Dissertations / Theses on the topic 'Cell respiration'

Plants produce a variety of secondary metabolites, e.g. to defend themselves against herbivores or to attract pollinating insects. Plant cell biotechnology offers excellent opportunities in order to use such secondary plant metabolites to produce goods with consistent quality and quantity throughout the year, and therefore to act independently from biotic and abiotic environmental factors. This article presents results of an extensive study of plant cell in vitro cultivation in a modern shake flask system with non-invasive online respiration activity monitoring unit. Comprehensive screening experiments confirm the successful transfer of a model culture (sunflower suspension) into the shake flask monitoring device and the suitability of this respiration activity monitoring unit as qualified tool for screening of plant in vitro cultures (sunflower and sage suspension). The authors demonstrate deviations between online and offline data due to varying water evaporation from different culture flask types. The influence of evaporation on growth-specific parameters thereby rises with increasing cultivation time. Furthermore, possibilities to minimize the impact of evaporation, either by adjusting the inlet air moisture or by measuring the evaporation in combination with an appropriate correction of the measured growth values, are shown.

Early development of O2 chemoreception and hypoxic responses under normoxic (150 mmHg) and chronically hypoxic (110 mmHg) conditions were investigated in Xenopus laevis from hatching to 3 weeks post fertilization. Development, growth, O2 consumption, ventilatory and cardiac performance, and branchial neuroepithelial cells (NEC) density and size were determined. At 3 days post fertilization (dpf), larvae started gill ventilation at a rate of 28 ± 4 beats/min and showed increased frequency to 60 ± 2 beats/min at a PO2 of 30 mmHg. Also at 3 dpf, NECs were identified in the gill filament buds using immunohistochemical methods. Lung ventilation began at 5 dpf and exhibited a 3-fold increase in frequency from normoxia to a PO2 of 30 mmHg. Hypoxic tachycardia developed at 5 dpf, causing an increase of 20 beats/min in heart rate, which led to a 2-fold increase in mass-specific cardiac output at a PO2 of 70 mmHg. At 10 dpf, gill ventilatory sensitivity to hypoxia increased, which was associated with the increase in NEC density, from 15 ± 1 to 29 ± 2 cells/mm of filament at 5 and 10 dpf, respectively. Unlike the elevated rate, cardiac and ventilatory volumes were independent of acute hypoxia. Despite increased cardioventilatory frequency, larvae experienced an average of 80% depression in during acute hypoxia. Chronic hypoxia (PO2 of 110 mmHg) decreased mass-specific cardiac performance before 10 dpf. In older larvae (10 to 21 dpf), chronic hypoxia decreased acute branchial and pulmonary hypoxic hyperventilation and increased NEC size. Collectively, these data suggest that larvae exhibit strong O2-driven acute hypoxic responses post-hatching, yet are still O2 conformers. All acute hypoxic responses developed before 5 dpf, and then the effects of chronic hypoxia started to show between 7 and 21 dpf. Thus, the early formation of acute hypoxic responses is susceptible to the environment and can be shaped by the ambient PO2.

De nombreuses observations montrent que la respiration cellulaire n'est pas corrélée à la concentration moyenne d'ADP (ou de paramètres dérivés tels que le rapport ATP/ADP ou le potentiel phosphate). (1) Nous rapportons qu'au sein de l'hépatocyte, l'ADP ne diffuse pas librement au travers de la membrane mitochondriale externe de sorte que la concentration cytosolique moyenne d'ADP ne reflète pas forcément la concentration d'ADP au contact des mitochondries. Il est possible que cette étape de diffusion de l'ADP puisse être modulable et participe au contrôle de la respiration in vivo. (2) Les mitochondries provenant de rats soumis à un régime alimentaire carencé en acides gras polyinsaturés ont une composition phospholipidique profondément modifiée. Ceci s'accompagne d'une modification des liens existant entre la force protonmotrice et la respiration, et entraîne une diminution du rendement de l'oxydation phosphorylante. (3) Notre équipe a montré que l'almitrine modifie la stchiométrie de l'ATPsynthase. Nous rapportons que la stchiométrie de l'ATPsynthase varie aussi en fonction de la concentration de nucléotides adényliques. (4) Les conséquences et la signification de ces phénomènes originaux sont discutés en termes de régulation du métabolisme énergétique cellulaire.

During development of type 1 and type 2 diabetes plasma insulin patterns are altered. Since the islet insulin release pattern has been implicated in this development, insulin secretion from single islets was studied and linked to the islet protein levels of uncoupling protein-2 (UCP2). Islets were isolated from NOD- and KKA^y- mice, GK- and GK-derived congenic rats, which are animal models of diabetes, and three human subjects with type 2 diabetes. At basal glucose (3 mM), insulin release from such islets was pulsatile and the amount released was comparable to that of control islets. When the glucose concentration was raised to 11 mM insulin release was essentially unchanged in islets isolated from older NOD- and KKA^y- mice, GK- and Niddm1i congenic rats, and NIDDM persons. In islets from Niddm1f congenic rats, younger NOD- and KKA^y-mice, control animals and normal human donors the secretion rate increased 2-9 fold when the glucose concentration was raised. This rise in secretion was manifested as increase of the amplitude of the insulin oscillations without affecting their frequency. Impaired glucose-induced insulin release was associated with reduction in glucose oxidation measured in NOD-islets, unaffected respiration measured in GK-islets and higher protein level of UCP2 measured in KKA^y-islets. When the UCP2 amounts in KKA^y-islets were reduced by culture to those of control islets, glucose-induced insulin secretion was essentially normalized. Our studies suggest that the deranged plasma insulin patterns observed in diabetes are related to decrease in the amplitude of insulin oscillations from the islets rather than loss of the oscillatory activity. This reduction of pulse amplitude may be related to impaired glucose metabolism and/or increased mitochondrial uncoupling.

Apenas muito recentemente estratégias genômicas e pós-genômicas têm sido utilizadas em estudos de espécies arbóreas importantes na silvicultura. Nos países tropicais, a exemplo do Brasil, as espécies de eucalipto são especialmente importantes nos plantios comerciais principalmente destinados à indústria de papel e celulose. O eucalipto é caracterizado por elevadas taxas de crescimento e alta adaptabilidade resultante da interação de mecanismos moleculares e processos metabólicos ainda pouco conhecidos. Nesse sentido, o presente trabalho teve como objetivo principal o estabelecimento de perfis transcricionais comparativos para folhas e para o xilema em formação de árvores de Eucalyptus grandis. grandis. O uso do método SAGE permitiu analisar 5864 genes dos quais 2247 (38%) puderam ser identificados. Foram encontrados 464 genes diferencialmente expressos, sendo 47 exclusivamente expressos na região cambial e 64 nas folhas. Além disso, as estratégias adotadas na identificação tags-genes permitiram que as SAGE tags fossem eficientemente utilizadas na diferenciação de isoformas gênicas tecido-específicas e de localização celular específica. Genes relacionados à fotossíntese, fotorrespiração e destoxificação celular foram preferencialmente encontrados na biblioteca SAGE de folhas, enquanto genes relacionados à síntese da parede celular, organização do citoesqueleto e respiração, foram preferencialmente expressos na biblioteca SAGE de madeira. Os níveis de expressão dos transcritos sugeriram a ocorrência de possíveis mecanismos de controle transcricional comum para grupos de genes funcionalmente relacionados e foram também utilizados para sugestão de genes e processos que representam alvos potenciais para o melhoramento do eucalipto.
Recently genomic and post-genomic strategies have been used to study important tree species in planted forests. In the tropical countries, like Brazil, Eucalyptus species are especially important in commercial plantations destined for the paper and cellulose industry. Eucalyptus species are characterized by their high growth rates and great adaptability resulting from the interaction between molecular mechanisms and metabolic processes that are still uncharacterized. Thus, the main goal of this work was the comparison of the transcriptional profiles from leaves and the cambial region of Eucalyptus grandis trees. The use of SAGE allowed the evaluation of 5864 expressed tags of which 2247 (38%) could be identified. 464 differentially expressed tags were indicated, of which 47 were exclusively expressed in the cambial region library and 64 in the leaf library. Furthermore, the strategies used in the tag mapping process allowed an efficient use of the SAGE tags to distinguish gene isoforms with tissue-specific and/or specific cell localizations. Genes related to photosynthesis, photorespiration and cellular detoxification were preferentially found in the SAGE leaf library, while genes involved in cell wall biosynthesis, cytoskeletal organization and respiration were preferentially expressed in the developing xylem. Transcript expression levels suggested the presence of a common transcriptional control for a few functionally related genes, for example, some lignin related genes. Importantly, transcript expression levels were also used for the identification of target genes and processes potentially useful in future Eucalyptus breeding programs.

Des cellules isolees d'erable (acer pseudoplatanus l. ) cultivees dans une solution nutritive complete ou depourvue de fer ont ete comparees afin d'etudier les impacts d'une deficience en fer sur la cellule vegetale non photosynthetique. En premier lieu, nous avons observe que l'absence de fer dans le milieu de culture ralentit le rythme des divisions cellulaires et impose un arret de croissance apres 1 a 1,5 generations. A l'issue de la phase de division, les cellules presentent une senescence rapide et meurent de facon prematuree. Dans le but de cerner les evenements responsables de ces perturbations physiologiques, nous avons explore quelques fonctions metaboliques majeures de la cellule d'erable: la synthese des grandes chaines polycarbonees insaturees et la respiration. Ainsi, l'analyse des acides gras insatures et des carotenoides a montre que la carence de fer bloque severement certains mecanismes membranaires de desaturation en cis et en trans respectivement: la desaturation du c18:2 en c18:3 par les desaturases microsomiales et/ou plastidiales; la desaturation du phytoene, precurseur des carotenoides, par la phytoene desaturase de l'enveloppe plastidiale. L'implication du fer dans ce dernier systeme n'etait pas connue jusqu'a present. Pendant l'installation de la carence de fer, la respiration basale des cellules reste normale. Toutefois, la vitesse de la respiration decouplee ne cesse de decroitre sous l'effet du dysfonctionnement progressif de la succinate deshydrogenase: seul le transfert des electrons via le complexe ii de la chaine respiratoire est en effet altere, malgre une depletion generalisee en centres fer-soufre et en cytochromes mitochondriaux. A l'issue de la phase de division, les mitochondries fonctionnent au maximum de leurs capacites sans pouvoir repondre integralement aux besoins energetiques du metabolisme. S'ensuit alors un effondrement de l'activite respiratoire, associe a une disparition totale des cretes de la membrane interne des mitochondries. La desorganisation de la fonction respiratoire sous l'influence de la carence de fer est probablement le facteur responsable de la mort des cellules d'erable

Nous avons étudié les effets du glycérol sur la croissance et le métabolisme des cellules végétales non chlorophylliennes, en utilisant des cellules isolées d'érable sycomore (Acer Pseudoplatanus, l. ) et d'Echinochloa. Le glycérol pénètre dans les cellules par diffusion. Il est ensuite phosphoryle en SN-glycérol-3-P qui alimente la respiration, via les réactions terminales de la glycolyse. La néoglucogenèse est impossible car le SN-glycérol-3-P est un inhibiteur compétitif de la glucose-6-phosphate isomérase. Ainsi, la synthèse de saccharose, d'amidon et de composes pariétaux s'arrête. De plus le cycle oxydatif des pentoses cesse d'être alimente, ce qui entraine un arrêt de la synthèse des pentoses et de la réduction des nitrates. Lorsque le glycérol est utilise comme seule source de carbone, la croissance des cellules est alors stoppée. Pourtant le seul fonctionnement de la respiration mitochondriale suffit à entretenir durant plusieurs semaines la survie des cellules, qui ne présentent aucun signe d'autophagie. Par contre, une carence de glycérol entrainera brutalement le processus autophagique. Par ailleurs, nous avons observe que le glycérol entraine l'accumulation d'oligopolysaccharides de types B-1,3 et de phosphohomoserine (un intermédiaire de la synthèse d'isoleucine, issu de l'aspartate) lorsque le milieu de culture contient du saccharose. En utilisant l'homosérine (dont nous avons caractérise le transport actif dans les cellules et la métabolisation) et les herbicides inhibant l'acétolactate synthase, nous avons montre que le site principal de régulation de la synthèse in vivo de l'isoleucine est situe au niveau de la thréonine déshydratase. Une observation a été mentionnée en annexe : l'induction de la respiration insensible au cyanure par les inhibiteurs de l'acétolactate synthase

L'exposition de cellules d'érable à des concentrations sublétales de cuivre (50 m) s'accompagne d'un arrêt de l'augmentation de la masse fraîche au bout du 5#e#m#e jour de culture. Cet arrêt de croissance est précédé par une diminution de la respiration couplée et découplée, avec diminution concomitante des marqueurs mitochondriaux (fumarase, cytochrome oxydase, cardiolipide). Le cuivre provoque ainsi une dilution progressive des mitochondries dans la population cellulaire, due à un arrêt de la division des mitochondries précédent l'arrêt de la division des cellules. Lorsque le cuivre est soustrait du milieu, la division des mitochondries, puis des cellules, sont relancées. Nous disposons ainsi d'un levier pour analyser le contrôle du nombre de mitochondries dans les cellules. Par ailleurs, nous avons montré que l'oxydase alternative est étroitement contrôlée par le niveau de cuivre dans le milieu de culture des cellules (activité et quantité de protéine). Les effets de l'excès de cuivre sur la photosynthèse ont été étudiés en utilisant des chloroplastes purifiés présentant un haut degré d'intégrité morphologique et fonctionnelle (épinard et pois). La teneur de 1. 8 m de cuivre donne une inhibition (ic50) de 50% du dégagement d'oxygène, alors que sur les thylacoides purifiés on observe un ic50 apparent de 54 m. Ceci suggère que le cuivre agit essentiellement sur les enzymes du cycle de Benson. Enfin, nous avons observé une interférence entre le cuivre et le manganèse au niveau de la photosynthèse des chloroplastes intacts, et pas au niveau des thylacoides. Les analyses par oxygraphie et par fluorescence modulée suggèrent qu'il y a une compétition entre le cuivre et le manganèse au niveau de leur transport à travers l'enveloppe plastidiale.

Le cerveau adulte des mammifères conserve sa capacité à générer de nouvelles cellules cérébrales à partir de cellules souches neurales (CSNs), principalement localisées dans deux régions cérébrales spécifiques, l'hippocampe et la zone sous-ventriculaire (SVZ). Ce processus, appelé neurogenèse, permet la formation de nouveaux neurones et de nouvelles cellules gliales (astrocytes et oligodendrocytes). Différents signaux contrôlent la prolifération et la différenciation des CSNs. Parmi ces signaux, les hormones thyroïdiennes (HTs) sont impliquées dans la prolifération des CSNs de la SVZ et dans la différenciation neuronale. À l’inverse des cellules différenciées, telles que les neurones ou les glies, les CSNs ont un fonctionnement – ou métabolisme – principalement basé sur la glycolyse et sur une faible respiration mitochondriale. Or l'évolution du métabolisme des CSNs peut influencer leur choix de destin cellulaire. Les HTs jouant un rôle important dans l'activation du métabolisme mitochondrial, j'ai testé l'hypothèse selon laquelle le choix du destin des CSNs de la SVZ adulte se ferait grâce à l'influence de la signalisation thyroïdienne sur l'activité mitochondriale. J'ai tout d'abord montré in vivo et in vitro que les HTs permettent la détermination des CSNs en précurseurs neuronaux dans la SVZ, tandis qu'une période d'hypothyroïdisme favorise la détermination gliale. La transthyrétine, protéine de liaison des HTs, est spécifiquement présente dans les cellules de la SVZ ayant un destin neuronal, alors que la désiodase de type 3, inactivatrice des HTs, est exprimée par les précurseurs oligodendrocytaires (OPCs), indiquant une activationdifférentielle de la signalisation thyroïdienne dans les deux lignages cellulaires. Par ailleurs j'ai pu observer que les cellules s'engageant vers un destin neuronal possèdent une plus grande activité mitochondriale que les OPCs. La présence d'HTs favorise de plus la respiration mitochondriale, ainsi que la production de dérivés réactifs de l'oxygène (ROS) issus de l'activité des mitochondries, dans les cellules de la SVZ. Un blocage des protéines de la chaîne respiratoire empêche les HTs de promouvoir la détermination neuronale, montrant la nécessité de l'activation mitochondriale pour l'engagement des CSNs en précurseurs neuronaux. On sait d'autre part que les modifications morphologiques (ou dynamiques) mitochondriales sont nécessaires à l'augmentation de la respiration. La division (ou fission) des mitochondries est en particulier essentielle à une bonne répartition intracellulaire de la production de l'énergie issue de la respiration, ainsi qu'à la migration cellulaire. Dans les cellules de la SVZ, j'ai montré que l'action des HTs permet l'activation de la protéine DRP1, médiatrice de la fission mitochondriale, et ce principalement dans les cellules du lignage neuronal. Les HTs favorisent donc la détermination des CSNs de la SVZ vers un destin neuronal grâce à l'activation de la respiration et de la fission mitochondriales
The adult mammalian brain maintains its capacity to generate new cells from neural stem cells (NSCs), mainly localized in two specific brain regions, the hippocampus and the sub-ventricular zone (SVZ). This process, named neurogenesis, results in the production of new neurons and new glial cells (astrocytes and oligodendrocytes). Several signals control NSCs proliferation and differentiation. Among those, thyroid hormones (THs) are involved in NSCs proliferation in the SVZ and in neuronal differentiation. NSC metabolism relies mainly on glycolysis associated with a low mitochondrial activity, whereas mature cells, like neurons and glia, preferentially use oxidative phosphorylation. Changes in NSC metabolism can impact cell fate. As THs play an important part in activating mitochondrial metabolism, I hypothesized that the influence of TH signaling on mitochondrial activity triggers NSC fate choice in the adult SVZ. First, I showed in vivo and in vitro that THs allow NSC determination in neuronal precursors, whereas a short hypothyroidism favors glial determination. Transthyretine, a TH binding protein, is specifically present in the SVZ cells having a neuronal fate, while type 3 deiodinase, a TH inhibitor, is expressed by oligodendrocyte precursor cells (OPCs). These results indicate that THs signaling isdifferentially activated in neuronal and glial cell lineages. I observed that cells adopting a neuronal fate display a greater mitochondrial activity when compared to OPCs, and that TH signaling favors mitochondrial respiration and ROS production in the SVZ cells. Inhibiting the mitochondrial respiratory chain prevents TH-mediated promotion of neuronal determination, proving the need of mitochondrial activation for NSC commitment toward a neuronal phenotype. Besides, it is also known that modifications of mitochondrial morphology (or mitochondrial dynamics) are required for the respiration to increase. Among mitochondrial dynamics, fission is crucial for a good intracellular repartition of energy production, and for cell migration. In the SVZ cells, I showed that, DRP1, the main inducer of mitochondrial fission, is activated by THs mainly in cells adopting a neuronal fate. Thus, THs favor NSC fate choice toward a neuronal phenotype through the activation of mitochondrial metabolism and mitochondrial fission in the adult mouse SVZ

Neuroepithelial cells (NECs) of the zebrafish gill filament have been previously identified as bimodal O2 and CO2/H+ sensors that depolarize in response to chemostimuli via inhibition of background K+ channels. To further elucidate the signaling pathway underlying CO2/H+ chemoreception in the NECs we employed microspectrofluorometric techniques to examine the effects of hypercapnia on [Ca2+]i and pHi. NECs increased their [Ca2+]i in response to acidic hypercapnia (5% CO2, pH 6.6) and isocapnic acidosis (normocapnia, pH 6.6), but not to isohydric hypercapnia (5% CO2, pH 7.8). The acid- induced increase in [Ca2+]i persisted in the absence of extracellular Ca2+, and Ca2+ channel blockers (Cd2+, Ni2+ and nifedipine). NECs exhibited a rapid and reversible drop in pHi in response to acidic hypercapnia and isohydric hypercapnia. Isocapnic acidosis also induced intracellular acidification within NECs, but it was less severe than the drop in pHi elicited by acidic hypercapnia and isohydric hypercapnia. The rate and magnitude of intracellular acidification was reduced by the CA-inhibitor, acetazolamide, without effect on the acid-induced increase in [Ca2+]i. Acetate was used to investigate the relationship between pHi and [Ca2+]i. Acetate induced intracellular acidification without augmentation of [Ca2+]i. The results of this thesis demonstrate that (1) extracellular acidification, but not CO2, is critical to the hypercapnia-induced increase in [Ca2+]i (2) the increase in [Ca2+]i is independent of the drop in pHi (3) the increase in [Ca2+]i is not mediated by the influx of Ca2+ across the plasma membrane.

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Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior
The improper disposal of nitrogen in receiving water courses causes problems such as toxicity to living beings through the consumption of oxygen to meet the nitrogen demand, eutrophication and nitrate contamination of aquifers. For this reason it is often necessary to be carried out complementary treatment of wastewater to eliminate or reduce the concentration of this compound in the wastewater. The objective of this study is to evaluate the biological removal of nitrogen compounds using submerged aerated and anoxic filters as post-treatment of an anaerobic system, with low cost and innovative technology, which in previous studies has shown high removal efficiency of organic matter and great potential biological nitrogen compounds removal. The simple design with perforated hoses for air distribution and filling with plastic parts proved to be very efficient in relation to organic matter removal and nitrification. The system presented, in the best stage, efficiency in converting ammonia to nitrate by 71%, and produced a final effluent concentration below 10 mg / L of NH3-N. In addition, carbon concentration was removed by 77%, producing final effluent with 24 mg/L COD. However, denitrification in anoxic filter was not effective even with the addition of an external carbon source. There was a reduction of up to 56% of nitrogen caused by the process of simultaneous nitrification and denitrification (SND). The high voids space presented by this type of support material coupled with direct aeration of the sludge, allows the respiration of biomass retained between the endogenous phase, increased cell retention time and sludge retention capacity, producing a final effluent with turbidity less than 5 UT and total suspended solids around 5.0 mg/L
A disposi??o inadequada do nitrog?nio em corpos receptores gera problemas como: toxicidade para seres vivos; consumo de oxig?nio do meio para atender a demanda nitrogenada; eutrofiza??o; e contamina??o dos aqu?feros por nitrato. Por esta raz?o ? muitas vezes necess?rio que seja realizado tratamento complementar dos esgotos para eliminar, ou reduzir, a concentra??o deste composto nas ?guas residu?rias. O objetivo deste trabalho ? avaliar a remo??o biol?gica dos compostos nitrogenados utilizando filtros aerados submersos como p?s-tratamento de um sistema anaer?bio, com tecnologia inovadora, de baixo custo, que em estudos anteriores demonstrou grande efici?ncia na remo??o de mat?ria org?nica carbon?cea e grande potencial na remo??o biol?gica de compostos nitrogenados. A forma simples como o sistema foi concebido, com mangueiras perfuradas para distribui??o do ar e preenchimento com pe?as pl?sticas - condu?te cortado - mostrou-se bastante eficiente em rela??o ? remo??o de mat?ria org?nica e na nitrifica??o. O sistema apresentou, na melhor fase, efici?ncia na convers?o de nitrog?nio amoniacal em nitrato de 71%, e produziu efluente final com concentra??o de N-NH3 inferior a 10 mg/L. Al?m disso, observou-se uma redu??o de 77% na concentra??o de carbono, produzindo efluente final com 24 mg/L de DQO. Contudo a desnitrifica??o no filtro an?xico n?o se mostrou eficiente mesmo com a adi??o de uma fonte externa de carbono. Mesmo assim observou-se redu??o de at? 56% do nitrog?nio causado pelo processo de Nitrifica??o e Desnitrifica??o Simult?neas (SND). O grande ?ndice de vazios apresentado por este tipo de material suporte aliado ? aera??o direta do lodo, permite que a respira??o da biomassa retida entre na fase end?gena, aumentado o tempo de reten??o celular e a capacidade de reten??o de lodo, produzindo um efluente final com turbidez inferior a 5 UT e SST em torno de 5,0 mg/L

Intense exercise is known to cause alterations in the psychophysiological status of an athlete. Monitoring the health and recovery of an athlete is imperative for the maintenance of performance and reduced fatigue and injury incidence. The physicality associated with select sports results in significant elevations and suppression of psychophysiological biomarkers that are often modulated by game-related impacts, intense training regimes and psychosocial aspects associated with the professional era. The aim of the studies outlined in this thesis were to determine the effectiveness of selected “stress” markers in several sports that result in significant “stress”, and quantify the level of acute and chronic “stress” following individual games and competitions to improve athlete management and recovery. Study one aimed at developing a new strong-cation exchange high performance liquid chromatography (SCX-HPLC) method for the detection and quantification of urinary pterins and creatinine in a body-building cohort completing high intensity resistance training. The method had an intra- and inter-assay variability of 3.04 % and 5.42 % respectively, with visibly clear peaks and no tailing. Urinary neopterin (NP) and 7,8-dihydroneopterin during a week of competitive natural body-building did not significantly change indicating no alteration in immune system function and oxidative stress. It did provide evidence for the use of specific gravity as a similarly reliable method for urine volume correction following exercise. Study two focused on a playoff game of elite amateur rugby. The time course changes of NP, cortisol, salivary immunoglobulin A (sIgA) and myoglobin in 11 elite amateur rugby players were measured up to 86 hours post-game. Cortisol increased 4-fold, myoglobin 2.85-fold, NP 1.75-fold and total NP 2.3-fold, all significant, whilst sIgA did not change. All markers returned to baseline within 17 hours providing valuable information for sample collection schedule optimization. Respiratory elastance was also measured by ventilation for the assessment of exercise induced lung inflammation/injury following the game (Chapter three). There was an increase in elastance in selected individuals that did not correlate with either global positioning system (GPS) or impact data. It was shown however, that a ventilator is capable of measuring respiratory changes in a conscious and healthy individual. Study three focused on the final three games of professional rugby in the 2013 ITM Cup. The acute and cumulative changes in the same four markers were analysed following three home games. There were significant increases in NP, total NP, cortisol and myoglobin along with significant suppression of sIgA (p < 0.05). Large intra- and inter-individual variation existed between players with changes associated with total impacts. Moreover, impact induced muscle damage may account for changes in oxidative status. Specific gravity (SG) was shown to be a more reliable marker for urine volume correction in comparison to creatinine; while some players showed signs of cumulative stress. Study four examined stress in a professional team throughout the 22 week 2014 Super 15 competition. Part one investigated changes in oxidative stress and muscle damage markers to solidify the muscle damage/oxidative status theory postulated in the previous study. Experimental evidence showed iron and myoglobin are separately capable of oxidizing 7,8-dihydroneopterin to NP in vitro. It was then identified that players who suffered the greatest muscle damage as a result of impacts also had the greatest change in oxidative status (NP). This evidence suggests rugby union induces significant alterations in oxidative status that may be exacerbated by the impact induced release of myoglobin. Part two measured urinary NT-proBNP during the last two consecutive home games to identify whether rugby union causes significant cardiovascular stress and if the pre to post-game change can be explained by GPS technology. Significant individualized elevations were observed in games one and two which did not correlate with any GPS measurements or impacts. Concentrations returned to normal ~ 36 hours post-game suggesting no permanent damage to cardiac muscle had occurred. The lack of correlation suggests GPS technology is not an accurate measure of cardiovascular stress in professional rugby union. Part three involved the measurement of cortisol, total NP and sIgA throughout the season to assess the degree of cumulative stress. Samples were taken at regular intervals ~ 36 hours post-game for 22 weeks. Extreme inter-individual variation was present. Select individuals showed continual elevation in immune system activation and psychophysiological stress, whilst others presented with a continual decline in immune system function. Collectively however, minor deviations from baseline in all markers were observed and participation in long distance travel did not significantly affect the psychophysiological status of the group. Together this suggests a season does not cause an accumulation in psychophysiological stress, although careful individual player analysis is warranted. Understanding rugby union positional demands is essential for training program specification and position specific development of players. Part four used GPS, video-analysis and biochemical analysis to identify positional demands in five regular season games. Forwards tended to be involved in more impacts and covered less distance, while backs covered more distance and carried the ball into contact more regularly. There was no difference in the psychophysiological status between positions indicating both aspects of stress (impacts and distance covered) may induce a similar response. Alternatively, individual biological variation may be solely responsible for this change suggesting careful consideration should be given when using traditional work-load measures such as GPS when quantifying “stress”. Part five assessed the effectiveness of varied recovery interventions. Total NP, cortisol, myoglobin and sIgA were measured pre- post- and ~ 36 hours post game to identify which intervention was most effective at returning players to a psychophysiological state that allowed for the resumption of normal training. Findings concluded the immediate post-game strategy employed by the team (cold bath, consumption of protein and carbohydrates, compression garments and eight hours sleep) seemed to provide the greatest psychophysiological improvement regardless of the “next-day” intervention. There was large inter-individual variation and players were still in a state of recovery ~ 36 hours post-game as indicated by the elevated total NP and sIgA concentrations. Study five had four aspects. Develop a new, cost-effective and simple reverse phase HPLC (RP-HPLC) method for the quantification of urinary myoglobin in a clinically relevant range, quantify the level of structural stress following a simulated mixed martial arts (MMA) contest, determine whether cold water immersion attenuates the level of inflammation and muscle damage following a contest, and whether this hypothesized attenuation may be explained by cryotherapy induced mononuclear cell activation suppression in vitro. The RP-HPLC method had an intra- and inter-assay variations from 0.32 - 2.94 %. Linearity was in the range of 5 – 1000 µg/mL which detected significant increases in urinary myoglobin following the MMA contest. Total NP was found to significantly increase following the contest and return to approximately pre-contest levels 24 hours later for the passive group only. Cold water immersion was further found to attenuate the total NP increase in the first two hours post-contest solidifying its use as a recovery technique following intense exercise, while cryotherapy significantly suppressed T-cell activation. This study provides a reliable and repeatable assay for muscle damage quantification in a clinically relevant range, evidence of the physicality associated with MMA, and indicates cold water immersion is a reliable recovery intervention that may impart its positive benefits through T-cell suppression. The data generated by these investigations highlights the necessity for individual physiological analysis. Group data often masks the extreme variation that exists in clinical and exercise trials where treatment and management of athletes is conducted for recovery and performance. Biochemical analysis provides an added sophistication of work-load and psychophysiological assessment that common technological methods cannot emulate. With a lack of correlation between the quantitative changes in specific non-overlapping biomarkers and GPS, video-analysis and questionnaires, it would seem pertinent to develop a non-invasive quantitative approach in elite sport to understand the level of exercise-induced psychophysiological stress for the precise management of athletes.

Un problème fondamental pendant l'évaluation in vitro de la toxicité de composés organiques volatils (COVs) est le manque de connaissance de l'évolution de la concentration des COVs à laquelle les systèmes vivants sont exposés au cours des études expérimentales. Ce travail présente un nouveau dispositif expérimental conçu pour étudier l'exposition des systèmes vivants aux COVs. Le dispositif est formé de deux compartiments séparés par une membrane hydrophobe poreuse et permet des durées relativement longues de manipulations sans restreindre la respiration cellulaire. Une modélisation théorique qui couple la conservation de masse et du moment entre les différentes phases et la respiration des cellules hybridomes (ATCC CRL-1606) au sein du dispositif a été développée. Le modèle permet de prédire l'évolution de la concentration des COVs, de l'oxygène et du dioxyde de carbone dans le dispositif. Les résultats simulés pour le transfert des COVs ont revélé une bonne concordance avec les résultats expérimentaux et ont montré que le type de membrane et son diamètre, le coefficient de partage des COVs et la hauteur de la phase liquide ont une influence significative sur l'évolution de la concentration de ceux-ci dans la phase liquide. Néanmoins la disponibilité de l'oxygène au niveau des cellules dépend principalement de la densité cellulaire initiale, de la vitesse spécifique de consommation de ce gaz et de la hauteur du liquide alors que les paramètres liés à la membrane ont une influence sur le contrôle du pH
A major problem during in vitro evaluation of the toxicity of volatile organic compounds (VOC) is the lack of knowledge of the evolution of the concentration of such compounds during the course of experimental studies with living systems. This work presents the design of a novel experimental device for the study of cell culture exposure to VOCs. The device is made of two compartments separated by a porous hydrophobic membrane and allows relatively long durations of handling without restricting cellular breathing. A theoretical modeling which couples mass and moment conservation between the different phases inside the device with the breathing kinetics of hybridoma cells (ATCC CRL-1606) was developed. The model allows predicting the evolution of the concentration of the VOCs, the oxygen and the carbon dioxide inside the device. The simulations of the mass transfer of the VOCs simulated presented a good agreement with experiments and showed that the type of membrane and its diameter, the VOCs partition coefficient and the height of the liquid phase have a significant influence on the evolution of their concentration in the liquid phase. Nevertheless, the availability of oxygen for the cells depends mainly on the initial cellular density, the specific kinetics of consumption of this gas and on the height of the liquid phase, whereas the parameters related to membrane have an influence on the control of the pH

RESUMO: O corpo carotídeo (CB) é um pequeno órgão sensível a variações na PaO2, PaCO2 e pH. As células tipo I (células glómicas) do corpo carotídeo, as unidades sensoriais deste órgão, libertam neurotransmissores em resposta às variações dos gases arteriais. Estes neurotransmissores atuam quer em recetores pré-sinápticos, localizados nas células tipo I, quer em recetores póssinápticos, localizados nas terminações do nervo do seio carotídeo, ou em ambos. A activação dos recetores pré-sinápticos modula a atividade do corpo carotídeo, enquanto que, a activação dos recetores pós-sinápticos, de carater excitatório, desencadeia um aumento da frequência de descarga das fibras do CSN, com subsequente despolarização dos neurónios do gânglio petroso, e posterior despolarização de um grupo específico de neurónios do centro respiratório central, desencadeando, como resposta final, hiperventilação. Estes recetores pré- e pós-sinápticos podem ser classificados em ionotrópicos ou metabotrópicos, estando os últimos acoplados a adenilatos ciclases transmembranares (tmAC). O mecanismo exato pelo qual as variações dos gases arteriais são detetadas pelo CB não se encontra ainda completamente elucidado, mas tem sido sugerido que alterações nos níveis de cAMP estejam associadas ao mecanismo de deteção de variações de O2 e CO2. Os níveis de cAMP podem ser regulados através da sua via de síntese, mediada por dois tipos de adenilatos ciclases: tmAC sensível aos eurotransmissores e adenilato ciclase solúvel (sAC)sensível a variações de HCO3/CO2, e pela sua via de degradação mediada por fosfodiesterases. A via de degradação do cAMP pode ser manipulada farmacologicamente, funcionando enquanto alvo terapêutico para o tratamento de patologias do foro respiratório (e.g. asma, hipertensão pulmonar, doença pulmonar obstructiva crónica e apneia do sono), que induzem um aumento da actividade do CB.O trabalho descrito nesta dissertação partiu da hipótese de que a actividade do CB é manipulada por fármacos, que interferem com a via de sinalização do cAMP, tendo sido nosso objectivo geral, investigar o papel do cAMP na quimiotransdução do CB de rato, e determinar se a actividade dos enzimas responsáveis pela via de sinalização do cAMP é ou não regulada por variações de O2/CO2. Assim, a relevância deste trabalho é a de estudar e identificar possíveis alvos moleculares (sAC, isoformas de tmAC e PDE) com potencial para serem usados no tratamento de patologias relacionadas com o controlo respiratório. A primeira parte do presente trabalho, centrou-se na caracterização farmacológica da PDE4 no CB e em tecidos não quimiorecetores (e.g. gânglio cervical superior e artérias carótidas), e na observação do efeito de hipóxia aguda na acumulação dos níveis de cAMP, induzidos pelos inibidores de PDE, nestes tecidos. A quantificação de cAMP foi efectuada por técnica imunoenzimática (EIA), tendo sido elaboradas curvas de dose-resposta para os efeitos de inibidores, não específicos (IBMX) e específicos para a PDE2 e PDE4 (EHNA, Rolipram e Ro 20-1724), nos níveis de cAMP acumulados, em situações de normóxia (20%O2/5%CO2) e hipóxia (5%O2/5%CO2). A caracterização das PDE no gânglio cervical superior foi aprofundada, utilizando-se a técnica de transferência de energia de ressonância por fluorescência (FRET) em culturas primárias de neurónios, na presença de inibidores não específicos (IBMX) e específicos para a PDE3 e PDE4 (milrinone e rolipram, respetivamente). Foram igualmente estudadas, através de RT-qPCR, as alterações na expressão de PDE3A-B e PDE4A-D, no gânglio cervical superior, em resposta a diferentes percentagens de oxigénio. Na segunda parte do trabalho investigou-se a via de síntese do cAMP no CB em resposta a variações na concentração de HCO3/CO2. Em concreto, o protocolo experimental centrou-se na caracterização da sAC, dado que a sua actividade é regulada por variações de HCO3/CO2. A caracterização da expressão e regulação da sAC, em resposta a variações de HCO3/CO2 ,foi efectuada no CB e em tecidos não quimioreceptores periféricos (e.g. gânglio cervical superior, petroso e nodoso) por qRT-PCR. A actividade deste enzima foi caracterizada indirectamente através da quantificação dos níveis de cAMP (quantificação por EIA), induzidos por diferentes concentrações de HCO3/CO2, na presença de MDL-12,33-A, um inibidore da tmAC. A expressão das isoformas da tmAC no CB e gânglio petroso foi determinada por RT-qPCR. Adicionalmente, estudámos a contribuição relativa da tmAC e sAC no mecanismo de sensibilidade ao CO2 no CB. Para o efeito foram estudadas as alterações: 1) nos níveis de cAMP (quantificado por EIA) na presença de diferentes concentrações de HCO3/CO2 e ao longo do tempo (5-30 min); 2) na ativação da proteína cinase A (PKA, FRET baseado em sensores) em células tipo I do CB; e 3) na frequência de descarga do CSN (registos) na presença e ausência de ativadores e inibidores da sAC,tmAC e PKA. Por último, foi caracterizada a expressão e actividade da sAC nos quimioreceptors centrais (locus ceruleus, rafe e medula ventro-lateral) através de técnicas de RT-qPCR e EIA. A expressão das isoformas da tmAC foi aprofundada no locus coeruleus através de RT-qPCR. Por fim, comparámos a contribuição da tmAC e sAC nos níveis de cAMP no locus coeruleus em condições de normocapnia e hipercapnia.O nosso trabalho teve os seguintes resultados principais: 1) PDE4 está funcional no corpo carotídeo, artérias carótidas e gânglio cervical superior de rato, embora a PDE2 só se encontre funcional neste último; 2) Os efeitos dos inibidores de PDE nos níveis de acumulação de cAMP foram exacerbados em situações de hipóxia aguda no CB e artérias carótidas, mas foram atenuados no gânglio cervical superior; 3) No gânglio cervical superior, diferentes tipos de células apresentaram uma caracterização específica de PDEs, sugerindo uma subpopulação de células neste gânglio com funções fisiológicas distintas; 4) Embora todas as isoformas de PDE4 e PDE3 estivessem presentes no gânglio, a PDE3a, PDE4b e a PDE4d foram as isoformas mais expressas. Por outro lado, incubações de gânglio cervical superior, em diferentes percentagens de oxigénio, não alteraram (não regularam) significativamente a expressão das diferentes isoformas de PDE neste órgão; 5) a sAC encontra-se expressa e funcional no CB e nos quimiorecetores centrais estudados (locus coeruleus, rafe e medula ventrolateral). A sAC apresenta maior expressão no CB comparativamente aos restantes orgãos estudados, exceptuando os testículos, orgão controlo. Variações de HCO3/CO2 de 0/0 para 24/5 aumentaram os níveis de cAMP no CB e quimiorecetores centrais, tendo sido o aumento mais significativo observado no CB. Concentrações acima dos 24mM HCO3/5%CO2 não induziram alterações nos níveis de cAMP, sugerindo que a actividade da sAC se encontra saturada em condições fisiológicas (normocapnia) e que este enzima não desempenha qualquer papel na deteção de situações de hipercapnia; 6) No CB, a expressão das isoformas tmAC1, tmAC4, tmAC6 e tmAC9 é mais elevada comparativamente à expressão da sAC; 7) Utilizamos diferentes inibidores da tmAC (MDL 12-330A, 500μM, 2’5’-ddADO, 30-300μM, SQ 22536, 200μM) e da sAC (KH7, 10-100μM) para estudar a contribuição relativa destes enzimas na acumulação do cAMP no CB. Tanto a tmAC como a sAC contribuem para a acumulação dos níveis de cAMP em condições de hipercapnia. Contudo, existe um maior efeito destes inibidores nas condições de 12 mM HCO3/2.5%CO2 do que em condições de normocapnia e hipercapnia, sugerindo um papel relevante destes enzimas na atividade do CB em situações de hipocapnia; 8) Não se observaram variações nos níveis de cAMP em resposta a diferentes concentrações de HCO3/CO2 ao longo do tempo (5-30 min). O efeito inibitório induzido por ddADO e KH7 foi sobreponível após 5 ou 30 minutos de incubação em todas as concentrações de HCO3/CO2 estudadas; 9) Por último, verificou-se um aumento na frequência da descarga do nervo do seio carotídeo entre as condições de normocapnia e hipercapnia acídica. Ao contrário do KH7 (10μM), o 2’5’-ddADO reduziu significativamente a frequência de descarga do nervo, quer em condições de normocapnia quer de hipercapnia acídica. Contudo, não se verificou aumento na frequência de descarga do nervo entre normocapnia e hipercapnia isohídrica, sugerindo que a sensibilidade à hipercapnia no CB é mediada por variações de pH. Em conclusão, os resultados decorrentes deste trabalho permitiram demonstrar que, embora os enzimas que medeiam a via de sinalização do cAMP possam ser bons alvos terapêuticos em condições particulares, a sua actividade não é específica para o CB. Os resultados sugerem ainda que o cAMP não é um mediador específico da transdução à hipercapnia neste orgão. Contudo, os nossos resultados demonstraram que os níveis de cAMP são mais elevados em condições fisiológicas, o que sugere que o cAMP possa ter uma função homeostática neste orgão. Por último, o presente trabalho demonstrou que os aumentos de cAMP descritos por outros em condições de hipercapnia, não são observáveis quando o pH se encontra controlado. ------------------ ABSTRACT: The work presented in this dissertation was aimed to establish how specific is cAMP-signaling pathways in the CB mainly in different CO2 conditions and how O2 concentrations alter/drives the manipulation of cAMP signaling in the CB. The experimental studies included in this thesis sought to investigate the role of cAMP in the rat CB chemotransduction mechanisms and to determine whether the enzymes that participate in cAMP signal transduction in the CB are regulated by O2/CO2. We characterized the enzymes involved in the cAMP-signaling pathway in the CB (sAC, tmAC, PDE) under different O2/CO2 conditions. Our results demonstrated that many of these enzymes are involved in CO2/O2 sensing and while they may be useful in treating conditions with alterations in CO2/O2 sensing,they will not be specific to chemoreception within the CB: 1) PDE4 is ubiquitously expressed in CB and non-chemoreceptor related tissues and their affinity to inhibitors change with O2 tensions in both CB and carotid arteries, and 2) sAC and tmAC are expressed in peripheral and central chemo- and non-chemoreceptor tissues and their effect on cAMP levels do not change between normocapnic and isohydric hypercapnic conditions. Our results provide evidence against a specific role of cAMP as a mediator for O2 and CO2 chemotransduction in the rat CB and emphasized the role of pH in CO2 sensitivity of the CB. Furthermore, our results demonstrate that cAMP levels are maintained higher under physiological conditions, supporting recent finding from our lab, which all together suggests that cAMP has a homeostatic function in this organ.

Dans ce travail de thèse, nous nous sommes intéressés aux rôles de l’hypoxie intermittente (HI) sur l’inflammation respiratoire. Dans un premier travail, nous avons caractérisé le profil inflammatoire des cellules épithéliales nasales humaines (CENH) en réponse à l’HI et mis en évidence une augmentation significative de la sécrétion d’IL-8, de PDGF AA, de VEGF et de gélatinases. L’IL-8 sécrétée était active comme en atteste le pouvoir chémotactique majeur des surnageants de CENH envers les neutrophiles. De plus, nous avons montré que l’HI per se était responsable d’une augmentation de la migration des neutrophiles et que l’addition d’IL-8 potentialisait cet effet, d’autant plus qu’elle agissait sur des neutrophiles de patients présentant un syndrome d’apnées/hypopnées du sommeil, déjà activés. Dans le second travail, nous avons mis en évidence une réponse spécifique des cellules musculaires lisses bronchiques à l’HI avec une sécrétion de VEGF et surtout une augmentation de leurs capacités de migration. De surcroît, nous avons montré que le surnageant des CENH soumises à l’HI induisait une augmentation majeure de ces capacités de migration, dépendant de la sécrétion de PDGFAA. Nos études montrent que l’HI induit une réponse inflammatoire des cellules résidentes de l’arbre trachéobronchique, cellules épithéliales et musculaires lisses. L’utilisation d’un modèle in vitro a permis d’isoler cette réponse tissulaire spécifique de l’influence d’une inflammation vasculaire et systémique. Ces résultats peuvent expliquer la neutrophilie observée dans les expectorations des apnéiques ainsi que les résultats fonctionnels respiratoires de ces patients
In present work, we addressed the role of Intermittent Hypoxia (IH) on respiratory inflammation. In a first study, we characterized the inflammatory profile of the human nasal epithelial cells (CENH) in response to IH and showed a significant increase of the IL-8, PDGF AA, VEGF and gelatinases secretion. The released IL-8 was active, as shown by the ability of supernatants to induce neutrophil migration. Furthermore, we showed that IH per se induced neutrophil migration and that IL-8 had an additive effect, especially on already activated neutrophils from patients suffering from sleep apnea/hypopnea syndrome (SAHS). In the second study, we showed a specific response of bronchial smooth muscle cells (BSMC) to IH with induction of VEGF secretion and an increase in migration capacities. In addition, supernatants of CENH exposed to IH induced a major increase of BSMC migration, dependent on PDGFAA secretion. Our studies show that IH leads to an inflammatory response of the resident tracheobronchial cells, both epithelial and smooth muscle cells. The use of an in vitro model allowed to isolate the specific tissular response from the influence of a vascular and systemic inflammation. These results can explain the neutrophilia observed in induced sputum of patients with SAHS, as well as the results of their pulmonary functional tests

Examines the regulation of hexose transport in a human fibroblast cell strain deficient in HADH CoQ reductase. Observations from this study "provide a basis in understanding the molecular mechanism(s) of transport regulation in human cells."

Commercial scale mammalian cell culture systems have many technical barriers: (i) accumulation of inhibitory waste products; (ii) sensitivity to low level of shear; and (iii) cellular oxygen demand in excess of bioreactor's capability to provide oxygen without damaging cells. Because of these problems, systematic approaches for providing sufficient oxygen transfer rates to large scale mammalian cell culture systems have yet to be adequately developed. This work, however, suggests a unique approach to this problem from a different point of view. A research program has been undertaken to determine if the metabolism of mammalian cells can be manipulated to minimize or eliminate the oxygen transfer capabilities required by the cells from a given bioreactor. The respiration inhibitor, rotenone, has been added to the cultures of a T cell fusion partner, Jurkat, at levels that completely inhibit respiration. Growth of the cells, at these concentrations of rotenone in completely defined media, did not occur unless the media was supplemented with at least 1 mM pyruvate or at least 1% fetal bovine serum. Respiration inhibited cells utilized much less glutamine, respiratory fuel, and concomitantly produces much less ammonia. They grew to one million cells per ml at near normal rate but the maximum cell density was 65% that of un-inhibited cultures. Proline and a minimum amount of glutamine (0.2 mM) were found to be essential for the growth of Jurkat cells in the presence of rotenone. Intracellular thiol content measured with flow cytometry technique was found to be lower for rotenone cultures, possibly due to the free radicals formation at higher oxygen concentration. With continuous reactor experiments, the fact that Jurkat cells grow without using oxygen in the presence of rotenone was firmly confirmed and more metabolic details were found. In addition to the increased glycolysis, respiration inhibited culture increased the utilization of arginine, asparagine, serine, histidine, and aspartic acid and increased the production of lysine, glycine, glutamic acid, and alanine. At steady state with 90 nM of rotenone, pulse inputs of proline and pyruvate increased the cell density up to 1.4 million cells per milliliter.

M.Tech.
Low-intensity laser irradiation (LILI), also known as photo-biostimulation, is a type of phototherapy which is based on the application of low power monochromatic and coherent light mostly in the wavelength range of 600 to 1 000 nm to injuries and lesions to stimulate healing. LILI has been shown to increase adenosine triphosphate (ATP) synthesis, cell proliferation (Silveira et al., 2007 and 2009; Hawkins and Abrahamse, 2006a) and collagen synthesis, and release of growth factors from cells (Eells et al., 2004). LILI has not been fully embraced and is mainly due to the fact that the biochemical mechanisms underlying the positive effects are not completely understood (Hamblin and Demidova, 2006). Though the mechanisms of photo-biostimulation on a variety of mitochondrial enzymes have been proposed and studied by different researchers, most of the mechanisms are based on oxygen consumption studies and lack direct experimental support (Chen et al., 2008). This project was designed to study the enzyme activity and expression of the mitochondrial respiratory chain complexes (I to V) post-irradiation with a wavelength of 660 nm and a fluence of 5 or 15 J/cm2 in isolated human skin fibroblast cells. Diabetes mellitus (DM) is a group of diseases marked by high levels of blood glucose resulting from defects in insulin production, insulin action or both. It is found worldwide and is estimated to affect 1.1% of the world population (World Health Organization, WHO, 2002). Estimates from 2009 by the International Diabetes Federation suggest that the number of adults with diabetes in the world will expand by 54%, from 284.6 million in 2010 to 438.4 million in 2030. The projected growth for sub-Saharan Africa is 98%, from 12.1 million in 2010 to 23.9 million in 2030 (Mbanya et al., 2010). Though DM is more common in industrialized countries, the trend is changing.

Bone has several crucial functions including mechanical support of movement, hematopoiesis, maintenance of mineral homeostasis, and energy regulation. Bone also undergoes continuous remodeling to maintain its structural integrity, which suggests it has strong respiration and energy consumption capability. It has been shown that during development, bones, in particular, osteoblasts, rely on glucose uptake for proper skeletal development. However, the effect of energy utilization on osteocytes’ function is currently unknown. Osteocytes are terminally differentiated osteoblasts and are deeply embedded into the mineralized matrix of bone. Previous studies have shown that PTH promotes bone anabolism, in part, by stimulating osteoblasts anaerobic glycolysis while suppressing glucose oxidation through the TCA cycle. In osteocytes, PTH suppresses Sost expression (the gene encoding a potent inhibitor of bone formation) by inducing HDAC4/5 nuclear translocation and MEF2C inhibition. Recently, Scriptaid, an HDAC complex inhibitor, has been shown to induce Mef2 expression and exercise-like adaptation in mouse muscles. In myocytes, Scriptaid disrupts the HDACs co-repressor complex and induces nuclear export of HDAC4/5 with MEF2 activation. This will subsequently increase the expressions of several genes related to energy utilization such as Glut4 and Pdk4. Thus we hypothesized that Scriptaid might regulate Sost and Glut4 expression in osteocytes. To investigate the effect of Scriptaid on osteocytes, we treated a mouse osteocytic cell line, Ocy454-12H, with Scriptaid. Unexpectedly, Scriptaid potently suppressed Sost, whereas it increased Glut4 expression. Scriptaid stimulated osteocyte respiration and glucose consumption rate. Mechanistically, Scriptaid treatment of Ocy454-12H induced nuclear translocation of Hdac5 whereas it did not affect Hdac4. Silencing of Hdac5 expression with shRNA increased Sost basal expression and blocked Sost suppression induced by Scriptaid. However, Glut4 up-regulation by Scriptaid was independent of the HDAC4/5-MEF2C pathway. Glut4 luciferase reporter assays demonstrated that two additional transcription factors binding sites, O/E&NF1 and C/EBPα, may mediate Scriptaid-induced Glut4 up-regulation. Taken together, these data demonstrate that in osteocytes Scriptaid suppresses Sost expression through regulating HDAC5-MEF2C signaling. However, Scriptaid increases Glut4 expression through Hdac5-independent mechanisms, and dependent on O/E&NF1 and C/EBPα.

Cell death in the mesocarp of berries occurs late in the ripening process, and may influence berry sensory attributes and water retention. There are cultivar-dependent correlations between mesocarp cell death and berry shrivel. Cell death is likely to be associated with yield losses of up to 30% for Shiraz due to berry shrivel, which concentrates sugars and potentially leads to high alcohol content in wine. The main objective of this thesis was to investigate the association between berry internal oxygen concentrations and berry cell death. Experiments were carried out at the Waite vineyards and at the SARDI research vineyard in Nuriootpa. Firstly, in Chapter 2, internal oxygen concentration ([O₂]) across the mesocarp was measured in berries from Chardonnay and Shiraz, both seeded, and Ruby Seedless, from the Waite vineyards, using an oxygen micro-sensor. Change of berry and seed respiration was investigated in Chardonnay. The lenticel density of Chardonnay and Shiraz berry pedicels (stem and receptacle) was assessed. We then tested the long-term effect of blocking pedicel lenticels on berry internal [O₂] profiles and cell death. Air spaces within the Chardonnay berries at different developmental stages were visualized using x-ray micro-CT. Second, in Chapter 3, a factorial trial of two irrigation regimes was applied in season 2014/2015 and a factorial trial of two irrigation regimes and two temperatures was applied in season 2015/2016, in Nuriootpa. Midday stem water potential, stomatal conductance and photosynthetic rate were measured to examine the efficiency of drought and canopy heating treatments. The oxygen micro-sensor was used to measure oxygen concentration in grapes and their respiration rate. Lastly, in Chapter 4, we tested the effects of overhead shading (2014/2015), rootstocks and kaolin application (both in 2016/2017), on Shiraz berry cell death and berry shrivel. In Chardonnay, Shiraz and Ruby Seedless grapes, steep [O₂] gradients were observed across the skin and [O₂] decreased toward the middle of the mesocarp. As ripening progressed, the minimum [O₂] approached zero in the seeded cultivars and correlated to the profile of cell death across the mesocarp. Seed respiration declined during ripening, from a large proportion of total berry respiration early to negligible at latter stages. [O₂] increased towards the central axis corresponding to the presence of air spaces visualised using x-ray micro-CT. These air spaces connect to the pedicel where lenticels are located that are critical for berry O₂ uptake as a function of temperature, and when blocked caused hypoxia in Chardonnay berries, ethanol accumulation and cell death. This work has been published in the Journal of Experimental Botany in February 2018. In Chapter 3, the results of a factorial field experiment comprising two thermal regimes (ambient and heated) and two irrigation regimes (irrigated and non-irrigated) are reported. Non-irrigation, in the first season, increased the rate of cell death relative to control irrigated Shiraz vines. In the second season, non-irrigation advanced the onset of cell death relative to the irrigated treatments independent of temperature. Non-irrigation treatments in the second season also decreased [O₂] within the berry mesocarp relative to the irrigated treatments. An association was established between mesocarp [O₂] and berry cell death. Berry respiration and total berry porosity were also found to decrease during berry ripening. This work has been submitted to the Australian Journal of Grape and Wine Research in February 2018. In Chapter 4, three preliminary trials were undertaken to investigate possible mitigation strategies for berry cell death and dehydration. Trial 1 tested the effect of overhead shading on berry dehydration, cell death and internal oxygen concentrations [O₂]. Trial 2 tested the effect of rootstocks having different drought tolerance on berry dehydration and cell death. Trial 3 tested the effect of kaolin spray, which has been proposed to reduce leaf and cluster temperature and transpiration. Overhead shading reduced the rate of increase in cell death and berry dehydration in Shiraz. This treatment also affected the progression of hypoxia in the berry. Shiraz on the drought tolerant 140 Ruggeri had significantly less cell death and berry dehydration than the less drought tolerant rootstocks (420 A and Schwarzmann). Kaolin spray application reduced berry shrinkage independently of either mesocarp cell death or cluster and leaf temperature. In summary, grape internal [O₂] declined during fruit development and was correlated with the profile of mesocarp cell death. Lenticels on the pedicel provided a pathway for O₂ diffusion into the berry and when covered to restrict O2 diffusion into the berry caused a large reduction in [O₂] in the centre of the berry, an increase in ethanol concentration and cell death. Differences in internal O₂ availability of berries between cultivars could be associated with seed development and differences in lenticel surface area. Higher rates of mesocarp cell death caused by water stress was also associated with hypoxia within grape berries. Rootstocks with different drought resistance properties can affect Shiraz berry dehydration and cell death. Kaolin can effectively reduce Shiraz dehydration after the peak berry mass was reached. The data generated in this study provides the basis for further research into the role of berry gas exchange on berry quality and cultivar selection for adapting viticulture to a warming climate. Understanding the association between berry internal oxygen status and berry shrivel and cell death, as well as the effect of strategies to mitigate cell death and berry shrivel, will provide researchers and growers with new insights in berry ripening and is the basis for future research on berry flavour development and yield optimization.
Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2018