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Mitochondrial mutations and heteroplasmy have been associated with disease states that result from inadequate cellular energy production. As mitochondrial DNA (mtDNA) encodes many of the polypeptides involved in oxidative phosphorylation (OXPHOS), mtDNA mutations may lower energy production which is required for cell division and sustained ATP synthesis. In order to test the relationship between mtDNA mutations and the rate of cell division, a mammary epithelial cancer cell line, MCF-7, is used as a model. Nine proliferate single cell clones have been isolated from MCF-7. Population doubling times of six single cell clones and the MCF-7 stock have been determined. Clones with distinctly different growth rates were selected for mutational analysis. Growth rates of these clones appeared to be different from each other. Using polymerase chain reaction (PCR) and DNA sequencing, three cases of heteroplasmy have been identified in the mitochondrial genes of the MCF-7 stock and four single cell clones (ATPase C9119T, ND6 T14300G, Cytb G15807A). Heteroplasmy present in the Cytb gene is differs between single cell clones. Differences between the growth rates may be indicative of metabolic variations in these single cell clones. The OXPHOS enzymes encoded by the mutated genes were quantified by standard enzymatic assays. The assays demonstrated significant differences in specific activity between the clones, but were not correlated with mitochondrial heteroplasmy. This thesis determines that the differences in specific activity observed between clones is of nuclear origin.

Mitochondria are the main sites for adenosine triphosphate (ATP) generation within most cells. Structural and functional alterations of mitochondria due to genetic abnormalities of mitochondria can cause respiratory chain dysfunction. In this study, the important role of mitochondria in energy metabolism was determined by comparing the effect of mitochondrial DNA (mtDNA) mutations on growth patterns and oxidative phosphorylation (OXPHOS) enzyme activities of six isolated clones (B5, B12, D4, D9, E1 and E8); as well as the effect of ATP supplement to culture using the slowest growing clone. The isolated clones had shown distinct growth pattern and morphology. The difference in proliferation rates among the clones was ascertained by the doubling times (B5=26.4h. B12=43.2h. D4=25.7h. D9=33.6h. E1=26.9h and E8=28.8h). The clone's slow growth rate was likely the result of mitochondrial mutations in the 16S rRNA gene, ND1, ND4, ND6 and COX III. Five heteroplasmic mutations were found in clone B12 (G2480T, C2513G, A2520T, C9527T and C14263G), one heteroplasmic mutation in clone D9 (A4137G) and one homoplasmic mutation in clone D4 (C11496). The mutations in clone B12 appeared to be deleterious to the cell by disrupting mitochondrial OXPHOS activities and reducing energy output. Additionally, extracellular ATP supplement to OXPHOS deficient clone B12 facilitated cell growth and enhances the gene expression. Increased expression of mtDNA-encoded respiratory chain complexes observed in clone B12 compared to clone D4 may reflect mitochondrial genomic adaptation to perturbations in cellular energy requirements. The stimulation of mitochondrial biogenesis may be a cellular response in compensation for defects in OXPHOS associated with mtDNA mutations. My data support the hypothesis that the variability in functional manifestations of mtDNA is attributed to the nature of the mutation, number of mutation and the gene specifically affected. These results will help to further our understanding of the relationship between mitochondrial mutation and cellular function.

Mitochondrial mutations and heteroplasmy have been associated with disease states that result from inadequate cellular energy production. As mitochondrial DNA (mtDNA) encodes many of the polypeptides involved in oxidative phosphorylation (OXPHOS), mtDNA mutations may lower energy production which is required for cell division and sustained ATP synthesis. In order to test the relationship between mtDNA mutations and the rate of cell division, a mammary epithelial cancer cell line, MCF-7, is used as a model. Nine proliferate single cell clones have been isolated from MCF-7. Population doubling times of six single cell clones and the MCF-7 stock have been determined. Clones with distinctly different growth rates were selected for mutational analysis. Growth rates of these clones appeared to be different from each other. Using polymerase chain reaction (PCR) and DNA sequencing, three cases of heteroplasmy have been identified in the mitochondrial genes of the MCF-7 stock and four single cell clones (ATPase C9119T, ND6 T14300G, Cytb G15807A). Heteroplasmy present in the Cytb gene is differs between single cell clones. Differences between the growth rates may be indicative of metabolic variations in these single cell clones. The OXPHOS enzymes encoded by the mutated genes were quantified by standard enzymatic assays. The assays demonstrated significant differences in specific activity between the clones, but were not correlated with mitochondrial heteroplasmy. This thesis determines that the differences in specific activity observed between clones is of nuclear origin.

Mitochondria are the main sites for adenosine triphosphate (ATP) generation within most cells. Structural and functional alterations of mitochondria due to genetic abnormalities of mitochondria can cause respiratory chain dysfunction. In this study, the important role of mitochondria in energy metabolism was determined by comparing the effect of mitochondrial DNA (mtDNA) mutations on growth patterns and oxidative phosphorylation (OXPHOS) enzyme activities of six isolated clones (B5, B12, D4, D9, E1 and E8); as well as the effect of ATP supplement to culture using the slowest growing clone. The isolated clones had shown distinct growth pattern and morphology. The difference in proliferation rates among the clones was ascertained by the doubling times (B5=26.4h. B12=43.2h. D4=25.7h. D9=33.6h. E1=26.9h and E8=28.8h). The clone's slow growth rate was likely the result of mitochondrial mutations in the 16S rRNA gene, ND1, ND4, ND6 and COX III. Five heteroplasmic mutations were found in clone B12 (G2480T, C2513G, A2520T, C9527T and C14263G), one heteroplasmic mutation in clone D9 (A4137G) and one homoplasmic mutation in clone D4 (C11496). The mutations in clone B12 appeared to be deleterious to the cell by disrupting mitochondrial OXPHOS activities and reducing energy output. Additionally, extracellular ATP supplement to OXPHOS deficient clone B12 facilitated cell growth and enhances the gene expression. Increased expression of mtDNA-encoded respiratory chain complexes observed in clone B12 compared to clone D4 may reflect mitochondrial genomic adaptation to perturbations in cellular energy requirements. The stimulation of mitochondrial biogenesis may be a cellular response in compensation for defects in OXPHOS associated with mtDNA mutations. My data support the hypothesis that the variability in functional manifestations of mtDNA is attributed to the nature of the mutation, number of mutation and the gene specifically affected. These results will help to further our understanding of the relationship between mitochondrial mutation and cellular function.

Mathematical models, that depict the dynamics of a cancer cell population growing out of the human body (in vitro) in unconstrained microenvironment conditions, are considered in this thesis. Cancer cells in vitro grow and divide much faster than cancer cells in the human body, therefore, the effects of various cancer treatments applied to them can be identified much faster. These cell populations, when not exposed to any cancer treatment, exhibit exponential growth that we refer to as the balanced exponential growth (BEG) state. This observation has led to several effective methods of estimating parameters that thereafter are not required to be determined experimentally. We present derivation of the age-structured model and its theoretical analysis of the existence of the solution. Furthermore, we have obtained the condition for BEG existence using the Perron-Frobenius theorem. A mathematical description of the cell-cycle control is shown for one-compartment and two-compartment populations, where a compartment refers to a cell population consisting of cells that exhibit similar kinetic properties. We have incorporated into our mathematical model the required growing/aging times in each phase of the cell cycle for the biological viability. Moreover, we have derived analytical formulae for vital parameters in cancer research, such as population doubling time, the average cell-cycle age, and the average removal age from all phases, which we argue is the average cell-cycle time of the population. An estimate of the average cell-cycle time is of a particular interest for biologists and clinicians, and for patient survival prognoses as it is considered that short cell-cycle times correlate with poor survival prognoses for patients. Applications of our mathematical model to experimental data have been shown. First, we have derived algebraic expressions to determine the population doubling time from single experimental observation as an alternative to empirically constructed growth curve. This result is applicable to various types of cancer cell lines. One option to extend this model would be to derive the cell cycle time from a single experimental measurement. Second, we have applied our mathematical model to interpret and derive dynamic-depicting parameters of five melanoma cell lines exposed to radiotherapy. The mathematical result suggests there are shortcomings in the experimental methods and provides an insight into the cancer cell population dynamics during post radiotherapy. Finally, a mathematical model depicting a theoretical cancer cell population that comprises two sub-populations with different kinetic properties is presented to describe the transition of a primary culture to a cell line cell population.

Submitted by Jaqueline Silva (jtas29@gmail.com) on 2016-09-08T19:58:04Z No. of bitstreams: 2 Dissertação - Gabriel Eliseu Silva - 2014.pdf: 1496066 bytes, checksum: 022a464ffaf53a95f5e7c2aba78aeff4 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Jaqueline Silva (jtas29@gmail.com) on 2016-09-08T19:58:15Z (GMT) No. of bitstreams: 2 Dissertação - Gabriel Eliseu Silva - 2014.pdf: 1496066 bytes, checksum: 022a464ffaf53a95f5e7c2aba78aeff4 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2016-09-08T19:58:15Z (GMT). No. of bitstreams: 2 Dissertação - Gabriel Eliseu Silva - 2014.pdf: 1496066 bytes, checksum: 022a464ffaf53a95f5e7c2aba78aeff4 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2014-08-21 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / (Dynamics of the shrub-tree community in three areas of cerrado sensu stricto in the municipality of Jataí, southwest of Goiás). The aim of the study was to assess the changes in shrub-tree community in a range of approximately three years, involving changes in the structure and vegetation composition with soil factors and the fire. The study was conducted in three areas of “cerrado sentido restrito”, using a ten plots of 20 × 50m in each area. All shrub-tree individuals with DAS (diameter at the heigh of the ground) ≥ 5 cm in the first survey (T1) were plaquetados, estimated their heights and identified their species (2009 in BAT and LAJ, and 2010 in FRP). The second survey (T2) was carried out in 2012 in the area of LAJ and in 2013 in the other two areas (BAT and FRP). T2 survivors were re-measured and those who have to enter the inclusion criteria (recruits) were measured and plaquetados. T1 in BAT, it was found 70 species, 853 individuals and basal area of 8.75 m2. T2 is sampled-63 species, 601 individuals and basal area of 9.12 m2. In the interval between the two surveys (4.16 years) nine species disappeared and two joined the community. The diversity index of Shannon and Pielou evenness were 3.26 and 0.77, respectively, for T1. For T2, only the diversity changed, dropping to 3.16. The average annual mortality rate was 11.55% and the recruitment 2.4%. In LAJ met 83 species, 1391 individuals and basal area of 16.82 m2 in T1 and T2 is already sampled-87 species, 1503 individuals and basal area of 18.65 m2. In the period of 3.58 years, eight species entered four disappeared in the community. The diversity index was 3.62 at T1 and T2 3.65. The average annual mortality rate was 2.27% and 4.13% of recruitment. In FARP met 77 species, 1863 individuals and basal area of 10.87 m2 in T1, T2 and already sampled to 75 species, 1395 individuals and basal area of 10.1 m2. In the period of three years, seven species have disappeared and five joined the community. The diversity index was 3,62 at T1 and T2 3,65. For the evenness values were 0,8 for both surveys. The average annual mortality rate was 10,37% and 1,73% of recruitment. The three cerrado narrow sense fragments altered the species richness, the number of individuals and basal area, but no significant changes in the diversity index between the first and the second survey. This may be related to variations in wealth, depending on the species with few individuals. The highest mortality found for BAT and FRP may be related to the presence of fire, while for the LAJ the largest recruitment is associated with absence of disturbance. The change in density and growth of individuals can still be conditioned to the soil characteristics. In LAJ and BAT the clay soil, while allowing the establishment and growth of forest species and individuals susceptible to fire, in BAT, it is believed that the fire has acted as limiting changes in the structure, preventing the density of vegetation; and LAJ, the increased density and increased basal area possibly occurred in the absence of fire. In the FRP, it is suggested that any fire and the sandy soil can contribute to the limitation on structural changes and the wealth of the community. Thus, in the LAJ greater rate of recruitment in relation to the recorded mortality, may possibly be related to the absence of fire and soil characteristics, providing tree compaction stage and a possible succession of the community, indicating the formation of a cerradão. Regarding the areas of BAT and FARP the highest mortality rates in relation to recruitment may ester associated with the presence of fire, preventing the thickening in the community and keeping the cerrado vegetation type narrow sense. / (Dinâmica da comunidade arbustivo-arbórea de três áreas de cerrado sentido restrito no município de Jataí, sudoeste de Goiás). O objetivo do trabalho foi verificar as mudanças ocorridas na comunidade lenhosa em um intervalo aproximado de três anos, associando as alterações na estrutura e na composição vegetacionais com os fatores edáficos e o fogo. O estudo foi realizado em três áreas de cerrado sentido restrito, utilizando dez parcelas de 20 × 50m em cada área, onde foram amostrados todos os indivíduos lenhosos com DAS (diâmetro a altura do solo) ≥ 5 cm no primeiro levantamento (T1), plaquetados, estimados suas alturas e identificados suas espécies (2009 no BAT e na LAJ, e 2010 na FRP). O segundo levantamento (T2) foi realizado em 2012 na área da LAJ e em 2013 nas outras duas áreas (BAT e FRP). Em T2 os sobreviventes foram remedidos e aqueles que passaram a entrar no critério de inclusão (recrutas) foram mensurados e plaquetados. Em T1 no BAT, encontrou-se 70 espécies, 853 indivíduos e área basal de 8,75 m2. Em T2 amostrou-se 63 espécies, 601 indivíduos e área basal de 9,12 m2. No intervalo entre os dois levantamentos (4,16 anos) nove espécies desapareceram e duas ingressaram na comunidade. O índice de diversidade de Shannon e a equabilidade de Pielou foram 3,26 e 0,77, respectivamente, para T1. Para T2, apenas a diversidade alterou, caindo para 3,16. A taxa média anual de mortalidade foi 11,55% e a de recrutamento 2,4%. Na LAJ encontrou-se 83 espécies, 1391 indivíduos e área basal de 16,82 m2 em T1, e já em T2 amostrou-se 87 espécies, 1503 indivíduos e área basal de 18,65 m2. No período de 3,58 anos, oito espécies ingressaram e quatro desapareceram na comunidade. O índice de diversidade em T1 foi de 3,62 e em T2 3,65. A taxa média anual de mortalidade foi de 2,27% e a de recrutamento 4,13%. Na FARP encontrou-se 77 espécies, 1863 indivíduos e área basal de 10,87 m2 em T1, e já em T2 amostrou-se 75 espécies, 1395 indivíduos e área basal de 10,1 m2. No período de 3,0 anos, sete espécies desapareceram e cinco ingressaram na comunidade. O índice de diversidade em T1 foi de 3,62 e em T2 3,65. Para a equabilidade os valores foram 0,8 para ambos os levantamentos. A taxa média anual de mortalidade foi de 10,37% e a de recrutamento 1,73%. Os três fragmentos de cerrado sentido restrito alteraram a riqueza de espécies, o número de indivíduos e área basal, porém não houve mudanças consideráveis nos índices de diversidade entre o primeiro e o segundo levantamento. Isso pode estar relacionado à variação da riqueza, em função das espécies que apresentaram poucos indivíduos. As maiores mortalidades encontradas para a BAT e FRP podem estar relacionadas à presença do fogo, enquanto para a LAJ o maior recrutamento esteja associado à ausência da perturbação. A alteração na densidade e o crescimento dos indivíduos podem estar condicionados ainda às características do solo. Na LAJ e no BAT o solo argiloso, apesar de possibilitar o estabelecimento e crescimento dos indivíduos de espécies florestais e sensíveis ao fogo, no BAT, acredita-se que o fogo tem atuado como limitante nas alterações da estrutura, impedindo o adensamento da vegetação; e na LAJ, o aumento da densidade e incremento em área basal, possivelmente ocorreu devido à ausência de queimadas. Já na FRP, sugere-se que o fogo eventual e o solo arenoso possam contribuir com a limitação nas alterações estruturais e na riqueza da comunidade. Assim, na LAJ a maior taxa de recrutamento registrada em relação à de mortalidade, possivelmente pode estar relacionada à ausência do fogo e às características do solo, proporcionando o adensamento arbóreo e um possível estágio sucessional da comunidade, indicando a formação de um cerradão. Em relação às áreas do BAT e da FARP as maiores taxas de mortalidade em relação às de recrutamento podem estar associadas à presença de queimadas, impedindo o adensamento na comunidade e mantendo a fitofisionomia de cerrado sentido restrito.