Dissertations / Theses on the topic 'Metabolic CD34+ cells'

Orientadores: Cristina Pontes Vicente, Patrícia Muniz Mendes Freire de Moura
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: As células progenitoras endoteliais (EPCs) estão envolvidas em neovasculogênese e na manutenção da homeostase vascular, sua deficiência pode ter papel na patogênese da hipertensão. Este estudo teve como objetivo analisar o perfil de expressão das EPCs circulantes e diferentes fatores de risco cardiovascular em mulheres hipertensas, na pré-menopausa, em comparação com mulheres normotensas saudáveis. Realizou-se um estudo caso-controle com 45 mulheres voluntárias, faixa etária de 30 a 50 anos (41 ± 6) no Ambulatório do Pronto Socorro Cardiológico de Pernambuco. As EPCs definidas como CD45-/CD34+/KDR+ foram coletadas em sangue venoso periférico e analisadas por citometria de fluxo. As mulheres foram classificadas como controles (CT) saudáveis normotensas com PAS (pressão arterial sistólica) < 130 mmHg e PAD (pressão arterial diastólica) < 85 mmHg (n=15), com hipertensão primária: a) Leve (HL) PAS=140-159mm Hg e PAD=90-99 mmHg (n=15) e b) Severa (HS) PAS > 180 mmHg e PAD > 110 mmHg (n=15). Os grupos foram entrevistados quanto aos hábitos de fumo, prática de exercícios físicos e Índice de Massa Corporal (IMC), sendo aferido o nível da PA em repouso. Realizou-se análise nos prontuários dos resultados dos exames séricos de colesterol total, lipoproteínas de alta densidade-colesterol (HDL-c), lipoproteínas de baixa densidade-colesterol (LDL-c), triglicerídeos e glicemia de jejum, no mês da coleta das amostras sanguíneas. Os resultados comprovaram redução significativa ao número de EPCs no HL (74%) e HS (88%) versus CT; e redução de 67% no HS versus HL, evidenciando relação inversa entre o número de células e o estágio da hipertensão. O grupo HS apresentou aumento de 49% de células CD45+ demonstrando padrão inflamatório e redução de 61% de CD45-/CD34+. Quanto aos níveis séricos verificou-se: HDL-c [HL (52±7); HS (48±5)]; LDL-c [HL (130±8); HS (143±15)]; triglicerídeos [HL (138±19); HS(153 ±40)]; glicemia de jejum [HL(95±7);HS(121±39)] e IMC [HL(31±4);HS(29±3)]; revelando que 67% das mulheres com hipertensão severa apresentavam síndrome metabólica (SM). O desenvolvimento da hipertensão e da SM foi diretamente correlacionado com a diminuição das EPCs. Portanto, a contagem de EPCs pode ser considerada um marcador biológico adequado para indicar a gravidade do estado hipertensivo em mulheres
Abstract: Endothelial progenitor cells (EPCs) are involved in neovasculogenesis and maintenance of vascular homeostasis and their impairment may have a role in the pathogenesis of hypertension. This study aimed analyzes the expression profile of circulating EPCs and different cardiovascular risk factors in hypertensive premenopausal women compared with healthy normotensive women. A case-control study was conducted enrolling 45 women volunteers, aged from 30- 50 years (41 ± 6) in Ambulatory of the Cardiologic Emergency Hospital of Pernambuco. EPCs numbers were determined by flow cytometry in peripheral blood as the CD45-/CD34+/KDR+ cells. The women were classified as healthy normotensive controls (CT) with SBP (systolic blood pressure) <130 mmHg and DBP (diastolic blood pressure) < 85 mmHg (n=15), and with essential hypertension; a) mild (MH), SBP=140-159 mmHg and DBP=90-99 mmHg (n=15); and b) severe (SH), SBP>180 mmHg and DBP>110 mmHg (n=15). The group were interviewed regarding smoking habits, physical exercise and body mass index (BMI), and measured the level of blood pressure at quiescent. An analysis in records of test results cholesterol, high density lipoprotein-cholesterol (HDL-c), low density lipoprotein-cholesterol (LDL-c), triglycerides and fasting glucose in the month of collection of blood samples. The results found a significant reduction in circulating EPCs numbers in MH (74%) and SH (88%) when compared to the CT and reduction of 67% in SH when compared to MH, an inverse relationship between the number of cells and the stage of hypertension. SH group showed an increase of 49% CD45+ cells demonstrating inflammation and reduction of 61% CD45-/ CD34+ cells. Regarding the biochemical serum was found: HDL-c [MH (52±7); SH (48±5)]; LDL-c [MH (130±8); SH (143±15)]; triglycerides [MH (138±19); SH (153±40)]; fasting glucose [MH (95±7); SH (121±39)] and BMI [MH (31±4); SH (29±3)]; revealing that 67% of women with severe hypertension had metabolic syndrome (MS). Development of hypertension and the parameters related to MS are directly correlated with a decrease of circulating EPCs. Therefore, the EPCs counting may be considered a suitable biological marker to follow up the evolution of the hypertensive state in women
Doutorado
Biologia Celular
Doutora em Biologia Celular e Estrutural

Les cellules stromales mésenchymateuses (CStroM) comprennent des cellules souches (CS) multipotentes capables de régénérer des tissus lésés par des agressions de type ischémique. Pourtant, une mortalité élevée des CStroM après transplantation est mise en évidence lors de leur prise de greffe. Par conséquent, explorer les stratégies visant à améliorer la viabilité des greffes cellulaires constitue le défi de la thérapie cellulaire. À cette fin, nous avons effectué des analyses fonctionnelles et métaboliques sur deux types de population différents contenant des CS somatiques : des CStroM et une population de cellules partenaires de la niche hématopoïétique, les CD34+.Les cellules CStroM ou CD34+ ont été cultivées dans des conditions d'anoxie (absence d'O2) et de type ischémique (anoxie/aglycémie, absenced'O2 et de glucose, AA) ou à 3% d’O2 correspondant à la concentration physiologique optimale, puis analysées.Les tests fonctionnels révèlent que les cellules CStroM et CD34+ présentent des propriétés de prolifération et de différenciation complètes en anoxie. Les analyses fonctionnelles de cellules individuelles et d'expression génique ont révélé que les CStroM et CD34+ sont non seulement maintenues dans un état d'AA, mais sont celles dans lesquelles les CS, ayant la capacité de prolifération et de différenciation la plus élevée, sont les plus enrichies. L'analyse métabolique multiparamétrique montre que la survie en anoxie est principalement soutenue par la glycolyse et le métabolisme des lipides. En revanche, l'homéostasie énergétique des CStroM dans la condition AA est partiellement assurée par l'activité mitochondriale anaérobie engageant particulièrement les complexes mitochondriaux I, III et l'ubiquinone. De plus, une accumulation importante de succinate dans cette condition pour les deux types de CS a été mise en évidence. Ceci est dû en partie à une inversion de la succinate déshydrogénase, qui à son tour est entraînée par le débordement de fumarate provenant de la dégradation des nucléotides puriques et par une activité de la navette malate-aspartate. Cependant, les principales voies contribuant à l'accumulation de succinate comprennent la stimulation du glucose/pyruvate induit par le glycogène, ainsi que le métabolisme du corps cétonique, des acides aminés et du propanoate qui fournissent du succinyl-CoA converti en succinate. De plus, la survie en ischémie des CStroM est liée au métabolisme des sulfides et à la consommation de H2S, ainsi qu’à une survie améliorée en présence des donneurs de H2S. L’oxydation du H2S médiée par la SQR entraîne le transport réverse des électrons au niveau du complexe mitochondrial I, en utilisant le glutathion comme accepteur d'électrons. L'analyse de l'utilisation des substrats énergétiques a montré que les cellules CD34+ en anoxie semblent utiliser majoritairement les oses simples afin d’alimenter la voie glycolytique et une diminution conséquente du métabolisme mitochondrial en comparaison à la condition à 3% d’O2. En revanche, en AA, les intermédiaires du cycle de Krebs sont utilisés de manière intensive pour fournir les coenzymes NAD/NADH.Nos résultats révèlent une grande flexibilité métabolique des populations de CStroM et CD34+ s'appuyant sur l’enrichissement en CS somatiques détecté en anoxie ou dans la condition mimant l’ischémie. Ainsi, contrairement aux cellules différenciées, les CS somatiques (mésenchymateuses et hématopoïétiques) ont la capacité de survivre dans des conditions d'anoxie et d'aglycémie en utilisant les voies énergétiques conservatrices évolutives existant chez les premiers eucaryotes vivant dans des zones anoxiques enrichies en sulfide. L’exploitation de ce conditionnement ex vivo dans des conditions imitant l’ischémie pourrait constituer une stratégie visant à améliorer la survie des CStroM implantées dans des tissus hypoxiques/ischémiques
Mesenchymal stromal cells (MStroC) comprise multipotent stem cells (SC) capable of regenerating tissues damaged by ischemic insults. However, high mortality of MStroC after transplantation is highlighted during their engraftment. Therefore, exploring strategies to improve the viability of cell grafts constitutes the challenge of cell therapy. To this end, we performed functional and metabolic analyzes on two different types of populations containing somatic SC: MStroC and a population of hematopoietic niche partner cells, CD34+.MStroC or CD34+ cells were cultured under conditions of anoxia (absence of O2) and ischemic type (anoxia/aglycemia, absence of O2 and glucose, AA) or at 3% O2 corresponding to the physiological optimal concentration, then analyzed.Functional assays reveal that MStroC and CD34+ cells exhibit complete proliferation and differentiation properties in anoxia. Functional analyzes of single cells and gene expression revealed that MStroC and CD34+ are not only maintained in an AA state, but are those in which SC, having the highest proliferation and differentiation capacity, are the most enriched. Multiparametric metabolic analysis shows that survival in anoxia is mainly supported by glycolysis and lipid metabolism. On the other hand, the energy homeostasis of MStroC in the AA condition is partially ensured by anaerobic mitochondrial activity particularly involving mitochondrial complexes I, III and ubiquinone. Furthermore, a significant accumulation of succinate in this condition for both types of SC was demonstrated. This is due in part to an inversion of succinate dehydrogenase, which in turn is driven by fumarate spillover from purine nucleotide degradation and malate-aspartate shuttle activity. However, major pathways contributing to succinate accumulation include glycogen-induced glucose/pyruvate stimulation, as well as ketone body, amino acid, and propanoate metabolism which provide succinyl-CoA converted to succinate. Furthermore, MStroC ischemia survival is linked to sulfide metabolism and H2S consumption, as well as improved survival in the presence of H2S donors. SQR-mediated H2S oxidation results in reverse electron transport at mitochondrial complex I, using glutathione as an electron acceptor. The analysis of the use of energy substrates showed that CD34+ cells in anoxia seem to mainly use simple sugars in order to fuel the glycolytic pathway and a consequent reduction in mitochondrial metabolism compared to the 3% O2 condition. In contrast, in AA, Krebs cycle intermediates are used intensively to provide the coenzyme NAD/NADH.Our results reveal a great metabolic flexibility of MStroC and CD34+ populations based on the enrichment of somatic SC detected in anoxia or in the condition mimicking ischemia. Thus, unlike differentiated cells, somatic SC (mesenchymal and hematopoietic) have the capacity to survive in conditions of anoxia and aglycemia using the evolutionary conservative energy pathways existing in early eukaryotes living in anoxic zones enriched in sulfide . Exploiting this ex vivo conditioning under conditions mimicking ischemia could constitute a strategy to improve the survival of MStroC implanted in hypoxic/ischemic tissues

Trichuris trichiura is a gastrointestinal dwelling nematode that infects almost 500 million people worldwide. T. muris occurs naturally in mice and is very closely related the human whipworm, making it a suitable model to dissect the immune response against the parasite. Studies using the Trichuris muris system have identified CD4+ T cells as dictators of the outcome of infection. In wild type mice, infection with a high dose of T. muris eggs leads to resistance and worm expulsion, which are dependent on a Th2 response and the secretion of type 2 cytokines especially interleukin (IL) 13. Chronicity is dependent on a Th1 response and occurs when mice are infected with a low dose of T. muris eggs. It is well established that metabolic changes are essential to promoting T cell activation and effector function. Moreover, during chronic infection the host immune system is continuously exposed to parasite antigen, which represents a metabolic challenge. This thesis has investigated the importance of T cell metabolism during response against T. muris. Data presented here show that low and high dose T. muris infections promote upregulation of the glycolytic pathway in CD4+ T cells. During later stages of chronic infection, CD4+ T cells displayed supressed glycolysis and mitochondrial respiration, and may be due to metabolic modulation imposed by the parasite. Leucine uptake via the amino acid transporter Slc7a5 was previously shown to be required for mTORC1 activation and for T cell effector function. Data presented here show that in early stages following a high dose T. muris infection, mice that lack Slc7a5 in T cells have delayed worm expulsion, impaired production of antibodies, and lower levels of IL-13. Their CD4+ T cells present reduced glycolytic rates when compared to cells from cohoused infected wild type mice. However, at later stages of infection, antibody, IL-13 and glycolytic levels were restored together with worm expulsion. CD4+ T cells from the early stage of infection showed reduced phosphorylation of mTOR, which suggested that impairment of function was mTOR dependent. Indeed, mice lacking mTOR in T cells fail to expel a high dose of parasites. They showed abrogation of IL-13 production, impairment in antibody class switching and their CD4+ T cells failed to upregulate glycolysis. Thus, this thesis shows that mTOR is essential for the proper functioning of T cells during T. muris infection and efficient amino acid transport plays a significant role. Taken together, these data show that metabolic orchestration of T cell function influences the capacity to effectively control helminth infection and that even subtle changes in T cell metabolic control can have a major effect on response phenotype.

Naturally-occurring CD4+Foxp3+ regulatory T cells (nTreg) play a central role in maintaining immune self-tolerance as well as modulating immunity towards pathogens. Pathogens may establish chronic infections in immunocompetent hosts by engaging nT reg in order to promote immunosuppression. The goal of the research described here is to test the hypothesis that nTreg modulate protective immunity to malaria, and consequentially affect susceptibility to the parasite. To investigate this question, the functional dynamics of CD4+Foxp3 + nTreg cells were evaluated in mice infected with blood-stage Plasmodium chabaudi AS. Adoptive transfer of nTreg to infected wild-type C57BL/6 (B6) mice or infection of transgenic B6 mice over-expressing Foxp3 resulted in increased parasitemia and reduced survival compared to control mice. Moreover, while resistant B6 mice exhibited decreased splenic nT reg frequencies at day 7 post infection, susceptible A/J mice maintained high numbers of nTreg at this time. Investigation of the effects of nTreg regulation on immune cell function in P. chabaudi AS-infected mice revealed that increased nTreg frequencies led to decreased malaria-specific lymphoproliferation and increased systemic levels of IL-10. Unlike B6 mice, increased splenic nTreg frequencies in infected A/J mice correlated with decreased effector T cell proliferation and IFN-gamma secretion, decreased B cell and NK cell proliferation as well as deficient IFN-gamma secretion by NK cells. Finally, nTreg proliferated within infected sites in both B6 and A/J mice, albeit to a greater extent in susceptible A/J mice. Altogether, these results demonstrate that nTreg suppressed anti-malarial immunity, and in turn promoted parasite growth and persistence.

Hintergrund: Die Funktion von Immunzellen hängt von einer konstanten und ausreichenden Energieversorgung ab, die über die OXPHOS in den Mitochondrien und die Glykolyse im Zytosol realisiert wird. Die wichtigsten Substrate dafür sind Sauerstoff und Glukose. Fragestellung: Bei schweren Erkrankungen oder in Entzündungsgebieten ist die zelluläre Energieversorgung stark beeinträchtigt, weil in der Mikroumgebung der Zelle Sauerstoff und Nährstoffe inadäquat bereitgestellt werden. Ziel war herauszufinden, ob und wie humane Immunzellen ihre Lebensfähigkeit und funktionellen Aktivitäten unter solchen Umständen aufrechterhalten. Methoden: Humane CD4+ T-Zellen und CD14+ Monozyten wurden durch MACS aus peripherem Blut gesunder Spender isoliert. Die Sauerstoffverbrauchsmessung mittels Clark-Elektrode war Maß der oxidativen Energiebildung, die mit Myxothiazol und Glukoseentzug gehemmt wurde. Die CD3/CD28-stimulierte T-Zell-Proliferation wurde durchflußzytometrisch mittels CFDA SE analysiert. Basierend auf dem Paraformaldehyd-Saponin-Prozedere wurde die Zytokinsynthese ebenfalls am FACS bewertet, nachdem die T-Zellen in Anwesenheit von Brefeldin A mit PMA/Ionomycin stimuliert wurden. Mit einem käuflichen Testsystem (FACS-Technik) wurde die monozytäre Phagozytose untersucht. Die HIF-1alpha-Expression wurde nach PMA-Ionomycin-Stimulation von Myxothiazol-behandelten T-Zellen auf mRNA- und Proteinebene gemessen. Ergebnisse: Bei Glukoseanwesenheit waren alle untersuchten Immunfunktionen trotz vollständig gehemmter OXPHOS unbeeinträchtigt. Erst bei gleichzeitigem Glukoseentzug, der per sé Proliferation und Phagozytose signifikant beeinträchtigte, waren sie signifikant vermindert. Es wird vermutet, daß T-Zellen die Energieverluste mit einem überschießenden Effekt ihres Sauerstoffverbrauchs und stark angetriebener Glykolyse kompensieren. HIF-1alpha ist dabei nicht entscheidend für die Umschaltung auf anaerobe Energiesynthese. Schlußfolgerung: Die Daten quantifizieren die Energieanforderungen der funktionellen Aktivität in hochgereinigten humanen Immunzellfraktionen. Es wurde nachgewiesen, daß sich Immunzellen unerwartet lange an eine massiv beeinträchtigte Energetik adaptieren können und ihre spezifischen Funktionen aufrechterhalten.
Background: The function of immune cells is dependent upon a constant and adequate supply of energy. Energy is formed via OXPHOS in the mitochondria and via cytosolic glycolysis. Oxygen and glucose are the main substrates for energy synthesis. Objective: In severe diseases or in inflamed areas cellular energy supply is significantly impaired due to inadequate supply of cellular microenvironment with oxygen and nutrients. The aim of this study was to answer the question, whether and how human immune cells maintain viability and functional activity under these circumstances. Methods: Human CD4+ T cells and CD14+ monocytes were isolated by MACS from peripheral blood of healthy donors. The extent of oxidative energy formation was determined via measurement of oxygen consumption using a Clark type electrode. Energy production was restricted in glucose-free cell culture medium and by gradually inhibited OXPHOS using myxothiazol. T cell proliferation was flow-cytometrically analysed using CFDA SE after stimulation with CD3 and CD28 antibodies. Cytokine synthesis was assessed by flow-cytometrical immunofluorescence and the paraformaldehyde-saponin procedure after stimulation of T cells with PMA/ionomycin in the presence of brefeldin A. Phagocytosis of monocytes was measured using a commercial test system (FACS technique). HIF-1alpha expression was assessed by semiquantitative PCR and immunoblot after the stimulation of myxothiazol treated T cells with PMA/ionomycin. Results: In glucose-containing medium all investigated immune functions were unaffected even under complete suppression of OXPHOS. Only when OXPHOS and glycolysis were simultaneously and almost completely suppressed a significant decrease was found. Glucose deprivation per se caused both a significantly reduced proliferation and phagocytosis. It is supposed, that T cells are able to compensate for an energy deficit by an excess of oxygen consumption and strongly induced glycolysis. However, HIF-1alpha was found to be not crucial for switching to anaerobic energy synthesis. Conclusion: These data quantify the energy requirement of functional activity in highly purified human immune cell fractions. An unexpectedly high adaptive potential of immune cells to maintain specific functions even under massively impaired energetic conditions could be demonstrated.

La famiglia di p53 comprende i tre fattori di trascrizione p53, p63 e p73. Il capostipite della famiglia, p53, è definito il “guardiano del genoma”, grazie alla sua abilità di arrestare il ciclo cellulare in presenza di danno al DNA, consentendone una adeguata riparazione e, nell’eventualità che tale danno non possa essere riparato, promuovere la morte cellulare per apoptosi. p53, dunque, agisce funzionalmente come un classico soppressore di tumore. Infatti, mutazioni con perdita di funzione colpiscono il gene p53 nel 50% circa dei tumori umani. Dopo circa vent’anni di studi intensivi su p53, arrivò, inaspettatamente, la scoperta dei due suoi “parenti” stretti, ad alta omologia di sequenza: p73 e p63. Il gene p63 codifica per le isoforme TAp63, dotate del dominio di trans-attivazione all’N-terminale e, grazie ad un secondo promotore, le isoforme ∆Np63, prive di tale dominio ma, comunque, in grado di trans-attivare i propri geni target attraverso un secondo dominio di trans-attivazione. Inoltre, meccanismi di splicing alternativo all’estremità 3’ terminale, generano isoforme C-terminali. Mentre l’isoforma TA “mima” la funzione oncosoppressiva del parente p53, ∆Np63 mostra una spiccata attività oncogenica, in particolare nei carcinomi a cellule squamose di diversa origine, dove risulta altamente espresso. Infatti, ∆Np63 esibisce una notevole abilità nel regolare la trascrizione di molti geni target coinvolti nelle principali caratteristiche biologiche del tumore, tra le quali crescita, evasione dall’apoptosi, migrazione, invasione ed angiogenesi. Nel seguente studio, sveliamo un nuovo programma trascrizionale orchestrato dall’isoforma oncogenica ∆Np63 e che vede come co-protagostista l’acido ialuronico e la sua attività pro-tumorale. L’acido ialuronico, uno dei maggiori costituenti della matrice extracellulare, si comporta da ligando di specifici recettori di membrana plasmatica, tra i quali il CD44, attivandoli ed “accendendo” pathways coinvolti nella crescita, migrazione, invasione e sopravvivenza cellulare. Come atteso, molti tipi tumorali sfruttano la segnalazione dell’acido ialuronico per promuovere la loro crescita, sopravvivenza ed, in fine, la loro chemioresistenza. In particolare, l’interazione tra l’acido ialuronico e il CD44 favorisce l’attivazione dei recettori tirosina chinasi, tra i quali l’EGFR, e dei loro pathways oncogenici. Inoltre, l’attivazione del CD44 da parte dell’acido ialuronico, promuove l’espressione dei trasportatori ABC ed il conseguente efflusso degli agenti chemioterapici presenti nella cellula. Nel nostro studio, mostriamo la sorprendente abilità di ∆Np63, in cellule tumorali, di regolare positivamente il signaling dell’acido ialuronico, agendo sui livelli dell’acido ialuronico e del suo principale recettore CD44. Infatti, ∆Np63 è in grado di indurre direttamente l’espressione della sintasi 3 dell’acido ialuronico (Has3), coinvolta nella sintesi dell’acido ialuronico e, reprimere quella delle ialuronidasi 1 e 3 (Hyal1 e 3), implicate nella degradazione dell’acido ialuronico, determinando un aumento dei livelli extracellulari di acido ialuronico. Inoltre, ∆Np63 direttamente transattiva il gene CD44, auementandone i livelli proteici. In accordo con tali evidenze in vitro, in diversi datasets di tumori primari di origine squamosa, troviamo che l’espressione di p63 correla positivamente con quella di Has3 e CD44. Conseguentemente, osserviamo un’attivazione dell’EGFR e un aumento dei livelli dei trasportatori ABC, guidati dall’asse ∆Np63/acido ialuronico. Infine, dimostriamo la capacità di ∆Np63 di promuovere la chemioresistenza in maniera dipendente dall’acido ialuronico. Infatti, la deplezione di p63, similmente a quella di Has3 e all’inibizione chimica della sintesi dell’acido ialuronico, riduce la resistenza delle cellule tumorali agli agenti chemioterapici. Inoltre, in data sets di tumori umani, la correlazione positiva tra l’espressione di p63 e Has3 risulta essere un fattore prognostico negativo sulla probabilità di sopravvivenza dei pazienti, suggerendo la crucialità dell’asse p63/acido ialuronico nella tumorigenesi guidata da p63 nei carcinomi squamosi di testa e collo.
p63 is a transcription factor belonging to the p53 family. Like other members of the p53 family, the TP63 gene is expressed as multiple isoforms arising by either alternative promoter usage or differential splicing events at the C-terminus. Particularly, the ΔN proteins, although lack the canonical transcriptional activation domain, are endowed with an alternative transactivation domain and promote cancer cell survival and tumor progression in squamous cell carcinomas (SCCs) of different origins. By combining RNA-seq approach with co-expression studies in human primary tumors, we identified and characterized the hyaluronan synthase 3 (Has3), the hyaluronidase 1 and 3 (Hyal1 and 3), and CD44 as novel ∆Np63 regulated genes. Has3 catalyzes the synthesis of hyaluronic acid (HA), while Hyal1 and Hyal3 catalyze the degradation of HA. CD44 is the main signal-transducing HA plasma membrane receptor. We found that in HNSCC cell lines of different origin, ∆Np63 silencing decreased Has3 expression and concomitantly increased the expression of Hyal1 and Hyal3, resulting in the inhibition of the extracellular level of HA. Mechanistically, we found that ∆Np63 directly bound to and activated the promoter of Has3 and, conversely, bound to the promoter and the 3’-terminal region of Hyal3 and 1, respectively, resulting in the repression of gene expression. Accordingly, in several primary squamous tumor datasets, we found that the expression of p63 was positively correlated with that of Has3. HA-mediated signaling is mediated through its interaction with plasma membrane receptors, including primarily CD44. We found that in tumor cells, ∆Np63 silencing decreased CD44 expression and, accordingly, the expression of p63 was positively correlated with that of CD44 in primary squamous tumor data sets. Mechanistically, we found that ∆Np63 directly bound to and activated the promoter of CD44. HA-CD44 interaction is important for mediating chemoresistance by sustaining the full activation of several tyrosine kinase receptors (EGFR, ErbB2 and IGFR) and promoting the expression of ABC drug transporters. We found that ∆Np63, through its action on HA metabolism and CD44 abundance, regulated the tyrosine kinase receptors activation and ABC drug transporter expression, mediating thus, tumor chemoresistance. Notably, in vitro ∆Np63-depletion, similar to Has3 silencing and HA-synthesis inhibition by 4-MU treatment, reduced cancer cell chemoresistance, indicating the ∆Np63 ability to promote chemoresistance in a HA-dependent manner. Moreover, in different human tumors data sets, the positive correlation between p63 and Has3 expression was a negative prognostic factor on HNSCC patient survival, suggesting that the ΔNp63/HA signaling axis is an important determinant of the p63-driven tumorigenesis.

Les lymphocytes T CD4+ Foxp3+ régulateurs (Treg) participent à la régulation de l’activité du système immunitaire et sont essentiel au maintien de la tolérance immune. Ces cellules sont ainsi bénéfiques lors de pathologies auto-immunes tandis que leur action anti-inflammatoire favorise la croissance tumorale. Comprendre le fonctionnement des Treg constitue donc un axe majeur pour le développement de nouvelles stratégies thérapeutiques applicables à ces deux types de pathologies. Au cours de ce travail, nous nous sommes intéressés au métabolisme des Treg étant donné que les informations disponibles à ce jour font encore l’objet de controverse. A l’inverse des lymphocytes T conventionnels (Tconv), qui utilisent un métabolisme de type anabolique sous le control de mTOR, les Treg sont aujourd’hui considéré comme des cellules présentant un métabolisme de type catabolique favorisé par l’AMPK. Afin de vérifier cette hypothèse, nous avons utilisés des modèles murins de délétion conditionnelle permettant la délétion génique d’AMPK ou mTOR, spécifiquement dans les Treg. L’étude de ces souris a permis de mettre en évidence un rôle jusqu’ici insoupçonné de mTOR dans la stabilité et la migration des Treg ainsi qu’une implication de l’AMPK dans la capacité des Treg à inhiber la réponse anti-tumorale. Ces travaux remettent ainsi en question les considérations actuelles sur le métabolisme des Treg et ouvrent la voie vers une meilleure compréhension du métabolisme de ces cellules à l’homéostasie et en condition pathologique
CD4+ Foxp3+ regulatory T cells (Treg) are able to control the activity of the immune system and are essential to maintain immune tolerance. These cells are thus beneficial in autoimmune diseases while their anti-inflammatory action promotes tumor growth. Understanding Treg biology is therefore a major axis for the development of new therapeutic strategies applicable to these two types of pathologies. In this work we investigated Treg metabolism as information available to date are still controversial. In contrast to conventional T cells (Tconv), which use anabolic metabolism under the control of mTOR, Treg are today considered as catabolic cells with a metabolism favored by AMPK. In order to test this hypothesis, we used conditional knock out mice allowing gene deletion of AMPK or mTOR, specifically in Treg. The study of these mice highlight an unsuspected role of mTOR in the stability and migration of Treg as well as an involvement of AMPK in the ability of Treg to inhibit anti-tumor response. This work puts in question current considerations on Treg metabolism and opens the way towards a better understanding of the metabolism of these cells at homeostasis and in pathological context

La susceptibilité des lymphocytes T CD4 (LT) à l'infection par VIH-1 est régulée par le métabolisme du glucose et de la glutamine. Cependant, les contributions relatives de ces nutriments à l'infection étaient peu connues lorsque j’ai débuté ma thèse. Au cours de mes travaux, j'ai identifié la glutaminolyse comme une voie majeure alimentant la phosphorylation oxydative (OXPHOS) dans les sous-populations de LT activés naïves et mémoires, et j'ai découvert que l'induction de ce réseau métabolique est nécessaire pour une infection optimale par VIH-1. J’ai constaté qu’en condition limitante en glutamine, l’α-kétoglutarate (α-KG), un intermédiaire du cycle TCA (TriCarboxylic Acid) issu de la glutaminolyse, constitue un facteur clé de l'infection des LT CD4 par VIH-1. L'ajout d’α-KG exogène induit une augmentation rapide du ratio OXPHOS/glycolyse et rend les LT naïfs et mémoires plus susceptibles à l’infection. Par ailleurs, l’inhibition du flux glycolytique du pyruvate vers le lactate induit une augmentation de l'OXPHOS et de l'infection des LT CD4 par VIH-1. En accord avec ces données, les LT CD4 infectés présentent une augmentation de la biomasse et de l'activité mitochondriale en comparaison à leurs homologues non infectés. Ces données identifient l'équilibre OXPHOS/glycolyse aérobie comme un élément clé de l'infection des LT par VIH-1.Afin de mieux appréhender les voies métaboliques régulant l'infection des LT par VIH-1, j'ai développé une approche complémentaire basée sur l’utilisation de shRNA ciblant spécifiquement les transporteurs de nutriments GLUT1, ASCT2 et CAT1, permettant notamment le transport du glucose, glutamine et arginine dans la cellule, respectivement. Ainsi, j’ai observé une diminution de la survie d’environ 80% des cellules shRNA+, témoignant ainsi de l’importance de ces transporteurs lors de l’activation de ces cellules. Cependant, la permissivité des LT CD4 à l’infection par VIH-1 est impactée différemment par la diminution de l’expression des transporteurs de nutriments. Conformément aux données présentées ci-dessus, l’inhibition de GLUT1 n'a pas eu d'impact significatif sur l'infection par VIH-1, tandis que l’inhibition de CAT1 a réduit de manière significative l’OXPHOS ainsi que l'infection par VIH-1 (de 35 %). Il est toutefois surprenant de constater que l’inhibition d’ASCT2 entraîne une augmentation de l'infection de 20 %. Cela était associé à une persistance significativement plus élevée des cellules T naïves dont l’expression d’ASCT2 était inhibée, par rapport aux LT mémoires. Ces données mettent ainsi en évidence l’importance relative de ces 3 transporteurs de nutriments dans la survie des LT naïfs par rapport aux LT mémoires et démontrent leur impact spécifique sur la permissivité de ces populations à l'infection par VIH-1.En conclusion, en utilisant deux approches complémentaires, mes travaux de thèse révèlent l'impact critique de l'état énergétique d'une cellule T CD4 sur sa susceptibilité à l'infection par VIH-1. Mes données mettent en évidence l'importance du métabolisme mitochondrial, avec un environnement riche en intermédiaires du cycle du TCA comme l’α-KG, dans la régulation de la sensibilité des LT à l'infection par VIH-1. En outre, l'expression des transporteurs de nutriments impacte différentiellement la sensibilité des LT naïfs et mémoires à l'infection par VIH-1. Ces études offrent donc de nouvelles perspectives utilisant le métabolisme pour le développement de stratégies thérapeutiques ciblées contre l'infection par VIH-1
The susceptibility of CD4 T cells to HIV-1 infection is regulated by glucose and glutamine metabolism, but the relative contributions of these nutrients to infection are not known. During my PhD, I identified glutaminolysis as a major pathway fueling oxidative phosphorylation (OXPHOS) in activated naïve as well as memory CD4 cell subsets, and found that induction of this metabolic network is required for optimal HIV-1 infection. Moreover, we determined that under conditions of attenuated glutaminolysis, the α-ketoglutarate (αKG) TCA (tricarboxylic acid) cycle intermediate is a rate-limiting step in infection; exogenous α-KG directly increased OXPHOS and rendered both naïve and memory CD4 T cells significantly more sensitive to infection. Furthermore, blocking the glycolytic flux of pyruvate to lactate resulted in an increased OXPHOS and a significantly augmented level of HIV-1 infection. In agreement with these data, infected CD4 T cells exhibited increased mitochondrial biomass and respiration as compared to their non-infected counterparts. These data identify the OXPHOS/ aerobic glycolysis balance as a major regulator of HIV-1 infection in CD4 T lymphocytes.In order to gain more insight into the metabolic pathways regulating HIV-1 infection in CD4 T cells, we developed a complementary approach to target upstream processes, specifically altering glucose (GLUT1), glutamine (ASCT2), and arginine (CAT1) transporter expression by lentiviral-mediated delivery of specific shRNAs. Testifying to the importance of these transporters, CD4 T cells with downregulated expression of either GLUT1, ASCT2 or CAT1 were negatively selected, resulting in a loss of approximately 80% of shRNA-transduced cells within 14 days. Notably, the permissivity of CD4 T cells to HIV-1 infection was differentially impacted by inhibition of specific nutrient transporters. Consistent with the data presented above, knockdown of GLUT1 did not significantly impact HIV-1 infection whereas knockdown of CAT1 significantly decreased both OXPHOS as well as HIV-1 infection (by 35%). Surprisingly though, ASCT2 knockdown resulted in a significantly augmented infection, by approximately 20%. Mechanistically, we found that this was associated with a markedly higher persistence of naïve, as compared to memory, T cells with downregulated ASCT2 levels. These data highlight differences in the relative importance of distinct nutrient transporters in the survival of naïve vs memory CD4 T cell subsets and demonstrate their specific impact on the sensitivity of these populations to HIV-1 infection.In conclusion, using two complementary approaches, my PhD research has revealed the critical impact of a CD4 T cell’s energetic state on its susceptibility to HIV-1 infection. My data identify the importance of mitochondrial metabolism, with an environment rich in TCA cycle intermediates such as α-KG, in regulating the susceptibility of CD4 T cells to HIV-1 infection. Furthermore, I find that nutrient transporter expression differentially impacts the sensitivity of naïve and memory CD4 T cells to HIV-1 infection. These studies therefore provide new prospects for the development of targeted metabolic therapeutic strategies against HIV-1 infection

Les leucémies aigües myéloïdes (LAM) sont les leucémies les plus fréquentes chez l'adulte, caractérisée par l'expansion clonale de myéloblastes immatures. Malgré un taux élevé de rémission complète après chimiothérapie conventionnelle d'induction, le pronostic est mauvais dans les LAM à cause de fréquentes rechutes. Les rechutes sont causées par une population leucémique chimiorésistante (CLR), pouvant être enrichies en cellules souches leucémiques quiescentes (CSL). Cependant, ces hypothèses n'ont jamais été testées directement in vivo. Mon travail de thèse a été initialement consacré à valider cette hypothèse in vivo. Nous avons d'abord développé une approche chimiothérapeutique cliniquement pertinente, en traitant avec de la cytarabine (AraC) des souris préalablement xénogreffées à partir de cellules primaires (PDX) de patients atteints de LAM pour caractériser leurs cellules résiduelles post-AraC. Cette population résiduelle et résistante n'est pas nécessairement enrichie en cellules immatures ou en cellules quiescentes et non plus en cellules initiatrices de leucémie. Les cellules résistantes à la chimiothérapie in vivo ont par contre conservé des mitochondries actives, présentent une oxydation accrue des acides gras et une signature génique spécifique, définissant un statut de phosphorylation oxydative élevé ("OxPHOS High"). Le traitement des lignées cellulaires de LAM "OxPHOS High" a démontré une résistance à la chimiothérapie in vivo contrairement aux lignées cellulaires avec un statut "OxPHOS Low". Cibler le statut "OxPHOS High" induit un changement énergétique vers le statut "Low OxPHOS" et améliore l'effet antileucémique de l'AraC.Dans la deuxième partie de mon doctorat, nous avons identifié l'ecto-nucléoside triphosphate diphosphohydrolase-1 CD39 (ENTPD1) surexprimée dans les CLR in vivo après AraC par analyses transcriptomiques des cellules résistantes in vivo. Par cytométrie en flux (FACS), nous avons confirmé que l'AraC augmente l'expression de CD39 à la surface cellulaire des lignées de LAM in vitro et in vivo ainsi que dans 24 modèles PDX. Nous avons également observé cette augmentation chez 50 patients après 35 jours de chimiothérapie intensive par rapport au diagnostic. Fait intéressant, une expression élevée de CD39 chez les patients atteints de LAM est associée à une mauvaise réponse à l'AraC in vivo. Nous avons en outre démontré que la voie de signalisation en aval de CD39 dépend de l'axe AMPc-PKA et son inhibition par H89 sensibilise les cellules de LAM à l'AraC en inhibant PGC1a, TFAM et la fonction OxPHOS mitochondrial. [...]
Acute myeloid leukemia (AML) is the most common adult leukemia characterized by clonal expansion of immature myeloblasts. Despite a high rate of complete remission after conventional front-line induction chemotherapy (e.g. daunorubicin or idarubicin plus cytarabine), the prognosis is very poor in AML. This unfavorable overall survival is caused by frequent relapses due to chemoresistant leukemic cell populations (RLCs) in AML. RLCs are thought to be enriched in quiescent leukemic stem/immature cells (LSCs). However, these hypotheses have never been tested directly in vivo. My PhD work was devoted to validate this hypothesis in vivo. We first developed a clinically relevant chemotherapeutic approach treating patient derived xenografts (PDX) with cytarabine (AraC) to characterize AraC residual cells. AraC-treated AML cells are not necessarily enriched for neither immature cells or quiescent (G0) cells or leukemic initiating cells (LICs). Rather chemotherapy resistant cells in vivo have high levels of reactive oxygen species (ROS), showed increased mitochondrial mass, and retained active mitochondria, consistent with a high oxidative phosphorylation (OxPHOS) status. AraC residual cells exhibited increased fatty acid oxidation and a high OxPHOS gene signature predictive for treatment response in PDX and patients. Treatment of High OxPHOS but not Low OxPHOS human AML cell lines demonstrated chemotherapy resistance in vivo. Targeting mitochondrial metabolism induced an energetic shift toward low OxPHOS status and markedly enhanced antileukemic effects of AraC in AML. In the second part of my PhD study, we identified ecto-nucleoside triphosphate diphosphohydrolase-1 CD39 (ENTPD1) overexpressed in RLCs in vivo after chemotherapy. We confirmed that AraC increased cell surface CD39 expression in AML cell lines in vitro and in vivo as well as in 24 diverse PDX models. We further observed this increase in 50 patients at 35-days post-intensive chemotherapy compared to their respective diagnosis. Interestingly, high CD39 expression on AML patients was associated with a worse response to AraC in vivo. We furthermore demonstrated that CD39 downstream signaling pathway was dependent on cAMP-PKA axis and its inhibition by H89 sensitized AML cells to AraC through the inhibition of PGC1a, TFAM and mitochondrial OxPHOS function.[...]

The prevalence of diabetes is approaching epidemic proportions worldwide. There is currently no cure for type 1 diabetes, and successful treatment requires constant monitoring of blood sugars and use of exogenous insulin to prevent hyperglycemia. Diabetes will be curable when pancreatic β-islet cells can be transplanted into diabetes patients without requiring long-term immunosuppression. This will require learning more about the induction of functional tolerance, a state that maintains the competence of the immune system to most antigens but protects graft-specific antigens from immune rejection, permitting transplantation. One known mechanism of peripheral tolerance is T cell anergy, a phenotype of hypo-reponsiveness in CD4+ T cells. The focus of this thesis is a description of factors shown to be specific to the induction and maintenance of T cell anergy, whose loss reverses the anergic phenotype, restoring the ability of the cells to proliferate in response to antigen. The first of these is Egr-2, a zinc-finger transcription factor, whose presence is required for the induction of anergy induced in T cell clones by TCR stimulation in the absence of costimulation. Egr-2 is shown to be important to anergy induction but not anergy maintenance. In contrast, a negative costimulation receptor, PD-1, is shown to be necessary for the maintenance of anergy. It is possible that learning more about the genetic factors that orchestrate T cell anergy will prove useful in the development of tolerance-based protocols for organ and tissue transplantation without the use of long-term immunosuppression.

碩士
國立臺灣大學
分子與細胞生物學研究所
102
The mechanism of cell growth is strictly regulated.Two phenomena of metabolic reprogramming are involved in cell growth, including Warburg effect and Glutamine-dependent anaplerosis, which are regulated by PI3K/AKT/mTOR pathway. Under growth factors stimulation, cellular protein levels of Hypoxia induce factor-1α (HIF-1α) and c-Myc are massively increased by mechanistics target of rapamycin complex 1 (mTORC1). Our data show that CDK4-cyclin D complex increases protein level of c-Myc. Furthermore, our results demonstrate that increased c-Myc causes activation of anaplerosis that sustains the activity of mTORC1 but not that of mTORC2. In Drosophila studies, it has been shown that the promotion of cell growth by CDK4-cyclin D complex is mediated through Prolyl hydroxylase (PHD). A recent study demonstrated that hydroxylation of Pyruvate kinase M2 isoform (PKM2) by PHD3 enhances recruitment of HIF-1α to hypoxia response elements (HREs) and results in increased expression of PKM2 and PHD3. Consistently, our study shows that treatment with Fascaplysin, the inhibitor of CDK4-cyclin D complex, decreases the protein level of PHD3 in HeLa cell. It appears that PHD3 is involved in the hydroxylation of PKM2, which leads to the positive feedback to protein level of PHD3 and promotes metabolic reprogramming in cell growth.

Recent evidence has shown that leptin, the adipose-tissue derived hormone, can influence T cell homeostasis and survival through the control of genes involved in these processes. Leptin deficient ob/ob mice and humans show reduced circulating number of T cells particularly in the CD4+ compartment. Here, we investigate in vivo how leptin controls survival and proliferation of normal and autoreactive CD4+ T cells. We have previously observed the impaired capacity of myelin-olygodendrocyte glycoprotein peptide (MOG35-55)-specific CD4+ T cells, to transfer experimental autoimmune encephalomyeleitis (EAE) in ob/ob mice. In vivo this process was associated with a progressive decline in dalyed-type hypersensitivity responses (DTH) and proliferation against the MOG35-55 peptide. Adoptive transfer experiments with pathogenic-MOG35-55-activated CD4+ T cells labeled with the fluorescent die 5-6-carboxyfluorescein succinimidylester (CFSE) into normal wild-type (WT), ob/ob and ob/ob-recombinant leptin (rleptin) treated mice, showed in ob/ob mice a reduction of the number of CFSE+CD4+ T cells at different time points as well as increased apoptosis rate and reduced Bcl-2 expression. Similar results were obtained after transferring into ob/ob mice homogeneous, antigen-specific T cells from pigeon cytochrome c (PCC) transgenic (AND-TCR) mice. Next, we explored the molecular targets possibly involved in this process and concentrated our attention on the AKT-mammalian-target of rapamycin (mTOR) signaling pathway. We observed a reduced expression of the AKT and of the downstream mTOR target, S6 ribonuclear protein (S6), in MOG35-55-specific T cells transferred into ob/ob mice. The involvement of the mTOR pathway was also confirmed by a reduced survival of the CD4+ T cells in WT mice after treatment with rapamycin and subsequent reduction in circulating leptin levels. These data highlight the role of leptin in the regulation of survival and proliferation of autoreactive T cells and link the metabolism to autoimmunity.

Understanding the mechanisms that control T cell function and differentiation is crucial to develop new strategies to modulate immune function and prevent autoimmune and inflammatory disease. The balance between effector (Teff; Th1, Th2 and Th17) and regulatory (Treg) T cells is critical to provide an appropriate, but not excessive, immune response and therapies to induce Treg or inhibit Teff are likely promising treatment strategies. It has recently become clear that T cell metabolism is important in both T cell activation and differentiation. T cells undergo a metabolic reprogramming upon activation and not all differentiated T cell subsets utilize the same metabolic fuels or programs.

These metabolic differences are not trivial, as T cell metabolism is tightly

regulated and dysregulation can lead to cell death or reduced immunity. An

understanding of the metabolic differences between Teff and Treg may lead to a new direction for treating inflammatory diseases by modulating the Teff:Treg balance through metabolic inhibition. Previous studies have shown that Teff express higher levels of the glucose transporter Glut1 than Treg, however the role of Glut1, and importantly, the cell-intrinsic role of glucose metabolism in T cell differentiation and inflammation was not previously examined. The work presented here examines the role of Glut1 in T cell differentiation. We show that effector CD4 T cells were dependent on Glut1 for proliferation and function both in vitro and in vivo. In contrast, Treg were Glut1-independent and capable of suppressing colitis in the absence of Glut1 expression.

Additionally, previous studies have shown broad metabolic differences between Teff and Treg, however the specific metabolic profiles of Teff and Treg are poorly understood. Here, Teff and Treg metabolism is examined to test if dependence on distinct metabolic pathways will allow selective targeting of different T cell populations. We show that pyruvate dehydrogenase kinase 1 (PDHK1) is differentially expressed in the T cell subsets and inhibition of PDHK1 selectively suppresses Th17 and promotes Treg differentiation and function. Because Teff and Treg have distinct metabolic profiles, we hypothesized that the Treg-­specific transcription factor FoxP3 may drive the Treg oxidative metabolic program. We therefore examined the role of FoxP3 in T cell metabolism and determined that FoxP3 promotes glucose and lipid oxidation and suppresses glycolytic metabolism. Importantly, we show that promoting glycolysis with transgenic expression of Glut1 inhibits Treg suppressive capacity. Together, these data suggest that FoxP3 drives an oxidative metabolic program that is critical to Treg function. Overall, this work examines the metabolic phenotypes and regulation of Teff and Treg and potential metabolic targets that could be used to treat autoimmune and inflammatory disease.

Dissertation

Tese de doutoramento em Bioquímica, na especialidade de Biologia Celular, apresentada ao Departamento de Ciências da Vida da Faculdade de Ciências e Tecnolologia da Universidade de Coimbra
A activação de linfócitos T, desencadeada por acoplamento do receptor de células T e por várias citocinas, pode apenas ocorrer após a célula gerar energia suficiente. A resposta às crescentes necessidades biossintéticas de uma célula T activada pode ser alcançada pelo aumento da disponibilidade de nutrientes e pela adopção de um metabolismo anabólico, permitindo optimizar a produção de ATP. Em muitos tipos de células diferentes, a função mitocondrial é melhorada pelas proteínas reguladoras de informação silenciosa (Sir), as quais se tratam de deacetilases de histonas dependentes de NAD+ e sensíveis a potencial redox. No entanto, desconhece-se o papel destas proteínas na regulação do metabolismo dos linfócitos T. Resveratrol é um fenol natural com propriedades anti-envelhecimento e anti-inflamatórias, efeitos atribuídos à capacidade deste polifenol em modular o metabolismo celular por indução da actividade da deacetilase Sirtuína 1 (Sirt1). O estudo aqui apresentado mostra que a expressão da proteína Sirt1 aumenta de forma significativa em linfócitos T CD4+ humanos após estimulação do receptor de células T (TCR) e que tratamento com resveratrol promove um acréscimo na actividade desta deacetilase. Esta amplificação da expressão de Sirt1 encontra-se também associada a um aumento na fosforilação dos resíduos de serina na sequência de consenso do sinal de localização nuclear (NLS), algo consistente com as minhas observações de que após activação das células T a expressão de Sirt1 é predominantemente nuclear. Curiosamente, apesar do aumento verificado na expressão de Sirt1, a activação dos linfócitos T encontra-se também associada a um incremento na acetilação dos factores de transcrição p53 e FoxO1, duas proteínas amplamente descritas na literatura como alvos da actividade de deacetilase de Sirt1. Ainda assim, pude verificar que tratamento com resveratrol resultava numa diminuição considerável dos níveis de acetilação de p53 e de FoxO1. De destacar, que o tratamento de linfócitos T CD4 com uma dose alta de resveratrol bloqueia a sinalização proximal ao TCR, resultando numa interrupção do ciclo celular na fase G0. Em contraste, linfócitos T tratados com uma dose baixa de resveratrol não só exibem um estado de activação normal aquando da iniciação do sinal TCR, como são capazes de amplificar as ciclinas e as quinases dependentes de ciclinas (Cdks) necessárias à normal progressão do ciclo celular. Além disso, linfócitos T activos tratados com uma baixa dose de resveratrol exibem elevada actividade metabólica, conforme demonstrado pela maior expressão do transportador de glucose Glut1, respectiva captação de glucose e da função mitocondrial. Além disso, pude ainda verificar que em comparação a linfócitos T activos controlo, os linfócitos T tratados com dose baixa de resveratrol exibem níveis de ATP intracelular mais elevados e um aumento da respiração mitocondrial. De salientar, porém, que apesar de exibirem uma capacidade metabólica notável, tratamento de linfócitos CD4 com uma dose baixa de resveratrol resulta numa paragem do ciclo celular na transição entre as fases G2/M, consequência da ocurrência de um stress genotóxico associado ao aumento da fosforilação da histona H2AX, um marcador de quebra da cadeia dupla de ADN e da helicase de manutenção de minicromossomas Mcm2. A fosforilação tanto da histona H2AX como da helicase Mcm2 encontram-se associadas ao fenómeno de stress replicativo e no decurso do meu estudo foi-me possível verificar que o bloqueio do ciclo celular na transição G2/M se deve à activação do guardião do genoma e regulador do ciclo celular, p53. Adicionalmente, pude também verificar que esta activação de p53 era mediada simultaneamente pela quinase ATR, um dos principais mediadores da reparação de ADN em resposta a um stress replicativo, e pela quinase AMPK, uma enzima conhecida por se associar a Sirt1 na regulação do equilíbrio energético das células. Em conclusão, se por um lado o tratamento de linfócitos T com resveratrol resulta num incremento metabólico e energético graças a um aumento na activade das enzimas Sirt1 e AMPK, por outro, acaba por afectar a integridade genómica levando à activação de p53 e, consequentemente, a um bloqueio na progressão do ciclo celular.
T lymphocyte activation, triggered by T cell receptor engagement and by various chemokines, can only occur when the cell has generated sufficient energy resources. The increased biosynthetic demands of an activated T cell can be met by increasing nutrient availability and adopting an anabolic metabolism, allowing an enhanced ATP generation. In many different cell types, mitochondrial function is enhanced by the Silent information regulator (Sir) proteins, NAD+-dependent redox-sensing histone deacetylases. However, the role of these proteins in regulating T lymphocyte metabolism is not known. Resveratrol is a natural polyphenol that possesses anti-aging and anti-inflammatory properties, being its effects attributed to its capacity to modulate cellular metabolism via induction of the Sirtuin1 (Sirt1) histone deacetylase. Here, I show that Sirt1 is highly upregulated following TCR stimulation of primary human CD4 T lymphocytes with resveratrol further augmenting Sirt1 expression and deacetylase activity. Sirt1 upregulation is also associated with phosphorylation of serine residues in the Nuclear Localization Signal (NLS) consensus, consistent with my observations that Sirt1 is predominantly nuclear following T cell activation. Despite the increase in Sirt1 expression, I find that T cell activation leads to an increased acetylation of p53 and FoxO1, two known Sirt1 targets. Not surprisingly, the TCR-induced acetylation of p53 and FoxO1 was significantly decreased following activation of the Sirt1 deacetylase with resveratrol. Importantly, high dose resveratrol blocked proximal TCR signaling and progression from the G0 phase of the cell cycle. In contrast, CD4 T lymphocytes treated with low dose resveratrol were strongly activated and upregulated all cyclins and Cyclin-dependent kinases (Cdks) required for cell division. Furthermore, these cells exhibited high metabolic activity as monitored by upregulation of the Glut1 glucose transporter, increased glucose uptake and enhanced mitochondrial function. De facto, when compared to control cells, activated T lymphocytes treated with low dose resveratrol show boosted mitochondrial respiration and higher intracellular ATP levels. Nevertheless, low dose resveratrol induced a G2/M genotoxic arrest, with augmented phosphorylation of the histone H2AX double-stranded DNA break marker and the minichromosome maintenance helicase Mcm2. Phosphorylation of both H2AX and Mcm2 is associated with the onset of replicative stress, and I was able to verify that the cell cycle exit observed in lymphocytes treated with low dose resveratrol was associated with the activation of p53. Furthermore, I found that this activation of p53 was mediated both by the serine/threonine-protein kinase ATR, which is involved in sensing DNA damage and activating the DNA damage response, and the AMP-activated protein kinase (AMPK), a fuel sensor known to interact with Sirt1 and share many of its targets. Thus, while resveratrol increased Sirt1 and AMPK activity, producing a positive energy balance in CD4 T cells, its negative regulation of DNA integrity resulted in the induction of a p53-dependent cell cycle checkpoint

碩士
中國醫藥大學
基礎醫學研究所碩士班
102
PART1: Metabolic syndrome is a group of risk factors that increase heart disease and other health problems. The risk factors for development of metabolic syndrome include hyperglycemia, hyperlipidemia, hypertension, low levels of HDL and high levels of triglyceride. Acute toxicity from accumulation of long-chain fatty acids lead to cardiovascular diseases, a phenomenon known as lipotoxicity. Lipoptoxicity leads to imbalance of energy metabolism and result in cardiac dysfunction and contractile dysfunction. Cardiomyocytes use fatty acid (50-70%) and glucose (20-30%) for ATP production. Fatty acids and glucose are uptake by cells through via cluster of differentiation 36 (CD36) and glucose transporter type 4 (GLUT4). In health cardiomyocyte, there are two metabolic pathways, one is muscle contraction pathway and the other one is insulin pathways. They affect CD36 and GLUT4 translocated to cell membrane. In addition, there is convincing evidence that when fatty acids accumulate, the pathway from Akt to CD36 would be inhibited. In this study, we aim to investigate the effect of CD36 and GLUT4 switch and metabolism. In the present study, heart-derived H9c2 cells and neonatal rat ventricular myocytes (NRVMs) were treated with palmitic acid for 24 h. Results showed that CD36 was decreased in cell membrane. Conversely, GLUT4 was accumulated. Palmitic acid also affects energy to switch from CD36 (fatty acid) to GLUT4 (glucose) in a time dependent manner and only affects CD36 protein level but not transcription level. On the other hand, exercise can increase level of high-density lipoprotein (HDL). We want to know whether HDL could inhibit palmitic acid-induced lipoptoxicity and energy switch. PART2:Metabolic syndrome is a group of risk factors that increase heart disease and other health problems. Recent study showed that acute toxicity from accumulation of long-chain fatty acids lead to cardiovascular diseases and result in heart failure, a phenomenon known as lipotoxicity. Lipoptoxicity leads to imbalance of energy metabolism. Autophagy is critical for cell survival during energy crisis state. Its dysregulation has been implicated in a wide variety of cardiovascular pathologies. AMPK related with autophagy. AMPK is an energy-sensing kinase which was activated by metabolism stress. It can regulate autophagy. Recent study showed that palmitic acid-induced autophagy in H9c2 cell, but mechanism still unclear. Our aim to know whether the palmitic acid-induced autophagy via AMPK regulated and the relationship with apoptosis. The H9c2 cardiomyoblast cells were treated with palmitic acid. The result showed that palmitic acid-induced lipoptoxicity and autophagy, and further, we founded that palmitic acid increased LC3 in autophagosome by puncta assay. In western blot, PT172AMPK and other autophagy-related proteins were increased. After added LY294002 and 3-MA, autophagy and apoptosis-related proteins were inhibited. Specially, after added BaFA1 which was lysosome inhibitor, cleavage caspase 3 was accumulated. The same result showed in lysotracker assay and flow cytometry. Taken together, our present results confirmed that palmitic acid-induced lipotoxicity and result in energy imbalance to activate AMPK, mTOR-indepentent signaling pathway which caused autophagic cell death. Recent study showed that high-density lipoprotein inhibited autophagy via PI3K/Akt/mTOR signaling pathway and had anti-apoptosis. The H9c2 cardiomyoblast cells were treated with different dosage of HDL. The result showed that HDL could inhibit palmitic acid-induced autophagy and autophagic cell death. After combined with BLT-1 which was inhibitor of HDL receptor SR-B1, we proved that HDL inhibited autophagy and autophagic cell death via PI3K/Akt/mTOR signaling pathway.