The overall objective of this thesis was to examine mitochondrial bioenergetics in muscle biopsies from humans with type 1 diabetes (T1D) to gain a deeper understanding of the cellular mechanism(s) underlying changes to skeletal muscle health reported in T1D, a phenotype we have referred to as ‘diabetic myopathy’. It was hypothesized that humans with T1D, compared to their matched counterparts without diabetes (control), would demonstrate significant deficiencies in muscle mitochondrial function and ultrastructure/content as determined by the gold-standard in vitro methodology: high-resolution respirometry and transmission electron microscopy, respectively. It was further hypothesized that sex differences would not exist in mitochondrial function with T1D, and mitochondrial deficiencies would be more dramatic at an earlier age with T1D. Adults with uncomplicated T1D and strictly matched controls (age, sex, BMI, self-reported physical activity levels) were recruited from surrounding university-dwelling communities. Site-specific deficiencies in mitochondrial respiration, H2O2 emission, and calcium retention capacity were found in young, physically active adults with T1D despite normal mitochondrial content. Further experiments revealed that muscle mitochondrial respiration in women and men differentially adapt to the T1D environment where men with T1D have lower complex II but higher complex I respiration compared to women with T1D, while women (irrespective of T1D) have lower ADP sensitivity. Women with T1D also demonstrated lower H2O2 emission compared to men with T1D. In contrast, despite a lower mitochondrial content in middle- to older-aged adults with T1D, mitochondrial respiration (normalized to content) was either normal or increased in adults with T1D compared to control, with observable differences between sexes. Overall, this research has demonstrated that despite being recreationally to physically active, adults with uncomplicated T1D with moderately well-managed glycemia demonstrate alterations in skeletal muscle mitochondrial function and ultrastructure, including differences between sexes. / Dissertation / Doctor of Science (PhD) / Type 1 diabetes (T1D) is a complex disease that still has no known cure. Current treatment focuses on managing blood sugar levels with exogenous insulin injections and frequent blood sugar checks. However, over time, people with T1D still develop serious complications that inevitably impact their quality of life and lifespan. A potential adjuvant therapy to prevent complications in T1D is improving the health of skeletal muscle through exercise given its role in stabilizing blood sugar/lipid levels and whole-body insulin sensitivity. However, this area continues to be severely understudied in the T1D population. Thus, this thesis examined skeletal muscle metabolic ‘health’ from adults with T1D who do not have major diabetes complications and manage their blood glucose moderately-well. Through a series of novel experiments, we found that young and middle- to older-aged adults with T1D have alterations in the metabolic engines of their muscles, and depending on biological sex, the alterations manifest as either heightened or degraded cellular function. These findings are the first to provide a comprehensive cellular investigation of the impact of T1D on the metabolic health of skeletal muscle in people with T1D and provide the foundation for future research examining skeletal muscle as an essential and early adjuvant therapy in this population.

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Purpose of the Review Mitochondrial dysfunction has long been proposed to play a crucial role in the pathogenesis of a considerable number of disorders, such as neurodegeneration, cancer, cardiovascular, and metabolic disorders, including obesity-related insulin resistance and non-alcoholic fatty liver disease (NAFLD). Mitochondria are highly dynamic organelles that undergo functional and structural adaptations to meet the metabolic requirements of the cell. Alterations in nutrient availability or cellular energy needs can modify their formation through biogenesis and the opposite processes of fission and fusion, the fragmentation, and connection of mitochondrial network areas respectively. Herein, we review and discuss the current literature on the significance of mitochondrial adaptations in obesity and metabolic dysregulation, emphasizing on the role of hepatocyte mitochondrial flexibility in obesity and NAFLD. Recent Findings Accumulating evidence suggests the involvement of mitochondrial morphology and bioenergetics dysregulations to the emergence of NAFLD and its progress to non-alcoholic steatohepatitis (NASH). Summary Most relevant data suggests that changes in liver mitochondrial dynamics and bioenergetics hold a key role in the pathogenesis of NAFLD. During obesity and NAFLD, oxidative stress occurs due to the excessive production of ROS, leading to mitochondrial dysfunction. As a result, mitochondria become incompetent and uncoupled from respiratory chain activities, further promoting hepatic fat accumulation, while leading to liver inflammation, insulin resistance, and disease’s deterioration. Elucidation of the mechanisms leading to dysfunctional mitochondrial activity of the hepatocytes during NAFLD is of predominant importance for the development of novel therapeutic approaches towards the treatment of this metabolic disorder.

Os modelos de evolução de vias metabólicas estão baseados em técnicas moleculares e bioinformática e nem sempre levam em consideração o contextos fisiológico e ecológico do organismo. Assim, tomando como plataforma o metabolismo de nitrogênio, procurou-se estabelecer uma hipótese evolutiva para o uso de α-aminoácidos por metazoários como fonte de nitrogênio. O objetivo é traçar essa história evolutiva, contextualizando fisiológica e ecologicamente as alterações que ocorreram no perfil de utilização desses compostos. Para traçar essa história evolutiva, recorreu-se a dados disponíveis na literatura partindo-se dos elementos moleculares/metabólicos que compõem o ciclo do nitrogênio e em qual contexto geológico e evolutivo se deu tal história. Os dados obtidos, reorganizados e reestruturados nesse novo contexto, permitiram conclusões originais no presente trabalho, a saber: (1) a capacidade de fixação de nitrogênio atmosférico foi um fator de seleção natural positiva na transição da atmosfera redutora para oxidante; (2) os organismos fixadores de nitrogênio são bem mais disseminados do que o admitido classicamente; (3) o produto final da fixação biológica de nitrogênio in vivo são α- aminoácidos, e foram um fator de pressão seletiva para os organismos incapazes de fixar nitrogênio; (4) os metazoários evoluíram posteriormente a esse cenário e seu aparato metabólico está mais adaptado para o aproveitamento líquido do nitrogênio obtido apenas na forma de α-aminoácidos. / Metabolic pathway evolution models are molecular and computational based, and do not take account the physiological and ecological contexts in which organisms are inserted. Thus using the nitrogen metabolism as a platform, an evolutionary hypothesis on the α-amino acids utilization by metazoans was proposed. The objective of the present work is to trace an evolutionary history of the nitrogen usage by metazoans taking account the profile changes on a physiological and ecological basis. In order to trace this evolutionary history, a scrutiny were performed in the specialized literature aiming at data about the molecular and metabolic elements which perform the nitrogen cycle and in which geologic and evolutive context has passed such history. The reorganization of obtained data in a new context allowed original conclusions in the present work as follows: (1) the capability of fixing the atmospheric nitrogen was a positive selection factor in the atmospheric condition transition from reductive to oxidant; (2) nitrogen fixing organisms are far most wide spread than classically admitted; (3) α-amino acids are the biological nitrogen fixation end product in vivo, and are a selective factor for non-fixing organisms; (4) metazoans evolved afterwards in these scenario and their metabolic apparatus is adapted to the nitrogen net utilization obtained in the α-amino acid form.

O objetivo deste trabalho foi estimar as exigências e a eficiência energética de vacas de corte lactantes e não gestantes, bem como do par vaca/bezerro. Posteriormente foi correlacionada a eficiência do par vaca/bezerro durante a lactação com a eficiência das mesmas vacas durante a terminação. Na fase de lactação os pares vaca/bezerro foram avaliados desde após o nascimento (17±5, d DP) até a desmama (210 d). Após a desmama foi conduzido o ensaio de terminação (67 d) e abate somente das matrizes. Foram avaliadas 10 vacas adultas ½ Caracu x Nelore (CN) e 10 ½ Angus x Nelore (AN), acasaladas com touros Red Angus (RA) e Canchim (CC), respectivamente. Na lactação o oferecimento da dieta (2,30 Mcal EM/kg e 12,4% PB) variou individualmente ao longo do período com objetivo de manter constante o peso das matrizes ao longo da lactação. A mesma dieta foi oferecida a vontade para a progênie. Durante o ensaio de terminação em confinamento a dieta (2,37 Mcal EM/kg e 10,5% PB) foi fornecida à vontade. A ingestão de matéria seca das matrizes foi de 92,4 g MS/kg PM0,75 (CV=3,2%; P=0,458). Vacas AN apresentaram produção de leite corrigida para gordura 12% superior (P<0,05). A concentração energética do leite (Mcal/kg) das vacas CN foi 9,4% superior (P<0,05). A eficiência (Mcal leite/Mcal EM ingerida) não foi diferente. A exigência de energia metabolizável de lactação (EMl) foi maior (P<0,05) para vacas AN em relação as CN, 90,1 e 74,2 (EMl, kcal EMl/kg PM0,75), respectivamente. Bezerros ½CC¼AN¼NE apresentaram ingestão de energia metabolizável (IEM) proveniente do leite 11,3% (P<0,05) superior. Bezerros ½RA¼CR¼NE foram 10,7% mais eficientes (g GP210d/Mcal IEM leite+dieta sólida). Não houve diferença para eficiência dos pares vaca/bezerro, contudo houve considerável variação individual, onde a eficiência média foi 35,3 g GP210d/Mcal IEM par (CV=11,5%). A eficiência do par foi positivamente relacionada ao ganho do bezerro (r=0,79; P<0,01) e negativamente relacionada à IEM do par (r=-0,58; P<0,05). Vacas CN ao abate apresentaram EGS 29% menor, AOL 13% maior e maior proteína no corpo vazio (P<0,05). Vacas AN apresentaram 14,7% maior proporção de órgãos internos relativo ao peso vazio e maior peso dos intestinos (P<0,01). As correlações fenotípicas demonstraram associação entre as eficiência de secreção do leite das vacas nas fase de lactação e de ganho de peso na fase de terminação (r=0,43; P=0,09). A eficiência de secreção de leite das vacas na lactação apresentou correlação com a eficiência do par (r=0,68; P<0,01). O consumo alimentar residual (CAR) das vacas na terminação foi correlacionado com o CAR das vacas em lactação (r=0,53; P<0,05) e com o CAR dos pares (r=0.62; P<0.01). Isso indica que vacas de corte mais eficientes durante a terminação são também mais eficientes no período de cria. / This study estimated the requirements and feed efficiency of non-pregnant lactating beef cows, as well as cow calf pairs. It also correlated the efficiency of the cows and their progeny during lactation to efficiency determined for the same cows in a finishing trial after weaning. In the lactating phase cow calf pairs were evaluated from just after birth (17±5, d) to weaning at 210 d. The finishing trial for the mature cows started after weaning and lasted 67 d after wich cows were slaughtered. Twenty adult cows were evaluated, 10 ½ Caracu x Nelore (CN) and 10 Angus x Nelore, bred to Red Angus (RA) and Canchim (CC) bulls, respectively. During lactation the diet (2.30 Mcal ME/kg and 12.4% CP) was offered in variable amounts to maintain individual live weight and condition score constant. The same diet was offered ad libitum to the calves. During the finishing phase cows were fed ad libitum (2.37 Mcal ME/kg e 10.5% CP). The dry matter feed intake of cows was 92,4 and SD 2,9 g DM/kg BW0,75. AN cows had fat corrected milk 12% higher compared to CN cows (P<0.05). However, milk energy content (Mcal/kg) was 9.4% higher (P<0.05) for CN cows. Efficiency of milk production (Mcal milk/Mcal ME intake) was not different. Metabolizable energy for lactation (MEl) was higher (P<0.05) for AN cows in comparison to CN, respectively 90.1 and 74.2 (MEl, kcal MEl/kg BW0,75). ME milk intake was 11.3% (P<0.05) higher for ½CC¼AN¼NE calves. Rates of gain were not different between calves, but ½RA¼CR¼NE were 10.7% more efficient (g LWG210d/Mcal MEI milk+solid diet). There was no difference in cow calf pairs efficiency, but there was a great individual variability with a mean of 35,31 g and SD of 4,08 g LWG210d/Mcal EM for the pair. Cow/calf pair efficiency was positively related to calf weight gain (r=0.79; P<0.01) and negatively to the pair metabolizable energy intake (r=-0.58; P<0.05). At slaughter CN cows presented backfat thickness 29% lower, longissimus muscle area 13% higher and higher empty body weight protein (P<0,05). AN cows had 14.7% higher relative internal organ weight and higher total intestine weight (P<0.01). The phenotypical correlations between efficiency indexes indicate moderate association between cow efficiency measured during lactation and in a short finishing period after weaning (r=0.43; P=0.09). Exist correlations between cow efficiency lactation and efficiency of the pair (r=0.68; P<0.01). Residual Feed Intake (RFI) during the finishing phase was moderately correlated to the RFI calculated during the lactation period for the cow calf pairs RFI (r=0.62; P<0.01) and the cows (r=0.53; P<0.05) indicated that beef cows more efficient in feed conversion during the finishing phase are more efficient during the lactation period.

Os modelos de evolução de vias metabólicas estão baseados em técnicas moleculares e bioinformática e nem sempre levam em consideração o contextos fisiológico e ecológico do organismo. Assim, tomando como plataforma o metabolismo de nitrogênio, procurou-se estabelecer uma hipótese evolutiva para o uso de &#945;-aminoácidos por metazoários como fonte de nitrogênio. O objetivo é traçar essa história evolutiva, contextualizando fisiológica e ecologicamente as alterações que ocorreram no perfil de utilização desses compostos. Para traçar essa história evolutiva, recorreu-se a dados disponíveis na literatura partindo-se dos elementos moleculares/metabólicos que compõem o ciclo do nitrogênio e em qual contexto geológico e evolutivo se deu tal história. Os dados obtidos, reorganizados e reestruturados nesse novo contexto, permitiram conclusões originais no presente trabalho, a saber: (1) a capacidade de fixação de nitrogênio atmosférico foi um fator de seleção natural positiva na transição da atmosfera redutora para oxidante; (2) os organismos fixadores de nitrogênio são bem mais disseminados do que o admitido classicamente; (3) o produto final da fixação biológica de nitrogênio in vivo são &#945;- aminoácidos, e foram um fator de pressão seletiva para os organismos incapazes de fixar nitrogênio; (4) os metazoários evoluíram posteriormente a esse cenário e seu aparato metabólico está mais adaptado para o aproveitamento líquido do nitrogênio obtido apenas na forma de &#945;-aminoácidos. / Metabolic pathway evolution models are molecular and computational based, and do not take account the physiological and ecological contexts in which organisms are inserted. Thus using the nitrogen metabolism as a platform, an evolutionary hypothesis on the &#945;-amino acids utilization by metazoans was proposed. The objective of the present work is to trace an evolutionary history of the nitrogen usage by metazoans taking account the profile changes on a physiological and ecological basis. In order to trace this evolutionary history, a scrutiny were performed in the specialized literature aiming at data about the molecular and metabolic elements which perform the nitrogen cycle and in which geologic and evolutive context has passed such history. The reorganization of obtained data in a new context allowed original conclusions in the present work as follows: (1) the capability of fixing the atmospheric nitrogen was a positive selection factor in the atmospheric condition transition from reductive to oxidant; (2) nitrogen fixing organisms are far most wide spread than classically admitted; (3) &#945;-amino acids are the biological nitrogen fixation end product in vivo, and are a selective factor for non-fixing organisms; (4) metazoans evolved afterwards in these scenario and their metabolic apparatus is adapted to the nitrogen net utilization obtained in the &#945;-amino acid form.

The peripheral skin microcirculation reflects the overall health status of the cardiovascular system and can be examined non-invasively by laser methods to assess early cardiovascular disease (CVD) risk factors, i.e. oxidative stress and endothelial dysfunction. Examples of methods used for this task are the laser Doppler flowmetry (LDF) and laser fluorescence spectroscopy (LFS), which respectively allow tracing blood flow and the amounts of the coenzyme NAD(P)H (nicotamide adenine dinucleotide) that is involved in the cellular production of ATP (adenosine triphosphate) energy. In this work, these methods were combined with iontophoresis and PORH (post-occlusive reactive hyperaemia) reactive tests to assess skin microvascular function and oxidative stress in mice and human subjects. The main focus of the research was exploring the nonlinear dynamics of skin LDF and NAD(P)H time series by processing the signals with the wavelet transform analysis. The study of nonlinear fluctuations of the microcirculation and cell energy metabolism allows detecting dynamic oscillators reflecting the activity of microvascular factors (i.e. endothelial cells, smooth muscle cells, sympathetic nerves) and specific patterns of mitochondrial or glycolytic ATP production. Monitoring these dynamic factors is powerful for the prediction of general vascular/metabolic health conditions, and can help the study of the mechanisms at the basis of the rhythmic fluctuations of micro-vessels diameter (vasomotion). In this thesis, the microvascular and metabolic dynamic biomarkers were characterised <i>in-vivo</i> in a mouse model affected by oxidative stress and a human cohort of smokers. Data comparison, respectively, with results from control mice and non-smokers, revealed significant differences suggesting the eligibility of these markers as predictors of risk associated with oxidative stress and smoke. Moreover, a relevant link between microvascular and metabolic oscillators was observed during vasomotion induced by α-adrenergic (in mice) or PORH (in humans) stimulations, suggesting a possible role of cellular Ca²⁺oscillations of metabolic origin as drivers of vasomotion which is a theory poorly explored in literature. As future perspective, further exploration of these promising nonlinear biomarkers is required in the presence of risk factors different from smoke or oxidative stress and during vasomotion induced by stimuli different from PORH or α-adrenergic reactive challenges, to obtain a full picture on the use of these factors as predictors of risk and their role in the regulation of vasomotion.