Tesis sobre el tema "Hexokinase"

ABSTRACT PURIFICATION AND CHARACTERIZATION OF HEXOKINASE ISOENZYMES FROM Rhizopus oryzae Dedeoglu, Didem MS., Department of Biotechnology Supervisor: Prof.Dr. Haluk Hamamci Co-supervisor: Dr. Seyda Aç
ar February 2007, 116 pages Glycolysis is the central metabolic pathway for living organisms. Its regulation is important for the yield of the end products which are industrially important. These end products, like lactic acid produced by Rhizopus oryzae, are industrially important. Rhizopus oryzae is a filamentous fungus producing lactic acid and ethanol. The lactic acid yield of R. oryzae is low (&
#61566
70 %) compared to that of lactic acid bacteria (&
#61502
95 %) still it is noteworthy because R. oryzae produces only the L (+) form of lactic acid which can be metabolized in the human body. The yield of an industrial process should be high for the feasibility of the production of a particular product. If a way can be found increase the flux through the glycolysis the yield of lactic acid may increase as well. Keeping this in mind we wanted to focus on the first step of glycolysis, hexokinase of R. oryzae. Hexokinase catalyzes the reaction that converts glucose to glucose-6-phosphate. In this study for the first time the two isoenzymes of hexokinase of R. oryzae were purified and characterized by biochemically and kinetically Hexokinase has two isoenzymes. The purified enzymes (isoenzymes1 &
isoenzymes2) obeyed Michealis-Menten Kinetics. The Km value of purified isoenzyme 1 is 0.16 mM and isoenzyme 2, 0.21 mM at pH 7.70 for glucose. The Km value of isoenzyme1 for fructose was 28.8 mM. Essentially isoenzyme 2 can not utilize fructose. None of the isoenzymes were inhibited by trehalose-6-phophate.The monomer moleculer weight of isoenzymes were estimated SDS PAGE analysis. There were two different values for molecular weight of isoenzmye 1
62.9 and 42.5 kDa and two values for isoenzyme 2
56.2 and 41.6 kDa

L'hexokinase II (HK-II) catalyse la réaction de phosphorylation du glucose en glucose-6-phosphate, permettant ainsi son utilisation. Le diabète de type 2 est caractérisé notamment par un défaut dans l'étape d'entrée/phosphorylation du glucose en réponse à l'insuline. HK-II a été préalablement identifié comme un des gènes anormalement régulés dans cette pathologie. J'ai tout d'abord étudié la régulation in vitro de l'expression du gène HK-II en réponse à l'insuline dans un modèle de cellules musculaires humaines. Nous avons montré que la région -369/-270 du promoteur HK-II confère l'effet transcriptionnel de l'insuline et implique le facteur de transcription SREBP-1. Dans une deuxième partie, une étude physiologique a permis de démontrer que SREBP-1, comme dans le foie et le tissu adipeux, représente un des médiateurs de l'effet transcriptionnel de l'insuline dans le muscle et confirme l'implication de ce facteur dans le contrôle de l'expression de gènes clés du métabolisme des glucides

Chez le fruit de tomate, les teneurs en sucres, acides aminés et acides organiques contribuent à ses caractéristiques gustatives et nutritionnelles et la richesse en protéines et en paroi végétale conditionne en partie sa texture. La connaissance et la compréhension de lka régulation du métabolisme du fruit de tomate sont essentielles pour en améliorer la qualité. Pour cela, pendant cette thèse, nous avons développé un outil de fluxomique, afin de quantifier des flux de carbone dans le métabolisme intermédiaire du péricarpe de fruit de tomate. Nous avons défini des conditions pour l'analyse des flux chez la tomate : des lamelles de péricarpe sont excisées et incubées dans une solution nutritive. La détermination d'une vingtaine de flux a été réalisée sur la variété Ailsa Craig, par marquage des métabolites avec du [1-13C]- et du [2-13C]-glucose dans des conditions d'état stationnaire isotopique et métabolique, après mesure d'enrichissements de composés par RMN du 1H et du 13C et optimisation du calcul des flux du réseau impliqué. Cette étude réalisée sur trois milieux de culture, le milieu MS/10 de référence, le milieu MS dix fois plus concentré, et le milieu MS*+Glutamine à la fois source de carbone et d'azote organique, a permis de tester la robustesse du réseau métabolique. L'application de cette méthodologie à des tomates surexprimant des hexokinases de levure (Y32) et d'Arabidopsis (HK4 et HK37) a permis de définir des perturbations du métabolisme sur HK4 : cette lignée présente une augmentation du cycle des hexoses-P et une réduction du flux de la voie des pentoses phosphates, ce qui pourrait expliquer une réduction de croissance observée in planta
In tomato, concentrations in sugars, amino acids and organic acides determine partly fruit's taste and nutritional characteristics. Moreover, proteins and cell wall determine mainly its texture. Metabolism knowledge and apprehension of its regulation in tomato fruit are essentials to ameliorate its quality. In this work, we have developed a fluxomic tool to quantify carbon fluxes in intermediary metabolism in tomato fruit pericarp. We have first defined conditions for flux analysis in tomato fruit : pericarp slices are excised and incubated in nutritive solution. Determination of about 20 fluxes were performed with Ailsa Craig, using labeling carbon experiments with [1-13C]- and [2-13C]-glucose. After metabolic and isotopic steady state, measurements of metabolite enrichments by 1H and 13C NMR were used to calculate fluxes of the metabolic network. This study, performed on three media, MS/10, the medium reference, MS ten more concentrated and MS*+Glutamine for carbon and organic source, allowed to test the network robustness. Fluxes analyses were then performed on tomato fruits over expressing yeast (Y32) and Arabidopsis hexokinase (HK4 and HK37). We showed metabolism perturbations in HK4 : the increase in the hexoses-P cycle and the decrease in the pentoses-P pathway. These modifications could explain the reduction of fruit development in this genetically affected plant

Hexokinase is the first enzyme in glycolysis, a major pathway for the generation of energy in all eukaryotes. Mammalian cells have four isoforms (I, II, III, IV) that have different tissue distribution and kinetic properties. Among all isoforms, human hexokinase II (hHKII) has been found to be implicated in many cancers with an increased expression which serves a dual role. First, it maintains the high glycolytic rate of malignant cells (Warburg effect) and second it prevents apoptosis when is bound to mitochondria. Trypanosoma brucei is a parasite that causes Human African Trypanosomiasis (HAT) and has two isoforms with extensive sequence similarity (98%), TbHKI (active form) and TbHK2 (inactive form). The bloodstream-form parasites (BSF) depend exclusively on glycolysis for their survival. The enzyme from both organisms is a validated target for drug-discovery against both cancer and HAT. The aim of the present study is the discovery of novel and specific inhibitors of the enzymes based on their structure. Structure-based drug discovery is commonly used in pharmaceutical companies to aid in the discovery of potent lead compounds. In silico studies were performed in this project using the known crystal structure of human hexokinase I and a model of TbHKI generated by the protein modelling tool Phyre2. The docking programs, AutoDock (AD) and AutoDock Vina (Vina), were chosen to perform the docking of ~3 million compounds to the target molecules and scoring functions calculated the predicted binding affinities of each compound. In total, 28 compounds were purchased to test on the target molecules. In the experimental part of the project, the two enzymes were cloned, expressed and purified. hHKII was successfully purified giving a high yield of active and pure protein. The protein was characterised using many biophysical methods to establish the oligomeric state, the homogeneity and the secondary structure. Crystallisation trials failed and for this reason, N and C domains of the hHKII were purified separately. Unfortunately, the domains also failed to crystallise thus SAXS data were collected and analysed to gain information of their shape at low resolution. A novel inhibition assay was developed and used to identify four weak inhibitors against full length hHKII. TbHKI was difficult to express in a soluble form as most of the protein was expressed in inclusion bodies. The purification resulted in a small amount of active protein that was used entirely for biochemical assays. Four compounds were purchased from the docking of the TbHKI model and one was found to inhibit the enzyme over 65% at 100 μM. Because the active site of both enzymes (hHKII, TbHKI) is well conserved the compounds from hHKII docking were also screened against the TbHKI. Four compounds were found to inhibit this enzyme while one of them was also an inhibitor for human isoform. The remaining three were specific for inhibition of TbHKI.

Une copie du gene de l'hexokinase est perdue dans les tumeurs astrogliales malignes du fait de la perte recurrente d'un chromosome 10. L'activite enzymatique totale de l'hexokinase est fortement diminuee par rapport a celle du cerveau normal, alors qu'un pourcentage eleve de son activite est associee a la mitochondrie. Le metabolisme energetique constitue une cible therapeutique des gliomes. Cette hypothese a ete confortee par l'effet de la lonidamine, agent anti-glycolytique, qui inhibe la croissance de gliomes humains xenogreffes sur souris nude ; cet effet etait d'autant plus important que la fraction mitochondriale d'hexokinase etait elevee. La co-localisation de la porine, proteine d'ancrage de l'hexokinase a la mitochondrie et des recepteurs peripheriques des benzodiazepines a conduit a emettre l'hypothese que la co-inhibition de ces deux cibles aurait un effet synergique inhibiteur sur le metabolisme et la survie des gliomes. Le diazepam, un ligand des recepteurs peripheriques des benzodiazepines, surexprimes dans les gliomes, amplifie les effets de la lonidamine in vitro et in vivo. L'adressage de l'hexokinase a la mitochondrie, est controle par le ph intracellulaire. En imposant des changements de ph intracellulaire, une majoration de l'ancrage de l'hexokinase est obtenue a ph basique avant meme que les parametres glycolytiques ne soient modifies. Le ph intracellulaire est generalement modifie dans les cellules tumorales vers des valeurs plus alcalines par rapport aux cellules normales. L'hypericine, agent photosensibilisant, produit des especes reactives de l'oxygene et acidifie le ph intracellulaire. La combinaison du stress oxydatif et de l'acidification induit par l'hypericine photoactivee a permis de montrer que la cible principale de cet agent est la mitochondrie ; les fonctions mitochondriales et l'activite de l'hexokinase liee a la mitochondrie etaient inhibees et ces effets induisaient la mort cellulaire rapide par apoptose. L'ensemble de cette these renforce le choix de l'hexokinase et de son environnement mitochondrial immediat comme cible therapeutique potentielle des gliomes et indique qu'une combinaison d'agents therapeutiques agissant sur differentes cibles mitochondriales pourrait entraver leur metabolisme energetique et les conduire a la mort.

The cytokine, interleukin-7 (IL-7), has essential growth activities that maintain the homeostatic balance of the immune system. Little is known of the mechanism by which IL-7 signaling regulates metabolic activity in support of its vital function in lymphocytes. We observed that IL-7 deprivation caused a rapid decline in ATP levels that were attributable to loss of intracellular glucose retention. To identify the transducer of the IL-7 metabolic signal, we examined the expression of three important regulators of glucose metabolism, the glucose transporter, GLUT-1, and two glycolytic enzymes, Hexokinase II (HXKII) and phosphofructokinase-1 (PFK1), using an IL-7-dependent T-cell line and primary lymphocytes. We found that in lymphocytes deprived of IL-7 loss of glucose uptake correlated with decreased expression of HXKII. Re-addition of IL-7 to cytokine deprived lymphocytes restored the transcription of the HXKII gene within 2 hours, but not that of GLUT-1 or PFK1. IL-7-mediated increases in HXKII, but not GLUT-1 or PFK-1, were also observed at the protein level. Inhibition of HXKII with 3-Bromopyruvate or specific siRNA decreased glucose utilization, as well as ATP levels, in the presence of IL-7, while over-expression of HXKII, but not GLUT-1, restored glucose retention and increased ATP levels in the absence of IL-7. This IL-7 mediated HXKII gene expression was abrogated with inhibition of JNK pathway. IL-7 also increased activation of AP-1 complex and DNA binding of JunD, a transcriptional complex thought to be negative regulator of proliferation. We found that over expression of HXKII caused cell cycle arrest and cell death, indicating that a potent IL-7 signal could produce negative growth signals. We conclude that IL-7 controls glucose utilization by regulating the gene expression of HXKII through activation of JNK-JunD pathway, suggesting a mechanism by which IL-7 supports bioenergetics that control cell fate decisions in lymphocytes.
Ph.D.
Burnett School of Biomedical Sciences
Medicine
Biomedical Sciences PhD

Mise au point d'un milieu optimise assurant un metabolisme oxidatif des sucres chez kluyveromyces fragilis. Mise en evidence de deux systemes enzymatiques limitant le metabolisme: transport des sucres et phosphonylation des hexoses. Obtentrion de mutants deficients en alcool deshydroenase et hexokinase

REDD1 contribue au dialogue entre le métabolisme énergétique et la masse musculaire.REDD1 est une protéine ubiquitaire et conservée qui est exprimée en réponse à de nombreux stress et pathologies associés à une atrophie du muscle squelettique, un paramètre corrélé à la mortalité des patients. REDD1 est connue pour inhiber la voie Akt/mTORC1 qui contrôle la synthèse des protéines (composants majoritaires du muscle), mais également d'autres macromolécules tels les ribosomes, les nucléotides ou le glycogène. Nos travaux montrent, grâce à un modèle murin, que REDD1 est capable d'une part d'inhiber la synthèse protéique ce qui conduit à l'atrophie du muscle, et d'autre part de réduire le stockage du glycogène musculaire. Cependant, sa délétion est responsable d'une augmentation du métabolisme basal, d'une réduction de la capacité d'exercice et d'une aggravation de l'atrophie musculaire en situation d'hypoxie. Ces altérations du métabolisme ne sont pas liées à un dysfonctionnement mitochondrial, mais associées à une moindre inhibition de la signalisation d'Akt et/ou mTORC1, tous deux responsables de l'activation de processus anaboliques couteux en énergie. Pris ensembles, ces résultats suggèrent que REDD1 agit comme modérateur de la dépense en ATP dans des situations de stress énergétique
REDD1 contributes to the crosstalk between energetic metabolism and skeletal muscle mass. REDD1 is a ubiquitous and conserved protein, which is expressed in response to numerous stresses and pathologies responsible of muscle atrophy, a parameter correlated with patient mortality. REDD1 is known to inhibit Akt/mTORC1 pathway which controls synthesis of proteins (the major component of muscle) and other macromolecules such as ribosome, nucleotide or glycogen. Our work shows on a mice model that REDD1 inhibits protein synthesis, leading to skeletal muscle atrophy, and reduces muscle glycogen storage. However, REDD1 deletion is responsible of an increase in basal metabolism, a reduction of exercise capacity and an exacerbation of hypoxia-induced skeletal muscle atrophy. These metabolic alterations are not associated with a mitochondrial dysfunction but rather with an hyper activation of the Akt/mTORC1 pathway which is responsible for the stimulation of energy demanding processes. Altogether, these results strongly suggest that REDD1 acts for moderating ATP demand in energetic stress conditions

The production, transport and assimilation of organic carbon ultimately drive the growth of plants. In this work, two enzymes, Sucrose Phosphate Synthase (SPS) and Hexokinase (HXK), prominent in their role of carbon production in the form of sucrose at the source, have been examined for their role at the sink, where carbon is assimilated. It has been postulated that the presence of sucrose-forming enzymes in the sink serves a function to reform sucrose from apoplastic cleavage or partake in a “futile” cycle of sucrose cleavage and such that small changes in metabolite enable large changes in sink carbon strength. In order to determine if SPS is involved in carbon sink strength, A. thaliana TDNA insertional lines and P. trichocarpa RNAi stem and developing xylem with decreased SPS transcript expression were analyzed. It was determined that loss of SPS transcript generally increases soluble sugars: sucrose, glucose and fructose, in the leaf and stem as well as starch in the leaf. Structural carbohydrates were generally unaffected and Klason soluble lignin decreased. Similarly, A. thaliana TDNA insertional lines with decreased HXK transcript expression were utilized to determine the role of HXK using stem tissue as a carbon sink model. Soluble sugars mainly increased in the leaf of athxk3 TDNA insertional line whereas starch increased in both leaf and stem of the same line. Interestingly, structural carbohydrate levels of the cell wall were perturbed in HXK TDNA insertional lines. The results were found to be consistent with the postulated roles of SPS and HXK that predict a function in sucrose formation from apoplastic cleavage, which allows for fine-tuning of major intracellular metabolites and adjustment of sink strength.
Forestry, Faculty of
Graduate

Le travail presente est relatif a la synthese et a l'etude du mode d'action d'inhibiteurs des quatre premieres enzymes de la glycolyse; ces inhibiteurs ont ete etudies a la fois sur des enzymes du mammifere ainsi que sur celles d'un parasite dont la glycolyse est un metabolisme essentiel, le trypanosome. Une premiere partie bibliographique fait une presentation de la glycolyse chez le mammifere et chez le trypanosome et montre le parti qui pourrait etre pris des differences de ce metabolisme entre l'hote et le parasite pour developper des molecules agissant specifiquement sur les secondes et qui donc seraient des precurseurs d'anti-parasitaires. La deuxieme partie decrit la synthese d'un certain nombre d'inhibiteur potentiels de l'hexokinase, deux grandes familles de composes pour la plupart originaux sont decrits: des analogues structuraux du glucose-6-phosphate d'une part, des derives du glucose portant des substituants a la position 2 d'autre part. Une etude cinetique faite a partir des composes les plus actifs a permis de montrer qu'un certain nombre d'entre eux etaient reconnus au niveau du site actif de l'hexokinase. La derniere partie est relative a la recherche d'inhibiteurs de type suicide de l'aldolase. Deux analogues du substrat fructose-1,6 diphosphate ont ete prepares, ces composes etant des precurseurs des motifs enone et sulfene et donc susceptibles de donner lieu a une inactivation irreversible de l'aldolase, un mecanisme d'action est etabli

Thesis (Ph.D.)--Boston University PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you.
Mitochondrial injury and apoptosis promote organ failure after ischemic acute kidney injury (AKI), a common cause of morbidity and mortality. In these studies, we propose that hexokinase (HK), mitofusin 2 (MFN2) and Bax, key mitochondrial associated proteins, modulate apoptotic cell death and organ function after ischemia. In the kidney, HKI and HKII isoforms both possess mitochondrial localization sequences. In vivo ischemia reduced murine proximal tubule HKII content and caused mitochondrial HKII dissociation. In cultured renal epithelial cells, expression of HKI or II significantly improved survival after ATP depletion, an in vitro model of ischemia, without preventing Bax activation or reducing mitochondrial fragmentation, a determinant of organelle sensitivity to injury. HKII over-expression increased mitochondrial associated HKII during stress and decreased mitochondrial Bax accumulation, a major cause of outer membrane permeabilization and apoptosis, suggesting that HK improves renal cell survival by antagonizing Sax-mediated injury. Deficiency of MFN2, a pro-fusion protein, caused mitochondrial fragmentation in primary proximal tubule cells without altering baseline or maximal oxygen consumption rate, or cell apoptosis. However, MFN2 deficiency significantly increased mitochondrial Bax accumulation and exacerbated mitochondrial outer membrane injury after stress. In the mouse, whole kidney MFN2 knockout caused severe mitochondrial fragmentation in renal epithelial cells. However, despite a small (20%) decrease in nephron number compared to littermate controls, newborn knockouts exhibited normal tubular and organ function. Surprisingly, proximal tubule specific MFN2 knockouts were also protected from renal ischemia. Although histologic injury scores as well as levels of apoptosis and necrosis, were similar, renal function and animal survival were significantly higher in proximal tubule specific MFN2 knockout mice at 24 and 48 hours post-ischemia. Interestingly, cortical oxidant stress was halved while cortical proliferation was nearly 4 times higher in proximal tubule knockouts compared to control, suggesting that MFN2 deficiency promotes organ recovery and survival after ischemia by enhancing proximal epithelial cell growth. While HK and MFN2 modulate Bax-mediated mitochondrial injury and apoptosis, "off-target" effects of MFN2 on renal cell proliferation ameliorate ischemia-reperfusion injury. These studies highlight the role of Bax-mediated mitochondrial injury in ischemic organ failure and suggest new targets for both attenuating injury and promoting organ recovery.

Human Rhinoviruses (HRVs) utilize Internal Ribosome Entry Sites (IRES) to drive viral protein synthesis. IRESs are specialized RNA elements present within the 5’ UTR of mRNAs that recruit ribosomes independently of the 5’ m7G cap structure. hnRNP A1 (heterogeneous nuclear ribonucleoprotein A1), a multifunctional RNA binding protein, is required for the IRES-dependent translation of many specific RNAs within the cell cytoplasm. The phosphorylation of hnRNP A1 is required for its cytoplasmic accumulation. I have identified and validated the role of HK2 in hnRNP A1 cellular localization by immunofluorescence microscopy, by analysis of HRV infection and by siRNA-based screening. These studies show that decreased HK2 protein levels lead to decreased cytoplasmic accumulation of hnRNPA1 during osmotic shock and HRV infection, to a decrease in HRV-infected cells and to decreased caspase activation in osmotically stressed and HRV-infected cells. Thus, HK2 may regulate hnRNP A1 cytoplasmic localization following HRV infection.

Chez le ray-grass anglais, espèce fourragère prédominante des prairies Normandes, il existe des transporteurs de saccharose (LpSUTs) responsables du transport latéral de ce composé pour permettre son chargement dans le phloème dans les tissus sources de carbone ou son déchargement dans les tissus puits. Ces transporteurs sont d’excellents candidats pour la gestion fine et efficace de la distribution des ressources glucidiques de la plante, notamment en réponse aux variations de l’environnement. Les caractéristiques biochimiques et la localisation tissulaire spécifique du transporteur LpSUT1 suggèrent qu’il est le principal responsable du transport latéral du saccharose.Nous avons cherché à moduler la disponibilité du saccharose en perturbant les relations sources et puits des tissus par des facteurs abiotiques (e.g. cycle nycthéméral) et biotiques (e.g. pulvérisation de bactéries au niveau de la phyllosphère). Dans ces deux conditions, l’expression du gène LpSUT1 dans les organes sources que sont les limbes est peu affectée, malgré une perturbation importante des teneurs en saccharose. L’apport de glucides, d’analogues structuraux et d’un inhibiteur de l’activité de l’hexokinase à des limbes excisés montre que le saccharose reste cependant un signal glucidique capable d’augmenter l’expression transcriptionnelle de LpSUT1. Le glucose est aussi un signal qui module négativement l’expression de LpSUT1 quand il est perçu par l’hexokinase, et positivement lorsqu’il est perçu indépendamment de cette dernière. Un excès de saccharose et de glucose apoplasmique pourrait être détecté au niveau membranaire ce qui régulerait positivement l’expression du gène LpSUT1 pour augmenter l’importation du saccharose vers le cytoplasme. A l’inverse, l’accumulation cytoplasmique de glucose provoquerait l’inhibition de l’expression du gène LpSUT1 pour y limiter l’accumulation de glucides.La régulation transcriptionnelle du gène LpSUT1 fait appel à des voies de signalisation complexes, sensibles en partie aux signaux glucidiques, mais d’autres LpSUTs et facteurs de régulation devront encore être identifiés pour mieux comprendre les enjeux du transport de saccharose dans la gestion du carbone chez le raygrass anglais
In the perennial ryegrass, the predominant forage species of Normand meadows, sucrose transporters (LpSUTs) are involved in the lateral transport of this compound to load sucrose in the phloem of the source tissues and its unloading in sink tissues. These transporters are excellent candidates for the tight and efficient management of the plant sugars, particularly in response to environmental variations. The biochemical properties and specific tissular localization of the sucrose transporter LpSUT1 suggest that it is the main actor of the lateral transport of sucrose.We induced a modulation of the sucrose availability in the tissues through the disruption of the source/sink relationships using abiotic (e.g. diel cycle) and biotic factors (e.g. spraying bacteria abundantly on the phyllosphere). In both conditions, LpSUT1 expression in source tissues like the leaf blades was not too much affected, despite the important disruption of the sucrose content. By supplying excised leaf blades with sugars, sugar analogs and an inhibitor of the hexokinase activity, we showed that sucrose was still a sugar signal which could up-regulate LpSUT1 expression. Glucose is also a signal for LpSUT1 down-regulation when perceived by the hexokinase and for its up-regulation when perceived in a way independent of the hexokinase. The excess of apoplastic sucrose and glucose could be detected at the plasmalemma which could induce LpSUT1 up-regulation to increase the sucrose import to the cytoplasm. Conversely, the cytoplasmic accumulation of glucose could downregulate LpSUT1 expression to limit the accumulation of cytoplasmic sugars.The transcriptional regulation of LpSUT1 involved complex signaling pathways partially responding to sugar signals, but other regulation factors should be identified to further comprehend the implications of the sucrose transport for the perennial ryegrass carbon management

Les biocapteurs sont des moyens d’analyse en plein essor à la fois rapides, sélectifs et peu coûteux applicables à des domaines extrêmement variés (environnement, santé, agroalimentaire,…). Dans ce type d’outil, un élément sensible de nature biologique (anticorps, enzyme, microorganisme, ADN…) doté d’un pouvoir de reconnaissance pour un analyte ou un groupe d’analytes est associé à un transducteur pouvant être de type électrochimique, optique ou thermique.Dans ce travail, nous nous sommes intéressés au développement de trois biocapteurs pour la détection de substances biologiquement actives. Le premier permet la détermination simultanée de l’adénosine triphosphate (ATP) et du glucose par ampérométrie, le deuxième celle de la créatine kinase, et le troisième est un biocapteur conductimétique pour la quantification de l’ATP. Dans les deux premiers biocapteurs, deux enzymes (l’hexokinase et la glucose oxydase) sont immobilisées à la surface de microélectrodes constituées d’un disque de platine. Le troisième biocapteur est basé sur l’immobilisation de l’hexokinase sur des microélectrodes interdigitées en or. L’immobilisation est réalisée dans tous les cas par co-réticulation des enzymes en présence d’albumine de sérum bovin à l’aide de glutaraldehyde. Les caractéristiques analytiques des biocapteurs ont été déterminées et différentes procédures ont été développées pour l’analyse d’échantillons réels. Les biocapteurs ont pu être appliqués avec succès à la quantification de l’ATP, du glucose et de la créatine kinase dans des préparations pharmaceutiques et du sérum sanguin
Biosensors are rapid, selective and inexpensive devices that combine a biological recognition element, the so-called bioreceptor (e.g. enzymes, antibodies, DNA or microorganisms) to a physical transducer (e.g. electrochemical, optical, thermal or piezoelectrical). They can be used to detect one specific analyte or one family of analytes for a wide range of applications (e.g. environment, food, health). In this work, the detection of biologically active substances was targeted. A biosensor system for simultaneous determination of adenosine triphosphate (ATP) and glucose, a biosensor for creatine kinase analysis, and a novel conductometric biosensor for ATP determination were developed. In the first two biosensors, two enzymes (hexokinase and glucose oxidase) were immobilized at the surface of platinum disc microelectrodes for amperometric detection. The third biosensor was based on hexokinase immobilized onto gold interdigitated microelectrodes for conductometric detection. In all cases, the enzymes were co-immobilized with bovine serum albumin by cross-linking using glutaraldehyde. Analytical characteristics of the biosensors were determined and different procedures were developed for real samples analysis. The biosensors could be successfully applied to the determination of ATP, glucose, and creatine kinase in pharmaceutical samples and blood serum

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemical Engineering, 2017.
Cataloged from PDF version of thesis.
Includes bibliographical references.
Selectivity is a very important consideration when designing cancer treatment. Therefore, targeting cancer-specific isoforms of key metabolic enzymes is a very promising strategy. Elevated glucose consumption and lactate secretion under aerobic conditions is a hallmark of many cancer cells. One of the major contributors to this highly glycolytic phenotype is hexokinase 2 isoform (HK2), which is significantly overexpressed in many tumors but not in normal adult tissue. Several small molecule inhibitors targeting HK have exhibited promising anticancer activity. However, these small molecule inhibitors cannot differentiate between HK isoforms, leading to severe side effects. Elucidating the differences between HK isoforms can guide us in designing isoform-specific inhibitors. Moreover, the complexity and robustness of cellular metabolism and the intricate mechanism underlying cancer development and progression make it very difficult to effectively treat cancer by targeting a single metabolic enzyme. Improved understanding of the metabolic effects of HK to cancer can guide us in identifying selective targets and designing effective combination therapies. We first studied the differential subcellular localizations of HKl and HK2 by selective permeabilization and cell fractionation. We then characterized the metabolic functions of HK for cell growth and proliferation using stable isotope labeling and metabolic flux analysis. Key findings were verified in a hepatocellular carcinoma cell line using low glucose feeding or HK inhibitor. These experiments identified differential HK enzymatic activity in different cell compartments, and discovered key metabolic pathways co-regulated with glycolysis via cellular redox status. These findings may provide guidance in designing cancer-specific inhibitors or combination therapy targeting multiple synergistic metabolic pathways.
by Zhe Zhang.
Ph. D.

OVCA is the most lethal gynecological cancer, due primarily to late diagnosis and chemoresistance (Canada, 2014; Society, 2014b). CDDP resistance is a major hurdle to successful therapy (MayoClinic, 2014). The mechanism of chemoresistance is multi-factorial including defects in apoptotic pathway and key tumor suppressor as well as dysregulation of metabolism (Borst et al., 2000; Galluzzi et al., 2012a; Siddik, 2003). Elevated aerobic glycolysis is a major source for fulfilling high energy demand of cancer, but the role of metabolic reprogramming and its regulatory mechanism in OVCA cells remain unknown. p53 is a key tumor suppressor involved in apoptosis and frequent defect of p53 (> 80%) exist in epithelial OVCA. HKII is a key metabolic enzyme involved in the first step of glycolysis and its frequent presence in the mitochondria (80% >) has been reported in multiple cancers. We demonstrate here that CDDP-induced, p53-mediated HKII down-regulation and mitochondrial p53-HKII interaction are determinants of chemosensitivity in OVCA. CDDP decreased HKII (mRNA abundance, protein level), altered its cellular localization and glycolysis in p53-wt chemosensitive OVCA cells, a response loss or attenuated in p53 deficient cells. HKII depletion sensitized chemoresistant cells to CDDP -induced apoptosis in a p53- dependent manner. In addition, p53 binds to HKII and facilitates its nuclear localization. Mechanistically, our data suggest that CDDP-activated p53 (phosphorylated p53; P-p53 Ser15) interacts with HKII in the nucleus for its regulation. Upon entry to the nucleus, P-p53(Ser15) transcriptionally regulates HKII by promoter binding, contributing to the regulation of HKII and aerobic glycolysis, eliciting apoptosis in chemosensitive OVCA cells. Conversely, this response is compromised in p53 defect chemoresistant cells. Using proximity ligation assay (PLA) in human OVCA cell lines and primary tumor cells and tumor sections from OVCA patients, we have demonstrated that nuclear HKII-P-p53(Ser15) intracellular trafficking is associated with chemosensitivity in vitro and in vivo. Furthermore, the nuclear HKII-P-p53(Ser15) interaction may be useful as a biomarker for chemosensitivity in multiple epithelial subtypes of OVCA.

Os canais seletivos a ânions dependente de voltagem (VDACs) são um grupo de proteínas, primeiramente identificadas na membrana mitocondrial externa, capazes de formar estruturas de poros hidrofílicos em membranas. As VDACs são conhecidas pela sua função essencial no metabolismo celular e nos estágios recentes de apoptose. Em mamíferos, foram identificadas três isoformas de VDACs (VDAC1, 2 e 3). Uma pesquisa proteômica, consistindo de eletroforese bi-dimensional seguida por western blotting com anticorpos anti-VDAC 1, anti-VDAC 2 e anti-VDAC 3 e espectrometria de massas com fonte de ionização/desorção à laser assistido por matriz e tempo de vôo foi utilizada para estudar a expressão das isoformas de VDAC em mitocôndrias cerebrais de aves, ratos e bois. Foi estudada a possibilidade que diferenças na expressão relativa das isoformas de VDAC possam ser um fator determinante da proporção espécie-dependente dos sítios de ligação da hexoquinase tipo A: tipo B nas mitocôndrias cerebrais. Os spots foram caracterizados, e a intensidade de sinal foi comparada entre os spots. VDAC1 e VDAC2 foram divididas dentro de múltiplos spots. A VDAC1 foi dividida em dois spots nos géis bi-dimensionais realizados com amostras de cérebros de ratos e bois, e três spots para cérebros de aves. A VDAC2 foi separada em três, cinco e dois spots para cérebros de ratos, bois e aves, respectivamente. Os resultados reportam uma heterogeneidade de carga das VDACs 1 e 2 nos cérebros analisados. A VDAC1 foi a mais expressa das três isoformas. Além disso, a expressão da VDAC1 mais VDAC2 foi muito maior em cérebros de aves e bois do que em cérebros de ratos. Mitocôndrias de cérebro de aves mostraram uma maior expressão de VDAC1 e menor de VDAC2. As mitocôndrias de cérebro bovino apresentaram os níveis mais altos de VDAC2. A VDAC3 não foi detectada nos cérebros das espécies estudadas.
The voltage dependent anion selective channels (VDACs) are a group of proteins first identified in the mitochondrial outer membrane that are able to form hydrophilic pore structures in membranes. VDAC are known to play an essential role in cellular metabolism and in the early stages of apoptosis. In mammals, three VDACs isoforms (VDAC1, 2, 3) have been identified. A proteomic approach, consisting of two dimensional electrophoresis, followed by western blotting with anti-VDAC 1, anti-VDAC 2 and anti-VDAC 3 and by matrix assisted laser desorption/ionization time of flight mass spectrometry was used to study the expression of VDAC isoforms in rat, bovine and avian brain mitochondria. We were studying the possibility that differences in the relative expression of VDAC isoforms may be a factor in determining the species-dependent ratio of type A: type B hexokinase binding sites on brain mitochondria. The spots were characterized, and the signal intensities among spots were compared. VDAC1 and VDAC2 were divided into multiple spots. VDAC1 was divided in two spots in two dimensional gels of rat and bovine brains and three spots in avian brains. VDAC2 was separated into three, five and two spots in rat, bovine and avian brains, respectively. The results report charge heterogeneity of VDACs 1 and 2 in the analyzed brains. VDAC1 was the most abundantly expressed of the three isoforms. Moreover the expression of VDAC1 plus VDAC2 was much higher in avian and bovine brains than in rat brains. Avian brain mitochondria showed the highest expression of VDAC1 and the lowest of VDAC2. Bovine brain mitochondria had the highest levels of VDAC2. No VDAC 3 was detected in studied species brains.

Activation of AMP-activated protein kinase (AMPK) results in the upregulation of several intracellular systems which help to prepare a cell for a high energy challenge. The magnitude of the AMPK response to a 10 min bout of exercise has been found to decrease in red quadriceps (RQ) following training, while putative AMPK roles seem to be maintained; specifically, the biogenesis of mitochondria and higher levels of hexokinase II and glucose transporter 4 (GLUT4). If the AMPK response to exercise is responsible in part for these adaptations, how can they be maintained if the AMPK response is attenuated? The purpose of this study was to determine whether phosphorylation of AMPK in RQ increases during 2-hr training bouts after rats have trained for 8 wks. Male Sprague-Dawley rats ran up to 30 m/min up a 15% grade, 2 hr/day for 8 wks. On the final bout of exercise, trained rats ran for 0 (TRC), 30 (TR1), or 120 min (TR2) up a 15% grade at 30 m/min. Red quadriceps (RQ), soleus, and white quadriceps (WQ) were immediately collected and frozen for analysis. Citrate synthase activity increased in RQ (79 ± 3 vs. 37 ± 4 µmol/g/min) and soleus (64 ± 4 vs. 35 ± 2 µmol/g/min) but not in WQ compared to non-trained controls. In trained rats, maximal increases in T-172 phosphorylation of AMPK occurred after 30 min of exercise (relative values = 1.29 ± 0.06 vs. 1.00 ± 0.06). AMPK phosphorylation did not change significantly in trained rats that ran for 2 hrs (1.31 ± 0.09) compared to rats that ran for 30 min. Similarly, maximal increases in AMPK activity in trained rats occurred after 30 min of exercise (pmoles/min/mg = 2.67 ± .05 vs. 1.09 ± .41) and AMPK activity did not change significantly in trained rats that ran for 2 hrs (2.79 ± .17) compared to rats that ran for 30 min. Previous studies demonstrated a 2−3 fold increase in AMPK activity in non-trained rats after 30 min of exercise at lower work rates. These results demonstrate that the AMPK response to exercise is attenuated even after two-hr bouts of exercise. This implies that the increase in mitochondrial oxidative enzymes, GLUT4, and hexokinase II may be maintained by signals other than the AMPK signaling system. The CREB signaling pathway is one such system. Western analysis of phospho-CREB (Ser133) showed a statistically significant increase in phospho-CREB content in trained rats relative to control. No change in phospho-CREB protein expression was observed between TRC, TR1, and TR2 rats. Significant increases of muscle phospho-CREB content in TRC relative to untrained rats suggest that CREB remains phosphorylated in trained rats even after 24 hrs of rest. Accordingly, chronically increased phospho-CREB in muscle of trained rats relative to controls may explain in part how increased levels of mitochondria are maintained in the face of reduced AMPK response. Alternatively, the attenuated AMPK response may still be above the threshold required for inducing adaptations to endurance training.

A VDAC é a proteína mais abundante da membrana mitocondrial externa. Possui diversas funções, tais como o controle da troca de metabólitos, através da membrana, e a participação no maquinário apoptótico. Estudamos o interactoma da VDAC com as proteínas mitocondriais neuronais do cérebro bovino e murino, a fim de compreender se a expressão diferenciada da VDAC1 e VDAC2 verificada entre essas células estão associadas às diferenças nas interações da VDAC. Os complexos proteicos foram analisados por 2D Blue Native SDS-PAGE e identificados via MALDI-TOF TOF usando o software Mascot e o banco de dados NCBInr. Foram identificados 27 e 46 spots em murino e bovino, respectivamente. Nós identificamos proteínas solúveis e incorporadas na membrana que não são participantes da fosforilação oxidativa, dentre elas a aldeido deidrogenase e muitas outras constituintes de complexos mitocondriais já conhecidos tão bem como novos, tais como a putative stomatin-like protein 2 complex e a switch-associated protein 70. Nossos resultados mostraram que os neurônios bovinos possuem mais complexos (5) contendo a VDAC do que em ratos (1), os quais indicam uma cinética diferencial de acoplamento e desacoplamento. Interessantemente, a lista contendo as proteínas identificadas inclui algumas proteínas conhecidas ou supostamente localizadas em compartimentos não-mitocondriais, por exemplo, a myc-induced nuclear antigen. O interactoma diferencial da VDAC entre as espécies bovina e murina, evidencia a presença de uma base comum, porém com diferentes ambientes estruturais, as quais podem ser a base da diferença entre os sítios de ligação A e B observados nas diferentes espécies.
The voltage dependent anion channel (VDAC) is the most abundant protein of outer mitochondrial membrane. VDAC controls metabolite exchange through this membrane and the apoptosis machinery. We studied the interactome of VDAC with mitochondrial proteins of neuronal cells from rat and bovine brain. We wished to understand if the differential expression of VDAC1 and VDAC2 verified between these cells was linked to differences in the VDAC interactions. Protein complexes were analyzed by 2D Blue Native SDS-PAGE and were identified by MALDI-TOF TOF using Mascot software against the NCBInr database. Number of 27 e 46 spots were identified from rat and bovine brain, respectively. We identified soluble and membrane-embedded non-OXPHOS proteins, among them aldehyde dehydrogenase, and many as constituents of known mitochondrial complexes as well as novel ones such as putative stomatin-like protein 2 complex and switchassociated protein 70. Our results showed that bovine neurons had more protein complexes (5) containing VDAC than rat cells (1), which indicates a differential kinetics of assembly or disassembly. Interestingly, the identification list included some proteins known or presumed to be localized to nonmitochondrial compartments, for example, myc-induced nuclear antigen. Our results support evidences of differential apoptotic and energetic mechanisms verified in these brains. The differential VDAC interactome between bovine and murine, support evidences of a common base, but whith different structural environment, which may be the basis of the difference between the binding sites A and B observed in these brains.

La période automno-hivernale est caractérisée, chez le pêcher, par un état de dormance des bourgeons végétatifs. Dans la sève xylémienne, la concentration de sorbitol diminue et celle des hexoses augmente durant la période de pré-débourrement des bourgeons. Une importante absorption de sorbitol et une forte activité des enzymes de dégradation du sorbitol caractérisent la période d'endodormance contrairement à la période de pré-débourrement. De plus, la zone de croissance du bourgeon absorbe majoritairement du glucose. Le transport de sorbitol est spécifiquement inhibé selon la concentration extracellulaire de glucose. Le mécanisme de la transduction du signal implique la voie dépendante de l'hexokinase (fortes concentrations de glucose). Les phosphatases ont un rôle dans la voie de transduction (faibles concentrations de glucose). L'hexokinase est impliquée durant la période d'endodormance tandis que la période de pré-débourrement associe à la fois l'hexokinase et les phosphatases

Les resultats presentes dans ce manuscrit s'inscrivent dans le cadre d'un theme de recherche concernant l'application de la voltametre cyclique a l'analyse de reactions catalysees par les enzymes. Ceci a permis d'approfondir la connaissance du mecanisme d'oxydation du d-glucose par la glucose oxydase avec pour mediateur des derives de ferrocene. Premierement, nous avons montre que la glucose oxydase demeure un catalyseur efficace meme dans un milieu de viscosite proche de celle de melanges de sucres industriels. Deuxiemement, lorsqu'une chiralite est introduite sur un substituant lateral du ferrocene, elle n'affecte pas la vitesse de la reaction de ce mediateur avec la glucose oxydase. Un edifice constitue de monocouches moleculaires successives de glucose oxydase biotinylee et d'avidine a ete construit a la surface d'une electrode de carbone vitreux. Nous avons synthetise l'enzyme biotinylee. Un protocole optimal d'immobilisation a ete elabore. Il apparait que si la compacite des constructions utilisant les interactions avidine-biotine est identique a celle trouvee dans le cas des constructions utilisant les interactions antigene-anticorps, la concentration superficielle en enzyme active dans une monocouche est nettement inferieure a celle trouvee avec les interactions antigene-anticorps. Toujours en utilisant l'assemblage avidine-biotine, nous avons reussi a greffer un systeme catalytique a enzyme et mediateur attaches a la surface de l'electrode. L'analyse quantitative de ce systeme montre que la mobilite du ferrocene lie controle la cinetique du processus catalytique. Enfin, nous avons immobilise un systeme bi-enzymatique en multimonocouches a la surface d'une electrode. La competition vis a vis du glucose des deux enzymes, la glucose oxydase et l'hexokinase en presence d'atp a ete mise en evidence. Nous avons synthetise l'hexokinase biotinylee. Nous sommes en mesure de maitriser et de prevoir la reponse en courant d'un tel systeme bi-enzymatique.

Este trabalho teve por finalidade a implementação e desenvolvimento de novos métodos para a análise de curvas de espalhamento de raios X a baixo ângulo por sistemas monodispersos. O resultado básico final deste trabalho foi a confecção de três programas de computador e suas aplicações em proteínas de interesse biológico. ELLFIT é um programa de computador que encontra o elipsóide cuja curva de SAXS melhor se ajusta a uma dada curva experimental. Para casos favoráveis este programa é capaz de determinar a dimensão máxima e algumas características básicas do formato da partícula. CRYSOL é um programa para a avaliação de curvas de espalhamento em solução para proteínas com estrutura atômica conhecida. O programa usa a expansão de multipolos para o cálculo rápido da promediação espacial e simula uma camada de hidratação ao redor da proteína. CRYSOL pode predizer a curva de SAXS de uma determinada proteína e compará-la com dados experimentais. HOMDIM é um programa para a determinação da posição das sub-unidades de um homodímero no caso de ser conhecida somente a estrutura da sub-unidade sozinha. Dada a curva experimental e a amplitude da sub-unidade, HOMDIM procura os parâmetros posicionais que descrevem o homodímero. Estes e outros programas foram aplicados a várias proteínas. O método da expansão de multipolos foi usado na determinação do envelope molecular de uma inibidora de ALPHA-amilase. O programa CRYSOL foi utilizado para resolver uma ambiguidade na estrutura quatemária cristalina da hexocinase e o programa HOMDIM para a proposição de um novo modelo para a estrutura quatemária da aspartato transcarbamilase no estado R em solução
This work was aimed at the implementation and development of new methods for solution scattering analysis of monodisperse systems. The basic final result of this work was the development of three programs and their applications to proteins of biological interest. ELLFIT is a computer program, which finds the elipsoid whose SAXS curve has the best fit to a given experimental curve. In favorable cases, this program is able to determine the maximum dimension and some basic characteristic of the particle shape. CRYSOL is a program for evaluating the solution scattering from, proteins of known structure. The program uses multipole expansion for fast calculation of the spherically averaged scattering pattern and takes into account the hydration shell. Given the atomic coordinates it can predict the solution scattering curve and compare it with the experimental scattering curve. HOMDIM is a program to determine the position of both subunits of a homodimer when only one sub-unit structure is known. Given the experimental curve of the homodimer and the subunit scattering amplitudes. HOMDIM searches for the positional parameters, which describe the homodimer. These and other programs were used to study several proteins. The multipole expansion method was used in the shape determination of an ALPHA-amylase inhibitor. The program CRYSOL was used to solve the ambiguity in the hexokinase quaternary crystal structures and the program HOMDIM was utilized for the quaternary structure modeling of the R-state of the aspartate transcarbamilase in solution

Hintergrund: Die Versorgung von Immunzellen mit Energie in Form von ATP ist Grundlage eines funktionstüchtigen Immunsystems. Diese wird durch die mitochondriale OXPHOS oder durch die zytosolische Glykolyse gewährleistet. Sauerstoff und Glukose stellen die Hauptsubstrate dieser Stoffwechselprozesse dar. Fragestellung: Unter pathologischen Bedingungen wie sie in Entzündungsgebieten herrschen, konnte ein relativer Sauerstoffmangel experimentell nachgewiesen werden. Ziel dieser Arbeit war es herauszufinden, in welcher Weise die Funktionen einer definierten Lymphozytenpopulation (CD4+) durch Sauerstoffmangel beeinflusst werden. Methoden: Nach Isolation von CD4+ Zellen aus peripherem Blut gesunder Spender, wurden definierte Zellmengen stimuliert und in einem mit einer Sauerstoffelektrode ausgestatteten Gefäß unter Luftabschluß inkubiert. Zu definierten Zeitpunkten wurden Proben zur ATP-Messung entnommen, sowie Protein- und RNA-Lysate hergestellt. Die Vitalität zu Anfang und zum Ende der Inkubation wurde mittels Propidium-Jodid-Färbung im FACS bestimmt. Aus gesammelten Überständen wurden mittels Multiplex-ELISA die Konzentrationen von IL-1beta, IL-2, IL-6, IL-8, IL-10, TNF-alpha und MCAF gemessen. Als Kontrollen dienten unter Normoxie inkubierte Aliquots der Zellsuspensionen. HIF-1alpha wurde mit Immunoblotting nachgewiesen. Transkriptionsänderungen von SOD1 und HK1 wurden durch SYBR-Green Real-Time-PCR quantifiziert. Ergebnisse: Stimulierte CD4+-Zellen von Normalspendern schütten unter dem Einfluss von Hypoxie vermehrt proinflammatorische und chemotaktisch wirksame Zytokine, sowie zur Differenzierung notwendige antiinflammatorische Zytokine aus. Die Verfügbarkeit von Glukose hat hierauf einen verstärkenden Effekt. Eine hypoxische Umgebung sorgt in Abhängigkeit von der Versorgung mit Glukose für eine Anpassung der zellulären Atmungsrate. Glukose ist für die Aufrechterhaltung eines konstanten ATP-Levels verantwortlich. Die glykolytische Energiegewinnung unter Hypoxie kompensiert den Ausfall der OXPHOS. Hypoxie führt bei stimulierten CD4+-Zellen bei freier Glukoseverfügbarkeit zu einer vermehrten Transkription des Hexokinase1-Gens. Glukosemangel bewirkt dagegen in hypoxischer Umgebung eine Transkriptionssteigerung des SOD1-Gens.
Background: The energy supply of immune cells in form of ATP is the cornerstone of a functional immune system. This supply is realized by either mitochondrial OXPHOS or cytosolic glycolysis. Oxygen and glucose present the main substrates in these metabolic processes. Objective: Relative shortness of oxygen could be determined experimentally under pathological conditions present in inflamed tissues. The aim of this study was to determine the extent of hypoxic influence on the cellular function of CD4+ lymphocytes. Methods: Human CD4+ cells were isolated from peripheral blood of healthy blood donors by MACS sorting. Following a defined protocol cells were stimulated and incubated in a sealed container with a Clark type electrode. Samples were taken for measurements of ATP content. RNA- and Protein lysates were made to quantify the transcription of SOD1 and HK1 by SYBR green RT-PCR and look for the presence of HIF-1alpha by immunoblot analysis respectively. Supernatants were used to measure the expression of IL-1beta, IL-2, IL-6, IL-8, IL-10, TNF-alpha and MCAF using a multiplex ELISA assay. Aliquots of cell supspensions incubated under normoxic conditions served as controls. Results / Conclusion: Under the influence of hypoxia stimulated CD4+ lymphocytes of healthy blood donors express proinflammatory and chemotactically active as well as anti-inflammatory cytokines important for cell differentiation. The availability of glucose leads to an increase of this effect. An hypoxic environment dependant on the availability of glucose leads to an adaptation of cellular respiration. Glucose deficiency provokes an increase in cellular oxygen utilization. The availability of glucose is responsible for a constant intracellular ATP level. This proves that in CD4+ lymphocytes glycolysis is capable of compensating for hypoxically impaired oxidative phosphorylation thus providing enough ATP to enable cellular function. Hypoxia under glucose provision leads to an increase in mRNA expression for HK1, a key enzyme of glycolysis. Lack of glucose under hypoxic conditions results in an increase in mRNA expression for SOD1. Glucose therefore serves in CD4+ cells as an agent of constant energy supply that leads to cell survival and an upkeep of a proinflammatory environment through cytokine expression.

Thesis (MSc)--Stellenbosch University, 2013.
ENGLISH ABSTRACT: In the present-day competitive global market, wine industries are constantly aiming to improve the wine-making process,including the role of yeast. The most commonly used wine yeast is Saccharomyces cerevisiae, which is able to produce high quality wines, but problem fermentations do sometimes arise. The occurrence of stuck and sluggish fermentations pose a serious problem leading to loss of productivity and quality. Although the precise mechanism leading to stuck fermentations is unknown, they are often correlated with high fructose to glucose ratios in the wine-must. S. cerevisiae is a glucophylic yeast, indicating its preference for consuming glucose over fructose. Both these hexose sugars are present in unfermented wine must, mostly in equal concentrations. As fermentation progresses, glucose is consumed at a faster rate than fructose, leading to an increase in the fructose to glucose ratio. Yeast are left with the undesirable fructose at the later stages of fermentation, when the environmental stresses on the yeast can lead to stuck or sluggish fermentation. This residual fructose can lead to undesirable sweetness, as fructose is about twice as sweet as glucose. Even with the extensive research into yeast metabolism, there is as yet no definitive explanation as to why yeasts ferment glucose faster than fructose. This study aimed to investigate the mechanism responsible for the faster consumption of glucose over fructose of a commercially used wine yeast strain S. cerevisiae VIN 13. The first two steps of sugar metabolism, uptake and phosphorylation, were investigated as the possible sites of discrepancy in fermentation rates. Enzyme rates and affinities for both glucose and fructose as substrates for the relevant enzymes were experimentally determined. These kinetic parameter values were used to improve an existing model of yeast glycolytic pathway to model wine fermentations. The feasibility of constructing and validating a kinetic model of wine fermentations were investigated, by comparing model predicted fluxes with experimentally determined fluxes. Another aspect of this study was an investigation into the effect of hexose sugar type on fermentation profiles. Wine fermentations were done with only one hexose sugar as carbon source to determine if it has an effect on the flux through metabolism. This work succeeded in the construction of a kinetic model that distinguished between glucose and fructose as carbon source. The glucose was consumed faster than fructose, with control lying in the hexose transport step. It was also established that fermentation prfiles of fermentations with only one sugar was the same for both one sugar type fermentations. Fermentation with either glucose or fructose as the sole carbohydrate source had the same specfic production and consumption rates as normal fermentations with both sugars. Construction of detailed kinetic models can aid in the metabolic and cellular engineering of novel yeast strains. By identifying the importance of hexose transport, and thus the glucophilic character of the yeast, in flux control, yeast transporters can be targeted for strain improvement. This may in turn lead to more effective fermentation practices for controlling problem fermentations, or to the development of novel strains that utilizes fructose in the same manner as glucose, and in so doing lower the risk of stuck or sluggish wine fermentation.
AFRIKAANSE OPSOMMING: In die hedendaagse kompeterende wynmark is wynmakers aanhoudend besig om die wynmaak proses te verbeter en dit sluit die verbetering van wyngis in. Die mees algemeenste gebruikte wyngis is Saccharomyces cerevisiae, omdat dit wyn van gehalte produseer, maar probleem fermentasies kom wel voor. Die verskynsel van vasval of stadige fermentasies kan lei tot die verlies van produksie en kwaliteit. Die oorsaak van probleem fermentasies is gewoontlik veelvoudig, maar die verhouding van glukose tot fruktose in die wyn-mos kan ongunstig raak om fermentasies te onderhou. S. cerevisiae is 'n glukofiliese gis, wat sy voorkeur om glukose bo fruktose te gebruik beskryf. Albei hierdie heksose suikers is teenwoordig in ongefermenteerde wyn-mos, meestal in gelyke hoeveelhede. Soos fermentasies vorder word glukose vinniger verbruik as fruktose wat lei tot 'n toename in die fruktose tot glukose verhouding. Die gis moet dus die fruktose in die later stadium van fermentasie gebruik wanneer die omgewings druk op die gis kan lei tot probleem fermentasies. Die oorblywende fruktose kan lei tot ongewenste soetheid aangesien fruktose twee keer soeter is as glukose. Selfs met die ekstensiewe navorsing met betrekking tot gis metabolisme is daar nog nie 'n verduideliking hoekom gis glukose vinniger as fruktose gebruik nie. Hierdie studie het beoog om die meganisme wat lei tot die vinniger verbruik van glukose oor fruktose te ondersoek vir 'n kommersieël gebruikte gis S. cerevisiae VIN 13. Die eerste twee stappe van suiker metabolisme, suiker opname en fosforilasie, was ondersoek as die moontlike punt van die verskil in fermentasie tempo. Ensiem snelhede en affiniteite vir beide glukose en fruktose as substrate vir die ensieme van belang was eksperimenteel bepaal. Hierdie waardes is gebruik om 'n bestaande model van gis glikolise aan te pas vir wyn fermentasies. Die uitvoerbaarheid van saamstel en valideer van 'n kinetiese model van wyn fermentasies was ondersoek, deur model voorspelde fluksie waardes met eksperimentele fluksie waardes te vergelyk. 'n Ander aspek van die studie was die ondersoek van die effek van heksose suiker tipe op fermentasie profiel. Wyn fermentasies is gedoen met slegs een heksose suiker as koolstof bron om te bepaal of dit 'n invloed het op die fluksie deur metabolisme. Hierdie werk het daarin geslaag om 'n kinetiese model saamtestel wat onderskei tussen glukose en fruktose as koolstof bron. Die glukose is vinniger verbruik as fruktose, met beheer gesetel in die heksose opname stap. Dit was ook vasgestel dat fermentasie profiele van fermentasies met slegs een suiker nie verskil het vir fermentasies met slegs fruktose of glukose. Fermentasies met slegs een suiker het dieselfde spesifieke produksie en konsumpsie tempo gehad as die normale fermentasie met albei suikers. Die konstruksie van 'n gedetailleerde kinetiese model kan gebruik word in die metaboliese en sellulêre ontwikkeling van nuwe gisstamme. Deur die ontdekking van die belangrikheid van heksose opname in fluksie beheer, wat lei tot die glukofiliese karakter van gis, kan gis opname geteiken word vir gis ontwikkeling. Dit mag om die beurt lei tot meer effektiewe fermentasie praktyk in die beheer van probleem fermentasies, of die ontwikkeling van nuwe stamme wat fruktose in dieselfde manier as glukose benut, en sodoende die risiko van vasval of stadige wyn fermentasies verlaag.
National Research Foundation
Post-graduate Merit Bursary

>Magister Scientiae - MSc
Knowledge of haematological 'normdata', of experimental animals, and the biological variables that affect it is essential in order to recognise variations from the normal. In addition, the haemopoietic system may be regarded in principle as good material for studies of the cellular events associated with ageing. These considerations, together with the well documented effects of age on various physiological processes, prompted an investigation into the effects of donor age on several blood parameters. Review of the literature revealed that age-related changes in blood parameters have been reported for several species, but the documentation thereof is incomplete, inconsistent and inconclusive in many respects. Blood samples from male Wistar rats of nine different biological ages, ranging from birth to 96 weeks of age, were analysed for haematological and biochemical parameters. These included the blood cell counts, erythrocytic indices, haemoglobin concentration, haematocrit, erythrocytic 2,3-diphosphoglycerate and adenosine triphosphate levels, and erythrocytic glucose 6-phosphate dehydrogenase and pyruvate kinase activities. Data was obtained which demonstrates that all blood parameters measured underwent significant, although not al~ays regular, age-related changes. These changes were found to be more marked during the first month of life than at any other period. Evidence is also presented to show that the depressed haemoglobin concentration during the early postnatal life may not imply a condition of 'physiologic anaemia' as was previously thought. Since the blood profile exhibits only slight changes from about 24 weeks of age, it does not seem that the haemopoietic system of the old rat deteriorates significantly as to constitute a limiting factor for the animal's life. However, the importance of taking an animal's age into account when blood parameters constitute experimental results is emphasised. The second phase of this study involved a detailed investigation of the effect of the animal's age on erythrocytes in particular. These cells have limited life-spans, and are often used as models in studies of cellular ageing. Special emphasis was therefore placed on comparing the relative effects of host and cellular ageing on the properties of these cells. Erythrocytes from rats between one and 48 weeks of age were separated into two populations by a modification of the conventional density gradient centrifugation technique. The two populations were assumed to differ in mean cell age and were analysed for erythrocytic indices, phosphate ester concentrations and the activities of glucose 6-phosphate dehydrogenase and pyruvate kinase. Evidence is presented to show that ageing rat erythrocytes exhibit a decrease in volume, phosphate ester content and enzyme activities while the cellular haemoglobin concentration increases. Differences in the mean cell age however, does not seem to account for the donor-age-related effects observed in the whole blood parameters. Rather, the significant differences found in the characteristics of similarly aged red cells, between variously aged donors, demonstrate that the biological age of the organism influences the red cells and probably the ageing thereof in vivo. The contribution of the changing status of the erythrocyte's environment of progressively older animals, to alterations which take place in the ageing red cell is discussed.

Le développement tumoral tel qu'il est récemment modélisé selon le concept des cellules souches cancéreuses (CSC) est un modèle statique dans lequel les CSC seraient les seules responsables de l'émergence, de la résistance aux traitements ainsi que de la récurrence tumorale. Cependant, la biologie du cancer est bien plus complexe et la plasticité des CSC suggère l'existence d'une conversion bidirectionnelle entre les CSC et les non-CSC. Cette thèse vise à élucider les mécanismes par le biais desquels l'autophagie, un processus d'auto-digestion, régit le destin des CSC mammaires et apporte une meilleure compréhension au processus de plasticité. Nos résultats soulignent l'implication de l'autophagie dans le remodelage métabolique en augmentant la glycolyse aux dépens de la phosphorylation oxydative et ceci est accompagné par l'émergence des CSC. En effet, nous montrons que l'Oncostatine M (OSM), une cytokine pro-inflammatoire de la famille de l'IL-6, régule l'autophagie et l'expression de l'hexokinase II (HK II). Cette enzyme, la première de la voie du métabolisme du glucose, est décrite pour jouer un rôle clé dans l'effet "Warburg". Nous montrons que l'invalidation de l'expression de HK II et PI3K/AKT prévient l'induction de la population CSC. De manière originale, nos résultats mettent en évidence un nouveau rôle pour l'autophagie qui confère, par acétylation, une protection à l'HK II contre la dégradation par le protéasome, permettant ainsi de maintenir une glycolyse accrue nécessaire pour l'émergence et le maintien des CSC
Tumor development as recently modelized according to the concept of cancer stem cells (CSCs) is a static model in which CSCs are the only ones responsible for emergence, resistance to treatment and tumor recurrence. However, the cancer biology is complex and the plasticity of CSCs suggests the existence of a bidirectional conversion between CSCs and non-CSCs. This thesis aims to elucidate the mechanisms by which autophagy, a process of self-digestion, governs the fate of breast CSCs and provides a better understanding of the process of plasticity. Our results highlight the involvement of autophagy in metabolic remodeling by increasing glycolysis at the expense of oxidative phosphorylation and this is accompanied by the emergence of CSCs. Indeed, we show that Oncostatin M (OSM), a pro-inflammatory cytokine of the IL-6 family, regulates autophagy and the expression of hexokinase II (HK II). This enzyme, the first of the glucose metabolism pathway, is described to play a key role in the 'Warburg' effect. Here we report that inhibition of HK II and PI3K / AKT prevent the induction of CSC population. Notably, the results presented in this thesis attribute to autophagy a new role which confers, by acetylation, a protection to HK II against the degradation by the proteasome, making it possible to maintain an increased glycolysis required for the emergence and maintenance of CSCs

Par RMN du phosphore-31, nous avons suivi la production de désoxy-2-glucose-6 phosphate dans les hématies intactes, incubées à 35°C, avec du désoxy-2 glucose utilisé à concentration saturante vis-à-vis de l'hexokinase. Parallèlement nous avons suivi la disparition en fonction du temps des 2,3 diphosphoglycerate et de l'ATP ainsi que la production des phosphates inorganiques. Ce travail nous a donc permis de mettre au point un protocole de mesure de la vitesse de phosphorylation du désoxy-2 glucose par l'hexokinase dans des hématies intactes. La variabilité interindividuelle de cette vitesse a été testée avec le sang de dix sujets sains de sexe masculin, elle n'est que de 30 %. Par contre pour des sujets de sexe féminin, nous avons observé des variations de 250 %. Toutefois une constance de cette vitesse est observée pour les femmes ménopausées et de plus pour les femmes soumises à un contraceptif oral, nous avons distingué une différentiation de la vitesse suivant la nature de la pilule. Aucune relation simple entre ce paramètre et les taux hormonaux mesurés dans le plasma n'a pu être établie, par contre ce paramètre montre une tendance à la corrélation avec la concentration en 2,3 DPG. Pour des sujets anémiés soumis à un traitement de dialyse, cette vitesse est environ 2 ou 3 fois plus élevée ce qui traduit un métabolisme du désoxy-2 glucose plus rapide qui est du à un taux élevé de réticulocytes chez ces sujets. La mesure des vitesses de phosphorylation pour des concentrations en sucre variable et non saturante, nous a permis de mesurer les paramètres cinétiques apparents (K'm et V'max) de l'hexokinase dans son milieu naturel
With 31 P NMR, we have followed the 2-deoxyglucose (2DG) production in human intact erythrocytes incubated at 35 °C with 2-deoxyglucose at saturating concentration towards hexokinase. At the same time we have followed time course of intracellular metabolites (2, 3-DPG, Pi, ATP). So this work has allowed us to establish a measurement protocol of the 2 DG phosphorylation rate by hexokinase in intact red blood cells. This rate parameter was tested on the blood of 10 healthy male subjects, his variation is only 30%. Nevertheless it shows 250% of variation for the blood of healthy female subjects. However a constancy of the rate was observed on menopausal women and differentiation of this parameter for women under oral contraceptive with respect to the contraceptive rapture. We haven't observed any relation between the hormonal plasma level and the rate, but it appear that this parameter may be related linearly with the concentration of 2,3-DPG. For anaemic subjects, this parameter is 2 or 3 times higher which translate a rapid 2DG metabolism related to reticulocyte high level for this subjects. The measurement of the phosphorylation rates for variable and no saturing concentrations, allows us to determine the apparent hexokinase kinetic parameters (K'm et V'max) in this natural milieu

Alzheimer’s Disease (AD) is a neurodegenerative disorder of the central nervous system. It is mainly sporadic, however, a little percentage of cases is inherited (Familial AD, FAD) and due to autosomal dominant mutations on three different genes, coding for Amyloid Precursor Protein (APP), Presenilin 1 (PS1) and Presenilin 2 (PS2). Presenilins, mainly localized at Endoplasmic Reticulum (ER) membranes, are the catalytic core of the ɣ-secretase complex, although several ɣ-secretase-independent activities of PSs, such as modulation of neurites outgrowth, apoptosis, autophagy, synaptic functions and regulation of Ca2+ homeostasis, have been described. Ca2+, a key intracellular second messenger, is involved in multiple cellular functionalities. Interestingly, alterations in Ca2+ homeostasis have been proposed as an early event in different neurodegenerative diseases, including AD. Notably, FAD-PS mutants have been reported to be directly involved in these dysregulations. In our lab, it has been previously showed that PS2 expression, both WT and, more potently, FAD mutants (such as PS2-T122R), but not PS1, decreases the ER Ca2+ content, mainly by inhibiting SERCA pump activity. Moreover, PS2 increases ER-mitochondria physical and functional coupling, favouring the process of ER to mitochondria Ca2+ transfer. However, due to its effect on ER [Ca2+], which results in a lower amount of available Ca2+ within the ER, its expression dampens mitochondrial Ca2+ rises upon cell stimulation. Based on the well-established role of Ca2+ on mitochondrial metabolism, here we investigate the possible effects on mitochondrial functionalities of the complex balance between alterations in ER Ca2+ content and increased ER-mitochondria coupling, induced by FAD-PS2 mutants expression. A neuroblastoma cell line (SH-SY5Y) grown in a medium containing galactose, as a substitute of glucose, has been used. This growth condition enhances mitochondrial metabolism and results in an excellent experimental protocol to visualize possible mitochondrial defects. Lower total cellular ATP levels were measured in FAD-PS2-T122R expressing cells, grown either in glucose- or galactose-containing medium, with the reduction more evident in the latter condition, thus suggesting possible mitochondrial defects induced by PS2 expression. In order to investigate how Ca2+ dysregulation induced by PS2 could influence mitochondrial metabolism, we stimulated mitochondrial ATP production inducing ER Ca2+ release, followed by mitochondria Ca2+ uptake, using both bradykinin, as a maximal IP3R stimulation, and Fetal Calf Serum (FCS), as a more physiological stimulus. In both conditions, a reduction in mitochondrial ATP production, measured by a mitochondrial luciferase-based ATP probe, has been observed in cells expressing FAD-PS2, but not PS1. The defects in ATP synthesis were observed in SH-SY5Y, MEF, HT22 cells and in cortical neurons from PS2-N141I transgenic (Tg) mice (PS2.30H), by employing FRET-based ATP probes (ATeam 1.03) specifically targeted to the mitochondrial matrix or the nucleus. We also evaluated the glycolytic flux in these cells, by both employing a cytosolic luciferase-based ATP probe and measuring the extracellular medium acidification, but we did not observed any difference in these two parameters in FAD-PS2 expressing cells, compared to controls. In order to understand the mechanism through which PS2 causes the observed mitochondrial dysfunction, we firstly considered the marked Ca2+ dysregulation induced by PS2 expression. We thus decided to modulate Ca 2+ handling in control cells, to mimic the ER Ca2+ depletion caused by PS2 expression. We used two different approaches: i) treating control cells with a SERCA pump inhibitor (Cyclopiazonic acid, CPA), to partially reduce the ER Ca2+ content, or ii) overexpressing a mutated-MICU1 (MICU1mut), a component of the mitochondrial Ca2+ uniporter complex. Although both approaches were able to reduce the capacity of control cells to produce ATP, for similar mitochondrial Ca2+ uptake in control and PS2-expressing cells, a lower mitochondrial ATP production in FAD-PS2 expressing-cells compared to CPA-treated or MICU1mut expressing controls was still observed. Taken together, these results suggest that part of the FAD-PS2-induced defects in mitochondrial metabolism is due to a reduced ER Ca2+ content and, consequently, mitochondrial Ca2+ uptake, negatively regulating the Ca2+-dependent mitochondrial metabolism. However, additional mechanisms, induced by FAD-PS2, are likely involved in mitochondrial dysfunctions. We thus evaluated the respiratory chain activity measuring the oxygen consumption rate (OCR): both basal and maximal OCR were reduced in FAD-PS2, but not in FAD-PS1, expressing cells. Moreover, a reduced mitochondrial ATP-linked respiration was measured in PS2-T122R expressing cells, while no difference was found in the proton leak. Since the expression levels of the ATP synthase and the respiratory chain complexes were not affected by FAD-PS2 expression, and isolated mitochondria from WT and PS2-N141I Tg mice did not reveal substantial differences in mitochondrial respiratory activity, we reasoned that the impairment in ATP production observed in intact cells is not due to defective mitochondria per se, but likely depends on the cellular environment. Importantly, for a proper mitochondrial metabolism, the right amount of substrates produced through glycolysis in the cytosol has to reach the mitochondrial matrix to support the TCA cycle and the respiratory chain activity. Hexokinase1 (HK1), the enzyme that catalyses the first step of glycolysis converting glucose to glucose 6-phosphate, seems to be involved in the modulation of the mitochondrial substrates import, since HK1 interaction/detachment with/from mitochondria can modulate mitochondrial substrates permeability. Firstly, we measured a reduced HK1-mitochondria co-localization in FAD-PS2 expressing SH-SY5Y cells, in FAD-PS2 patient-derived fibroblasts and in primary cortical neurons from FAD-PS2-N141I Tg mice, compared to controls. By mimicking the FAD-PS2 effect on HK1-mitochondria interaction treating control cells with Clotrimazole, a drug capable to detach HK1 from mitochondria, a reduced mitochondrial ATP production was measured; however, the impairment on ATP production induced by clotrimazole was less marked than that caused by FAD-PS2 expression. These results indicate that, although the detachment of HK1 from mitochondria plays a pivotal role in causing mitochondrial defects upon FAD-PS2 expression, the PS2-induced Ca2+ dysregulation, described above, may additionally contribute to the overall mitochondrial impairment. These results have been confirmed also by a genetic approach. We down-regulated the expression of endogenous HK1, by specific siRNAs, and we rescued HK1 protein level by over-expressing siRNA-resistant full-length- (FL-HK1) or truncated- (Tr-HK1) HK1. This latter protein lacks the mitochondrial binding domain, but still conserves the catalytic activity. We found that, upon endogenous HK1 silencing, mitochondrial ATP production is strongly reduced. Interestingly, while the re-expression of FL-HK1 was able to completely rescue the reduced ATP production, the Tr-HK1 was unable to do it, again confirming that the detachment of HK1 from mitochondria is involved in the mitochondrial impairment caused by FAD-PS2. Related to HK1 and its role in the regulation of mitochondrial substrates permeability, an increase in the cytosolic amount of pyruvate was measured in FAD-PS2 expressing cells, compared to controls, employing a cytosolic FRET-based pyruvate probe, Pyronic. Importantly, by pharmacologically blocking mitochondrial pyruvate carrier (MPC), the protein responsible for mitochondrial pyruvate uptake, with two different drugs, UK5099 and Pioglitazone, no differences were anymore detected between control and FAD-PS2 expressing cells, suggesting that FAD-PS2 is acting on this pathway. Overall, we have showed that FAD-PS2 mutants decrease cellular ATP levels, in particular mitochondrial ATP production, by two different mechanisms: 1) causing Ca2+ dysregulation, mainly decreasing the ER Ca2+ content, and thus the amount of Ca2+ available for mitochondrial Ca2+ uptake; 2) inducing the detachment of HK1 from mitochondria, likely affecting the availability of substrates (i.e., pyruvate) for mitochondria. Further experiments will be aimed at: i) evaluate the impact of the PS2-dependent strengthened ER-mitochondria coupling on the reported mitochondrial defects; ii) defining the molecular mechanism through which FAD- PS2 mutants affect HK1 intracellular distribution; iii) evaluate the impact of these alterations on the onset/progression of the AD phenotype.
La malattia di Alzheimer è un disturbo neurodegenerativo del sistema nervoso centrale. È, principalmente, una malattia sporadica; tuttavia in una piccola percentuale di casi è ereditata e dovuta a mutazioni autosomiche dominanti in tre diversi geni, che codificano per la Proteina Precursore dell’Amiloide (APP), per Presenilina1 (PS1) e per Presenilina2 (PS2). Le preseniline, principalmente localizzate nella membrana del reticolo endoplasmatico (RE), costituiscono la porzione catalitica del complesso enzimatico della ɣ-secretasi. Le stesse, oltre ad essere fondamentali per l’attività di questo complesso enzimatico, hanno molte funzioni che sono indipendenti dalla ɣ-secretasi; tra queste, la modulazione della crescita dei neuriti, dell’apoptosi, dell’autofagia, delle funzioni sinaptiche e dell’omeostasi del Ca2+. Il Ca2+ è un secondo messaggero intracellulare fondamentale, coinvolto in molteplici funzionalità cellulari; alterazioni dell'omeostasi del Ca2+ sono state proposte come eventi precoci in diverse malattie neurodegenerative, tra cui la malattia di Alzheimer. In particolare, è stato dimostrato che mutazioni in PS2 associate a forme familiari di Alzheimer (FAD) sono direttamente coinvolte in queste alterazioni. Nel nostro laboratorio è stato precedentemente dimostrato che l'espressione di PS2, sia della forma WT ma soprattutto delle forme mutate associate a FAD (come PS2-T122R), ma non di PS1, riduce il contenuto di Ca2 + nel RE principalmente inibendo l'attività della pompa SERCA. PS2, inoltre, aumenta la vicinanza fisica e funzionale di RE e mitocondri, favorendo il processo di trasferimento di Ca2+ tra i due organelli; tuttavia, a causa del suo effetto sulla [Ca2+] nel RE, che ha come conseguenza una minore quantità di Ca2 + disponibile per il rilascio nel citosol, la quantità di Ca2 + che entra nei mitocondri, dopo stimolazione, è ridotta. Sulla base del ruolo fondamentale svolto dal Ca2 + nella regolazione del metabolismo mitocondriale, nel lavoro presentato in questa tesi abbiamo esaminato i possibili effetti sulla funzionalità mitocondriale del complesso equilibrio tra alterazioni del contenuto di Ca2+ nel RE e l’aumento della vicinanza tra RE e mitocondri, indotti dall’espressione di forme mutate di PS2 legate a FAD. Per svolgere questo studio abbiamo utilizzato una linea cellulare di neuroblastoma (SH-SY5Y), cresciuta in un terreno contenente galattosio, invece di glucosio. Infatti, le cellule cresciute in un terreno che contiene galattosio aumentano il metabolismo mitocondriale, rendendo così questo protocollo sperimentale ottimale per evidenziare eventuali difetti mitocondriali. In cellule esprimenti FAD-PS2-T122R, cresciute in un terreno contenente glucosio o galattosio, sono stati misurati livelli totali di ATP cellulare minori rispetto a quelli di cellule di controllo. La riduzione di questo parametro era più evidente in cellule cresciute in terreno contenente galattosio, suggerendo possibili difetti mitocondriali indotti da PS2. Per studiare come la deregolazione del Ca2+, causata dall'espressione di PS2, possa influenzare il metabolismo mitocondriale, abbiamo indotto il rilascio di Ca2+ dal RE, a cui segue un aumento di Ca2+ nei mitocondri che conseguentemente stimola la produzione di ATP mitocondriale. A tal fine abbiamo utilizzato sia bradichinina, come stimolo massimale del recettore IP3, sia siero fetale di vitello (FCS), contenente fattori che inducono una stimolazione più fisiologica dello stesso recettore. In entrambe le condizioni, è stata osservata una riduzione nella produzione di ATP mitocondriale, misurata utilizzando luciferasi (in particolare la sonda mitocondriale), in cellule esprimenti FAD-PS2, ma non in cellule che esprimevano FAD-PS1. I difetti nella sintesi di ATP sono stati osservati in cellule SH-SY5Y, MEF, HT22 e in neuroni corticali di topi FAD-PS2-N141I (Tg, PS2.30H), utilizzando anche sonde per l’ATP basate su FRET (ATeam 1.03), contemporaneamente espresse nella matrice mitocondriale e nel nucleo. Abbiamo anche valutato se l’espressione di FAD-PS2 potesse influenzare la glicolisi; per fare questo, abbiamo espresso in cellule una luciferasi citosolica, per valutare l’ATP prodotta nel citoplasma, e abbiamo misurato l’acidificazione del mezzo extracellulare, come indice di glicolisi. Per entrambe i parametri, non abbiamo osservato alcuna differenza tra cellule esprimenti FAD-PS2 o di controllo. Per comprendere il meccanismo attraverso il quale PS2 causa la disfunzione mitocondriale osservata, data la nota deregolazione dell’omeostasi del Ca2+ indotta da PS2, abbiamo innanzitutto deciso di simulare la deplezione di Ca2+ nel RE causata dall’espressione di PS2 nelle cellule di controllo. Abbiamo usato due approcci diversi: da un lato abbiamo trattato le cellule di controllo con un inibitore della pompa SERCA (acido ciclopiazonico, CPA) per ridurre il contenuto di Ca2+ nel RE, dall’altro abbiamo sovraespresso una forma mutata di MICU1 (MICU1mut). In entrambi i casi abbiamo ottenuto una riduzione nell’entrata di Ca2+ nel mitocondrio, mimando perfettamente il difetto causato dall’espressione di FAD-PS2. Come atteso, il trattamento con CPA e l'overepressione di MICU1mut riducono notevolmente la produzione di ATP rispetto alle cellule di controllo non trattate. Ciononostante, a parità di Ca2+ che entra nel mitocondrio in cellule esprimenti o meno FAD-PS2, abbiamo misurato una minore produzione di ATP mitocondriale in cellule esprimenti forme mutate di PS2, rispetto ai controlli trattati con CPA o esprimenti MICU1mut. Tali risultati suggeriscono che i difetti nel metabolismo mitocondriale indotti dall’espressione di FAD-PS2 solo almeno in parte riconducibili alla riduzione del contenuto di Ca2+ nel RE, e quindi al suo ingresso nei mitocondri. Tuttavia, sono probabilmente coinvolti meccanismi aggiuntivi nelle disfunzioni mitocondriali osservate. Abbiamo, quindi, valutato l'attività della catena respiratoria misurando la velocità nel consumo di ossigeno (OCR). E’ stato così possibile osservare che sia il consumo di ossigeno a basale che il massimo consumo di ossigeno sono ridotti in cellule esprimenti FAD-PS2, ma non FAD-PS1. Inoltre, in cellule esprimenti PS2-T122R è stata misurata una riduzione della respirazione mitocondriale legata alla produzione di ATP. Tuttavia, poiché i livelli di espressione dell’ATP sintasi e dei complessi della catena respiratoria non variano, in seguito all’espressione di PS2, e dato che misure di respirazione in mitocondri isolati da topi WT e PS2-N141I Tg non hanno rivelato differenze sostanziali, la riduzione nella produzione di ATP osservata in cellule intatta non è verosimilmente dovuta ad un’alterazione intrinseca nell'attività della catena respiratoria. Questo suggerisce che i difetti riscontrati possano dipendere dall'ambiente cellulare, piuttosto che da un difetto intrinseco degli stessi mitocondri. Per un corretto metabolismo mitocondriale, la giusta quantità di substrati prodotti nel citoplasma attraverso la glicolisi deve raggiungere la matrice mitocondriale per supportare il ciclo di Krebs e l'attività della catena respiratoria. L’esochinasi 1 (HK1), enzima che catalizza la prima reazione della glicolisi, convertendo il glucosio in glucosio 6-fosfato, sembra anche modulare l’ingresso dei substrati nei mitocondri, poiché l'interazione/distacco di HK1 con/dai mitocondri può modulare la permeabilità mitocondriale ai substrati. Abbiamo misurato una riduzione nella co-localizzazione tra HK1 e mitocondri in cellule SH-SY5Y esprimenti FAD-PS2, in fibroblasti da pazienti FAD con mutazioni in -PS2 e in neuroni corticali da topi transgenici FAD-PS2. Il trattamento di cellule di controllo con clotrimazolo, una sostanza nota per avere la capacità di indurre il distacco di HK1 dai mitocondri, si è rivelato capace di ridurre la colocalizzazione tra HK1 e mitocondri a un livello simile a quello causato da PS2, mimandone così l'effetto. In seguito a questo trattamento, cellule di controllo mostravano una ridotta produzione di ATP mitocondriale, rispetto a cellule non trattate; tuttavia, l'effetto del clotrimazolo sulla produzione di ATP era meno evidente rispetto alla diminuzione causata dall'espressione di FAD-PS2. Questo significa che, anche se il distacco di HK1 dai mitocondri svolge un ruolo importante nel determinare i difetti mitocondriali osservati in seguito a espressione di FAD-PS2, la disfunzione nell’omeostasi del Ca2+, descritta in precedenza, contribuisce anch’essa alla diminuzione complessiva dell’attività mitocondriale. Questi risultati sono stati confermati anche con un approccio genetico. Abbiamo abbattuto l'espressione di HK1 endogena, mediante specifici siRNAs e abbiamo sovra-espresso la forma intera di HK1 (FL-HK1) o la forma tronca di HK1 (Tr-HK1), proteina ques’ultima che manca del dominio di legame mitocondriale ma che presenta ancora l'attività catalitica. Il silenziamento della proteina endogena causa una notevole riduzione nella produzione di ATP mitocondriale; la ri-espressione di FL-HK1 è in grado di recuperare completamente il difetto nella produzione di ATP, mentre quella di Tr-HK1 no. Questi risultati confermano nuovamente che il distacco di HK1 dai mitocondri è coinvolto nella manifestazione dei difetti mitocondriali osservati in seguito all’espressione di FAD-PS2. Relativamente a HK1 e al suo ruolo nella regolazione della permeabilità mitocondriale ai substrati, in cellule esprimenti PS2 è stato misurato un aumento nella quantità di piruvato nel citoplasma . È importante notare come il blocco farmacologico della proteina responsabile del trasporto del piruvato all’interno del mitocondrio (MPC) con due diversi farmaci, UK5099 e Pioglitazone, annulli le differenze tra le cellule esprimenti FAD-PS2 e i controlli, indicando che l'espressione di FAD-PS2 agisce anche su questa via metabolica. In questo lavoro, abbiamo mostrato che forme mutate di PS2 legate a FAD diminuiscono i livelli cellulari di ATP, in particolare la produzione di ATP mitocondriale, con due diversi meccanismi: 1) causando una deregolazione dell’omeostasi del Ca2+, principalmente diminuendo il contenuto di Ca2+ nel RE, e quindi il conseguente ingresso di Ca2+ nel mitocondrio; 2) inducendo il distacco di HK1 dai mitocondri, influenzando così la disponibilità di substrati (per es., piruvato) per i mitocondri. Ulteriori esperimenti saranno finalizzati a: i) valutare l'impatto dell’aumento della vicinanza tra RE e mitocondri causato dall’espressione di PS2 sui difetti mitocondriali riportati; ii) definire il meccanismo molecolare attraverso il quale FAD-PS2 induce il distacco di HK1 dai mitocondri; iii) valutare l’eventuale impatto di queste alterazioni nella progressione del fenotipo AD.