Hydrothermal systems may have been more widespread in the Hadean due to a greater heat flux. To investigate this possibility, and unravel the mechanism(s) by which the pH of high-temperature vent fluids become acidic and what produces their distinctive black colour, reactions between model seawater and mafic/ultramafic rock were conducted. Results indicated that ancient, medium to high temperature (150-300°C), alkaline hydrothermal fluids would have precipitated carbonates, brucite, and calcite upon re-mixing with cold, slightly alkaline seawater and may have predominated in the Hadean. Acid pH was effected by the loss of magnesium from seawater and calcium loss from mafic rock. Black-smokers were unlikely in the Hadean as the ocean was probably acidic due to high levels of CO2. Water-rock reaction models were constructed to test the possibility that simple amino acids could have been generated in early hydrothermal fluids, and to see how pH and redox conditions affect their distribution (cf. Amend and Shock, 1998). Though concentrations of amino acids produced were negligible, amino acids were stable in low-temperature, alkaline, and reduced hydrothermal fluids and may have concentrated in the colloidal sieve comprising a hydrothermal mound. An extension of the experiment to determine if glycine could be condensed to higher carbon number amino acids (alanine, valine, leucine) under hydrothermal conditions, indicated that condensation may be 'pulled' by a decrease of H2O activity of the fluid. In conclusion, this study improved on previous environmental and reactant constraints by simulating the generation of inorganic prebiotic reactants from the local geochemical hydrothermal environment. Consequently, the quantity of chemical species such as hydrogen and sulfide available for organic synthesis were limited by the local geochemical settings in the model, whereas others have, often admittedly, used reactants in higher concentrations than were probably available when Life emerged.

Panic Disorder (PD) is characterized by unexpected, recurrent panic attacks, which are not restricted to certain situations, medication or stimuli. Like other anxiety disorders, PD is a multifactorial disorder and develops through the interaction of genetic and environmental risk factors. Despite an estimated heritability of up to 48%, no distinct genetic mechanism could be revealed yet. A dysregulation of the stress response has been shown in patients with PD and several studies could find an association of components of the corticotropin-releasing factor (CRF) system with PD. The corticotropin releasing hormone receptor 1 (CRHR1) is the main receptor of CRF in the brain and thus a crucial regulator of cerebral CRF signaling. Recent genetic studies found an association of certain CRHR1 single nucleotide polymorphisms (SNPs) with PD and other anxiety disorders. Among the associated CRHR1 SNPs, rs17689918 showed further evidence in a multilevel study regulating CRHR1 gene expression in panic-relevant brain regions and affecting brain activation in fMRI experiments, as well as flight behavior in a behavioral avoidance task (Weber et al, 2015). Here, we aimed to investigate the underlying neurogenetic and neurobiological mechanisms, by which the rs17689918 risk allele affects CRHR1 gene expression and receptor function, and its putative function in the pathophysiology of PD. Due to its intronic position and the predicted change of splicing regulatory elements by the risk allele of rs17689918, the expression of alternative spliced CRHR1 isoforms was investigated using quantitative real-time PCR (qPCR) in a human post-mortem brain tissue sample. Of eight known CRHR1 isoforms, expression of three CRHR1 isoforms and the CRHR1-IT1-CRHR1 readthrough transcript variant 5 – all expressing the seven transmembrane domains needed for functional receptors – was analyzed. Subsequently, electrophysiological assays were developed to measure the receptor activity of differentially expressed CRHR1 isoforms via co-expressed Kir2.3 potassium channels in vitro. In a second approach, possible epigenetic regulation of CRHR1 expression by rs17689918 was investigated by analyses of DNA methylation patterns of a CpG Island within the CRHR1 promoter region, firstly in a case-control sample for PD and secondly in a healthy control sample, separated in high and low anxious individuals. To investigate a possible gene × epigene × environment interaction, the impact of early life stress by means of childhood trauma was evaluated via the childhood trauma questionnaire (CTQ). Finally, consequences of differential DNA methylation of the CRHR1 promoter region on gene expression were investigated by luciferase-based reporter gene assays in vitro. The expression of CRHR1β was significantly decreased in amygdalae and midbrains of risk allele carriers. The expression of CRHR1-IT1-CRHR1 readthrough transcript variant 5 was significantly increased in forebrains and midbrains of risk allele carriers. All other analyzed isoforms showed no differences in expression between non-risk and risk allele carriers of rs17689918. The electrophysiological recordings of membrane potential showed an activation of Kir2.3 channels by CRHR1β in contrast to an inconsistent mix of activation and inhibition of Kir2.3 by the main isoform CRHR1α. DNA methylation of the CRHR1 promoter region was significantly reduced in panic disorder patients, as well as in high anxious individuals of an independent healthy control sample, but no direct relation to the rs17689918 risk allele could be discerned. However, the combination of carrying the risk allele, low DNA methylation and high CTQ scores lead to increased sum scores in the Beck Anxiety Inventory (BAI) in healthy individuals. Functional analyses revealed an activation of gene expression by decreased DNA methylation of the promoter region in vitro. Our results revealed that rs17689918 regulates CRHR1 function by increasing the expression of alternative transcript variants with altered function. Our analyses of DNA methylation revealed decreased methylation as a new risk factor for panic disorder and high anxious behavior, which in combination with other risk factors like childhood trauma and the rs17689918 risk allele might further increase cognitive and somatic anxiety symptoms. This supports the role of CRHR1 as a plasticity gene of anxiety behavior, i.e. a gene that is highly regulated by epigenetic or post-transcriptional mechanisms in response to environmental stressors. By its role in CRF signaling, the dysregulation of CRHR1 might extensively affect the stress response and contribute to the pathophysiology of stress-related disorders like PD. The understanding of the underlying mechanisms, especially the genetic and epigenetic regulation, would however enhance CRHR1 as a target of improved future therapeutics for PD and other anxiety disorders. / Die Panikstörung manifestiert sich durch unerwartete, wiederkehrende Panikattacken, welche sich nicht auf bestimmte Situationen, Medikationen oder Stimuli zurückführen lassen. Bei der Panikstörung handelt es sich, wie bei allen psychischen Erkrankungen, um eine sogenannte multifaktorielle Erkrankung, d.h. sie entwickelt sich aus einem Zusammenspiel von genetischen Faktoren und Umweltfaktoren. Trotz einer geschätzten Heritabilität von bis zu 48% ist bisher kein eindeutiger genetischer Mechanismus bekannt, der zur Entwicklung einer Panikstörung führt. Mehrere Studien konnten eine Fehlregulation der Stressantwort bei Patienten mit Panikstörung feststellen. Dabei konnten mehrfach Polymorphismen in Genen des Corticotropin-Releasing Faktor (CRF) Systems mit Panikstörung assoziiert werden. Insbesondere der Hauptrezeptor von CRF im Gehirn, der Corticotropin Releasing Hormon Rezeptor 1 (CRHR1), konnte in mehreren Studien mit Panikstörung und anderen Angsterkrankungen assoziiert werden. In einer kürzlich erschienenen Studie wurde gezeigt, dass der CRHR1 Einzelnukleotid-Polymorphismus rs17689918 die Genexpression von CRHR1 in Gehirnregionen reguliert, die auch in Angsterkrankungen eine Schlüsselrolle spielen. Zusätzlich zeigten Risikoallelträger eine veränderte Gehirnaktivierung in fMRT Experimenten und ein verändertes Fluchtverhalten in einem Verhaltenstest (Weber et al, 2015). In der vorliegenden Studie wurden die zugrundeliegenden neurogenetischen und neurobiologischen Mechanismen untersucht, anhand derer das Risikoallel von rs17689918 die CRHR1 Genexpression und Rezeptorfunktion beeinflusst, und welche Rolle diese in der Pathophysiologie der Panikstörung spielen. Aufgrund der Position von rs17689918 im Intron von CRHR1 und der in silico berechneten Änderung der Erkennungssequenz für Spleißregulatoren durch das Risikoallel von rs17689918 wurde die Expression alternativer Spleißformen von CRHR1 mittels quantitativer real-time PCR in humanen post-mortem Gehirnproben analysiert. Insgesamt wurde die Expression von vier CRHR1 Isoformen und die Expression der CRHR1-IT1-CRHR1 readthrough Transkriptvariante 5 analysiert. Für funktionelle Analysen wurden elektrophysiologische Assays entwickelt, um durch die Messung der Aktivität von ko-exprimierten Kir2.3 Kaliumkanälen die Rezeptoraktivität der CRHR1 Isoformen in vitro bestimmen zu können. Zusätzlich wurde eine mögliche epigenetische Regulation der CRHR1 Genexpression durch rs17689918 untersucht. Hierfür wurden DNA Methylierungsmuster eines Abschnittes einer CpG-Insel im Promoterbereich des CRHR1 Gens in einer Fall-Kontroll-Stichprobe für Panikstörung und einer weiteren gesunden Kontrollstichprobe – unterteilt in eine hoch-ängstliche und eine niedrig-ängstliche Gruppe – analysiert. Um mögliche Gen-Epigen-Umweltinteraktionen zu untersuchen, wurde zusätzlich der Einfluss belastender Lebensereignisse in frühen Lebensabschnitten mittels dem Childhood Trauma Questionnaire (CTQ) erfasst. Die Funktionalität von differenziell methylierten Abschnitten der CRHR1 Promoterregion wurde mittels Luziferase-basierten Reportergenassays in vitro untersucht. Die Ergebnisse der Expressionsanalyse zeigen eine signifikant verminderte Expression der Isoform CRHR1β in Amygdala- und Mittelhirnproben von Risikoallelträgern. Gleichzeitig war CRHR1-IT1-CRHR1 Transkriptvariante 5 in Vorderhirn- und Mittelhirnproben von Risikoallelträgern signifikant höher exprimiert. Alle anderen Isoformen zeigten keinen Expressionsunterschied zwischen Risiko- und Nicht-Risikoallelträgern von rs17689918. Elektrophysiologische Messungen des Membranpotentials fanden eine Aktivierung der ko-exprimierten Kir2.3 Kanäle durch Isoform CRHR1β, im Gegensatz zu einer inkonsistenten Regulation aus Aktivierung und Inhibition der Kanäle durch die Hauptvariante CRHR1α. Patienten mit Panikstörung zeigten eine geringere DNA-Methylierung der CRHR1 Promoterregion im Vergleich zu gesunden Kontrollen. Gleichzeitig haben hoch-ängstliche gesunde Probanden eine geringere DNA Methylierung der CRHR1 Promoterregion als weniger ängstlichen Probanden. Allerdings konnte kein Einfluss von rs17689918 auf die DNA-Methylierung gefunden werden. Probanden mit einer Akkumulation von Risikofaktoren wie dem Risikoallel von rs17689918, geringer DNA-Methylierung und hohen CTQ-Werten erreichen außerdem höhere Summenwerte im BAI. Die funktionellen Analysen zeigten eine Aktivierung der Genexpression infolge einer geringen DNA-Methylierung der differenziell methylierten CpG-Stellen. Die Ergebnisse zeigen, dass rs17689918 die Funktion von CRHR1 durch eine Verschiebung der Expression zu alternativen, weniger oder nicht-funktionellen Spleißvarianten steuert. Zusätzlich zeigen die Analysen, dass eine verringerte DNA-Methylierung der CRHR1 Promoterregion ein Risikofaktor für Panikstörung und erhöhtes Angstverhalten ist, welcher in Kombination mit weiteren Risikofaktoren wie Kindheitstraumata oder dem rs17689918 Risikoallel ängstliche Verhaltenszüge begünstigt. Dies unterstützt die Hypothese, dass CRHR1 die Funktion eines sogenannten „Plastizitätsgens“ für ängstliches Verhalten und Angsterkrankungen hat, d.h. ein Gen dessen Expression durch epigenetische und posttranskriptionale Modulation in Reaktion auf Umwelteinflüsse reguliert wird. Durch seine wichtige Funktion im CRF-Signalweg könnte eine Fehlregulation von CRHR1 einen weitreichenden Einfluss auf die humane Stressantwort haben und somit auch zur Pathophysiologie von stress-bedingten Erkrankungen wie Panikstörung beitragen. Das Verständnis der zugrundeliegenden Mechanismen, besonders der genetischen und epigenetischen Regulation, würde dazu beitragen CRHR1 als möglichen Ansatzpunkt zukünftiger Therapien für Panikstörung und anderen Angsterkrankungen zu erschließen.

T.A. Arias ... [et al.]. / Includes bibliographical references (leavse 13-14).

En aquest treball s’ha optimitzat el procés de producció de lipases de Rhizopus oryzae amb el fenotip Muts de Pichia pastoris mitjançant la utilització de substrats mixtes establint les condicions de procés més favorables. S’utilitza un sistema de 48 milibioreactors de 12 ml de volum amb l’objectiu d’optimitzar el procés de selecció de l’estratègia de cultiu més efectiva. Quan els cultius es realitzen utilitzant els fenotips Muts i Mut+ de P. pastoris sota promotors induïbles (AOX i FLD) s’obté un creixement deficient. La principal problemàtica recau en el sistema de condensació que presenten els milibioreactors a la part superior. S’utilitza el promotor GAP utilitzant glicerol i glucosa com a substrats, amb el que es demostra la viabilitat del sistema de 48 milibioreactors pel seguiment de bioprocessos. Posteriorment davant les avantatges que presenta la utilització de substrats mixtes en soques de fenotip Muts sota el promotor AOX, s’utilitzen sorbitol o glicerol juntament amb el metanol per observar l’efecte del cosubstrat sobre la producció de ROL. A l’utilitzar el sorbitol es realitzen cultius en semicontinu mantenint la concentració de metanol fixada en 0.5, 2 i 4 g l-1 i es manté la μ fixada en 0.01 i 0.02 h-1. S’observa que la concentració de metanol residual en el medi de cultiu és el paràmetre clau per l’optimització de la producció de ROL, trobant-se els valors màxims quan aquesta es troba fixada a 2 g l-1. A l’augmentar o bé disminuir aquesta concentració, la productivitat disminueix considerablement (2 vegades). Finalment es valida un sistema SIA per a l’anàlisi de sorbitol en línia. El sorbitol es considera un cosubstrat excel·lent però presenta una baixa μmax, fent així que el procés de producció sigui menys efectiu, si es compara amb una font de carboni alternativa. S’utilitza el glicerol com a cosubstrat, el que permetrà treballar a μ 5 vegades superiors. Es realitzen cultius en semicontinu mantenint la concentració de metanol fixada en 2 g l-1 (valor optimitzat prèviament). En aquest treball es realitzen diferents cultius on la μ es manté fixada en 0.1, 0.05 i 0.02 h-1. Es conclou que per evitar la repressió del promotor AOX la relació establerta entre les μ ha de ser inferior a 6 (μGli μMet-1), degut que al superar aquest valor s’observa una clara repressió del promotor AOX. A continuació, es decideix realitzar el canvi d’escala de les condicions més productives. Es realitzen varis cultius utilitzant el reactor a un volum inicial de treball de 30 l. Després d’estimar un favorable canvi d’escala es reprodueixen les condicions obtingudes en el millor dels processos. Durant aquest primer cultiu s’obtenen valors de productivitat volumètrica i específica 2.8 vegades inferiors als obtinguts prèviament en 5 l. Es realitzen experiments per comprovar la mescla del metanol al reactor UD50 mostrant un gradient màxim de 0.5 gMet l-1 a les diferents alçades. Es realitza una modificació al sistema d’addició del metanol, addicionant-lo de forma submergida a mitja alçada del reactor. Posteriorment els resultats milloren en un 48%, en comparació a l’addició pel capçal del reactor. Finalment es treballa amb una construcció genètica del fenotip Muts de P. pastoris que inclou la coexpressió del gen HAC1. Es treballa amb 2 soques, la primera coexpressa el gen HAC1 de forma induïble sota el promotor AOX i la segona ho realitza de forma constitutiva sota el promotor GAP. Quan s’utilitza la soca que coexpressa el gen de forma induïble, es troben millores significatives (augment de 1.5 vegades de la productivitat) al realitzar una estratègia amb substrats mixtes (sorbitol i metanol) a les condicions prèviament optimitzades. Quan s’utilitza la soca que coexpressa el gen de forma constitutiva, no es presenten millores en el procés de producció. / The objective of this thesis was to optimize the production of Rhizopus oryzae lipase in fed-batch mixed substrate conditions with a Pichia pastoris Muts strain. A milibioreactors bloc of 48 reactors with 12 ml working volume was tested to optimize the culture conditions under fed-batch strategy. When Mut+ and Muts phenotypes were used under AOX and FLD promoters, no satisfactory growth was observed, related with a deficiency presented by the cooler system allowing a lose of methanol of 0.5 gMet l-1 h-1. However, when Pichia pastoris under the constitutive promoter GAP was used, satisfactory growth was observed obtaining similar values than the ones obtained at higher volumes (5 l). In conclusion the 48 milibioreactors bloc represents an excellent tool to optimize the ROL production under GAP promoter, although was not satisfactory under AOX and FLD promoters. Subsequently to the advantage of using mixed substrates in place of using methanol as a sole carbon source, some experiments were done to optimize the ROL production under fed-batch conditions using sorbitol and glycerol as cosubstrats. When sorbitol was used the specific growth rate was maintained on 0.01 i 0.02 h-1 by a preprogrammed exponential feed. Methanol set-point concentration was maintained on 0.5, 2 i 4 g l-1 controlled by a predictive algorithm. Lipolytic activity, yields, productivity and specific productivity, but also specific growth, consumption and production rates were analyzed showing that the best values were reached when methanol concentration was fixed on 2 g l-1 showing an important reduction when this set-point was increased or decreased, independently of the specific growth rate used. The experiments showed that the key parameter was the set-point of methanol. On the other hand the specific growth influence was not representative. Finally a SIA analyser of sorbitol was tested. Sorbitol is considered an excellent cosubstrat but this low μmax in comparison with other possible cosubstrats, assures low productions. Different experiments using glycerol as cosubstrat were made at a constant methanol set-point of 2 g l-1, at three different glycerol feeding rates to assure a constant specific growth rate of 0.02, 0.05 and 0.1 h-1, by means of a pre-programmed exponential feeding rate strategy. The best production values were reached at the lowest specific growth rate tested. With Muts phenotype glycerol could not increase the productivity of the process comparing with sorbitol due to inhibitory effect on methanol consumption and recombinant lipase production. The specific growth relation must be fixed lower than 6 (μGli μMet -1), to avoid AOX promoter repression. The scale-up of the most effective process was done using a Biostat UD50 (30 l working volume). Methanol concentration was fixed on 0.02 h-1 and the specific growth rate was fixed on 0.02 h-1 by a sorbitol pre-programmed exponential feed. An encouraging scale-up was assumed (better KLa and similar geometry), although the first feed-batch cultivations under this conditions did not reproduced the values obtained under lab scale (5 l). Problems were related to the unsuccessful distribution (maximal gradient was 0.5 gMet l-1) of the substrates along the different height of the reactor. Methanol addition was submerged to the half height of the reactor to improve substrate distribution along the reactor. Feed-batch cultivation was done, and ROL production values were increase about 48%, in comparison with result obtained when methanol was added by the reactor headspace. And important increase was obtained, but is still necessary further research to reproduce values obtained in lab-scale. Finally a new Pichia pastoris strain construction co-expressing constitutively and inductively HAC 1 gene was used to optimize ROL production. Feed- batch cultivations were done using methanol and sorbitol as cosubstrats under the same conditions previously optimize (methanol concentration on 2 g l-1 and μ on 0.02 h-1). When inductively co-expression was used important improvements were obtained on productivity and specific productivity. On the other hand, when constitutively coexpression was used, inferior production values were obtained in comparison with results obtained with the non engineered strain.

L'aterosclerosi és una malaltia progressiva que es caracteritza per una acumulació de lípids en el subentoteli vascular. Evidències observacionals suggereixen que la peroxidació lipídica, la resposta inflamatòria i els processos trombogènics tenen un paper important en la patogènesi de l'aterosclerosi i les seves complicacions clíniques. Diversos estudis han demostrat l'existència de LDL oxidades, d'alguns dels seus components oxidats, de nivells elevats de citocines proinflamatòries i del principal inductor de la trombosi, el factor tissular, en plasma de pacients d'alt risc cardiovascular i en plaques ateroscleròtiques humanes on també s'ha observat acumulació de cèl·lules inflamatòries. Malgrat aquests coneixements no es disposa d'evidències directes que relacionin la peroxidació lipídica amb la inflamació. Un fet remarcable en aquest àmbit és el potencial del PPAR en l'aterosclerosi. El PPAR és un receptor nuclear diana de les tiazolidinadiones, fàrmacs utilitzats en el tractament de la diabetis mellitus tipus 2. Aquests fàrmacs mostren un efecte global antiateroscleròtic en models animals d'aterosclerosi i en estudis clínics amb humans. A nivell in vitro s'ha demostrat que aquests fàrmacs poden tenir un paper antiinflamatori important. A més, PPAR s'expressa de forma elevada en plaques ateroscleròtiques on colocalitza amb lípids oxidats. Recentment s'ha suggerit que la LDL oxidada podria ser una font de lligands endògens del PPAR tot i que encara no s'han establert. Els estudis que es presenten en aquesta tesi doctoral engloben nous coneixements de la peroxidació lipídica en la patogènesi de l'aterosclerosi i en concret en la inflamació i la trombosi. I donen suport a la teoria que la modificació oxidativa dels lípids té un paper important en la progressió de la malaltia aterotrombòtica. Els resultats que s'exposen en aquesta tesi doctoral demostren que el 2,4-decadienal, un aldehid apolar producte final de l'oxidació dels àcids grassos poliinsaturats majoritaris de les LDL, contribueix a la coneguda citotoxicitat de la LDL oxidada en cèl·lules musculars llises humanes. L'efecte citotòxic observat no és promogut per apoptosi cel·lular donat que no es produeix la fragmentació del DNA característica d'un estat apoptòtic. El 2,4-decadienal i l'hexanal, un altre aldehid apolar, a concentracions no citotòxiques aturen el creixement cel·lular i disminueixen els nivells de mRNA dels gens p53 i c-myc que estan involucrats en la regulació de l'apoptosi. Aquests resultats suggereixen que la citotoxicitat dels aldehids podria explicar-se per un fenomen de necrosi cel·lular. D'altra banda ambdós aldehids augmenten els nivells de mRNA i de proteïna del factor tissular. Nosaltres suggerim que aquest efecte podria ser a través de l'activació del factor de transcripció AP-1 format pel complex cFos-cJun prèvia síntesi de novo de c-Fos. Aquest efecte es reverteix amb un tractament previ amb simvastatina, un fàrmac hipolipemiant amb propietats antioxidants i antitrombòtiques. Pel que fa a la implicació de PPAR en l'aterosclerosi, 1) hem identificat dos elements de resposta PPAR en el promotor del gen del TNF humà que suggereixen una implicació directa de PPAR en la inflamació. Aquest estudi aporta més dades al coneixement del mecanisme d'acció de les tiazolidinadiones i descriu una nova via de modulació de l'expressió del TNF; 2) proposem el 2,4-decadienal com a candidat a mediador de PPAR en la placa d'ateroma donat que els resultats obtinguts demostren que activa PPAR. Augmenta la translocació de PPAR del citosol al nucli i augmenta la transcripció regulada per PPAR; 3) aportem evidències que descartarien l'efecte proateroscleròtic (augment de CD36) no desitjable de les tiazolidinadiones en pacients diabètics tipus 2. Aquests pacients tenen nivells elevats d'àcids grassos lliures circulants i les dades obtingudes demostren que la darglitazona, una tiazolidinadiona, en presència d'àcids grassos lliures no altera l'expressió de CD36 ni en monòcits ni en macròfags humans i no modifica el contingut lipídic dels macròfags. / Atherosclerosis is a progressive disease characterized by an accumulation of lipids in the subendothelial wall. Experimental evidence suggest that lipid peroxidation, inflammatory response and thrombogenic processes play an important role in the pathogenesis of atherosclerosis and its clinical complications. Oxidized LDL particles, some of their oxidative compounds, elevated levels of proinflammatory cytokines and tissue factor (the main inducer of thrombosis) have been detected in plasma of patients with high cardiovascular risk. It has also been reported the presence of all these components in human atherosclerotic plaques where also has been observed an accumulation of inflammatory cells. However, no direct evidence exist about a direct relation between lipid peroxidation and inflammation. A recent interesting finding is the possible role of PPAR in atherosclerosis. PPAR is the nuclear receptor target of thiazolidinediones, drugs used in the treatment of non insulin dependent diabetes mellitus. These drugs have shown a net antiatherosclerotic effect in animal models and in clinical studies in humans. In vitro experiments have shown that thiazolidinediones may have an important antiinflammatory action. PPAR is overexpressed in atherosclerotic plaques where colocalizes with oxidized lipids. Recently, oxidized LDL has been pointed as an important source of PPAR ligands.The work presented in this thesis shows new knowledge of the lipid peroxidation role in the pathogenesis of atherosclerosis emphasized in the action on inflammation and thrombosis. In summary, the obtained results support the theory that lipid oxidative modification plays an important role in the progression of atherothrombotic disease.Results show that the apolar aldehyde 2,4-decadienal, an end product of the oxidation of the main polyunsaturated fatty acids present in LDL, contributes to the well-known toxicity of oxidized LDL in human smooth muscle cells. The cytotoxic effect observed may not be promoted by an apoptotic state because the characteristic DNA ladder is not induced. 2,4-decadienal and hexanal, another apolar aldehyde, at non cytotoxic concentrations produce cell growth arrest and decrease p53 and c-myc mRNA levels, genes involved in the regulation of apoptosis. These results suggest that the cytotoxicity of aldehydes may be explained by cell necrosis.On the other hand, both aldehydes increase tissue factor mRNA and protein levels. We suggest that this effect may be produced through the activation of the transcription factor AP-1 formed by the complex cFos-cJun and by previous synthesis of c-Fos. This effect is prevented by a pretreatment with simvastatin, a hypolipidemic drug with antioxidant and antithrombotic properties.Concerning PPAR implication in atherosclerosis, 1) we have identified two PPAR response like elements in the human TNF promoter which suggest a direct link between PPAR and inflammation. This study adds more data in the knowledge of the mechanism of action of thiazolidinediones and describes a new pathway to modulate TNFexpression; 2) we propose that 2,4-decadienal may be a PPAR mediator in the atheromatous plaque based on our results that show PPAR activation by 2,4-decadienal. 2,4-decadienal increases PPAR translocation from the citosol to the nucleus and increases the transcription regulated by PPAR; 3) we also show evidence that the non desirable proatherosclerotic effect (CD36 induction) of thiazolidinediones would not be produced in type 2 diabetic patients. These patients have high levels of circulating fatty acids and our results show that darglitazone, a thiazolidinedione, in the presence of fatty acids neither alters CD36 expression in human monocytes and macrophages nor modifies macrophage lipid content.

Macrophages require presence of M-CSF to survive and proliferate. Incubation of bone marrow macrophages with soluble factors (LPS or IFN-gamma) or insoluble factors such as extracellular matrix proteins (Decorin, FN) macrophage proliferation was blocked. Moreover, pretreatment of macrophages with IFN-gamma protects from apoptosis induced by several stimuli. Inhibition of p21Waf with antisense oligonucleotides or using null mice showed that the induction of p21Waf by IFN-gamma mediate this protection. Thus, IFN-gamma makes macrophages unresponsive to apoptotic stimuli by inducing p21Waf and arresting the cell cycle at the G1/S boundary. The insoluble factors, decorin and fibronectin, inhibit macrophage proliferation through p27kip1 expression and the modification of ERK activity. Decorin treated macrophages but not fibronectin protect from apoptosis mechanism that require p21Waf expression. Decorin enhances the IFNgamma-induced expression of IA-alpha and IAß MHC class II genes. Moreover, it increases the IFN-gamma or LPS-induced expression of inducible NO synthase, TNF-alpha, IL-1ß, and IL-6 genes and the secretion of these cytokines. Using a number of extracellular matrix proteins, we found a negative correlation between adhesion and proliferation. However, the effect of decorin on macrophage activation is explained by its ability to block the binding of autocrine-produced TGFß on the surface of macrophages.These soluble and insoluble factors modulate the cell response through the interaction with surface receptors. Cell surface receptors of the integrin family are important regulators of the cell behavior. ß5 cytoplasmic domain has been reported to control cell migration and proliferation. Certain postadhesion are regulated through a pathway that requires both avß5 and PKC activity.Extensive data have been reported on the use of phage libraries to identify ligands. The large molecular diversity represented in phage peptide libraries facilitates the identification of motifs that map to protein interaction sites. Here we introduced an approach based on phage display technology to identify molecules that specifically interact with the cytoplasmic of the ß5 integrin subunit. We showed that a peptide that mimics annexin V binds to the cytoplasmic domain and triggers apoptosis. Annexin V is a cytosolic signaling protein known to inhibit PKC activity, and we demonstrated that annexin-V only binds to active form of PKC. Induction of apoptosis by this peptide is modulated by growth factors and by PKC antagonist. Caspase activity and the expression of ß5 integrin are also required.Caspases play an important role on apoptosis. XIAP functions as a caspase inhibitor and is a member of the inhibitors of apoptosis (IAP) family of proteins. All of the members of the IAP have been shown to inhibit programmed cell death. The human IAP family members bind to caspase 3 and caspase 7 with inhibitory constant values. We have selected peptides from a phage display library by using recombinant full-length human XIAP. A consensus motif was recovered from two independent screenings by using different libraries. Phage displaying variations of the consensus sequence bound specifically to the BIR2 domain of XIAP but not to other IAPs. Protein-protein interaction assays revealed that caspase-3 and -7 blocked the binding of the XIAP-binding phage to XIAP, indicating that this peptide targets a domain within XIAP that is related to the caspase-binding site. We also demonstrated that an internalizing version of the XIAP-binding peptide identified in our screenings could induce apoptosis in leukemia cells.Using a new approach for the screening by phage display technology we also characterize cells surface receptors in endothelial cell activation and proliferation. The molecular diversity in human blood vessels remains largely unexplored. We developed a selection method in which peptides that home to specific vascular beds are identified after administration of a peptide library. These data represents a step toward the construction of a molecular map of human vasculature and may have broad implications for development of targeted therapies.

Mutationstests werden in vitro und in vivo durchgeführt. Insbesondere die phänotypselektiven Mutationstests sind meist beschränkt auf die Detektion von Mutationen im Exon und gegebenenfalls in Promotorregionen. Um zunächst die Datenlage zu den üblicherweise verwendeten in vitro Mutationstests zu erweitern und somit eine Bewertung der zu untersuchenden Substanz zu erleichtern, sollte eine Methode zur Erfassung des Mutationsspektrums etabliert und im Rahmen der Untersuchung des mutagenen Potentials des Lebensmittelinhaltsstoffes Irilon angewendet werden. Es wurde eine Methode entwickelt, welche die Sequenzierung eines jeden einzelnen im Hypoxanthin-Guanin-Phosphoribosyltransferase-Test enstandenen 6-Thioguanin-resistenten Mutanten erlaubt und somit auch Rückschlüsse auf Mechanismen der Mutationsentstehung zulässt. Im Rahmen der Untersuchung zum mutagenen Potential des Lebensmittelinhaltsstoffes Irilon, wurde zwar kein Unterschied in der Mutantenfrequenz, jedoch sehr wohl ein mit steigenden Deletionen und sinkenden Basenpaarsubstitutionen verändertes Mutationsspektrum detektiert. Die Auswertung des Mikrokerntests unterstützte die Annahme, dass Irilon Chromosomenmutationenen induziert. Zudem wies Irilon ein starkes aneugenes Potential auf. Im Gegensatz zu den phänotypselektiven Mutationstests weisen genotypselektive Tests hingegen theoretisch keine Limitierungen hinsichtlich der zu untersuchenden Zielsequenz und der Organwahl auf. Ein Vertreter der genotypselektiven Tests ist der Random Mutation Capture Assay, der 2005 von Bielas und Loeb für das Intron 6 des humanen TP53-Gens publiziert wurde. Ein weiteres Ziel dieser Arbeit war es zu untersuchen ob die Technik des Random Mutation Capture Assays auf die Ratte übertragbar und ob bzw.unter welchen Bedingungen die Bestimmung von spontanen und induzierten Mutationsfrequenzen in verschiedenen Zielsequenzen möglich ist. Deshalb wurden zunächst das für das Tumorsuppressor Protein 53 kodierenden Gen p53, die für die 18S ribosomale RNA kodierenden DNA und das mitochondriale Cytochrom b Gen als Zielsequenzen gewählt und deren Eignung für die Anwendung im Random Mutation Capture Assays geprüft. Für jede Zielsequenz wurden alle für die Durchführung des Random Mutations Capture Assays benötigten molekularen Werkzeuge unter optimierten PCR-Bedingungen hergestellt und verifiziert. Für die Quantifizierung der Gesamtkopiezahl wurde je Zielsequenz eine spezifische Echtzeit-PCR-Methode entwickelt, welche TaqMan®-Sonden-basiert ist. Nach Optimierung der PCR-Bedingungen wurden je Zielsequenz Wiederfindungen im angestrebten Bereich von ca. 90-100% mit Schwankungen von maximal 20% erreicht. Ausgenommen hiervon war die für die 18S ribosomale DNA kodierende Zielsequenz. Eine Änderung der Echtzeit-PCR-Bedingungen führte zu keiner praktikablen Methode. Daher war diese Zielsequenz, welche trotz geringer DNA-Mengen versprach mehr DNA Kopien zu erhalten und somit die Bestimmung von geringen Mutationsfrequenzen zu erleichtern, nicht im Random Mutation capture Assay anwendbar. Für die Wahl einer DNA-Isolierungsmethode wurden 5 Methoden hinsichtlich einer für die Mutationsfrequenz-Bestimmung ausreichenden Kopiezahlausbeute, der Reinheit und des Kosten-/Zeitaufwands verglichen. Mit zwei der fünf Methoden wurde aus 100 mg Gewebe die höchste nukleären Kopienzahl isoliert, ausreichend um Mutationsfrequenzen im Bereich 1-2*10-7/bp zu bestimmen. Um jedoch die erwarteten Mutationsfrequenzen im Bereich von 1-3*10-8/bp (Intron) bzw. 2-3*10-9/bp (Exon) zu detektieren, wären 2-3 g Gewebe bzw. 3 mg DNA notwendig. Auf Grund der anatomischen Organgewichte wäre die Durchführung des nukleären Random Mutation Capture Assays somit auf vereinzelte Organe wie Leber, Dünndarm und Gehirn beschränkt. Zudem bestanden mit der Hybridisierung und dem Uracil-DNA-Glycosylase-Verdau zwei zusätzliche kritische Punkte, welche zu einer Minimierung der Kopiezahl oder einer fehlerhaften Einschätzung der Mutationsfrequenz führen können. Aus diesen Gründen wurde eine Entwicklung des Random Mutation Capture Assays für die Zielsequenz im p53-Gen verworfen. Die Kopiezahlausbeuten der mitochondrialen DNA waren ab 50 mg Gewebeeinsatz bei jeder der 5 untersuchten Methoden ausreichend zur Bestimmung einer angestrebten Spontanmutationsfrequenz zwischen 6-100*10-7/bp. Bei Gewebemengen unter 50 mg erwies sich die Aufarbeitung mit DNAzol® auf Grund zu niedriger Kopiezahlausbeuten als ungeeignet. In dieser Arbeit wurde nachfolgend die Phenol-Chloroform-Extraktion nach Vermulst et al (2008) verwendet. Im Rahmen der Etablierung der PCR zur Erfassung der Anzahl mutierter Kopien (Mutations-PCR) wurde ein Mutanten-Standard zur Anwendung als Positivkontrolle in PCR und Agarose-Gelelektrophorese hergestellt, verifiziert und fluorimetrisch quantifiziert. Wiederfindungsexperimente bestätigten, dass mit der etablierten Mutations-PCR eine einzelne Kopie amplifizier- und detektierbar ist. Um eine Auswertung einer Sequenzierung hinsichtlich Anzahl der Mutanten als auch der Sequenz an sich zu gewährleisten, wurde der akzeptierte Bereich an detektierten 1-19 (80 Reaktionen) gesetzt. Nachfolgend wurde in der gesunden Leber von männlichen und weiblichen Ratten erfolgreich die mitochondriale Spontanmutationsfrequenz mit dem entwickelten Random Mutation Capture Assay bestimmt. Diese betrug innerhalb einer mitochondrialen DNA-Lösung 3,2 ± 3,1 *10-6/bp (Median 2,7). Die Mutationsfrequenzen von 3 unabhängigen mitochondrialen DNA-Lösungen -isoliert aus demselben Organpulver- betrugen durchschnittlich 11,5 ± 8,6 *10-6/bp (Median 8,0) und waren somit ca. 3-mal höher. Ein Vergleich zwischen den Mutationsfrequenzen der männlichen und weiblichen Tiere resultierte in mitochondrialen Mutationsfrequenzen zwischen 1,6-34,4 *10-6/bp (männlich) und 3,0-12,9 *10-6/bp (weiblich), wobei zwischen männlichen und weiblichen Tieren kein statistischer Unterschied bestand (Mann-Whitney-Test; p<0,05). Um zu prüfen, ob die Mutationsraten bestimmt mit dem mitochondrialen Random Mutation Capture Assay und einem phänotypselektiven Mutationstest zu gleichem Maße auf ein mutagenes Potential hinweisen, wurde als nächstes der phänotypselektive Hypoxanthin-Guanin-Phosphoribosyltransferase-Test für normale Nierenepithelzellen der Ratte (NRK-Zelllinie) entwickelt. Nach einer 24 h Inkubation mit 0,1 µM 4-Nitrochinolin-1-oxid, einem bekannten Adduktbildner, stieg die Mutationsfrequenz im Exon des Hypoxanthin-Guanin-Phosphoribosyltransferase-Gens um den Faktor 5 im Vergleich zur Lösemittelkontrolle an. Mit Hilfe des entwickelten Random Mutation Capture Assays wurde in der DNA -isoliert zum Zeitpunkt der Selektion- eine dreifache Steigerung der Mutationsfrequenz im mt-Cytb-Gen detektiert. Somit war mit beiden Tests eine Erhöhung der Mutationsfrequenz in der gleichen Größenordnung detektierbar, wobei der phänotypselektive Mutationstest sensitiver war. Nachdem die Mutations-PCR ca. 1,5 Jahren angewendet wurde, stieg innerhalb von 4 Monaten unabhängig von der verwendeten Templatkonzentration sowohl die Häufigkeit der detektierten Schmierbanden als auch die des DNA hang up an. In 7 Mutations-PCRs, welche nach diesen Phänomenen nur mit Blindwerten durchgeführt wurden, lag der Anteil an detektierten DNA-Schmierbanden pro Mutations-PCR zwischen 25,0% und 38,8%, der des DNA hang up zwischen 17,5% und 48,8%. Das war häufiger als in Reaktionen mit Templat; ein Hinweis dafür, dass das Vorliegen von Templat Nebenreaktionen zu einem gewissen Grad verdrängte und dass die unspezifische Amplifizierung am Mastermix der Mutations-PCR lag. Eine Änderung von chemischen oder physikalischen Parametern innerhalb der PCR-Reaktion führte zu keiner Reduktion der Nebenprodukte. Somit war der für das mitochondriale Cytochrom b-Gen entwickelte Random Mutation Capture Assay nicht robust gegenüber Nebenreaktionen und ist daher nicht für einen routinemäßigen Einsatz geeignet. Zusammenfassend war eine Entwicklung der Primer und der molekularen Werkzeuge des Random Mutation Capture Assays vom Mensch auf Ratte mit allen drei gewählten Zielsequenzen möglich. Im Rahmen der Experimente zeigte sich, dass die Kopiezahl-PCR der Zielsequenz in der 18S ribosomale RNA kodierenden DNA nicht praktikabel und eine Bestimmung der Mutationsfrequenzen für das Tumorsuppressor Protein 53 kodierenden Gen p53 nur unter Berücksichtigung einer eingeschränkten Organauswahl möglich war. Für die Zielsequenz des mitochondrialen Cytochrom b Gens war der Random Mutation Capture Assay durchführbar. Allerdings erwies sich die Mutations-PCR als instabil. Folglich ist eine Bestimmung von Mutationsfrequenzen mit dem Random Mutation Capture Assay in Rattus norvegicus nur sehr begrenzt möglich. / Mutation tests are performed in vitro and in vivo. Especially, the phenotype-selective mutation tests are often limited to the detection of mutations in the exon and possibly promoter regions. In order to increase the number of data on the commonly used in vitro mutation tests and thus to facilitate an assessment of the substance to be examined, a method for detecting the mutational spectra should be established and used within the investigation of the mutagenic potential of the food ingredient irilone. A method which allows the sequencing of each individual 6-thioguanine-resistant mutant formed in the hypoxanthine-guanine phosphoribosyltransferase assay has been developed, making it possible to draw conclusions about the mechanism of mutation formation. Although no difference in the mutant frequency was detected in the study on the mutagenic potential of the food ingredient irilone, an alterd mutation spectrum with increasing deletions and decreasing base pair substitutions was detected. The evaluation of the micronucleus test supported the assumption that irilone induces chromosome mutations. In addition, irilone had a strong aneugene potential. In contrast to the phenotype-selective mutation tests, genotype-selective tests theoretically have no limitations regarding the target sequence or organ selection. One representative for a genotype-selective test is the Random Mutation Capture Assay, published in 2005 by Bielas and Loeb for the intron 6 of the human TP53 gene. Thus, the next aim of this work was to investigate whether the technique of Random Mutation Capture Assay is transferable to the genome of the rat and whether or more specifically under which conditions the determination of spontaneous and induced mutation frequencies in different target sequences is possible. Therefore, the p53 tumor suppressor protein p53 gene, the 18S ribosomal RNA encoding DNA and the mitochondrial cytochrome b gene were selected as target sequences. Then their suitability for use in the Random Mutation Capture Assay was analyzed. For each target sequence, all molecular tools needed to perform the Random Mutation Capture Assay were prepared and verified under optimized PCR conditions. For the quantification of the total copy number, a specific TaqMan® real-time PCR method was developed for each target sequence. After optimization of the PCR conditions, recoveries in the desired range of about 90-100% with variations of a maximum of 20% were achieved per target sequence., except the target sequence coding for the 18S ribosomal DNA. Changing the real-time PCR conditions did not lead to any practicable method. Therefore, this target sequence, which promised to obtain more DNA copies despite lower DNA levels and thus facilitate the determination of low mutation frequencies, was not applicable in Random Mutation Capture Assay. To make a choice regarding a DNA isolation method, 5 methods were compared with respect to the copy number, purity and the cost / time effort, sufficient for the mutation frequency determination. With two of these five methods it was possible to isolate a sufficient number of nuclear copies from 100 mg of tissue to determine mutation frequencies in the range 1-2*10-7 / bp. However, to detect the expected mutation frequencies in the range of 1-3*10-8 / bp (intron) and 2-3*10-9 / bp (exon), 2-3 g of tissue or 3 mg of DNA would be necessary. Due to the anatomical organ weights, the implementation of the nuclear Random Mutation Capture Assay would be limited to individual organs such as liver, small intestine and brain. In addition, hybridization and uracil-DNA glycosylase digestion gave rise to two additional critical points that could lead to a minimization of copy number or a misjudgment of the mutation frequency.For these reasons, development of the Random Mutation Capture Assay for the target sequence in the p53 gene was discarded. Using at least 50 mg of tissue, the copy number yields of mitochondrial DNA were sufficient to determine a desired spontaneous mutation frequency between 6-100*10-7 / bp in each of the 5 investigated methods. For tissue levels below 50 mg, preparation with DNAzol® was unsuitable due to low copy number yields. In this work, the phenol-chloroform extraction according to Vermulst et al (2008) was used. During the establishment of the PCR detecting the number of mutated copies (mutation PCR), a mutated standard used as a positive control in PCR and agarose gel electrophoresis was prepared, verified and fluorometrically quantified. Recovery experiments confirmed that a single copy can be amplified and detected using the established mutation PCR. In order to ensure a sequencing evaluation with respect to the number of mutants as well as the sequence itself, the accepted range of detected mutants was set to 1-19 (80 reactions). Next, the mitochondrial spontaneous mutation frequency was successfully determined in healthy livers of male and female rats using the developed Random Mutation Capture Assay. The mutations frequency of a mitochondrial DNA solution was 3.2 ± 3.1 * 10-6 / bp (median 2.7). The mutation frequencies of 3 independent mitochondrial DNA solutions isolated from the same organ powder were on average 11.5 ± 8.6 * 10-6 / bp (median 8.0) and thus were about 3 times higher. A comparison between the mutation frequencies of the male and female animals resulted in mitochondrial mutation frequencies between 1.6-34.4 * 10-6 / bp (male) and 3.0-12.9 * 10-6 / bp (female). There was no statistical difference between male and female animals (Mann-Whitney test, p <0.05). To test whether the mutation rates determined by the mitochondrial Random Mutation Capture Assay and a phenotype-selective mutation test indicate a mutagenic potential to the same extent, the phenotype-selective hypoxanthine-guanine phosphoribosyltransferase test for normal renal epithelial cells of the rat (NRK cell line) was developed. After a 24 h incubation with 0.1 μM 4-nitroquinoline-1-oxide, a known adduct former, the mutation frequency in the exon of the hypoxanthine-guanine phosphoribosyltransferase gene increased by a factor of 5 compared to the solvent control. Using the developed Random Mutation Capture Assay, a threefold increase in the mutation frequency in the mitochondrial Cytochrome b gene was detected in the DNA isolated at the time of selection. Thus, both tests showed an increase in the mutation frequency of the same order of magnitude, with the phenotype-selective mutation test being more sensitive. After the mutation PCR was applied for about 1.5 years, the frequency of the detected smear bands as well as that of the DNA hang up increased regardless of the template concentration used. In 7 mutation PCRs, which were performed after these phenomena only with blank values, the proportion of detected DNA smear bands per mutation PCR was between 25.0% and 38.8%, that of the DNA hang up between 17.5% and 48.8%. This was more often than in reactions with template -an indication that the presence of template displaced side reactions to a certain extent and that the non-specific amplification was due to the master mix of the mutation PCR. Changing chemical or physical parameters within the PCR reaction did not result in a reduction of the by-products. Thus, the Random Mutation Capture Assay developed for the mitochondrial cytochrome b gene was not robust to side reactions and is therefore not suitable for routine use. In summary, it was possible to develop the primers and the molecular tools of Random Mutation Capture Assay with all three selected target sequences of the rat genome. The experiments showed that the copy number PCR of the target sequence in 18S ribosomal RNA-encoding DNA was impractical and determination of the mutation frequencies for the tumor suppressor protein 53-encoding gene p53 was only possible with limited organ selection. For the target sequence of the mitochondrial cytochrome b gene, the Random Mutation Capture Assay was feasible. However, the mutation PCR proved unstable. Consequently, determination of mutation frequencies with the Random Mutation Capture Assay in Rattus norvegicus is very limited.

El objetivo de este estudio es la generación ex-vivo de eritrocitos a partir de la maduración y diferenciación de células CD34+ purificadas de sangre de cordón umbilical. El desarrollo de la terapia celular y la generación de diferentes tipos celulares a partir de células madre ha abierto el campo a la producción de diferentes componentes sanguíneos, entre ellos los eritrocitos, a partir de células madre hematopoyéticas (células CD34+). Existen diferentes fuentes de células CD34+, siendo el cordón umbilical una fuente de fácil acceso y donde éstas presentan una mayor capacidad de expansión. La necesidad de incrementar la tasa de expansión y el paso de enucleación celular son los puntos clave para hacer realidad la posibilidad de generar sangre en el laboratorio que sea equiparable a una unidad de sangre donada. Se han diseñado diferentes estrategias para generar eritrocitos a partir de células CD34+ de sangre de cordón umbilical. Estas estrategias se han basado en la utilización de diferentes medios de cultivo, citocinas, concentraciones celulares y estrategias de cultivo. Para el paso final de enucleación se ha pasado de un co-cultivo sobre capa estromal de células mesenquimales a un cultivo en suspensión celular. Se ha puesto a punto una metodología de cultivo consistente en la combinación de un medio específico con estrategias de alimentación que permiten la expansión y diferenciación de células CD34+ de cordón umbilical a eritrocitos maduros. Todavía son necesarios más ajustes para incrementar la tasa de expansión y optimizar la fase de maduración final. / The aim of this study was the in vitro generation of red blood cells (RBC) from CD34+ cells purified from human umbilical cord blood, offering an alternative to classical transfusion products. Within the hematopoietic stem cell sources, umbilical cord is readily available and it has a greater expansion capacity. Such increase of the expansion capacity and the terminal differentiation efficiency are the key points in the generation of blood in the laboratory. We have tested different methodologies to generate erythrocytes from CD34+ cells from human umbilical cord blood. The parameters investigated related with the increase of the expansion capacity involved the use of different culture media, cytokines, cell seeding density and dilution steps. For the terminal differentiation and enucleation steps, the stromal layer co-culture was switched into a suspension culture with erythropoietin. The erythroid progenitors were characterized by the expression of the surface markers CD45, CD36, CD235 (glycophorin A) and CD71 (transferrin receptor). Enucleated cells were discriminated from nucleated cells by labeling the genetic content. May-Grünwald-Giemsa staining was used for morphological analyses. A culture methodology that combines a specific medium formulation and feeding strategy has been successfully developed in bioreactors allowing the expansion and differentiation of CD34+ cord blood to mature red blood cells. Further improvements are still needed in order to increase the expansion rate and to optimize terminal differentiation step.

Esta Tesis estudia la caracterización de diferentes receptores purinérgicos, principalmente P2X7, en células presentadoras de antígeno de la estirpe mieloide mononuclear fagocítica (monocitos, macrófagos peritoneales y macrófagos derivados de médula ósea). En concreto, se determina como la señalización purinérgica afecta a la diferenciación de los macrófagos, la producción de eicosanoides y de TNF-α, en estados tanto pro-inflamatorias y como de inmunosupresión. Para ello, se emplearon diferentes estímulos celulares; señales de patógenos, principalmente lipopolisacarido (LPS) bacteriano, señal que activa a los macrófagos a través de los receptores tipo toll 4; y señales de daño celular, principalmente ATP y adenosina. Siendo los objetivos concretos de esta Tesis: 1) Estudiar cómo afecta la señalización purinérgica a la diferenciación de los macrófagos; 2) Analizar la relación entre la activación del receptor P2X7 en macrófagos y la producción de eicosanoides; 3) Determinar cómo el receptor P2X7 regula la liberación del factor de necrosis tumoral (TNF)-α en macrófagos extenuados. La metodología empleó macrófagos peritoneales humanos y monocitos de sangre periférica de donantes sanos y pacientes con un proceso séptico severo de origen abdominal; muestras de ratones de la cepa C57BL/6 y deficientes para los receptores a estudio (P2rx7-/- y P2rx4-/-) que se emplearon para estudios tanto in vivo como in vitro. En la mayoría de ensayos se emplearon macrófagos derivados de médula ósea de ratón. Los diferentes macrófagos fueron sometidos a distintos protocolos de estímulo según el objetivo abordado: diferentes dosis de LPS para estudiar la respuesta ante situaciones inflamatorias, o polarización de macrófagos a M1 con LPS e interferon-o a M2 con interleuquina (IL)-4, para en todos los casos estimular con un nucleótido. De los estudios in vitro se analizó la cantidad de IL-1β, TNF-α, prostaglandina (PG) E2, tromboxano A2 y leucotrieno B4 liberada al medio mediante la técnica ELISA, la muerte celular cuantificando la actividad de lactatodehidrogenasa en los sobrenadantes celulares; la viabilidad celular mediante ensayos de reducción meatabólica del bromuro de 3-(4,5- dimetiltiazol-2-ilo)-2,5-difeniltetrazol; la cuantificación de la expresión relativa de los genes que codifican para los diferentes receptores y marcadores citoquímicos de interés, empleando la técnica de PCR-transcriptasa reversa a tiempo real, la cuantificación de marcadores de membrana específicos de macrófagos mediante citometría de flujo, y finalmente la actividad de la enzima convertidora de TNF-α (TACE) y la actividad catepsina B, mediante técnicas fluorométricas. Los estudios in vivo realizados en ratón analizaron la actividad del receptor P2X7 tras la administración intraperitoneal de LPS, cuantificando IL-1β y TNF-α en lavados peritoneales empleando la técnica ELISA, la presencia de ATP extracelular en peritoneo se cuantificó mediante bioluminiscencia, y la medición de la temperatura rectal en un modelo inducido de sepsis mediante una sonda térmica. Resultados y conclusiones: En esta Tesis se demuestra que la señalización purinérgica es capaz de reducir el número de macrófagos diferenciados de médula ósea, resultando en una regulación positiva de ciertos marcadores M2, derivando en un fenotipo específico de macrófago. En los macrófagos maduros, la activación de P2X7 induce la liberación del mediador lipídico PGE2, siendo P2X7 importante en la respuesta febril, un signo clásico asociado con el proceso inflamatorio. Además, la señalización mediante el receptor P2X7 en macrófagos M1 extenuados es capaz de controlar la liberación clásica de TNF-α. Estos resultados tienen implicaciones clínicas, ya que el receptor P2X7 emerge como una diana terapéutica prometedora para la fiebre y su potenciación durante inmunosupresión podría aumentar la inmunidad en los procesos sépticos. / This Thesis studies the characterization of different purinergic receptors, mainly P2X7, analyzing their functions in antigen presenting cells of mononuclear phagocytic myeloid lineage (monocytes, peritoneal macrophages and bone marrow derived macrophages). Precisely, the Thesis determine how purinergic signaling affects macrophage differentiation, eicosanoids and tumor necrosis factor (TNF)-α release, during both proinflammatory and immunosuppressive conditions. Therefore, cells were treated with bacterial products, mainly lipopolysaccharide (LPS), which specifically activates Toll-like receptor 4, and signals of cell damage, mainly ATP and adenosine. Objectives: 1) To study how purinergic signaling affects macrophage differentiation; 2) To analyze the association between the activation of P2X7 in macrophages and the production of eicosanoids; 3) To identify how P2X7 receptor regulates TNF-α release during immune extenuation. Methods: The experiments were performed using peritoneal human macrophages and peripheral blood monocytes of healthy subjects and patients with severe sepsis; mice samples from strain C57BL/6 and the knockout for the genes encoding the receptors to study (P2rx7-/- and P2rx4-/-), these mice were used for both in vivo and in vitro studies. Mice bone marrow derived macrophages were used in almost all the experiments of this thesis. The different macrophages were stimulated with different protocols to address the different objectives: different doses of LPS to mimic inflammatory conditions; LPS and interferon- to polarize macrophages to M1 and with interleukin (IL)-4 to polarize to M2. The amount of IL-1β, TNF-α, prostaglandin (PG) E2, thromboxane A2 and leukotriene B4 released, were measured by ELISA; cell death quantification was measured by the activity of lactate dehydrogenase in cells supernatants; macrophage viability was measured by the metabolic reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; relative expression of genes encoding for the desired receptors and markers was quantified by real-time reverse-transcription PCR; the quantification of specific macrophage membrane markers was analyzed by flow cytometry; the converting TNF-α enzyme (TACE) and cathepsin B activity were measured by fluorometric techniques. In vivo mouse studies to study P2X7 receptor activity after intraperitoneal administration of LPS was analyzed by ELISA, measuring IL-1β and TNF-α in peritoneal lavage; detection of extracellular ATP in peritoneum was quantified by bioluminescence; rectal temperature was measured in a sepsis model. Results and Conclusions: this Thesis demonstrates that purinergic signaling is able to reduce the number of differentiated macrophages from bone marrow, which display upregulation of certain M2 markers, resulting in a specific phenotype of macrophage. In mature macrophages, ATP activating P2X7 receptor induces the release of the lipid mediator PGE2, being P2X7 important for the febrile response, a classical signal associated with the inflammatory process. Furthermore, P2X7 receptor signaling in extenuated M1 macrophages is able to control the classical release of TNF-α. These findings have important clinical implications, since P2X7 receptor emerges as a promising target for fever, however its potentiation during immunosuppression could boost immunity in septic processes.

The natural daily cycles of light and dark have played a fundamental role in shaping the development of an adaptive intrinsic clock mechanism which allows organisms to coordinate the function of multiple organs by setting the correct circadian timing of cellular processes ensuring proper homeostasis. In mammalian skin, homeostasis is maintained by epidermal stem cells (epSCs). EpSCs localize to specialized niches where they undergo cycles of quiescence and proliferation. Several pathways are known to play essential roles in epSC function; however, how are these pathways spatiotemporally coordinated, and why not all stem cells within the niche behave in the same manner, is still poorly understood. We have analyzed the role of the molecular circadian clock in fine-­‐tuning the behavior of epidermal stem cells. Using a fluorescent circadian reporter mouse model, we demonstrate that the dormant epidermal stem cell compartment contains two co-­‐existing populations of stem cells in different clock states. Global comparative transcriptome analysis indicated that each clock population corresponds to a distinct predisposition state of response towards stem cell activating and dormancy cues. We provide evidence that the core circadian transcription factors BMAL1 and CLOCK bind to regulatory elements in the promoters of several of these stem cell homeostatic genes, thus being directly responsible for creating these two stem cell clock states. Unbalancing this clock driven equilibrium of epSCs in vivo resulted in progressive changes in the response of stem cells to activating or dormancy cues, which led to a progressive premature tissue aging, and a significant reduction in the development of cutaneous squamous cell carcinomas. Thus, our results indicate that the molecular clock machinery fine-­‐tunes the spatiotemporal behavior of epidermal stem cells within their niche, and that perturbation of this mechanism affects tissue homeostasis and the predisposition to neoplastic transformation. / Los ciclos naturales de luz y oscuridad han sido determinantes en el desarrollo de un reloj molecular intrínseco que permite coordinar la función de múltiples órganos para mantener la homeostasis global del organismo. La homeostasis del compartimento queratinocítico de la piel depende de una población de células troncales adultas epidermales (epSCs). Las epSCs están localizadas en nichos específicos y especializados desde dónde responden a las necesidades de repoblación celular del tejido mediante la alternancia de fases de quiescencia y proliferación. Varias rutas de señalización regulan el comportamiento de las epSCs; sin embargo, aún no entendemos bien porqué no todas las epSCs se comportan de la misma manera dentro de un mismo nicho troncal, y cómo están coordinadas a nivel espacio-­‐temporal. Hemos analizado el impacto del ritmo circadiano sobre las función de las epSCs. Mediante un ratón reportero fluorescente del ritmo circadiano hemos demostrado que el nicho troncal quiescente contiene dos poblaciones de epSCs en diferentes fases de su reloj molecular. El análisis comparativo global del transcriptoma de ambas poblaciones indicó que las dos poblaciones corresponden a dos estados opuestos de predisposición a responder a estímulos de activación y quiescencia. Mostramos resultados que demuestran que los factores de transcripción circadianos Bmal1 y Clock regulan directamente la expresión de genes que regulan el comportamiento de las epSCs. La arritmia in vivo en las epSCs resultó en una pérdida progresiva de la homeostasis tisular, un envejecimiento prematuro y una reducción significativa en el desarrollo de tumores escamosos de piel. Por lo tanto, nuestros resultados indican que la maquinaria del reloj molecular permite a las epSCs a anticiparse y coordinar su respuesta a estímulos locales del nicho, lo que constituye un mecanismo esencial para su correcta función en el tejido