Submitted by LUÍS LENIN VICENTE PEREIRA null (luislenin@gmail.com) on 2017-03-02T15:07:34Z No. of bitstreams: 1 Tese Luis Lenin Vicente Pereira.pdf: 2894913 bytes, checksum: 01d77fed0e0eb6b5cfb7979e52829667 (MD5) / Approved for entry into archive by LUIZA DE MENEZES ROMANETTO (luizamenezes@reitoria.unesp.br) on 2017-03-08T13:17:27Z (GMT) No. of bitstreams: 1 pereira_llv_dr_sjrp.pdf: 2894913 bytes, checksum: 01d77fed0e0eb6b5cfb7979e52829667 (MD5) / Made available in DSpace on 2017-03-08T13:17:27Z (GMT). No. of bitstreams: 1 pereira_llv_dr_sjrp.pdf: 2894913 bytes, checksum: 01d77fed0e0eb6b5cfb7979e52829667 (MD5) Previous issue date: 2017-02-17 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A subordem Heteroptera possui sete infraordens com aproximadamente 80 famílias. A maioria ocorre em todos os continentes (exceto Antártica) e algumas ilhas. Além dos Heteroptera terrestres, há os aquáticos e semi-aquáticos que são amplamente distribuídos, e surpreendem por sua capacidade de habitar uma extraordinária variedade de ecossistemas, sendo encontrados em habitats de água doce e marinho, e variada faixa de altitude entre 0 e 4.700 m. Estudos sobre aspectos ultraestruturais da espermatogênese e, especificamente, a estrutura do espermatozoide em Heteroptera ainda são escassos, por este motivo o objetivo do presente estudo foi o de analisá-los, por meio de cortes semifinos corados com azul de toluidina ou impregnados por íons prata, e cortes ultra-finos analisados em microscopia eletrônica de transmissão, utilizando testículos de machos adultos das famílias Belostomatidae, Gelastocoridae, Gerridae, Mesoveliidae, Notonectidae e Veliidae. Após a análise ultraestrutural da espermatogênese foi possível determinar que o padrão flagelar do axonema é de 9+9+2 para todas as espécies analisadas sendo, portanto, o padrão para essa subordem, as mitocôndrias durante a espermatogênese assumem diferentes morfologias, sendo que inicialmente as mitocôndrias se unem formando o complexo mitocondrial e, posteriormente, se divide em dois derivados mitocondriais que estão posicionados bilateralmente em relação ao axonema. Os derivados mitocondriais apresentaram tamanhos diferentes para as espécies B. amnigenus (Notonectidae) e R. c. crassifemur (Gerridae) e para as demais espécies o tamanho foi semelhante. As células germinativas possuem em seu citoplasma o acúmulo de um material denominado corpo cromatóide estando localizado próximo ao núcleo. Com relação ao comportamento nucleolar da espécie Martarega brasiliensis foi observado de um a quatro corpúsculos nucleolares em células de Prófase I comprovando uma grande atividade sintética das células nessa fase da divisão celular. Células em Metáfase I apresentaram regiões organizadoras nucleolares na região telomérica de um dos autossomos. Ainda, nessa espécie, foi possível observar, em Anáfase I, vários corpúsculos nucleolares persistindo até a fase de Telófase I. Todas as ultraestruturas descritas nas espécies analisadas foram semelhantes às descritas na literatura para Heteroptera, corroborando as características sinapomórficas dessa subordem sendo elas: a) a presença de duas pontes que ligam o material intertubular do axonema flagelar às cisternas achatadas que aderem aos lados internos dos derivados mitocondriais; b) padrão flagelar do axonema de 9+9+2 e c) ausência de corpos acessórios. / The suborder Heteroptera has seven infraorders with approximately 80 families. Most occur in all continents, except Antarctica and some islands. In addition to terrestrial Heteroptera, there are also widely distributed aquatic and semi-aquatic species. This suborder have adapted to live in an extraordinary variety of ecosystems as freshwater and marine habitats and at altitudes ranging from 0 m to 4,700 m. The research concerning the ultrastructural aspects of spermatogenesis is a large and growing field of study, however, in the case of Heteroptera, research is still scarce. For this reason, the aim of this study was to analyze the ultrastructures and spermatogenesis through semi-thin sections stained with toluidine blue or silver ions (Ag-NOR) and ultrathin sections examined in transmission electron microscopy, using testes of adult males ofthe following families: Belostomatidae, Gelastocoridae, Gerridae, Mesoveliidae, Notonectidae and Veliidae. After ultrastructural analysis of spermatogenesis, it was possible to determine that the flagellar pattern of the axoneme is 9+9+2 for all species, being therefore, the pattern for this suborder. As spermatogenesis progresses, the mitochondria begins to cluster and concentrate on only one side of the cell. Then, the mitochondria combine to form a single mitochondrial complex, which subsequently divides into two mitochondrial derivatives. They are positioned on opposite sides of the axoneme. The mitochondrial derivatives presented different sizes for the species B. amnigenus (Notonectidae) and R. c. crassifemur (Gerridae) and for the other species the size was similar. The germ cells have in their cytoplasm the accumulation of a material denominated the chromatoid body, being located near the nucleus. Regarding the nucleolar behavior, M. brasiliensis showed nucleus in prophase I composed by the nucleolus and nucleolar corpuscles that varied from one to four, emphasizing that this insect has great synthetic activity during meiosis. The analysis of cells in metaphase I, showed that M. brasiliensis presents nucleolar organizing region (NOR) in at least one autosome. Furthermore, was not observed the phenomenon of nucleolar persistence. All the ultrastructures described in the analyzed species were similar to those described in the literature for Heteroptera, corroborating the synapomorphic characteristics of this suborder, being them: a) two opposite bridges in the axoneme connect the flattened cisterns adherent to the internal side of each mitochondrial derivative to the intertubular material; b) flagellar pattern of the axoneme of 9+9+2; c) accessory bodies are absent all along the flagellum.

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Mitochondria are essential organelles for most eukaryotic cells, containing intricate networks of numerous proteins. These include, among others, complexes I-IV of the electron transport chain. Being at the crossroads of the tricarboxylic acid cycle and the respiratory chain, mitochondrial complex II plays a key role in cellular metabolism. The protein complex, also known as succinate dehydrogenase, is capable of not only succinate oxidation and electron transfer but also contributes to the production of reactive oxygen species. Mitochondrial complex II consists of four subunits, SDHA-D, and four dedicated protein assembly factors SDHAF1-4 that participate in complex II biogenesis. Mutations and epigenetic modulations of genes coding for succinate dehydrogenase subunits or assembly factors are associated with pathological conditions such as neurodegenerative diseases, or may result in tumor formation. However, inborn complex-II-linked mitochondrial pathologies are rather understudied, compared to diseases with causative errors of other mitochondrial complexes, presumably due to the fact that none of complex II subunits is encoded in the mitochondrial genome. Recent studies have shown that impairment of mitochondrial complex II function or assembly leads to accumulation of alternative assembly forms...

Mitochondrial dysfunction has been implicated for a wide range of human diseases. One of the major biosynthetic processes in human mitochondria is the biogenesis of Iron-Sulfur (Fe-S) clusters which primarily involves in electron transfer reactions during oxidative phosphorylation (OXPHOS). Defects in Fe-S cluster biogenesis process leads to mitochondrial dysfunction and that eventually results in various human mitochondrial disorders. One of the major mitochondrial disorders associated with Fe-S cluster biogenesis impairment is exercise intolerance disorder ISCU myopathy, which is a result of loss of function of Fe-S cluster scaffold protein ISCU. Our biochemical results using yeast model system and HeLa cells lines suggests that ISCU Myopathy results in defective Fe-S cluster biogenesis in mitochondrial compartment. As a result, electron transport chain (ETC) complexes demonstrate significant reduction in their redox properties, leading to loss of cellular respiration. Furthermore, in ISCU Myopathy, mitochondria display enhancement in iron levels and reactive oxygen species, thereby causing oxidative stress leading to impairment in the mitochondrial functions. On the other hand, in mammalian mitochondria, the initial step of Fe-S cluster assembly process is assisted by NFS1-ISD11 complex, which delivers sulfur to the scaffold protein ISCU during Fe-S cluster synthesis. In humans, loss of ISD11 function leads to development of respiratory distress disorder, Combined Oxidative Phosphorylation Deficiency 19 (COXPD19). Our study maps the important ISD11 amino acid residues critical for in vivo Fe-S cluster biogenesis. Importantly, mutation of these critical ISD11 residues to alanine leads to its compromised interaction with NFS1, which results in reduced stability and enhanced aggregation of NFS1 in the mitochondria. Moreover, our findings highlight that, COXPD19 associated R68L ISD11 mutant displays reduced affinity to form a stable sub-complex with NFS1, thereby fails to prevent NFS1 aggregation, resulting impairment of Fe-S cluster biogenesis. The prime affected machinery is the ETC complex which demonstrates compromised redox properties, causing diminished mitochondrial respiration in COXPD19 patients. In summary, our findings provide compelling evidence that respiration defect due to impaired biogenesis of Fe-S clusters in ISCU myopathy patients, leads to manifestation of complex clinical symptoms. Additionally, our study highlights the role of ISD11 protein in Fe-S cluster biogenesis and maps the surface residues of ISD11 protein that are involved in interaction with sulfur donor protein NFS1. Moreover, we have demonstrated the molecular basis of disease progression of COXPD19 as a result of R68L ISD11 mutation.