The lactate level assesment in various body fluids plays an important role in the diagnostics of mitochondrial disorders in children. However, the interpretation of lactate level is often difficult due to its unspecificity and variability even in particular mitochondrial disorders. Three specific aims have been stated in this PhD Thesis: 1. To analyse the role of lactate examination in the differential diagnosis between children with mitochondrial disorders and children with other diseases. 2. To study the lactate level differences in various mitochondrial syndromes. 3. To characterise the clinical and laboratory data of neonates with mitochondrial disorders and to suggest new diagnostic algorhytms. Clinical and laboratory data from patients hospitalized in the Department of Pediatrics were collected. Laboratory methods were provided in the cooperation with the Mitochondrial laboratory of the Department of Pediatrics and Institute of Inherited Metabolic Disorders. The study with lactate levels in 107 patients documented that brief seizures lasting less than 2 minutes did not increase lactate concentration in the CSF. CSF-lactate was a relialable marker in differential diagnosis in the children with mitochondrial disorders against children with epilepsy. 2. The severity of particular phenotype is more...

Annotation: Powdery mildew Erysiphe alphitoides is one of the most important pathogenic fungus infecting pedunculate oak Quercus robur in Europe. As the identification of this species with morphologic markers is unreliable, the molecular markers development is necessary. In this work, we developed primers for PCR amplification of cytochrome b gene fragment. The results show high level of intraspecific variability of this mitochondrial gene and revealed the indistinct taxonomy relations within E. alphitoides s. lat group.

As abelhas da tribo Meliponini (abelhas sem ferrão) estão amplamente distribuídas pelas regiões tropicais do planeta, tendo um importante papel na polinização, sendo o gênero Melipona o que contém o maior número de espécies. A espécie Melipona marginata é uma das menores e mais ancestrais do grupo, e a exemplo de outras espécies nidifica em ocos de árvore. M. marginata, assim como outras espécies do gênero Melipona, vêm sofrendo com a destruição do seu ambiente natural, pelo desmatamento, sendo, aparentemente mais exigente que outras espécies quanto ao tamanho do fragmento florestal para se manter, devido a isso é encontrada apenas em fragmentos maiores, mais antigos e menos perturbados. Tendo em vista a perda de hábitat e o pouco conhecimento da biologia dessa espécie, este trabalho pretende analisar populações de M. marginata, através da técnica PCR+RFLP do DNA mitocondrial e marcadores microssatélites, visando contribuir para entendimento da estrutura populacional de M. marginata. Foram analisados 54 ninhos, provenientes dos estados de MG, SP, PR, SC e RS. Oito regiões do DNA mitocondrial foram amplificadas, e posteriormente digeridas com 12 enzimas de restrição. Foram detectados 14 haplótipos, sendo apenas um compartilhado. A população de SP apresentou o maior número de haplótipos. Os testes estatísticos demonstraram que as populações estão estruturadas e isoladas, não havendo fluxo gênico entre as populações. Já nas análises dos microssatélites foram analisados 4 locos, apresentando alta variabilidade genética, onde também foi verificado que as populações se encontram estruturadas. Os resultados obtidos podem ser explicados principalmente pela redução da área de floresta, mas, podem se dever a eventos antigos em conseqüência de mudanças climáticas ocorridas durante as últimas glaciações. / The tribe Meliponini (stingless bees) is present in all tropical regions of the world and has an important role in pollination. The genus Melipona has the highest number of species in the tribe. The specie Melipona marginata is considered the most ancestral within the genus, and like other species builds the nests in hollow of trees. Unfortunately several bee species have suffered with the devastation of their natural environment. M. marginata seems to be very dependent on the size of the forest fragment, being found only in the biggest, oldest and consequently more preserved ones. In view of the habitat loss and few biological knowledge about this specie, this research intended to analyse M. marginata populations molecularly, through mitochondrial DNA PCR-RFLP and microsatellite data., Fifty four colonies were analyzed, from MG, SP, PR, SC and RS states. Eight mitochondrial DNA regions were amplified and screened with 12 restriction enzymes. Fourteen haplotypes were verified and among them just one was shared. SP population showed the highest number of haplotypes. Statistic tests showed that the 5 populations were genetic structured and isolated, therefore not presenting gene flow. The 4 microsatellites loci analyzed showed high genetic variability. The statistics analysis indicated that the 5 populations are structure and isolated. These results can be explained mainly because the decrease of forest leading to population isolation, however we can not discard the hypothesis that this current scenario is a consequence of climatic changes occurred during the last glaciations.

Orientador: Edi Lucia Sartorato / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-13T04:42:57Z (GMT). No. of bitstreams: 1 DeMoraes_VanessaCristineSousa_M.pdf: 3761242 bytes, checksum: 82ef24aca3f38d5f5533f00365873b09 (MD5) Previous issue date: 2009 / Resumo: A perda auditiva é a mais comum das deficiências sensoriais da população em geral. A surdez congênita ocorre em cerca de 1 em cada 1000 nascidos vivos, dos quais aproximadamente 50% têm origem hereditária nos países desenvolvidos. A perda auditiva não-sindrômica pode ser causada por mutações em genes nucleares e genes mitocondriais (mtDNA). Mutações no mtDNA foram associadas ao uso de aminoglicosídicos e à surdez não-sindrômica em muitas famílias no mundo todo. No entanto, o background genético do indivíduo influencia a expressão fenotípica destas mutações patogênicas. Dessa forma, foi proposto que os genes nucleares modificadores modulam a manifestação fenotípica da mutação mitocondrial A1555G no gene MTRNR1. Ambos os genes nucleares modificadores TRMU e MTO1 codificam uma proteína mitocondrial altamente conservada, a qual acredita-se estar envolvida na modificação do tRNA. Estudos propuseram que o TRMU humano, assim como o gene nuclear MTO1 poderiam modular a manifestação fenotípica da surdez associada a mutações mitocondriais. O objetivo deste trabalho foi elucidar a contribuição de mutações mitocondriais, de mutações em genes nucleares modificadores e a exposição aos aminoglicosídeos no fenótipo da perda auditiva. Nossos achados sugerem que o background genético dos indivíduos pode desempenhar um papel importante na patogênese da surdez associada à mutação mitocondrial e ao uso de aminoglicosídeos. / Abstract: Hearing loss is the most prevalent sensorial deficit in the general population. Congenital deafness occurs in about 1 in 1000 live births, of which approximately 50% has hereditary cause in development countries. Non-syndromic deafness can be caused by mutations in both nuclear and mitochondrial genes. Mutations in mtDNA have been associated with aminoglycoside-induced and non- yndromic deafness in many families worldwide. However, the nuclear background influences the phenotypic expression of these pathogenic mutations. Indeed, it has been proposed that nuclear modifier genes modulate the phenotypic manifestation of the mitochondrial A1555G mutation in the MTRNR1 gene. The both putative nuclear modifiers genes TRMU and MTO1 encoding a highly conserved mitochondrial related to tRNA modification. It has been hypothesizes that human TRMU and also MTO1 nuclear genes may modulate the phenotypic manifestation of deafnessassociated mitochondrial mutations. The aim of this work was to elucidate the contribution of mitochondrial mutations, nuclear modifier genes mutations and aminoglycoside exposure in the deafness phenotype. Our findings suggest that the genetic background of individuals may play an important role in the pathogenesis of deafness-associated with mitochondrial mutation and aminoglycoside-induced. / Mestrado / Genetica Animal e Evolução / Mestre em Genética e Biologia Molecular

Uma das razões que fazem de Saccharomyces cerevisiae um organismo modelo é o grau de conservação dos mecanismos celulares que existe entre esta levedura e eucariotos superiores. Porém, mesmo após 21 anos do seqüenciamento do seu genoma, ainda existem mais de 600 ORFs com função desconhecida. Neste trabalho, selecionamos quatro delas para o estudo detalhado. MRPL34 (YDR115w) está presente na subunidade maior do ribossomo mitocondrial de levedura e apresenta similaridade com o gene L34 de E. coli e MRP-L34 de humanos. O mutante Δmrpl34 apresenta DNA mitocondrial (mtDNA) instável e para estudá-lo foram gerados alelos sensíveis à temperatura (ts). Com os ensaios de síntese protéica mitocondrial in vivo foi possível identificar clara diminuição da síntese de proteínas do mutante condicional. Mrpl34p foi identificada no extrato ribossomal, conforme esperado. A desestruturação da subunidade maior do ribossomo mitocondrial, utilizando os mutantes ts, nos forneceu indícios sobre intermediários existentes no seu processo de montagem. Verificamos que a porção N-terminal da proteína é responsável pelo endereçamento à mitocôndria. YPR116w também apresenta alta instabilidade do DNA mitocondrial, desta forma, mutantes termossensíveis foram utilizados nos experimentos. Uma das estratégias utilizadas visou a busca de parceiros genéticos. Verificamos que ylr091wp aumenta a estabilidade do mtDNA de ts- ypr116w, sugerindo atividade supressora. Também averiguamos que o alelo ts-ypr16w apresenta menor quantidade de tRNA mitocondrial, através de ensaios de Northen blot. Duas das ORFs escolhidas (YDL119c e YOR022c) tiveram sua caracterização inicial publicada em 2016, refletindo a importância deste tipo de pesquisa. Vimos que a proteína codificada por YDL119c está localizada na membrana interna da mitocôndria e que o mutante Δyor022c apresenta quantidades reduzidas de cardiolipina, quando crescido à 37ºC. / One of the reasons that turn Saccharomyces cerevisiae a model organism is the degree of conservation of cellular mechanisms that exist between this yeast and higher eukaryotes. However, even after 21 years of sequencing their genome, there are still more than 600 ORFs with unknown function. In this work, we selected four of them for the detailed study. MRPL34 (YDR115w) is present in the major subunit of the yeast mitochondrial ribosome and bears similarity to the L34 gene of E. coli and MRP-L34 from humans. The Δ mrpl34 mutant shows unstable mitochondrial DNA (mtDNA) and to study it, temperature sensitive alleles (ts) were generated. With the mitochondrial protein synthesis assays in vivo, it was possible to identify a clear decrease in the protein synthesis of the conditional mutant. Mrpl34p was identified in the ribosomal extract as expected. The disassembly of the major subunit of the mitochondrial ribosome, using the ts mutants, provided us some clues about intermediates in its assembly process. We have verified that the N-terminal portion of the protein is responsible for addressing the mitochondria. YPR116w also shows high mitochondrial DNA instability, in this way, thermosensitive mutants were used in the experiments. One of the strategies used was the search for genetic partners. We verified that ylr091wp increases the stability of ts-ypr116w mtDNA, suggesting suppressor activity. We also found that the ts-ypr16w allele has a smaller amount of mitochondrial tRNA, through Northen blot assays. Two of the chosen ORFs (YDL119c and YOR022c) had their initial characterization published in 2016, reflecting the importance of this type of research. We have seen that the protein encoded by YDL119c is located on the inner membrane of the mitochondria and that the Δyor022c mutant presents reduced amounts of cardiolipin when grown at 37 ºC.

Os membros da sub-família Toxoplasmatinae conhecidos são Hammondia hammondi, Toxoplasma gondii, Neospora hughesi, Neospora caninum, Hammondia heydorni e Besnoitia spp. Os cães (e provavelmente outras espécies de canídeos) são hospedeiros definitivos de N. caninum e H. heydorni. Os oocistos destas espécies de coccídios são morfologicamente indistinguíveis de forma que o diagnóstico coprológico diferencial entre os dois agentes é virtualmente impossível, se utilizadas metodologias convencionais de diagnóstico. Situação análoga é verificada com os gatos (e outras espécies de felídeos) com relação à infecção por T. gondii e H. hammondi. O objetivo deste trabalho foi propor a reconstrução filogenética de protozoários pertencentes à sub-família Toxoplasmatinae pela análise de seqüências de nucleotídeos de genes mitocondriais e de apicoplasto. Foram empregadas seqüências gênicas de CytB mitocondrial e de dois genes de apicoplasto, o gene codificador da subunidade beta de RNA polimerase DNA dependente (RpoB) e o gene codificador de proteína caseinolitica (ClpC). Pelas análises filogenéticas e de variabilidade nucleotídica e de aminoácidos, verifica-se que a espécie H. heydorni é eqüidistante de todas as outras espécies de toxoplasmatineos. Os posicionamentos relativos dos gêneros Toxoplasma, Neospora e Hammondia nas árvores filogenéticas não foram congruentes em todas as reconstruções, pois dependendo dos táxons que são empregados como grupos externos, as topologias das reconstruções variam e os clados formados são estatisticamente pouco suportados. Assim, a reconstrução de topologias produzindo com ramos curtos que derivam nós de baixo suporte estatístico, somado à eqüidistância evolutiva entre os táxons avaliados (Neospora spp., H. heydorny e T. gondii) permite supor que uma politomia consistente explicaria a evolução para estes organismos, ou seja, a resolução para o posicionamento relativo entre estes táxons poderia ser resultado de evolução radiada. Os genes de organelas mostraram-se mais conservados em relação aos genes nucleares. Embora os genes de apicoplasto possam ser mais conservados que genes nucleares, eles parecem ter relações entre substituições não sinônimas e substituições sinônimas consideravelmente superiores àquelas de genes nucleares e mitocondriais, o que pode indicar que os produtos gênicos estejam sendo submetidos a pressão seletiva positiva. No caso dos genes mitocondriais e nucleares, é possível supor que os mesmos estejam submetidos à pressão seletiva negativa, indicando que as substituições tendem a ser deletérias aos organismos e por isso as mudanças nos produtos gênicos devam ser menos freqüentemente registradas. Ainda, a variabilidade em sítios não sinônimos é consideravelmente superior para seqüências de apicoplasto em relação às demais, particularmente no caso das seqüências RpoB. Também em termos de variabilidade em sítios não sinônimos, percebe-se que as seqüências de genes de apicoplasto de H. heydorni são tão distintas das de T. gondii quanto de N. caninum. Nas análises realizadas com genes de apicoplasto, é marcante a divergência entre as duas linhagens de H. heydorni. Vale ressaltar que as diferenças genotípicas entre as duas linhagens de H. heydorni são maiores que as diferenças entre as duas espécies reconhecidas de Neospora, indicando que as duas linhagens de H. heydorni poderiam ser classificadas como duas espécies distintas, se apenas critérios de evolução molecular fossem considerados. / The known members of the sub-family Toxoplasmatinae are Hammondia hammondi, Toxoplasma gondii, Neospora hughesi, Neospora caninum, Hammondia heydorni and Besnoitia spp. Dogs (and probably other species of dogs) are definitive hosts of N. caninum and H. heydorni. The oocysts of coccidia of these species are morphologically indistinguishable and the coprological differential diagnosis between the two agents is virtually impossible if used conventional methods of diagnosis. Similar situation is observed with the cats (and other species of felids) with respect to T. gondii and H. hammondi. The objective of this study was to propose a phylogenetic reconstruction of protozoa belonging to the subfamily Toxoplasmatinae by analyzing nucleotide sequences of mitochondrial genes and apicoplast. We used gene sequences of cytochrome b and two apicoplast genes, the gene encoding the beta subunit of DNA dependent RNA polymerase (RpoB) and the gene encoding caseinolitic protein (ClpC). From the phylogenetic analysis and the analysis of nucleotide and amino acids variability, was shown that the species H. heydorni is equidistant from all other species of toxoplasmatineos. The relative positions of the genera Toxoplasma, Neospora and Hammondia in the phylogenetic trees were not congruent in all reconstructions, because the topologies of the reconstructions varies according to the taxons that are used as outgroups and clades are poorly supported statistically. Thus, reconstructions of topologies with short branches that derive to poorly statistical supported nodes, coupled with the evolutionary equidistance between taxa the assessed (Neospora spp. H. heydorni. and T. gondii) suggests that a consistent polytomous evolution would explain the evolution within this group of organisms, namely the the relative placement of these taxa could be the result of a radiated evolution. The genes of organelles were more conserved than nuclear genes. Although the apicoplast genes may be more conserved than nuclear genes, they have the ratio between non-synonymous substitutions and synonymous substitutions considerably higher than those of nuclear and mitochondrial genes, which may indicate that the gene products are being subjected to positive selective pressure. In the case of nuclear and mitochondrial genes, it is possible to assume that they are subject to negative selective pressure, indicating that the substitutions are likely to be harmful to organisms and therefore changes in gene products to be less frequently recorded. Still, the variability in non-synonymous sites is considerably higher for sequences of apicoplast in relation to others loci, particularly in the case of RpoB sequences. Also in terms of variability in non-synonymous sites, it is observed that the sequences of apicoplast genes of H. heydorni are as different from those of T. gondii as the N. caninum. The analyzes of apicoplast genes revealed a striking divergence between the two strains of H. heydorni. It is noteworthy that the genotypic differences between the two strains of H. heydorni are greater than the differences between the two species of Neospora, indicating that the two strains of H. heydorni could be classified as two distinct species; if solely criteria of molecular evolution were considered.

The role of mitochondria is multidimensional and ranges in vast areas, including apoptosis, cellular response towards stress, metabolism, which is regulated by a plethora of proteins, acting together to maintain cellular and organellar homeostasis. In spite of the presence of mitochondrial DNA, most of the mitochondrial proteins are nuclear encoded and translocated inside the organelle through dedicated translocases present on outer and inner membrane of mitochondria. To fulfil the cellular energy demand, mitochondria efficiently generate ATP by oxidative phosphorylation, and thus are considered as "power house of cell." There occurs a transfer of electrons from various oxidizable substrates to oxygen, which is achieved by a series of redox reactions with generation of water as a byproduct. This process is coupled with ATP synthesis, involves five protein-complexes present in the inner mitochondrial membrane. During this process, it generates extremely reactive intermediate species of oxygen as a byproduct collectively referred as Reactive Oxygen Species (ROS) through partial reduction of oxygen. These intermediate metabolites of oxygen include superoxide anion (O2-º), H2O2 and highly reactive hydroxyl radicals (OHº). Although ROS are produced by different cellular sources, such as widely expressed and evolutionary conserved NADPH Oxidases, xanthine oxidase, cyclooxygenases, lipoxygenases and cytochrome P450 enzymes but mitochondria are one of the major contributors of cellular ROS. Earlier, reactive oxygen species were considered as harmful but for past few decades, the role ROS has been appreciated as signalling molecules. Because of their high reactivity, these species can cause redox mediated modifications to cellular components and thus have an ability to participate in signalling process. The regulation of signalling pathway by ROS is governed by either alterations in cellular redox conditions or by oxidative modifications of certain residues in proteins, which are involved in signalling cascades. Reactive Oxygen Species can modify amino acid residues, interact with Fe-S clusters or other metal complexes and induce dimerization of proteins to alter protein structure and function. ROS causes modifications to critical amino acids, mainly by oxidation of cysteine residues, where oxidation of sulfhydryl group (-SH) of a single cysteine residue leads to formation of sulfenic (-SOH), sulfinic (-SO2H), sulfonic (-SO3H), or S-glutathionylated (-SSG) derivatives. Thus, by incorporating these modifications, ROS affects the function of proteins, thereby modulating the cellular signalling process. On the other hand, the accumulation of higher level of reactive oxygen species may damage cellular components causing oxidative stress. Therefore, it is necessary to maintain the ROS levels and regulation of intracellular redox homeostasis depends upon a complex network of antioxidant molecules. These antioxidants range from low molecular weight glutathione to large proteins like glutathione peroxidases. Cell has an array of antioxidants with different subcellular locations. Superoxide Dismutase which catalyzes dismutation of superoxides and converts them to H2O2, localizes in cytosol, mitochondrial intermembrane space and extracellular matrix. Different isoforms of Glutatione Peroxidases (GPx) and Peroxiredoxins (Prx) are located in cytosol as well as in mitochondria and scavenge H2O2 by using glutathione (GSH) and thioredoxin (Trx) respectively, as co-factors. During this peroxidase activity of GPx and Prx, GSH and Trx get oxidized and recycled back to the reduced form by Glutathione Reductase (GR) and Thioredoxin Reductase (TR) correspondingly, with the help of NADPH. Thus, GPx system (GPx, GR, GSH and NADPH) and Prx system (Prx, Trx, TR and NADPH) helps in maintenance of redox balance by scavenging H2O2. Catalase is present in peroxisomes for the catalytic degradation of H2O2. Along with Thioredoxin, glutaredoxin (Grx) also reduces protein disulphides and maintains the redox homeostasis. Although, reactive oxygen species are important for normal physiological process, oxidative stress caused by imbalanced ROS levels is thought to be involved in progression of many disorders. However, in most of the diseases, the role of ROS is not yet clear. Elevated oxidative stress is observed with insulin resistance and progression of type II diabetes mellitus, and the resultant high glucose levels alter mitochondrial physiology, leading to the fragmentation of organelle. However, on contrary it has also been observed that ROS improves insulin sensitivity. ROS is directly involved in progression of neurodegenerative disorders, which are characterized by oxidative stress mediated neuronal loss. Interestingly, in case of cancer ROS plays a differential role. At moderately higher levels, ROS helps cancer cells to detach from the matrix and thus assist in metastasis but the higher accumulation of ROS leads to oxidative stress mediated cell death. Thus, cancer cells have an enhanced expression level of antioxidants to maintain the optimum ROS concentration for their survival and proliferation. The role of ROS in cellular signalling and progression of diseases highlights the importance of redox regulation. Mitochondria being the major source of ROS, harbours various redox regulators such as a mitochondrial permeability transition pore (mPTP), inner membrane anion channel (IMAC), Ca++ ions, etc. In addition, certain proteins like Hsp31/DJ1 class also translocate into the organelle in a stress dependent manner to maintain redox homeostasis. These proteins are encoded by the nuclear genome and translocated in the organelle, suggesting the importance of mitochondrial import machinery in regulation of redox balance. Another such example is MIA pathway of protein import, where MIA40 regulates ROS indirectly by catalyzing folding of disulfide containing proteins such as SOD-1 in a redox coupled process. However, under most cases, the physiological disorders lead to uncontrolled production of reactive oxygen species, thereby overloading the cellular antioxidant defence machinery. The failure of the antioxidant machinery leads to enhanced disease progression. Under such disease conditions where the upheaval of redox homeostasis leads to the accumulation of ROS, artificial antioxidants can be used to protect cells against oxidative damage. Artificial systems such as Cyclodextrins, metal complexes, porphyrins, polymers, supramolecules and biomolecules such as nucleic acids, catalytic antibodies and proteins, have been created to mimic the structures and functions of natural enzymes through various approaches. In the present thesis, we have elucidated the role of two mitochondrial proteins, which are part of mitochondrial import motor, as redox regulators and the effect of artificial antioxidants in maintenance of redox homeostasis under stress. A detailed description on importance of ROS in cellular signalling and disease progression has been included in Chapter I, which gives a preface for the work mentioned in this thesis. Chapter II to chapter V elucidates the main objectives of the present thesis, which are: 1. Identification of novel human mitochondrial regulators of redox homeostasis • Role of NEF in redox sensing (Chapter II) • Evolved function of J-like protein in ROS regulation (Chapter III) 2. Characterization of potential artificial antioxidants as redox therapeutics • Organo-selenium compounds as potential artificial antioxidants (Chapter IV) • Use of nanoparticles as a natural antioxidant mimics (Chapter V) Chapter II: Mitochondrial Hsp70 (mtHsp70) plays a critical role for the import of the precursor proteins. The import activity of mtHsp70 is attributed by cyclic binding and release of precursor proteins which in turn is regulated by co-chaperones J-proteins and nucleotide exchange factor (NEF). The affinity for substrate is governed by the binding of ADP or ATP at the N-terminal nucleotide binding pocket of mtHsp70. The affinity for substrate is higher in ADP bound state as compared to ATP bound state. mtHsp70 by its ATPase activity hydrolyze ATP (low-affinity state) to ADP (high-affinity state), which is replaced back to ATP by NEF thus maintaining the mtHsp70 cycle for protein import. In the present study, we have biochemically and functionally characterized GrpEL1 and GrpEL2 as a nucleotide exchange factor for mtHsp70. We observed that like their yeast ortholog Mge1, both the mammalian NEFs interacts with mtHsp70 and exchange ADP from ATP to maintain the cycle of mtHsp70. Interestingly, we observed that both the NEFs are part of human mitochondrial import motor and are recruited at the import motor as hetero-subcomplex. The formation of GrpEL1-EL2 hetero-subcomplex is important to maintain the stability of both the NEFs. In this study, we have elucidated that the interplay between the two NEFs governs organellar response towards oxidative stress. Chapter III: Redox imbalance generates multiple cellular damages leading to oxidative stress mediated pathological conditions such as neurodegenerative diseases, diabetes, ageing and cancer progression. Therefore, maintenance of ROS homeostasis is most important, that involves well-defined antioxidant machinery. In the present chapter, we have identified for first time a component of mammalian protein translocation machinery, Magmas, to perform a critical ROS regulatory function. Magmas overexpression has been reported in highly metabolically active tissues, cancer cells and tissues of developmental origin that are prone to oxidative damage. We found that Magmas regulates cellular ROS levels by controlling its production as well as scavenging. Magmas promotes cellular tolerance towards oxidative stress by enhancing antioxidant enzyme activity, thus preventing induction of apoptosis and damage to cellular components. Magmas enhances the activity of ETC-complexes, causing reduced ROS production. Our results suggest that J-like domain of Magmas is essential for maintenance of redox balance. The function of Magmas as an ROS sensor was found to be independent of its role in protein import, underlying its dual role in human mitochondria. The unique ROS modulatory role of Magmas is highlighted by its ability to increase cellular tolerance to oxidative stress even in yeast model organism. The cyto-protective capability of Magmas against oxidative damage makes it an important candidate for future investigation in therapeutics of oxidative stress related diseases. Chapter IV: The dysregulation of antioxidant machinery in oxidative stress mediated disorders lead to accumulation of excess ROS, highlighting the importance of artificial antioxidants. For the therapeutics of oxidative stress related disorders, artificial antioxidants have been used as combination redox therapy. In order to realize potent biocompatible antioxidants with minimum toxicity, we have utilized two approaches – synthesis of organic compounds and nanoparticle based enzyme mimetics. We have synthesized novel isoselenazoles with high glutathione peroxidase (GPx) and peroxiredoxin (Prx) activities, which provide remarkable cytoprotection to human cells, mainly by exhibiting antioxidant activities in the presence of cellular thiols. The cytotoxicity of the isoselenazoles is found to be significantly lower than that of ebselen, which is being widely clinically evaluated by several research groups for the treatment of reperfusion injuries and stroke, hearing loss, and bipolar disorder. The compounds reported in this study has the potential to be used as therapeutic agents for disorders mediated by reactive oxygen species.. Chapter V: Nanomaterials with enzyme-like properties have attracted significant interest, although limited information is available on their biological activities in cells. Here, we show that V2O5 nanowires (Vn) functionally mimic the antioxidant enzyme, glutathione peroxidase by using cellular glutathione as a co-factor. Although a bulk V2O5 is known to be toxic to the cells, the property is altered when converted into a nanomaterial form. The Vn nanozymes readily internalize into mammalian cells of multiple origins (kidney, neuronal, prostate, cervical) and exhibit robust enzyme-like activity by scavenging the reactive oxygen species, when challenged against intrinsic and extrinsic oxidative stress. The Vn nanozymes fully restore the redox balance without perturbing the cellular antioxidant defense, thus providing an important cytoprotection for biomolecules against harmful oxidative damage. Based on our findings, we envision that biocompatible Vn nanowires can provide future therapeutic potential to prevent ageing, cardiac disorders and several neurological conditions, including Parkinson’s and Alzheimer’s disease.

Abstract A mitochondrial disorder may be one of the rare aetiologies of occipital stroke. Clinical and molecular analysis has suggested that 10% of young patients with occipital stroke have a mitochondrial disorder and 6% harbour the mutation 3243A>G in mitochondrial DNA (mtDNA), causing the MELAS syndrome. To identify other possible mtDNA mutations involved, we studied mtDNA genotypes in patients who had suffered an occipital stroke and in whom the common pathogenic mutations in mtDNA had been excluded. Since one systematic way of comparing mtDNA sequences is through phylogenetic analysis, a phylogenetic network for the Finnish mtDNA haplogroup U was constructed and used to identify differences in mtDNA between patients and controls. The usefulness of conformation sensitive gel electrophoresis (CSGE) for analysing differences within the coding sequence of mtDNA was also estimated. We studied mtDNA genotypes of 29 patients with occipital stroke. The aetiology of the stroke was assessed using the criteria of the Baltimore-Washington Cooperative Young Stroke Study, and migraine was diagnosed in 18 patients according to the International Headache Society criteria. Moreover, we studied the mtDNA genotypes of 42 patients with migraine and a total of 480 population controls who reported that they themselves and their mothers were healthy with respect to common clinical manifestations of mtDNA disease. The mtDNA haplogroups were detected by restriction fragment analysis and the mtDNA structures of 14 patients with occipital stroke and 43 subjects belonging to haplogroup U were examined by CSGE. The data acquired by CSGE were then used to construct a phylogenetic network for the Finnish mtDNA haplogroup U. We found CSGE to be a highly sensitive and specific method for screening mutations and polymorphisms in mtDNA. The sequence data on the 43 subjects belonging to the mtDNA haplogroup U were used to construct a phylogenetic network, which was found to be an unambiguous tree with few homoplasies that pointed to several previously unidentified common polymorphisms. The major branch of the network was U5, which seemed to be quite specific to the Finns. Branches representing haplogroups U2, U4, U7 and K could also be detected. Restriction fragment analysis of the patients with occipital stroke revealed that all those with migraine as a probable aetiology belonged to the mtDNA haplogroup U, suggesting that this genotype confers a risk of occipital stroke. In addition to the five patients with migrainous stroke, we analyzed the complete mtDNA coding sequences of nine other patients with occipital stroke belonging to haplogroup U by CSGE. Analysis of the phylogenetic network revealed an association of migrainous stroke with mtDNA haplogroup U5. Furthermore, the distribution of the mtDNA genotypes in the patients with stroke differed from that found in the controls. Four patients harboured potentially pathogenic mutations. CSGE proved to be an effective method for use in mitochondrial genetics, enabling us to construct an unambiguous network for the Finnish haplogroup U. Similar phylogenetic networks are required for the purposes of both medical genetics and population genetics. Such networks were found to be helpful in deciding between a rare polymorphism and a pathogenic mutation in patients with occipital stroke. Likewise, they enabled more detailed comparisons to be made between and within populations and allowed more accurate phylogenetic relationships to be determined.

Amyotrophic Lateral Sclerosis (ALS) is a rare and fatal neurodegenerative disorder resulting in the loss of motor neurons from the spinal cord and frontal cortex. The patterns of neurodegeneration, affected regions, age of onset, and time course of disease progression are all highly variable between and within variants of the disease. Familial ALS (fALS), inherited versions of ALS due to genetic changes, accounts for between 5-20% of all ALS cases, while the rest are sporadic, with either no causative mutation identified or no familial history of ALS. Recently, the discovery of C9orf72 hexanucleotide repeat expansions have been identified as one of the most common causes of familial ALS, with some patients presenting with dual phenotypes of ALS and frontotemporal dementia, leading to new hypotheses about the nature of neurodegenerative diseases. Despite the continued discovery of new ALS causative genes, little is known about the pathogenesis of the disease. While almost all variants include the presence of intracellular protein inclusions, the site of the protein plaques and involved proteins varies between genetic and phenotypic variants of this disease. Due to the lack of clear pathogenic mechanisms, several hypotheses have been developed to explain the process of neurodegeneration. Autophagy, the process of self-eating, leading to destruction of damaged or excess proteins and organelles, has been implicated as being altered in ALS. Multiple variants have demonstrated altered mitochondrial morphology and cellular energetic dynamics, which could explain previous observations that implicate the process of apoptosis in cellular death. Many of the involved proteins in ALS have functional roles for intracellular, nucleocytoplasmic, and axonal transport of various proteins or RNA. These three competing hypotheses are currently the most prominent hypotheses in the pathogenesis of ALS, and have largely been considered as separate and competing in past research. Is there a chance that the true pathogenesis leading to neuronal destruction via apoptosis involve all three hypotheses? Altered protein and RNA transport dynamics could lead to changes in cellular stress responses or overload autophagy pathways, leading to exacerbated cellular stress responses, leading to alterations in mitochondrial morphology and eventually cell death via apoptosis.

The primary objective of this proposal was to determine whether mitochondrial oxidative stress and variation in a particular mtDNA lineage contribute to the risk of developing cortical dysplasia and are potential contributing factors in epileptogenesis in children. The occurrence of epilepsy in children is highly associated with malformations of cortical development (MCD). It appears that MCD might arise from developmental errors due to environmental exposures in combination with inherited variation in response to environmental exposures and mitochondrial function. Therefore, it is postulated that variation in a particular mtDNA lineage of children contributes to the effects of mitochondrial DNA damage on MCD phenotype. Quantitative PCR and dot blot were used to examine mitochondrial oxidative damage and single nucleotide polymorphism (SNP) in the mitochondrial genome in brain tissue from 48 pediatric intractable epilepsy patients from Miami Children’s Hospital and 11 control samples from NICHD Brain and Tissue Bank for Developmental Disorders. Epilepsy patients showed higher mtDNA copy number compared to normal health subjects (controls). Oxidative mtDNA damage was lower in non-neoplastic but higher in neoplastic epilepsy patients compared to controls. There was a trend of lower mtDNA oxidative damage in the non-neoplastic (MCD) patients compared to controls, yet, the reverse was observed in neoplastic (MCD and Non-MCD) epilepsy patients. The presence of mtDNA SNP and haplogroups did not show any statistically significant relationships with epilepsy phenotypes. However, SNPs G9804A and G9952A were found in higher frequencies in epilepsy samples. Logistic regression analysis showed no relationship between mtDNA oxidative stress, mtDNA copy number, mitochondrial haplogroups and SNP variations with epilepsy in pediatric patients. The levels of mtDNA copy number and oxidative mtDNA damage and the SNPs G9952A and T10010C predicted neoplastic epilepsy, however, this was not significant due to a small sample size of pediatric subjects. Findings of this study indicate that an increase in mtDNA content may be compensatory mechanisms for defective mitochondria in intractable epilepsy and brain tumor. Further validation of these findings related to mitochondrial genotypes and mitochondrial dysfunction in pediatric epilepsy and MCD may lay the ground for the development of new therapies and prevention strategies during embryogenesis.