Todas as adaptações adquiridas através do treinamento físico são reversíveis durante a inatividade. Reduções significativas no consumo máximo de oxigênio (VO2max) são observadas dentro de duas a quatro semanas de destreinamento. Por outro lado, as consequências do destreinamento sobre o tecido adiposo são pouco estudadas. O objetivo foi investigar os efeitos de destreinamento físico sobre o metabolismo e celularidade do tecido adiposo periepididimal. Métodos e Resultados: Ratos Wistar machos, com idade de 6 semanas, foram divididos em 3 grupos: treinado (T) durante 12 semanas; destreinados (D), (treinados por 8 semanas e destreinados por 4 semanas), e sedentário (S) pareados por idade. O treinamento consistiu em sessões de esteira rolante (1h/dia, 5d/semk, 50 60% da capacidade máxima). A análise morfométrica do tecido PE revelou diferenças significativas entre os grupos. A área seccional dos adipócitos do grupo D foi significativamente maior que a T e S (3474 µm2 ± 68,8 µm2 vs 1945,7 µm2 ± 45,6 µm2 vs 2492,4 µm2 ± 49,08 µm2, respectivamente). Em comparação com as células dos animais do grupo T as células do D apresentaram 48% de aumento na capacidade de realizar a lipogênese, espontaneamente ou insulina estimulada. Já lipólise basal não se alterou. A redução de 15% na apoptose foi observada nos grupos T e D em relação ao S. Algumas expressões de genes foram alterados em D vs S: a adiponectina aumentou 3 vezes e PPAR-gama aumentou 2 vezes. O gene do Pref-1 foi 3 vezes maior em T vs S. Estes resultados sugerem fortemente que a adipogênese foi estimulada neste grupo. Conclusões: o destreinamento causa aumento significativo no tamanho dos adipócitos e na capacidade lipogênica. Como a apoptose celular na gordura PE foi reduzida em D e T, estes resultados sugerem que as alterações do tecido adiposo após destreinamento podem ser potencialmente obesogenicas. / All adaptations acquired through physical training are reversible during inactivity. Significant reductions in maximal oxygen uptake (VO2Max) are observed within two-four weeks of detraining. Conversely, the consequences of detraining on adipose tissue are poorly known. The objective was to investigate the physical detraining effects on metabolism and cellularity of rat periepididymal adipose tissue. Methods and Results: Male Wistar rats, ageing 6 weeks, were divided in 3 groups: trained (T) for 12 weeks; detrained (D), (trained for 8 weeks and detrained for 4 weeks), and age-matched sedentary (S). Training consisted in treadmill running sessions (1h/day, 5d/week, 50 60% of the maximal capacity). The morphometric analysis of PE tissue disclosed significant differences between the groups. The adipocyte sectional area of group D was significantly bigger than T and S (3474 µm2 ± 68,8 µm2 vs 1945,7 µm2 ± 45,6 µm2 vs 2492,4 µm2 ± 49,08 µm2, respectively). Compared to T the cells of D animals showed 48% increased ability to perform: lipogenesis, either spontaneously or insulin stimulated and isoproterenol-stimulated lipolysis. Basal lipolysis did not change. A 15% reduction in apoptosis was observed in groups T and D in relation to S. Some gene expressions were changed in D vs S: adiponectin (3-fold up) and PPAR-gamma (2-fold up). PREF-1 gene was 3-fold higher in T vs S. These results strongly suggest that adipogenesis was stimulated in this group. Conclusions: Detraining causes significant increase in adipocyte size and lipogenic capacity. As PE fat cell apoptosis was reduced in D and T, these results suggest that adipose tissue changes following detraining can potentially be obesogenic.

<p>This thesis describes the use of a new technology, the Cytosensor^® microphysiometer, in the in vitro evaluation of cytotoxic drugs, using the lymphoma cell line U-937 GTB and primary cultures of tumour cells from patients as model systems. The method was specifically applied to study the metabolic effects of the novel cyanoguanidine N-(6-(4-chlorophenoxy)hexyl)-N’-cyano-N’’-4-pyridylguanidine, CHS 828, currently in phase I/II clinical trials. </p><p>The Cytosensor^® measures metabolic effects as changes in the rate of extracellular acidification of cells exposed to a drug by perfusion. A number of standard cytotoxic drugs were found to produce typical and reproducible acidification response patterns during observation times up to 20 h. There seemed to be a relationship between a decrease in acidification and cytotoxicity, measured in the fluorometric microculture cytotoxicity assay (FMCA), after 20-24 h of continuous drug exposure.</p><p>In U-937 cells, CHS 828 induced a cytotoxic effect characterised by a steep concentration-response relationship followed by a plateau. After 24 h of incubation the DNA and protein synthesis were turned off. CHS 828 was found to produce a rapid and prolonged increase in extracellular acidification and lactate production similar to that of the structurally related mitochondrial inhibitor m-iodobenzylguanidine (MIBG). The CHS 828 induced acidification was observed in cell lines as well as in cells from various tumour types from patients and probably originates from increased glycolytic flux. The effects may be secondary to block of oxidative phosphorylation in the mitochondria, but the relevance of the early acidification is not clear. CHS 828 seemed to induce a late, at approximately 15 h, inhibition of the glycolysis followed by loss of ATP and subsequent cell death. After exposure to MIBG the loss of ATP and cell death occurred earlier and in parallel. The effects of CHS 828 were not found to resemble those of the structurally related polyamine biosynthesis inhibitor methylglyoxal-bis(guanyl-hydrazone) (MGBG). Thus, CHS 828 may represent a new and, thus, interesting mode of cytotoxic action worthwhile for further development.</p><p>In combinatory studies, a synergistic interaction was demonstrated between CHS 828 and the non-toxic drug amiloride. Additive-to-synergistic effects were also seen between CHS 828 and the bioreductive cytotoxic drug mitomycin C. In U-937 cells as well as in tumour cells from patients, CHS 828 demonstrated synergistic interactions in combination with melphalan and etoposide. </p><p>It is concluded that measurement in the Cytosensor^® microphysiometer of early cellular metabolic changes is a feasible and potentially valuable complement to more conventional methods used in the evaluation of anticancer agents. </p>

This thesis describes the use of a new technology, the Cytosensor® microphysiometer, in the in vitro evaluation of cytotoxic drugs, using the lymphoma cell line U-937 GTB and primary cultures of tumour cells from patients as model systems. The method was specifically applied to study the metabolic effects of the novel cyanoguanidine N-(6-(4-chlorophenoxy)hexyl)-N’-cyano-N’’-4-pyridylguanidine, CHS 828, currently in phase I/II clinical trials. The Cytosensor® measures metabolic effects as changes in the rate of extracellular acidification of cells exposed to a drug by perfusion. A number of standard cytotoxic drugs were found to produce typical and reproducible acidification response patterns during observation times up to 20 h. There seemed to be a relationship between a decrease in acidification and cytotoxicity, measured in the fluorometric microculture cytotoxicity assay (FMCA), after 20-24 h of continuous drug exposure. In U-937 cells, CHS 828 induced a cytotoxic effect characterised by a steep concentration-response relationship followed by a plateau. After 24 h of incubation the DNA and protein synthesis were turned off. CHS 828 was found to produce a rapid and prolonged increase in extracellular acidification and lactate production similar to that of the structurally related mitochondrial inhibitor m-iodobenzylguanidine (MIBG). The CHS 828 induced acidification was observed in cell lines as well as in cells from various tumour types from patients and probably originates from increased glycolytic flux. The effects may be secondary to block of oxidative phosphorylation in the mitochondria, but the relevance of the early acidification is not clear. CHS 828 seemed to induce a late, at approximately 15 h, inhibition of the glycolysis followed by loss of ATP and subsequent cell death. After exposure to MIBG the loss of ATP and cell death occurred earlier and in parallel. The effects of CHS 828 were not found to resemble those of the structurally related polyamine biosynthesis inhibitor methylglyoxal-bis(guanyl-hydrazone) (MGBG). Thus, CHS 828 may represent a new and, thus, interesting mode of cytotoxic action worthwhile for further development. In combinatory studies, a synergistic interaction was demonstrated between CHS 828 and the non-toxic drug amiloride. Additive-to-synergistic effects were also seen between CHS 828 and the bioreductive cytotoxic drug mitomycin C. In U-937 cells as well as in tumour cells from patients, CHS 828 demonstrated synergistic interactions in combination with melphalan and etoposide. It is concluded that measurement in the Cytosensor® microphysiometer of early cellular metabolic changes is a feasible and potentially valuable complement to more conventional methods used in the evaluation of anticancer agents.

Todas as adaptações adquiridas através do treinamento físico são reversíveis durante a inatividade. Reduções significativas no consumo máximo de oxigênio (VO2max) são observadas dentro de duas a quatro semanas de destreinamento. Por outro lado, as consequências do destreinamento sobre o tecido adiposo são pouco estudadas. O objetivo foi investigar os efeitos de destreinamento físico sobre o metabolismo e celularidade do tecido adiposo periepididimal. Métodos e Resultados: Ratos Wistar machos, com idade de 6 semanas, foram divididos em 3 grupos: treinado (T) durante 12 semanas; destreinados (D), (treinados por 8 semanas e destreinados por 4 semanas), e sedentário (S) pareados por idade. O treinamento consistiu em sessões de esteira rolante (1h/dia, 5d/semk, 50 60% da capacidade máxima). A análise morfométrica do tecido PE revelou diferenças significativas entre os grupos. A área seccional dos adipócitos do grupo D foi significativamente maior que a T e S (3474 µm2 ± 68,8 µm2 vs 1945,7 µm2 ± 45,6 µm2 vs 2492,4 µm2 ± 49,08 µm2, respectivamente). Em comparação com as células dos animais do grupo T as células do D apresentaram 48% de aumento na capacidade de realizar a lipogênese, espontaneamente ou insulina estimulada. Já lipólise basal não se alterou. A redução de 15% na apoptose foi observada nos grupos T e D em relação ao S. Algumas expressões de genes foram alterados em D vs S: a adiponectina aumentou 3 vezes e PPAR-gama aumentou 2 vezes. O gene do Pref-1 foi 3 vezes maior em T vs S. Estes resultados sugerem fortemente que a adipogênese foi estimulada neste grupo. Conclusões: o destreinamento causa aumento significativo no tamanho dos adipócitos e na capacidade lipogênica. Como a apoptose celular na gordura PE foi reduzida em D e T, estes resultados sugerem que as alterações do tecido adiposo após destreinamento podem ser potencialmente obesogenicas. / All adaptations acquired through physical training are reversible during inactivity. Significant reductions in maximal oxygen uptake (VO2Max) are observed within two-four weeks of detraining. Conversely, the consequences of detraining on adipose tissue are poorly known. The objective was to investigate the physical detraining effects on metabolism and cellularity of rat periepididymal adipose tissue. Methods and Results: Male Wistar rats, ageing 6 weeks, were divided in 3 groups: trained (T) for 12 weeks; detrained (D), (trained for 8 weeks and detrained for 4 weeks), and age-matched sedentary (S). Training consisted in treadmill running sessions (1h/day, 5d/week, 50 60% of the maximal capacity). The morphometric analysis of PE tissue disclosed significant differences between the groups. The adipocyte sectional area of group D was significantly bigger than T and S (3474 µm2 ± 68,8 µm2 vs 1945,7 µm2 ± 45,6 µm2 vs 2492,4 µm2 ± 49,08 µm2, respectively). Compared to T the cells of D animals showed 48% increased ability to perform: lipogenesis, either spontaneously or insulin stimulated and isoproterenol-stimulated lipolysis. Basal lipolysis did not change. A 15% reduction in apoptosis was observed in groups T and D in relation to S. Some gene expressions were changed in D vs S: adiponectin (3-fold up) and PPAR-gamma (2-fold up). PREF-1 gene was 3-fold higher in T vs S. These results strongly suggest that adipogenesis was stimulated in this group. Conclusions: Detraining causes significant increase in adipocyte size and lipogenic capacity. As PE fat cell apoptosis was reduced in D and T, these results suggest that adipose tissue changes following detraining can potentially be obesogenic.

Proteins from the Bcl-2 family are now for over 30 years widely studied mainly for their key role in apoptosis, a principal mode of regulated cell death. In the last ten years Bcl-2 proteins were also linked to the regulation of cellular signaling, mainly cellular metabolism and respiration. In this study we aimed to analyze non-apoptotic function of Bcl-2 proteins by their genetic elimination using the CRISPR-Cas12a approach and by the subsequent analysis of mitochondrial respiration, glycolysis and metabolic profiling. Our results confirmed that Bcl-2 proteins can modulate the level of mitochondrial respiration. The elimination of anti-apoptotic proteins Bcl-2, Bcl-XL and Mcl-1 decreased high respiration of cells lacking pro-apoptotic proteins Bax and Bak to the levels observed in parental U87-MG glioblastoma cells. Therefore, the loss of anti-apoptotic Bcl-2 proteins has greatly impacted mitochondrial respiration and it points to their role in a regulation of oxidative phosphorylation.

Thesis / Doctor of Philosophy (PhD)

Nutrient sensing kinases sense available nutrients and regulate cell activity accordingly. Three of these enzymes are AMP regulated kinase (AMPK, or Snf1 in yeast), PAS kinase, and target of rapamycin (TOR), are conserved from yeast to man and have overlapping function. AMPK and Snf1 are important in sensing when nutrient status in the cell is low and down regulating energy consuming pathways. PAS kinase is required for glucose homeostasis in the cell, and responds to glucose levels. TOR senses nutrients such as amino acids and upregulates cell growth pathways primarily through protein synthesis. Due to the varying nature of these enzymes, cross talk is expected in order for the cell to properly regulate cellular metabolism and growth in response to energy and nutrient availability. Previous studies have shown that activation of yeast PAS kinase under nutrient stress conditions requires the presence of Snf1. The aim of this thesis is to determine whether Snf1 directly phosphorylates and activates PAS kinase through both in vivo and in vitro approaches. PAS kinase was found to require Snf1 for both activation and phosphorylation in vivo. In vitro kinase assays were also performed to confirm a direct phosphorylation event. The results from this study support the direct phosphorylation and activation of PAS kinase by Snf1, linking cellular energy status to glucose allocation.

L’agrégation protéique joue un rôle clé dans la dégénérescence cellulaire et est notamment reliée à de nombreuses maladies humaines en lien avec le vieillissement telles que les maladies d’Alzheimer et Parkinson ou encore la maladie du prion. Chez la bactérie Escherichia coli, l’accumulation de dommages sous forme d’agrégats protéiques et leur ségrégation asymétrique au pôle ont permis de démontrer des signes de vieillissement chez cette bactérie. Cette thèse s’est concentrée sur l’étude de la dynamique spatiale des agrégats protéiques in vivo chez la bactérie E. coli. Les agrégats protéiques peuvent être classifiés comme corps d’inclusion dont on dit souvent qu’ils sont amorphes ou comme amyloïdes dont le niveau de structuration est très élevée par la présence de nombreux feuillets β. Combinant une double approche théorique et expérimentale, basée sur la modélisation et la microscopie time-lapse et microfluidique, nous avons étudié le mécanisme gouvernant le mouvement des agrégats protéiques et la transmission verticale d’agrégats de type prionoide sur plusieurs dizaines de générations. Nos résultats indiquent clairement que les agrégats protéiques sont régis par un mouvement Brownien de diffusion avec un coefficient de diffusion dépendant de la taille de la molécule. L’étude de protéinopathie amyloïde a démontré l’existence de lignages propageant deux types d’agrégats : globulaire ou en forme de "comet-like". Les lignées présentant les agrégats sous forme globulaire indiquent une augmentation de la taille des agrégats jusqu’à inhibition de la division cellulaire tandis que la forme "comet-like" est moins préjudiciable à la croissance. Nous avons également observé à faible fréquence des lignées avec un changement de type d’agrégat. A partir d’un agrégat gobulaire, des agrégats "comet-like" peuvent naître. / Protein aggregation plays a key role in cell decline and leads to several human disease linked to ageing like Alzheimer or Parkinson disease and prion disease. In Escherichia coli bacteria, ac- cumulation of damaged proteins and their asymmetric segregation allowed to show ageing signs. This thesis is focused on the in vivo spatial dynamics of protein aggregates in E. coli. Protein aggregates can be classified as inclusion bodies and they are amorphous or amyloid with a high order level due to β sheets. Combining a double theoretical and experimental approach, based on modeling and time-lapse and microfluidic microscopy, we studied the mechanism governing the motion of protein aggregates and the long-term vertical transmission of prionoid aggregates for about 10 generations. Our results show clearly that Brownian diffusion governs the motion of protein aggregates and the diffusion coefficient depends on the molecule size. The amyloid proteinopathy study shows the existence of lineages propagating two kind of aggregates : globular or comet-like. Lineages maintaining globular aggregates present an increase of the aggregate size until inhibition of the growth rate while comet-like aggregates are mildly detrimental to growth. We observed also at low frequency in some lineages the presence of both aggregates and a switch between them. Glo- bular foci give born to comet-like aggregates.

L'ingénierie tissulaire intervient pour restaurer le tissu osseux. Parmi les traitements possibles, l'utilisation d'un bioréacteur à perfusion permet l'amplification in vitro de cellules souches ou osseuses prélevées chez le patient avant réimplantation. La contrainte de cisaillement générée par l'écoulement stimule mécaniquement les cellules et amplifie la production tissulaire. Cette technique souffre cependant de sa conception empirique et doit à présent être optimisée. L'objectif de cette thèse est l'étude et la modélisation de la croissance tissulaire et de la prolifération cellulaire à l'échelle du bioréacteur. En particulier, il s'agit de comprendre l'impact de l'écoulement sur la formation du tissu. Pour cela, une double approche de modélisation et d'expérimentation a été adoptée. Des expériences de culture cellulaire ont permis de mettre au jour la prolifération préférentielle des cellules près des parois du bioréacteur comme conséquence de l'hétérogénéité du support, et l'évolution de la morphologie du tissu. Un modèle prédisant le devenir des cellules ainsi que la croissance tissulaire à l'échelle du bioréacteur est proposé. L'aspect multi-échelles du problème est pris en considération et les procédures d'homogénéisation sont menées à bien grâce à la méthode de prise de moyenne volumique. / Bone tissue engineering aims at restoring bone tissues. Among the possible treatments, the use of a perfusion bioreactor allows the amplification in vitro of the patient bone or stem cells prior to implantation. The advantage of using such bioreactors is two-fold: in addition to greatly improving species transport, tissue production is enhanced. Although promising, this technique suffers from its empirical conception and now needs to be optimized. The purpose of this thesis is to study and model tissue growth and cell proliferation under a fluid flow of culture medium at the scale of the bioreactor. In particular, we wish to understand the impact of fluid flow on tissue formation. To this end, a double approach of experimentation and modeling has been adopted. Cell culture experiments in a perfusion bioreactor highlighted the preferential cell proliferation in the parietal region as a consequence of the heterogeneity of the scaffold, and the evolution of the tissue morphology. A model for predicting the cell's fate along with tissue growth at the scale of the bioreactor is proposed. The hierarchy of the system is considered and the upscaling procedures are carried out with the Volume Averaging Method.

Disulfide bridges play a significant role in protein-folding as well as en- zyme activity and thus regulate many intra- and extracellular processes. Sulfhydryl oxidase QSOX1 forms S-S bridges de novo, modulating the activity of its substrates and thus directly or indirectly influences vital cel- lular processes. The first part of this thesis focuses on characterization of the role of QSOX1 in cancerogenesis, using breast cancer cell lines (MCF7, MDA-MB-231) and pancreatic cancer cell line (Panc-1), while the second part emphasizes the regulation of QSOX1 expression by different oxygen concentrations. To study the effect of QSOX1 on proliferation of triple-negative cancer cells MDA-MB-231, two genetically modified cell lines - QSOX1-overexpressing and QSOX1 knockout cell lines - were constructed. While increased QSOX1 protein levels do not have a significant effect, the absence of QSOX1 leads to a decreased cellular growth. Lack of QSOX1 also results in visible change in cellular morphology. QSOX1 knockout cells can be mostly characterized as more round-shaped with less noticeable or completely missing lamellipo- dia. This finding is with agreement with to-date literature suggesting that QSOX1 is important not only for cellular proliferation but also for migration and invasiveness. While authenticating the theory of...