Indiana University-Purdue University Indianapolis (IUPUI) / Because colorectal cancer (CRC) is the fourth most common cancer and the second leading cause of cancer death in the US, identifying biomarkers that might inform disease prevention and early diagnosis is of great public health importance. Mitochondria are key cytoplasmic organelles containing an independent genome, i.e., mitochondrial DNA (mtDNA). It has been increasingly recognized that mtDNA copy number (mtDNAcn) is a biomarker for mitochondrial function and cellular oxidative stress. To date, the few studies that have assessed associations between mtDNAcn and CRC outcomes have yielded inconsistent findings. Further, no epidemiologic study has examined the relationship between insulinemic potential of lifestyle and mtDNAcn. Therefore, in this dissertation, three studies were conducted using data from the Nurses’ Health Study and the Health Professionals Follow-Up Study. First, the association between pre-diagnostic leukocyte mtDNAcn and CRC risk was studied in a nested casecontrol study (324 cases/658 controls). Lower mtDNAcn was significantly associated with increased risk of CRC and proximal colon cancer. That inverse association remained significant among individuals with ≥ 8 years’ follow-up since blood collection, suggesting that mtDNAcn might serve as a long-term predictor of CRC risk. Second, possible associations of pre-diagnostic mtDNAcn with overall and CRC-specific survival were examined among 587 CRC patients. MtDNAcn was not significantly associated with survival overall or in subgroups by cancer location, grade, or stage. Among current smokers, there was an inverse association between one standard deviation (SD) decrease in mtDNAcn and increased overall death risk. Among patients diagnosed at or before 70.5 years of age and those with anti-inflammatory diets, reduced mtDNAcn was associated with lower CRC-specific death risk. Lastly, the cross-sectional association between empirical lifestyle index for hyperinsulinemia (ELIH) and mtDNAcn was investigated among 2,835 subjects without major chronic diseases (cancers, diabetes, and cardiovascular diseases). A significant inverse association was found: least-squares means ± SD of mtDNAcn z-score decreased dramatically across ELIH quintiles. Overall, the findings from this dissertation will contribute to the evaluation of mtDNAcn as a potential biomarker for CRC risk and prognosis, and inform future interventions designed to reduce the insulinemic potential of lifestyle factors to preserve mitochondrial function. / 2022-04-06

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.

Sepsis is defined as potentially fatal systemic inflammation, caused by an infection. It is the leading cause of ICU mortality and the 10th leading cause of death in the United States. Several models exist to mimic this disorder, and have demonstrated differential mortality rates between the models as well as the individual animals. Previous studies have shown that elevated levels of plasma mitochondrial DNA (mtDNA) correlated with mortality in septic patients, and cell-free mitochondrial DNA can elicit toll-like receptor mediated immune responses similar to LPS-mediated septicemia. However, the role of mtDNA in the pathophysiology sepsis is still unknown. The focus of this study was to create sepsis in a mouse model using the murine Cecal Ligation and Puncture (CLP) model, and measure plasma mtDNA levels. After CLP was performed on experimental mice, blood plasma was collected 24 hours later. Elevated amounts of circulating mtDNA were detectable in the plasma using real time PCR and cytochrome B2 as a marker of mitochondria. These data were correlated with plasma IL-6 levels, which were used to predict mortality within 5 days of CLP to stratify mice into two populations of those predicted to live or die following the procedure. We also aimed to investigate the effect of mtDNA and mitochondrial debris on naïve mouse macrophages in an in vitro study of the regulation of inflammatory cytokines interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), and interleukin-1 beta (IL-1β). In order to observe the effects of mtDNA on murine macrophages, mitochondria was purified from mouse liver and used to stimulate these cells alongside positive control, LPS. Stimulation with mtDNA and mitochondrial debris resulted in increased levels of TNF-α mRNA in lysed cells as well as their surrounding media as compared to control cells, as well as increased transcript half life as measured over four hours post stimulation with transcription inhibitor actinomycin D. The increases in mRNA half-life elicited by mtDNA were comparable to those observed after LPS addition. Stimulation also caused increased binding of TNF-α mRNA to the RNA binding protein, AUF1, as measured by immunoprecipitation of RNA-protein complexes and assayed for TNF-α binding by PCR. These results demonstrate that mitochondrial damage-associated molecular patterns regulate TNF-α mRNA expression at the post-transcriptional level through AUF1, an mRNA destabilizing factor. This is a novel mechanism that likely contributes to sepsis pathophysiology, and demonstrates the involvement of the mitochondrial fission and fusion balance and its regulation in the sepsis innate immune response.

Made available in DSpace on 2014-06-11T19:30:57Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-06-30Bitstream added on 2014-06-13T21:01:41Z : No. of bitstreams: 1 paneto_gg_dr_arafcf_parcial.pdf: 173215 bytes, checksum: 2ba141316e57f525b9ed10acf6c7e979 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / A identificação humana por meio da análise de DNA utiliza o perfil genético de um indivíduo baseado na combinação de diversos marcadores que são herdados de seus progenitores. Estes marcadores são geralmente diferenças nas sequências de DNA nuclear entre os indivíduos (polimorfismos). Em alguns casos, entretanto, a análise do DNA nuclear não pode ser aplicada. Isso ocorre quando o DNA da amostra apresenta-se degradado ou em casos onde o material biológico não apresenta o DNA nuclear. Nestes casos, a análise do DNA mitocondrial (DNA mt) é o método de escolha. Entretanto, em um mesmo indivíduo podem existir populações de DNA mt diferentes, fenômeno denominado heteroplasmia. Este trabalho teve como objetivo o estudo de polimorfismos presentes no DNA mt de indivíduos residentes na Grande São Paulo para aplicação na Identificação Humana. Para isso, foi sequenciada toda a região hipervariável do DNA mt de 160 indivíduos. Além disso, o SNP 3010 foi analisado por discriminação alélica (PCR em tempo real) para estudo do aumento do poder discriminatório quando os indivíduos não puderam ser diferenciados pela análise da região hipervariável. Foi desenvolvido, também, uma reação de SNaPshot em multiplex contendo 42 SNPs que permitiram a classificação das amostras em haplogrupos do DNA mt. Por fim, foram analisadas 100 amostras de cabelo dos mesmos indivíduos para o estudo da frequência de heteroplasmia na região HV3 do DNA mt. De um total de 160 amostras, 144 haplótipos diferentes foram encontrados quando analisamos toda a região hipervariável do DNA mt; 131 haplótipos foram únicos. O SNP 3010 foi suficientemente discriminatório para conseguir distinguir indivíduos que apresentavam o mesmo haplótipo em dois dos treze casos que apresentavam mais de um indivíduo com o mesmo haplótipo / The human identification by DNA analysis uses the genetic profile of an individual based on the combination of diverse markers that are inherited of its ancestors. These markers are generally differences in sequences of nuclear DNA between individuals (polymorphisms). In some cases, however, the analysis of the nuclear DNA cannot be applied. It occurs when the DNA sample is degraded or in cases which the biological material does not content nuclear DNA. In these cases, the mitochondrial DNA (mtDNA) analysis is the choice method. However, inside one individual can exist different mtDNA populations, phenomenon called heteroplasmy. The aim of this work was to study mtDNA polymorphisms in individuals residents in São Paulo metropolitan area for application in Human Identification. For this, we have sequenced the entire hypervariable region of mtDNA for 160 individuals. Furthermore, the SNP 3010 was analyzed by allelic discrimination (Real-Time PCR) to study the increase of the discriminatory power when individuals could not be differentiated by analysis of the hypervariable region. It was developed also, a SNaPshot multiplex reaction containing 42 SNPs that allowed the classification of samples in mtDNA haplogroups. Finally, 100 hair samples, from the same individuals, were analyzed for the study of heteroplasmy frequency in the HV3 region of mtDNA. Among 160 samples, 144 different haplotypes were found when the entire hypervariable region of mtDNA was analyzed; 131 haplotypes were unique. The SNP 3010 SNP was able to distinguish individuals who had the same haplotype in two of thirteen cases with more than one individual with the same haplotype. Our population was classified in 46.6% of African, 27.3% of European, 25.5% of Native American and 0.6% Asian origin using the SNaPshot panel of 42 SNPs in multiplex

Maintaining mitochondrial genome sequence integrity is essential for preserving normal mitochondrial function. Several human diseases have been associated with heteroplasmic mitochondrial genome mutations, but few genetic systems can simultaneously represent pathogenic mitochondrial genome evolution and inheritance. The nematode Caenorhabditis briggsae is one such model. Natural C. briggsae isolates are globally-distributed and phylogenetically grouped into three distinct clades, with isolates exhibiting varying levels of a large-scale mtDNA deletion, nad5∆. Furthermore, a small subset of clade II isolates exhibits putative compensatory mutations that may reduce the risk of deletion formation and accumulation in those populations. In this thesis, the author characterizes the dynamics of nad5∆ heteroplasmy levels during both development and transmission in several C. briggsae natural isolates, including two containing putatively protective compensatory mutations (C+). For all isolates tested, nad5∆ heteroplasmy levels increased across nematode development, with L1 (first larval stage) exhibiting the lowest deletion load for all but one isolate that exhibited highly variable nad5∆ levels, while the increase was slowest and overall nad5∆ levels remained relatively low in C+ isolates. These results support previous work suggesting that nad5∆ is a selfish element and demonstrate the protective nature of compensatory mutations in inhibiting mtDNA deletion accumulation. In nad5∆ inheritance assays, C+ isolates displayed a strong pattern of reversion to wildtype mtDNA levels that was not seen in isolates lacking compensatory mutations (C-). These assays also showed that nad5∆ inheritance was not well predicted by total maternal nad5∆ proportion in either C+ or C- isolates; offspring nad5∆ levels were generally much lower than maternal levels, consistent with some form of negative selection operating between generations. Assays of both maternal somatic and gonadal tissues had slightly more power to predict offspring deletion levels than did assays of whole-worm maternal samples; this result likely points to variance in deletion levels originating from an untested parental tissue type present within the whole-worm samples. This thesis provided deeper insights into the patterns of mtDNA deletion transmission and age-associated dynamics. It was the first project of its type to survey mutation dynamics and heteroplasmy levels of a naturally-occurring large-scale mtDNA deletion. Thus, this work serves to further develop C. briggsae for use as an experimental model of human mtDNA deletion dynamics and mitochondrial dysfunction.

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Abstract not available

<p>Leber´s hereditary optic neuropathy (LHON), a disease affecting vision, is caused by several point mutations in mitochondrial DNA. Mutations leading to a defect NADH ubiquinone oxidoreductase protein will affect the respiratory chain and cause a disturbed ATP production. It is still unknown why this defect leads to the degeneration of retinal ganglion cells and cells in the opticus nerve as well as demyelination of axons in these areas. Analysis of mitochondrial DNA is an important tool in the diagnosis of the disease. At the present time analysis is based on cleavage by restriction enzymes, which only detects two of the most frequent mutations: m.3460G>A and m.11778G>A. This is far too few considering that more than 30 mutations are known to be associated with LHON. Therefore a new analysis method is requested. Here we describe a method based on the sequencing of the mitochondrial genes MT-ND1, MT-ND4 and MT-ND6, which will detect more than 15 different point mutations associated with the disease. To validate the analysis, DNA from 31 patients with LHON symptoms were sequenced; of these 10 were found to be positive for a LHON mutation. This result indicates that the sequencing analysis will be more effective in diagnosis of LHON than restriction enzymes.</p> / <p>Lebers hereditära optikus neuropati (LHON) är en sjukdom som beror på genetiska förändringar i arvsmassan som leder till att cellens energiomsättning rubbas. Detta gör att nervceller i ögat och synnerven bryts ned vilket leder till en synnedsättning. En patient som drabbas av LHON har inga symptom fram till dess att synen börjar försämras. Sjukdomsförloppet går snabbt och på bara några veckor är patienten ofta helt blind. Diagnostik av LHON idag utgörs av flera undersökningar av öga och synfält. Diagnosen bekräftas av en analys av arvsmassan som finns i mitokondrien, cellens energifabrik. Här beskriver vi en ny förbättrad analysmetod baserad på DNA sekvensering, dvs. bestämning av baserna i mitokondriella arvsmassan. För att utvärdera analysen har vi undersökt 31 patienter med misstänkt LHON - av dessa visade sig 10 bära på en sjuklig förändring. Resultatet visar att sekvensering med fördel kan ersätta den tidigare analysmetoden då fler sjukliga förändringar kan påvisas och utförandet av analysen är mer användarvänligt.</p>

The marsh rice rat (Oryzomys palustris) is a semi-aquatic rodent endemic to the southeastern United States. Unlike most terrestrial small mammals, the marsh rice rat can easily disperse over water and has a close association with wetlands. These specialized traits have likely greatly shaped the genetic structure and diversity within this species. I studied genetic patterns within the marsh rice rat to understand how this species' specialized ecology, as well as the geologic and climatic history of the southeastern United States, affected the genetic structuring within this species. The phylogeography of many species in the southeastern United States has been studied and concordant geographic patterns of genetic variation exist among many of these species. Researchers have hypothesized that the biogeography of the southeastern United States has been influenced by the Pleistocene glacial cycles, producing similar genetic patterns within unrelated species. I first examined genetic patterns within the marsh rice rat at the macro scale of phylogenetics. This nominal species actually represents two cryptic species; populations in the eastern and western regions of its range are genetically divergent. I also identified three subspecies, in contrast to the six morphological subspecies historically recognized. The silver rice rat in the Lower Florida Keys and the Sanibel Island rice rat from Sanibel Island Florida are both subspecific taxa. Only one mainland marsh rice rat subspecies exists. I then studied the phylogeographic patterns within the marsh rice rat and determined that geographic patterns of genetic variation in this species are not concordant with the phylogeographic patterns uncovered in most other species of the southeastern United States. The genetic structuring within the marsh rice rat has been influenced not only by the geologic and climatic history of this region, but also by the species' semi-aquatic adaptation. I also studied genetic patterns at a micro scale by estimating present levels of gene flow and genetic diversity within populations. Gene flow is a contemporary factor in maintaining levels of genetic diversity within populations of the marsh rice rat. From the macro scale of phylogenetics to the micro scale of population genetics, the genetic structure of the marsh rice rat has been shaped by past climatic history and by this species' specialized ecology.

Leber´s hereditary optic neuropathy (LHON), a disease affecting vision, is caused by several point mutations in mitochondrial DNA. Mutations leading to a defect NADH ubiquinone oxidoreductase protein will affect the respiratory chain and cause a disturbed ATP production. It is still unknown why this defect leads to the degeneration of retinal ganglion cells and cells in the opticus nerve as well as demyelination of axons in these areas. Analysis of mitochondrial DNA is an important tool in the diagnosis of the disease. At the present time analysis is based on cleavage by restriction enzymes, which only detects two of the most frequent mutations: m.3460G&gt;A and m.11778G&gt;A. This is far too few considering that more than 30 mutations are known to be associated with LHON. Therefore a new analysis method is requested. Here we describe a method based on the sequencing of the mitochondrial genes MT-ND1, MT-ND4 and MT-ND6, which will detect more than 15 different point mutations associated with the disease. To validate the analysis, DNA from 31 patients with LHON symptoms were sequenced; of these 10 were found to be positive for a LHON mutation. This result indicates that the sequencing analysis will be more effective in diagnosis of LHON than restriction enzymes. / Lebers hereditära optikus neuropati (LHON) är en sjukdom som beror på genetiska förändringar i arvsmassan som leder till att cellens energiomsättning rubbas. Detta gör att nervceller i ögat och synnerven bryts ned vilket leder till en synnedsättning. En patient som drabbas av LHON har inga symptom fram till dess att synen börjar försämras. Sjukdomsförloppet går snabbt och på bara några veckor är patienten ofta helt blind. Diagnostik av LHON idag utgörs av flera undersökningar av öga och synfält. Diagnosen bekräftas av en analys av arvsmassan som finns i mitokondrien, cellens energifabrik. Här beskriver vi en ny förbättrad analysmetod baserad på DNA sekvensering, dvs. bestämning av baserna i mitokondriella arvsmassan. För att utvärdera analysen har vi undersökt 31 patienter med misstänkt LHON - av dessa visade sig 10 bära på en sjuklig förändring. Resultatet visar att sekvensering med fördel kan ersätta den tidigare analysmetoden då fler sjukliga förändringar kan påvisas och utförandet av analysen är mer användarvänligt.