The genus Geosmithia contains 11 described and several tens of undescribed species of fungi living nearly exclusively in galleries of subcorticolous insects, especially bark beetles. In this work, a phylogenetic analysis of the genus was made using DNA sequences of four protein-coding genes, namely TEF-1, RPB2, Mcm7 and Tsr1. The analysis has confirmed that ecological strategies of these fungi (such as association with conifers or broad leaved trees or symbiosis with ambrosia beetles) have evolved several times in this genus. 51 species are recognized based on the obtained phylogenetic tree according to Genealogical Concordance Phylogenetic Species Recognition. I have also tested utility of the above mentioned genes to serve as "barcode" for identification of closely related Geosmithia species.

A malária causa complicações envolvendo diversos órgãos, inclusive o fígado, onde se sabe que ocorrem inflamação e dano hepáticos, os quais contribuem para a severidade da doença. A interleucina (IL)-1 α é uma citocina pró-inflamatória que pertence à família da IL-1 e que pode ser produzida e liberada tanto por células não-hematopoiéticas, como hematopoiéticas em contextos como dano tecidual, infecção e doenças autoimunes em diversos órgãos. O presente estudo tem como objetivo avaliar a produção e o papel da IL-1 na imunopatogênese e na proteção durante a malária experimental. A produção da IL-1 foi investigada durante a inflamação e necrose no fígado em camundongos C57BL/6 infectados com o estágio eritrocítico do Plasmodium chabaudi. A fonte de IL-1 no fígado dos camundongos infectados foi determinada por imunofluorescência e citometria de fluxo. Camundongos IL1A-/- foram utilizados para avaliar o papel da IL-1 neste contexto. Durante a infecção aguda com o P. chabaudi, a inflamação hepática e o desenvolvimento das lesões necróticas são acompanhados pelo aumento nos níveis de IL-1 no fígado, que ocorre de forma independente do inflamassoma NLRP3 (Nod-like receptor protein 3). Os neutrófilos foram identificados como sendo a fonte de IL-1 α no fígado dos camundongos C57BL/6 infectados. De forma sistêmica, a deficiência em IL-1 α resultou numa diminuição da perda de peso e da hipotermia causados pela malária, mas teve um efeito menos significativo no controle da parasitemia. No fígado, a ausência de IL-1 reduziu o número de células TUNEL+, atenuando a necrose induzida pela infecção. A melhora no dano hepático nos camundongos IL1A-/- infectados está associada com uma menor resposta inflamatória em comparação aos camundongos C57BL/6 infectados, incluindo uma menor produção do TNF- α (Tumor necrosis factor alpha), citocina associada à apoptose de hepatócitos e, consequentemente, à necrose do tecido hepático durante a malária causada pelo P. chabaudi. Apesar de exercer um importante papel na imunopatogênese do dano e inflamação do fígado durante a fase eritrocítica da infecção pelo P. chabaudi, a ausência da IL-1 α não impediu a proteção conferida pela presença das formas eritrocíticas contra a reinfecção com esporozoítos. Em conclusão, neutrófilos produzem IL-1 α no fígado durante a fase aguda da malária causada pelo P. chabaudi. Esta citocina amplifica a resposta inflamatória à infecção e promove a necrose hepática, assim como exacerba a perda de peso e a hipotermia. / Malaria causes complications involving several organs, including the liver, where there are inflammation and damage that contribute to the disease severity. Interleukin (IL)-1 is a pro-inflammatory cytokine from the IL-1 family that can be released by non-hematopoietic or hematopoietic cells during tissue damage, infection and autoimmune diseases in different organs. The present study aims to evaluate the production and the role of IL-1 in the immunopathogenesis and in the protection during experimental malaria. IL-1 production was assessed during hepatic inflammation and necrosis in C57BL/6 mice infected with blood stages of Plasmodium chabaudi. The source of IL-1 in the liver of infected mice was determined by immunofluorescence and flow cytometry analyses. IL1A-/- mice were used to assess the role of IL-1 in this context. During acute P. chabaudi infection, hepatic inflammation and development of necrotic lesions were accompanied by an increase in IL-1 levels in the liver, which occurred independently of the Nod-like receptor protein 3 (NLRP3) inflammasome. Neutrophils were identified as the source of IL-1 α in the liver of infected C57BL/6 mice. Systemically, IL-1 deficiency resulted in reduction of weight loss and hypothermia caused by P. chabaudi malaria, but had minor effect on parasitemia control. In the liver, the absence of IL-1 reduced the number of TUNEL+ cells and attenuated the necrotic process induced by infection. The amelioration of liver damage in infected IL1A-/- mice was associated with lower inflammatory response compared to infected C57BL/6 mice, in particular with a decrease in tumor necrosis factor alpha (TNF- α) production, which has been directly implicated in the apoptosis of hepatocytes and, in consequence, the necrosis of the liver tissue during P. chabaudi malaria. Despite the important role in liver damage and inflammation immunopathogenesis during the blood-stage P. chabaudi infection, the absence of IL-1 α did not impair the protection conferred by blood stages against sporozoite reinfection. This study shows that neutrophils produce IL-1 α in the liver during acute P. chabaudi malaria. This cytokine amplifies the inflammatory response to infection and promotes liver necrosis, as well as exacerbates the weight loss and hypothermia.

<p>One of today’s most widely spread health problems is urinary incontinence, affecting 60-80% of the US population from age 15 and up. Treatment based on the possibility to implant a scaffold seeded with the patients’ own urine-derived stem cells, hUSC, to regenerate the damaged muscle tissue, would prove effective. A main challenge in regenerating new tissue from cell-seeded scaffolds is the limited cell survival due to insufficient oxygen diffusion to the center of the scaffold. Ways of enhancing cell survival, and thereby, proliferation and differentiation, is by hypoxic preconditioning of the cells or implantation in an oxygen-release material. Hypoxic preconditioning has shown to enhance proliferation as well as the expression of vascular endothelial growth factor, VEGF, in for example human bone marrow derived stem cells, hBMSC. VEGF is involved in the establishment of vasculature structures and an upregulation of its expression may therefore help promote quicker angeogenisis, increasing the oxygen supply and the cell survival. Oxygen-release materials have shown to enhance cell survival and growth both <em>in vitro</em> and <em>in vivo</em>.<em></em></p><p>This study aims to investigate the effect of hypoxia on hUSC, during 9 days of hypoxic culturing (2.0% ± 0.1% O₂) with and without oxygen-release material (PLGA 75:25 with 5 w% CPO) <em>in vitro</em>. hBMSC, and human smooth muscle cells, hSMC, have been used as control groups. Cell proliferation, morphology, differentiation, production of VEGF, and expression of hypoxia inducible factor HIF-1α have been studied.</p><p>According to the results, combining hypoxic preconditioning of hUSC with implantation in oxygen-release material could be an effective way to regenerate muscular tissue. Hypoxic preconditioning enhanced cell proliferation, production of VEGF, and HIF-1α expression. The increase of VEGF and HIF-1α would promote vascularization when implanted. The oxygen-release material showed possible promotion of cell differentiation, which would augment the hUSCs’ myogenic differentiation, while supplying oxygen until the tissue’s vascular structure has been established.</p>

One of today’s most widely spread health problems is urinary incontinence, affecting 60-80% of the US population from age 15 and up. Treatment based on the possibility to implant a scaffold seeded with the patients’ own urine-derived stem cells, hUSC, to regenerate the damaged muscle tissue, would prove effective. A main challenge in regenerating new tissue from cell-seeded scaffolds is the limited cell survival due to insufficient oxygen diffusion to the center of the scaffold. Ways of enhancing cell survival, and thereby, proliferation and differentiation, is by hypoxic preconditioning of the cells or implantation in an oxygen-release material. Hypoxic preconditioning has shown to enhance proliferation as well as the expression of vascular endothelial growth factor, VEGF, in for example human bone marrow derived stem cells, hBMSC. VEGF is involved in the establishment of vasculature structures and an upregulation of its expression may therefore help promote quicker angeogenisis, increasing the oxygen supply and the cell survival. Oxygen-release materials have shown to enhance cell survival and growth both in vitro and in vivo. This study aims to investigate the effect of hypoxia on hUSC, during 9 days of hypoxic culturing (2.0% ± 0.1% O2) with and without oxygen-release material (PLGA 75:25 with 5 w% CPO) in vitro. hBMSC, and human smooth muscle cells, hSMC, have been used as control groups. Cell proliferation, morphology, differentiation, production of VEGF, and expression of hypoxia inducible factor HIF-1α have been studied. According to the results, combining hypoxic preconditioning of hUSC with implantation in oxygen-release material could be an effective way to regenerate muscular tissue. Hypoxic preconditioning enhanced cell proliferation, production of VEGF, and HIF-1α expression. The increase of VEGF and HIF-1α would promote vascularization when implanted. The oxygen-release material showed possible promotion of cell differentiation, which would augment the hUSCs’ myogenic differentiation, while supplying oxygen until the tissue’s vascular structure has been established.

Made available in DSpace on 2016-06-02T20:19:13Z (GMT). No. of bitstreams: 1 2912.pdf: 5230025 bytes, checksum: e6829f4e9fed0f66d6055d44a7e1c4f0 (MD5) Previous issue date: 2010-03-26 / Universidade Federal de Sao Carlos / Skin flaps are widely used in plastic surgery, mainly in reconstruction surgeries (transference of skin graft, pre-made tissues). After the surgery one of the major complication is ischemia, which may cause necrosis of the flap. Several features have been studied with the aim of increasing the viability flaps. Among these features, the low laser therapy is an alternative treatment, since it can promote an increase in microcirculation and vascular neoformation. However, there are discrepancies in the literature of the parameters employed in the use of laser, especially the fluence used in treatment . The aim of this study was to investigate the effects of 2 different laser wavelengths (660nm e 780nm) at 30 and 40J/cm2, on the viability of skin flap in rats evaluated by the paper template, vessels blood counting, activity of matrix metalloproteinase-2 (MMP-2), evaluation of plasma levels of NO, and expression hypoxiainducible Factor 1_. Sixty male animals Wistar were used in this study and they were distributed into the following groups (n=12 each group): control group, group irradiated with 660nm, at 30J/cm2; group irradiated with 660nm, at 40J/cm2 group irradiated with 780nm, at 30J/cm2, and group irradiated with 780nm, at 40J/cm2. The skin flap was performed on the back of all animals studied, with a plastic sheet interposed between the flap and the donor site. The animals received laser irradiation immediately after surgery and within 4 days, using the technical point of contact, on 24 points on the skin surface and around it. On the seventh postoperative day was evaluated the percentage of necrotic area and collected samples of tissue for histological analysis, zymography and protein expression by Western blotting, animals were euthanized by exsanguination, and blood were evaluated plasma levels of NO. The data obtained from the evaluation by the method of the paper template showed no increase in the viability of skin flaps after laser treatment. For biochemical analysis we found that the laser modulates the activity of MMP-2 and expression of HIF-1_ and induced an increase in the number of vessels especially in the groups irradiated with 40J/cm2 Measurement plasma level of NO did not differ between groups. Molecular analysis showed that the application of laser parameters used here, although it stimulated angiogenesis by modulating HIF-1_ and activity of MMP-2 was not able to improve the viability of skin flaps. Thus, we conclude that to find the beneficial effects of laser therapy we need to understand their mechanisms of action and know the best parameters to use. / Os retalhos cutâneos são amplamente utilizados na cirurgia plástica, principalmente na reconstrutiva. Após o procedimento operatório, uma das principais complicações é a isquemia, podendo ocasionar a necrose do retalho. Vários recursos têm sido estudados com o intuito de aumentar a viabilidade desses retalhos. Dentre esses recursos, o laser de baixa intensidade é uma alternativa de tratamento, uma vez que pode promover um aumento da microcirculação e da neoformação vascular. Entretanto, existem discrepâncias na literatura em relação aos parâmetros empregados no uso do laser de baixa intensidade, principalmente das fluências utilizadas nos tratamentos. Este estudo teve como objetivo verificar o efeito de 2 comprimentos de onda diferentes (660nm e 780nm), com fluências de 30 e 40J/cm2, na viabilidade do retalho cutâneo randômico em ratos, avaliados pelo método do gabarito de papel, contagem dos vasos sanguíneos, atividade da metaloproteinase de matriz -2 (MMP-2), avaliação dos níveis plasmáticos de NO, e expressão do fator induzível por hipóxia (HIF-1_). Sessenta ratos da linhagem Wistar foram usados nesse estudo, sendo distribuídos em 5 grupos (n=12): grupo controle, grupo irradiado com 660nm a 30J/cm2; grupo irradiado com 660nm a 40J/cm2, grupo irradiado com 780nm a 30J/cm2, e grupo irradiado com 780nm a 40J/cm2. O retalho cutâneo foi realizado no dorso dos animais com dimensões de 10 X 4cm e uma barreira plástica foi interposta entre o retalho e o leito doador. Os animais receberam irradiação laser imediatamente após a cirurgia e nos 4 dias seguintes, utilizando-se a técnica pontual em contato em 24 pontos distribuídos sobre e ao redor do retalho. No sétimo dia pósoperatório foi avaliado a porcentagem de área de necrose e coletado amostras de tecido para análise histológica, zimografica e de expressão protéica por western blotting, os animais foram eutanaziados por exsanguinação, e no sangue foram avaliados os níveis plasmáticos de NO. Os dados obtidos a partir da avaliação pelo método de gabarito de papel mostraram que não houve aumento na viabilidade dos retalhos cutâneos após tratamento com laser. Pelas análises bioquímicas observamos que o laser modulou a atividade de MMP-2 e a expressão de HIF-1&#61537;, bem como induziu o aumento no número de vasos especialmente nos grupos irradiados com 40J/cm2 independente do comprimento de onda utilizado. A medida do NO circulante não apresentou diferença entre os grupos. A análise molecular mostrou que a aplicação do laser com os parâmetros aqui utilizados, embora tenha estimulado a angiogênese por modular HIF-1&#61537;&#61472;e a atividade de MMP-2 não foi capaz de melhorar a viabilidade dos retalhos cutâneos. Assim, concluímos que para encontrarmos os efeitos benéficos e seguros da laserterapia precisamos compreender seus mecanismos de ação e conhecer os melhores parâmetros de utilização.

La radiothérapie thoracique peut induire le développement de pneumopathies aiguës et de fibroses. La dysfonction du système vasculaire participe au développement de lésions radiques. Dans l'intestin, un KO endothélial de PAI-1 protège les souris de la fibrose radique. Le premier objectif de ce projet est d'explorer le rôle de PAI-1 dans l'apparition de la fibrose radique pulmonaire. L'irradiation thoracique de souris à 17 Gy altère sévèrement le parenchyme pulmonaire et l'analyse histologique révèle que l'invalidation de PAI-1 aggrave les lésions à 2 et 13 semaines. Cette invalidation ne protège donc pas les animaux des dommages radiques pulmonaires. L'organisation en parallèle du poumon permet d'envisager une tolérance à de fortes doses par fraction sur des petits volumes. Des irradiations en conditions stéréotaxiques ont donc été réalisées chez la souris. Les analyses histologiques montrent une déstructuration alvéolaire et un fort infiltrat inflammatoire au niveau de la zone cible. Un œdème est observable dans l'ensemble du poumon ipsilatéral deux semaines après irradiation. Le poumon ipsilatéral est également affecté par des altérations de structure, tel un épaississement des septa alvéolaires. Ces bouleversements se traduisent également au niveau transcriptomique. A la vue de l'ensemble de ces altérations, un test à l'effort a été réalisé pour évaluer l'impact potentiel sur la fonction pulmonaire. Les résultats mettent en évidence une diminution des performances des animaux. Les analyses sont à approfondir mais elles démontrent l'importance de s'intéresser aux tissus sains situés hors du volume cible mais recevant des fractions variables de la dose délivrée. / Radiation-induced endothelial dysfunction is known to participate to the development of normal tissue damage. PAI- is implicated in the phenotypic changes of irradiated endothelial cells and KOendo mice are protected from radiation damage to the gut. Whole thorax of PAI-1 KOendo and floxed mice were exposed to 17 Gy. Histological analyzes showed that PAI-1 KOendo induces a worsening of injuries at 2 and 13 weeks. Consequently, contrary to the gut no protection from radiation-induced lung damage is observed in PAI-1 KOendo mice. Our second aim was to study the effects of a single high dose stereotactic irradiation on pulmonary tissues. Histological analyzes and scanner imaging show important injuries on the targeted volume. An ipsilateral edema can also be observed 2 weeks after irradiation. Ipsilateral lung is moreover importantly damaged. A thickening of alveolar septa is notably observable. A transcriptomic analysis show important similarities between tissues from the ipsilateral lung and the focal lesion. As really highly damages have been observed in both scanner and histological analyzes, we decided to perform forced physical activity test on treadmill. A drastic decrease of maximal distance traveled has been observed from two weeks. These experiments highlighted a deficiency in respiratory function and all of these results show the importance of non-targeted irradiated pulmonary volume in the development of radiation-induced fibrosis. Effect of an endothelium-specific deletion of HIF-1α has been investigated in this model of stereotactic irradiation. Only few differences have been observed between KOendo and control mice. Experiments are still ongoing.

The P2X7R is a functionally distinct member of the P2X non-selective cation channels and has been implicated in the initiation of immune responses. One of the most extensively characterised immune responses of the receptor is to signal the rapid aggregation of the inflammasome complex and signal the release of IL-1β. These investigations have focused in providing direct comparisons of P2X7R-driven IL-1 responses between DC and mouse macrophages (peritoneal macrophages [PMΦ] and bone marrow derived macrophages [BM-MΦ]). Expression of the P2X7R has been identified in all three populations both at the transcriptional (P2X7A variant) and protein levels. Activation with lipopolysaccharide (LPS) (2h) induced a rapid dose dependent release of IL-6 but not of IL-1β in BM-DC. Rapid (2h) IL-1β release required both LPS priming and ATP activation. Both signals were also required for IL- 1β release in mouse ΒΜ-ΜΦ and PMΦ, however, at comparatively markedly lower levels. Furthermore, like with IL-1β, LPS did not induce IL-1α release in BM-DC. Interestingly, subsequent challenge with ATP evoked IL-1α release in BM-DC alone, with little or no detectable levels observed in activated BM-MΦ. This rapid IL-1β release (but not IL-6) was potently inhibited in both macrophages and DC with a P2X7R-specific inhibitor (A-740003) providing evidence that is predominantly a P2X7R-driven process. Treatment with A-740003 also potently inhibited IL-1α release from BM-DC suggesting that the ATP-P2X7R and caspase-1 activation might have a role in the release of the cytokine. Expression of gain-of-function P2X7K and loss-of- function P2X7J splice variants has been identified in both BM-DC and BM-MΦ, at the level of transcription. The possibility that a differential baseline or LPS-induced expression (at the transcriptional level) of P2X7J and P2X7K variants accounts for the diverse cytokine responses observed in BM-DC and BM-MΦ was also explored. However, the levels of expression for the various splice variants of interest (P2X7K and P2X7J) were found to be similar between the two cell types. The results of these investigations identify some subtle but intriguing differences in the mechanism of P2X7R activation and IL-1 release between DCs and macrophages. Purinergic signalling is increasing being implicated in the regulation of immune responses both in potentiating or suppressing inflammation. However, further work is required to decipher how the dynamic interplay between different purines can influence the immune activation of different cell types and indeed different cell subsets.

L’adaptation de l’organisme à son environnement est essentielle à sa survie. L’homéostasie énergétique permet l’équilibre entre les apports, les dépenses et le stockage d’énergie. Un surplus calorique important dérègle ce processus et mène au développement du syndrome métabolique caractérisé, entre autres, par une obésité, un diabète de type II, des maladies cardiovasculaires et des dyslipidémies. La ghréline participe au maintien de l’équilibre énergétique durant le jeûne en stimulant la production de glucose par le foie et le stockage lipidique dans le tissu adipeux. Le coactivateur transcriptionnel PGC-1alpha, surexprimé en situation de jeûne, est impliqué dans l’induction de la production de glucose par le foie et l’oxydation des acides gras. Notre hypothèse est que ces deux acteurs clés du métabolisme énergétique constituent un axe de régulation commun. Dans cette étude, nous montrons que la ghréline participe à la régulation de PGC-1alpha. Son récepteur GHS-R1a, possédant une forte activité constitutive, est également impliqué de façon indépendante au ligand. GHS-R1a réduit l’activité transcriptionnelle de PGC-1alpha tandis que l’ajout du ligand inverse modérément cette action. L’effet de GHS-R1a corrèle avec l’acétylation de PGC-1alpha qui est fortement augmentée de façon dose-dépendante. La stabilité de PGC-1alpha est également augmentée par le GHS-R1a indépendamment de l’ubiquitine. La ghréline diminue la capacité de PGC-1alpha à lier PPARbeta, un récepteur nucléaire partenaire de PGC-1alpha. De plus, la ghréline réduit, de façon ligand-dépendante, la capacité de coactivation de PGC-1alpha sur PPARbeta dans les hépatocytes. L’ensemble de ces résultats identifie PGC-1alpha comme cible du signal de la ghréline et suggère un axe de régulation ghréline/PGC-1alpha/PPARbeta.Une meilleure compréhension de cet axe de régulation va permettre la mise en évidence de nouvelles cibles thérapeutiques pour faire face aux pathologies associées au syndrome métabolique. / The adaptation of an organism to its environment is essential to its survival. Energy homeostasis is defined as the balance between intakes, expenses and storage of energy. An excess of calories disrupts this process and leads to the development of the metabolic syndrome that is characterized by obesity, type II diabetes, cardiovascular diseases and dyslipidemia. During fasting, ghrelin participates in the maintenance of energy balance by stimulating hepatic production of glucose and lipid storage in adipose tissue. The transcriptional coactivator PGC-1alpha is overexpressed in the liver during fasting and is involves in the induction of the hepatic glucose production and fatty acid oxidation. Our hypothesis is that these two key performers in the energy metabolism constitute a common axis control. In this study, we show that ghrelin plays a role in the regulation of PGC-1alpha. The ghrelin receptor GHS-R1a is also involved because of its strong constitutive activity in absence of ligand. We found that GHS-R1a inhibited PGC-1alpha transcriptional activity whereas adding ghrelin to cells moderated this effect. PGC-1alpha activation by GHS-R1a correlated with a dose-dependent increase of PGC-1alpha acetylation. The stability of PGC-1alpha was also increased by ghrelin receptor in a manner involving the ubiquitin-independent proteasome pathway. Ghrelin decreased the ability of PGC-1alpha to bind to PPARbeta, one of its nuclear receptor partners. Furthermore, ghrelin decreased the ability of PGC-1alpha to coactivate PPARbeta in a ligand-dependent manner in hepatocytes. Together, these results identify PGC-1alpha as a metabolic target of GHSR-1a signaling and defines a new regulatory axis involving ghrelin/PGC-1alpha/PPARbeta in hepatocytes. A better understanding of this regulation axis will provide novel aspects in therapeutic targeting of diseases associated with the metabolic syndrome.

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.

Die vorliegende Arbeit beinhaltet die Generierung und Evaluation von Natürlichen Killerzellen, die EGFRvIII-positive und SDF-1alpha sekretierende primäre Glioblastomzellen aufspüren, erkennen und effizient abtöten können. Die Kombination der gelenkten Zytotoxizität mit einer optimierten Migration von Effektorzellen des Immunsystems wird auf Grundlage der in dieser Arbeit gewonnenen Daten als ein vielversprechender Ansatz für eine zukünftige Therapie des primären Glioblastoms vorgeschlagen.