria estat l'origen de l'expansió cap al sud durant el Pleistocè Superior d'un grup de poblacions distribuïdes actualment a la vessant atlàntica ibèrica, i també hauria servit de base per a l'expansió cap al nord d'altres grups de truita durant interglacials anteriors.A la vessant atlàntica de la peninsula Ibèrica, l'estructura poblacional es troba associada a la xarxa hidrogràfica i es determinen fins a cinc unitats poblacionals: les truites dels rius Cantàbrics, les del Miño, les del Duero, les del Tajo i les del Guadalquivir. Les poblacions del Guadalquivir pertanyerien a un grup d'influència mediterrània. Els marcadors d'al·lozims i de DNA mitocondrial es troben fortament correlacionats en aquesta vessant, on apunten cap als mateixos grups de poblacions. Per contra, els rius de la vessant mediterrània haurien estat colonitzats pels llinatges Adriàtic i Mediterrani i s'hauria produït una intensa intergradació secundària entre aquests llinatges durant els períodes glacials a partir de l'expansió de les poblacions retingudes a les capçaleres durant els interglacials. Els grups de hibridació, l'aïllament i la deriva en el període interglacial fa que els grups de poblacions identificats pels marcadors d'al·lozims i de DNA mitocondrial no coincideixin. / The analyses performed in one hundred and ten brown trout (Salmo trutta) populations that cover its native European distribution, shows that the phylogenetic pattern is associated with the three major basins occupied by the species: Ponto-Caspian, Atlantic and Mediterranean. This differentiation is related with basin isolations during the Quaternary. The origin of the species is placed in the Ponto-Caspian region, in clear agreement with biogeographic models that postulates the Asian origin of European ichthyofauna. Further divergence occurred in the mid-lower Pleistocene generated the actual lineages: Atlantic and Duero in the Atlantic basin, Adriatic, Mediterranean and Marmoratus lineages in the Mediterranean rivers and the Danubian lineage in the Ponto-Caspian area.Pleistocene glacial periods have deeply modified the distribution range of brown trout, mainly in the Atlantic basin, where four major glacial refugia have been proposed: (i), at the East side of the ice sheet, (ii), in Central Europe, (iii), around the English Channel and (iv), around the Bay of Biscay. Only the three first seem to be involved in the recolonization of North Europe at the end of the last glacial period. The fourth glacial refugia, placed in Southern France and the Cantrabrian Sea area, would have been the origin of a southern expansion during Late Pleistocene. Nowadays, those trout populations are distributed in the Atlantic basin of the Iberian Peninsula. This glacial refugia could also be involved in older recolonizations of North-Europe previous to last glacial process.Brown trout population structure in areas at the Atlantic basin of the Iberian Peninsula is associated with river network. Thus, five trout population groups could be determined: trout from the Cantabrian Rivers, from the Miño River, from the Duero River, from the Tajo River and trout populations from the Guadalquivir basin. These last populations show a strong Mediterranean influence. In this basin, comparisons involving allozyme and mitochondrial DNA data are strongly correlated and points towards the same population groups. The rivers in the Mediterranean basin have been colonized by Adriatic and Mediterranean lineages, and strong secondary intergradations are reported among them during glacial periods. Population groups identified by allozyme and mitochondrial DNA do not agree as a consequence of this intergradation, as well as isolation and genetic drift during interglacial periods.

El trabajo realizado trata de estudiar la diversidad del genoma mitocondrial humano en poblaciones humanas de diferentes áreas geográficas que habían sido hasta ahora poco o nada estudiadas. Los grupos de poblaciones humanos estudiados en este trabajo esta formado por las poblaciones del oeste del Mediterráneo, de l'África sub-Sahariana, de la Isla de La Reunión, y de l'Asia. Cada una de estas poblaciones pertenecen a un entorno geográfico diferente y han padecido diferentes y numerosos movimientos de poblaciones que han modulado su composición genética. El análisis de diferentes polimorfismos del genoma mitocondrial han permitido entender los factores poblacionales, tal como la migración, la mezcla genética, la deriva genética, los efectos fundadores, y inferir la historia d la poblaciones bajo estudio, La metodología utilizada incluye diferentes tipos de técnicas adaptadas a los diferentes tipos de polimorfismos estudiados. La técnica aplicadas fueron la secuenciación, el análisis de fragmentos y la técnica de SNaPshot. Los resultados obtenidos han aportado un conocimiento nuevo de las poblaciones que han modulado la diversidad genética de los grandes grupos humanos a nivel continental pero también a un nivel mas regional.

La seqüenciació del genoma humà ha marcat una fita important en la història de la biologia. Com a conseqüència, la genètica i la genòmica han experimentat un progrés enorme. Això ha permès un millor coneixement tant de les causes genètiques de malalties humanes, com del per què de les diferències comunes entre individus. Com a sistemes complexos que som tots els éssers vius, hem de considerar el paper que tenen les interaccions entre les diferents parts del genoma a l'hora d'especificar el resultat final, és a dir, el fenotip. Igualment, podem dir que el genoma conté un conjunt d'instruccions, però que la forma en què aquestes es porten a terme depèn, també de contingències ambientals i històriques. Per tant, la naturalesa de les instruccions genètiques no és completament determinista en tots els casos, si bé hi ha una sèrie de processos en què sí que es compleix aquesta perfecta relació entre herència i expressió final. Aquesta mateixa situació es presenta amb certes alteracions genètiques i amb el desenvolupament de patologies, la qual cosa facilita enormement el diagnòstic precoç i obre les possibilitats per a la teràpia genètica. Però la gran majoria de fenotips, incloent-hi moltes condicions d'interès per a la medicina, tenen una base complexa, és a dir, no existeix "el gen" que determina el caràcter de forma unívoca, sinó que aquest és el resultat de l'acció simultània de molts gens, no tots amb la mateixa participació, juntament amb l'efecte de l'ambient. Aquesta tesi doctoral va arrencar en aquest punt, tenint com a objectiu l'aprofundiment en les bases genètiques d'un tipus de sordesa lligada a mutacions al vi Preface DNA mitocondrial i de la qual se'n tenien evidències de la implicació tant de factors ambientals com diversos factors genètics. D'altra banda, els tests basats en l'ADN són un dels primers usos comercials i d'aplicació mèdica d'aquests nous descobriments de la genètica. Aquests tests poden ser utilitzats per al diagnòstic de malalties, confirmació diagnòstica, informació del pronòstic, així com del curs de la malaltia, confirmar la presència de malaltia en pacients assimptomàtics i amb diferents graus de certesa, predir el risc de futures malalties en persones sanes i en la seva descendència. Aquest és l'objectiu final, i sovint encara utòpic, de la recerca en biomedicina: una millor comprensió del procés biològic, que derivi en un millor tractament i prevenció de la malaltia. Aquesta tesi també ha volgut contribuir humilment en aquest aspecte. Durant aquests anys s'han recollit centenars de mostres de famílies sordes, amb la finalitat de donar un "diagnòstic" de la causa genètica. Poques vegades ho hem conseguit, però en qualsevol cas, si això alguna vegada ha ajudat a algú d'alguna manera, ja em dono per satisfeta.

The lizard Podarcis carbonelli is an endangered species endemic to the Iberian Peninsula. One location where this species occurs is at the Berlengas Natural Preserve, an Atlantic archipelago off the coast of Portugal. These island populations are geographically separated from nearby mainland populations. The fundamental question is, are these insular individuals distinct from the mainland populations? Four localities were chose for comparison: two island populations and two nearby coastal populations. We assessed this question using three distinct approaches: molecular, morphological and physiological approach. We sequenced the 12S RNA, the mtDNA Control Region and the 7th intron of the !-fibrinogen gene and determined genetic diversity values as well as several parameters of population structure and differentiation. Individuals from these populations were also measured for several biometric characters and their blood lactate concentration was sampled. There was no genetic variation in both the mtDNA regions analyzed. The nuclear intron revealed high levels of genetic variation, with islands having in general lower values than the mainland regions. The four populations sampled had low levels of divergence; the populations of Berlenga and Peniche were the most distinct and the populations of Farilhão and Baleal were the most similar from the four populations sampled. Morphometric analyses revealed a different pattern of similarity among populations with the population of Farilhão being the only population statistically distinct from all other populations based on mass and SVL. Furthermore, island populations were in general more similar to each other than to mainland populations, with the exception of Berlenga males which in size are more similar to the Peniche males. The analysis of the blood lactate concentration revealed that the population of Peniche has significantly lower blood lactate levels than the populations of Farilhão and Berlenga. The lack of genetic differentiation found in the populations under study is most likely due to the recent divergence of these populations. Furthermore, the genetically most different populations (Berlenga and Peniche) are not the most distinct in terms of morphology, particularly the males. This suggests that genetic drift, the most likely mechanism behind the genetic differentiation seen, is not responsible for the morphological differences observed. The morphological differences seen can be attributed to: a possible difference in age of the individuals in each population; mechanisms of natural selection that are favoring specific phenotypes in each of the populations, or phenotypic plasticity. The differences in blood lactate levels found between the population of Peniche and the island populations can be attributed to differences in predatory pressure or home range size. It is suggested that the island populations are closely monitored due to their likely isolation, low mtDNA diversity and possible higher predatory pressure than initially predicted.

In the present study, haplogroup composition of five native Turkish sheep breeds, (Karayaka, Akkaraman, G&ouml / k&ccedil / eada, Dagli&ccedil / , Morkaraman) and two sheep breeds from neighboring countries (Herik from Iran, samples from Azerbaijan) were determined by single strand length polymorphism (SSCP) analysis of mitochondrial DNA (mtDNA) NADH dehydrogenase subunit 4 (ND4) region and restriction fragment length polymorphism (RFLP) analysis of mtDNA control (CR) region. Results of the SSCP and RFLP approaches were found to be 96,82% consistent. Most of the 3,18% inconsistency was due to unidentified band patterns of 9 individuals. SSCP method could identify haplogroups A, B and C, but not D and E. Similarly RFLP method could identify haplogroup A, B and possibly D, but not E and C. Data of the present study were compared with those of the previous studies to test the robustness of results under different samplings and were found to be homogenous with a previous study with similar sampling strategy. Neighbor joining tree, principal component analysis (PCA), Delaunay network analysis and analysis of molecular variance (AMOVA) were employed to analyze the haplogroup frequencies and breeds were separated in four groups according to the genetic barriers between breeds from different geographical locations. Strongest differentiation was present between two groups which were eastern breeds (Morkaraman, Herik-Iran and Azerbaijan) and western breeds (G&ouml / k&ccedil / eada, Akkaraman, Karayaka and Dagli&ccedil / ). Additionally, Azerbaijan was proposed as the entrance point of the haplogroup A and the Iran was proposed as the entrance point of haplogroup C to Anatolia with the Spearman rank correlation test.

Human mitochondrial DNA (mtDNA) is located in discrete DNA-protein complexes, so called nucleoids. These structures can be easily visualized in living cells by utilizing the fluorescent stain PicoGreen®. In contrary, cells devoid of endogenous mitochondrial genomes (ρ0 cells) display no mitochondrial staining in the cytoplasm. A modified restriction enzyme can be targeted to mitochondria to cleave the mtDNA molecules in more than two fragments, thereby activating endogenous nucleases. By applying this novel enzymatic approach to generate mtDNA-depleted cells the destruction of mitochondrial nucleoids in cultured cells could be detected in a time course. It is clear from these experiments that mtDNA-depleted cells can be seen as early as 48 h post-transfection using the depletion system. To prove that mtDNA is degraded during this process, mtDNA of transfected cells was quantified by real-time PCR. A significant decline could be observed 24 h post-transfection. Combination of both results showed that mtDNA of transfected cells is completely degraded and, therefore, ρ0 cells were generated within 48 h. Thus, the application of a mitochondrially-targeted restriction endonuclease proves to be a first and fast, but essential step towards a therapy for mtDNA disorders.