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.

Inadequate metabolic conditions in solid tumors lead to the formation of quiescent cancer cells that are suspended in a transient cell cycle arrest. When conditions change, quiescent cancer cells can re-enter the cell cycle and cause recurrence. Drug screening efforts have revealed mitochondrial oxidative phosphorylation as a unique metabolic dependency in quiescent cancer cells. The anthelmintic drug nitazoxanide is an inhibitor of oxidative phosphorylation and preferentially active against quiescent cancer cells in multicellular tumor spheroids.  In this thesis, we employed current and developed new models of quiescent cancer cells and applied live cell imaging for improved preclinical evaluation of cancer drugs in hepatocellular and colorectal carcinoma cell lines. As part of this work, a new assay to measure mitochondrial membrane potential in three-dimensional cell models was developed, an application of the JC-1 assay, and we demonstrated that the preferential activity against quiescent cancer cells of nitazoxanide is shared by two kinase inhibitors: sorafenib and regorafenib. The sensitivity of quiescent cancer cells to nitazoxanide, sorafenib, and regorafenib correlated with the disruption of the mitochondrial membrane potential. Nitazoxanide and sorafenib, in combination, caused an additive decrease in viability, mitochondrial membrane potential, and colony regrowth capacity.  Furthermore, we developed a quiescent hollow fiber assay and implemented an improved analysis using live cell imaging and adenosine triphosphate analysis. Hypoxia and cancer cell quiescence were enriched in hollow fiber macrocapsules over time, and the culture conditions affected nitazoxanide sensitivity. Additionally, we used basement membrane extract gel to support cell growth in hollow fiber macrocapsules and implanted macrocapsules in mice. We observed that the in vivo environment was favorable to cell growth. Through this characterization of the quiescent hollow fiber assay, we were able to outline important paths for future research.

This thesis investigates the molecular evolutionary and macroevolutionary consequences of flight loss in insects. Chapter 2 tests the hypothesis that flightless groups have smaller effective population sizes than related flighted groups, expected to result in a consistent pattern of increased non-synonymous to synonymous ratios in flightless lineages due to the greater effect of genetic drift in smaller populations. Chapter 3 tests the hypothesis that reduced dispersal and species-level traits such as range size associated with flightlessness increase extinction rates, which over the long term will counteract increased speciation rates in flightless lineages, leading to lower net diversification. The wide-spread loss of flight in insects has led to increased molecular evolutionary rates and is associated with decreased long-term net diversification. I demonstrate that the fundamental trait of dispersal ability has shaped two forms of diversity—molecular and species—in the largest group of animals, and that microevolutionary and macroevolutionary patterns do not necessarily mirror each other. / Generously funded by NSERC with a Canada Graduate Scholarship and the Government of Ontario with an Ontario Graduate Scholarship to T. Fatima Mitterboeck; NSERC with a Discovery Grant to Dr. Sarah J. Adamowicz

[EN] Post-transcriptional modification of the wobble uridine (U34) of a tRNA set is an evolutionary conserved process, produced by homologous proteins from the MnmA/MTU1, MnmE/GTPBP3 and MnmG/MTO1 families. Mutations in the human genes MTU1 and GTPBP3 or MTO1 produce acute infantile liver failure, and hypertrophic cardiomyopathy and lactic acidosis, respectively, which usually cause lethality in the first months of life. It is assumed that the primary cause of these diseases is the lack of the modifications introduced by the MTU1 protein in position 2 (tiol) and GTPBP3 and MTO1 proteins (taurinomethylation) in position 5 at U34 in a subgroup of mt-tRNAs. Nevertheless, the molecular mechanisms underlying these diseases (and other diseases associated with such modifications) are not clear. The reason why the typical defects of oxidative phosphorylation (due to impaired mitochondrial translation) produce such wide range of phenotypes is still unknown. Our hypothesis sustains that the mitochondria-nucleus retrograde signaling pathways triggered by the hypomodification at position 2 and 5 of U34 are different, and that each nuclear response is modulated by the genetic and epigenetic programs of cells and organisms. In this work, we have used the nematode Caenorhabditis elegans as a model organism to study the effects of inactivating the homologue proteins to MTU1, GTPBP3 and MTO1, which we have named as MTTU-1, MTCU-1 and MTCU-2, respectively. We have proved that these nuclear encoded proteins are located in mitochondria and are involved in U34 modification of mt-tRNAs. The mtcu-1 and mtcu-2 mutants show a reduction in fertility, while the mttu-1 mutant shows a reduction in fertility and a lengthening of the reproductive cycle (both phenotypes are thermosensitive). The phenotypes exhibited by the mttu-1, mtcu-1 and mtcu-2 mutants support our hypothesis, in which the mttu-1 single mutation, on the one hand, and the mtcu-1 and mtcu-2 single mutations, on the other hand, trigger different retrograde signaling pathways which produce specific nucear expression. Thus, a nuclear dependent phenotypic trait (as transcription or mt-tRNAs stability) and the expression of nuclear genes as ucp-4, hsp-6, hsp-60 and other genes involved in mitochondrial metabolism show a differential pattern in both group of mutants. hsp-6 and hsp-60 genes (UPRmt markers) are downregulated in mttu-1 single mutant, which could be related to fertility and reproductive cycle thermosensitivity. The three single mutants exhibit reduced expression of glycolysis and ß-oxidation genes (usually more drastic in the mttu-1 mutant), an induction of a glutaminolysis marker, and an induction of the ucp-4 gene, which encodes a transporter of the succinate to the mitochondria. Due to all three single mutants display a mild OXPHOS dysfunction, we propose that the observed changes in the expression of genes involved in the mitochondrial metabolism reveal a TCA cycle reprogramming aimed to compensate the reduction of acetil-CoA (coming from glycolysis and fatty acid oxidation) though the activation of anaplerotic pathways characterized by the succinate import to mitochondria by UCP-4 and the incorporation of 2-oxoglurate from glutaminolysis. We also analyze the effects of the simultanous suppression of modifications at positions 2 and 5 of U34 in C. elegans. The double mutant mtcu-2;mttu-1 displayed a severe OXPHOS dysfunction and a 5-fold higher AMP/ATP ratio, which was associated with embryonic lethality, developmental arrest in primary larval stages, penetrant sterility in adults and extended lifespan. This lifespan extension is modulated by signaling pathways which depend on AMPK (specifically on AAK-1 catalitic subunit) and steroid hormones, through DAF-9 and DAF-12 proteins. This work shows the important gene reprogramming related to mitochondrial metabolism in response to U34 hypomodification of mt-tRNAs, and shows new connexions between signaling pathways that extend lifespan. / [ES] La modificación post-transcripcional de la uridina de tambaleo (U34) de ciertos tRNAs es un proceso conservado evolutivamente, realizado por proteínas homólogas de las familias MnmA/MTU1, MnmE/GTPBP3 y MnmG/MTO1, y biológicamente relevante. De hecho, mutaciones en los genes humanos MTU1 y GTPBP3 o MTO1 causan fallo hepático infantil agudo y cardiomiopatía hipertrófica infantil, respectivamente, que producen letalidad durante los primeros meses de vida. Se asume que la causa primaria de estas enfermedades es la ausencia de las modificaciones introducidas por la proteína MTU1 en la posición 2 (tiol) y las proteínas GTPBP3 y MTO1 (taurinometil) en la posición 5 de la U34 en un grupo de mt-tRNAs. Se desconocen los mecanismos subyacentes y las razones por las que el déficit de OXPHOS resultante en todos los casos (atribuido a alteraciones de la traducción mitocondrial de proteínas) produce fenotipos tan diversos. Nuestra hipótesis es que la señalización retrógrada mitocondria-núcleo promovida por la hipomodificación de los mt-tRNAs en 2 ó 5 de la U34 es diferente y la respuesta nuclear viene modulada por el programa genético y epigenético de células y organismos. Hemos utilizado el nematodo C. elegans como modelo para estudiar los efectos producidos por la inactivación de las proteínas homólogas de MTU1, GTPBP3 y MTO1 a las que hemos denominado MTTU-1, MTCU-1 y MTCU-2. Hemos comprobado que estas proteínas, codificadas por el núcleo, son de localización mitocondrial y están implicadas en la modificación de la U34 de los mt-tRNAs. Los mutantes mtcu-1 y mtcu-2 presentan una reducción en su fertilidad y, en el caso del mutante simple mttu-1, fenotipos asociados a termosensibilidad. Los fenotipos exhibidos por los mutantes mttu-1, mtcu-1 y mtcu-2 sustentan la hipótesis de que la mutación mttu-1, y las mutaciones mtcu-1 y mtcu-2 promueven señales retrógradas diferentes que producen patrones de expresión nuclear específicos. Así, un rasgo fenotípico dependiente de genes nucleares (como lo es la transcripción y/o estabilidad de los mt-tRNAs) y la expresión de genes nucleares como ucp-4, hsp-6, hsp-60 y otros implicados en el metabolismo mitocondrial muestran un patrón diferente en los dos grupos de mutantes. Los genes hsp-6 y hsp-60 (marcadores de la UPRmt) están regulados a la baja en el mutante mttu-1. Los tres mutantes simples exhiben una reducción en la expresión de genes de la glicólisis y de la ß-oxidación de los ácidos grasos, una inducción en un marcador de glutaminolisis y una inducción en el gen ucp-4 (mayor en mttu-1) implicado en el transporte de succinato a la mitocondria. Dado que los tres mutantes simples presentan una disfunción OXPHOS relativamente suave, proponemos que los cambios de expresión en genes que modulan el metabolismo mitocondrial revelan una reprogramación del ciclo del TCA que compensa la disminución en el aporte de acetil-CoA procedente de glicólisis y oxidación de ácidos grasos con la activación de rutas anapleróticas del ciclo del TCA (importe de succinato a la mitocondria por UCP-4 y aporte de ¿-cetoglutarato procedente de la glutaminolisis). También analizamos los efectos de la anulación simultánea de las modificaciones en las posiciones 2 y 5 de la U34. El doble mutante mttu-1;mtcu-2 presenta una disfunción OXPHOS severa, con una ratio AMP/ATP 5 veces superior al control, que resulta en letalidad embrionaria, detención del desarrollo en estadios larvarios tempranos y esterilidad completa en los adultos que presentan, por otra parte, una longevidad unas dos veces superior a la cepa control. Este incremento de la longevidad está modulado por rutas de señalización que dependen de la subunidad catalítica AAK-1 (AMPK), y de hormonas esteroideas (proteínas DAF-9 y DAF-12). El trabajo muestra la importante reprogramación de genes relacionados con el metabolismo mitocondrial en respuesta a la hipomodificación de la U34 de los mt-tRNAs y / [CAT] La modificació post-transcripcional de la uridina de balanceig (U34) de certs tRNAs és un procés conservat evolutivament realitzat per proteïnes homòlogues a les de les famílies MnmA/MTU1, MnmE/GTPBP3 i MnmG/MTO1 i biològicament relevant. De fet, mutacions en els gens humans MTU1 i GTPBP3 o MTO1 causen fallada hepàtica infantil aguda i cardiomiopatia hipertròfica infantil amb acidosis làctica, respectivament, que produïxen letalitat durant els primers mesos de vida. S'assumix que la causa primària d'aquestes malalties és l'absència de les modificacions introduïdes per la proteïna MTU1 a la posició 2 (tiol) i per les proteïnes GTPBP3 i MTO1 (taurinometil) a la posició 5 de la U34 en un grup de mt-tRNAs. Es desconeixen els mecanismes subjacents en estes malalties i les raons per les quals el dèficit de la OXPHOS resultant en tots els casos (atribuït a alteracions de la traducció mitocondrial de proteïnes) produïx fenotips tan diversos. La nostra hipòtesi és que la senyalització retrògrada mitocondria-nucli promoguda per la hipomodificació dels mt-tRNAs en 2 o 5 de la U34 és diferent i la resposta nuclear en cada cas es dependent del programa genètic i epigenètic de cèl¿lules i organismes. Hem utilitzat el nematode C. elegans com a organisme model per a estudiar els efectes produïts per la inactivació de les proteïnes homòlogues de MTU1, GTPBP3 i MTO1 a les que hem denominat MTTU-1, MTCU-1 i MTCU-2. Hem comprovat que aquestes proteïnes, codificades pel nucli, són de localització mitocondrial i estan implicades en la modificació de la U34 dels mt-tRNAs. Els mutants mtcu-1 i mtcu-2 presenten una reducció en la seua fertilitat i, en el cas del mutant mttu-1, fenotipus associats a termosensibilitat. Els fenotipus exhibits pels mutants mttu-1, mtcu-1 i mtcu-2 sustenten la hipòtesi que la mutació mttu-1, i les mutacions mtcu-1 i mtcu-2 promouen senyals retrògrads diferents que produïxen patrons d'expressió nuclears específics. Així, un tret fenotípic dependent de gens nuclears (com ho és la transcripció i/o l'estabilitat dels mt-tRNAs) i l'expressió de gens nuclears com ucp-4, hsp-6, hsp-60 i altres implicats en el metabolisme mitocondrial mostren un patró diferent en els dos grups de mutants. Els gens hsp-6 i hsp-60 (marcadors de la UPRmt) estan regulats a la baixa en el mutant mttu-1. Els tres mutants simples exhibixen una reducció en l'expressió de gens de la glicòlisi i de la ß-oxidació dels àcids grassos, una inducció en un marcador de glutaminolisi i una inducció en el gen ucp-4 (major en el mutant mttu-1) implicat en el transport de succinat a la mitocondria. Atés que els tres mutants simples presenten una disfunció OXPHOS relativament suau, proposem que els canvis d'expressió en gens que modulen el metabolisme mitocondrial revelen una reprogramació del cicle del TCA que compensa la disminució en l'aportació d'acetil-CoA procedent de la glicòlisi i de l'oxidació d'àcids grassos amb l'activació de rutes anaplerótiques del cicle del TCA (importació de succinat a la mitocondria per UCP-4 i aportació de ¿-cetoglutarat de la glutaminolisi). També s'analitzen els efectes de l'anul¿lació simultània de les modificacions en 2 i 5 de la U34. El doble mutant mttu-1;mtcu-2 presenta una disfunció OXPHOS severa, amb una ràtio AMP/ATP 5 vegades superior al control, que resulta en letalitat embrionària, detenció del desenvolupament en estadis larvaris primerencs, esterilitat completa en els adults i una longevitat unes 2 vegades superior al control. Aquest increment de la longevitat està modulat per rutes de senyalització que depenen de la subunitat catalítica AAK-1 (AMPK), i d'hormones esteroidees (a través de les proteïnes DAF-9 i DAF-12). En resum, aquest treball mostra per primera vegada a nivell d'un animal model la important reprogramació de gens relacionats amb el metabolisme mitocondrial en resposta a la hipomodificació de la U34 dels mt-tRNAs i / Navarro González, MDC. (2016). Caenorhabditis elegans as a research tool to study mitochondrial diseases associated with defects in tRNA modification [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/61978 / TESIS

F1FO-ATPsynthase is a key enzyme in energy metabolism of the cell. Its deficit is caused usually by mutations in two structural genes MT-ATP6 and MT-ATP8 encoded by the mitochondrial DNA or in nuclear genes ATPAF2 and TMEM70 encoding the biogenesis factors and structural gene ATP5E. Deficiency of the F1FO-ATPsynthase leads to progressive and serious phenotype affecting organs with high energy demands. The first symptoms usually occurs in neonatal age and prognosis of the disease is fatal. Mutations in these genes result in both qualitative and quantitative defects of the F1FO-ATPsynthase. The study of molecular bases of mitochondrial disorders including F1FO-ATPsynthase deficiency uses large number of biochemical and molecular-genetic methods to determine a proper diagnosis which is essential for the symptomatic therapy and genetic counselling in affected families. The aim of the diploma thesis was to characterise the F1FO-ATPsynthase deficiency in isolated mitochondria from the lines of cultured cells by the determination oligomycin- sensitive ATP-hydrolytic activity of the F1FO-ATPsynthase, enzymatic activities of the respiratory chain complexes and to analyse changes in the steady-state levels of the representative subunits and whole complex of the F1FO-ATPsynthase in comparison with controls. 3...

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High-throughput drug screening (HTS) in live cells is often a vital part of the preclinical anticancer drug discovery process. So far, two-dimensional (2D) monolayer cell cultures have been the most prevalent model in HTS endeavors. However, 2D cell cultures often fail to recapitulate the complex microenvironments of in vivo tumors. Monolayer cultures are highly proliferative and generally do not contain quiescent cells, thought to be one of the main reasons for the anticancer therapy failure in clinic. Thus, there is a need for in vitro cellular models that would increase predictive value of preclinical research results. The utilization of more complex three-dimensional (3D) cell cultures, such as multicellular tumor spheroids (MCTS), which contain both proliferating and quiescent cells, has therefore been proposed. However, difficult handling and high costs still pose significant hurdles for application of MCTS for HTS. In this work, we aimed to develop novel assays to apply MCTS for HTS and drug evaluation. We also set out to identify cellular processes that could be targeted to selectively eradicate quiescent cancer cells. In Paper I, we developed a novel MCTS-based HTS assay and found that nutrient-deprived and hypoxic cancer cells are selectively vulnerable to treatment with inhibitors of mitochondrial oxidative phosphorylation (OXPHOS). We also identified nitazoxanide, an FDA-approved anthelmintic agent, to act as an OXPHOS inhibitor and to potentiate the effects of standard chemotherapy in vivo. Subsequently, in Paper II we applied the high-throughput gene-expression profiling method for MCTS-based drug screening. This led to discovery that quiescent cells up-regulate the mevalonate pathway upon OXPHOS inhibition and that the combination of OXPHOS inhibitors and mevalonate pathway inhibitors (statins) results in synergistic toxicity in this cell population. In Paper III, we developed a novel spheroid-based drug combination-screening platform and identified a set of molecules that synergize with nitazoxanide to eradicate quiescent cancer cells. Finally, in Paper IV, we applied our MCTS-based methods to evaluate the effects of phosphodiesterase (PDE) inhibitors in PDE3A-expressing cell lines. In summary, this work illustrates how MCTS-based HTS yields potential to reveal and exploit previously unrecognized tumor-specific vulnerabilities. It also underscores the importance of cell culture conditions in preclinical drug discovery endeavors.