The switch to unleaded fuels in the 1970s and the high air pollution in areas of high population density due to traffic particularly since the 1990s required the use of alternative fuel additives to achieve an improvement of the combustion. The utilization of oxygenated hydrocarbons as antiknock additives and so-called oxygenates provided a more complete and efficient combustion with simultaneously less harmful and polluting emissions. These include the synthetic ethers methyl tert-butyl ether (MTBE), ethyl tert-butyl ether (ETBE), tert-amyl methyl ether (TAME) and tert-amyl ethyl ether (TAEE). MTBE has a particular position as within some years it became the dominant oxygenate worldwide. Since then, over 100.000 leakages, most often in close proximity to gas stations, resulted in just as many oxygenate-contaminated sites of soil and groundwater within few years. The high water solubility of these ethers leads to an especially fast and extensive spread of the contamination plumes. Ether-contaminated groundwater has a turpentine-like taste that is noticed already in really low concentrations. Thus, such water can no longer serve as drinking water and requires a counter-measure. The chemical parameters of oxygenates decrease the efficiency of otherwise successfully applied techniques such as adsorption or aeration. In addition the ethers proved recalcitrant against microbial attack. The search for microorganisms that could degrade these synthetic oxygenates indeed resulted in the enrichment of many isolates. The majority of these isolates oxidize the ethers in a cometabolic manner either partially or completely to CO2. However, only few cultures are capable of independent growth on these oxygenates. These include the beta-proteobacteria Methylibium petroleiphilum PM1 and Aquincola tertiaricarbonis L108, of which the latter is of particular interest for the present work. Strain L108 is characterized by good growth on MTBE and is presently the only known isolate which is able to mineralize ETBE, TAME and TAEE at similar rates. This work examined the seemingly particularly well adapted oxygenate ether metabolism of strain L108, that was formerly isolated from an aquifer highly contaminated with MTBE. Via diverse deletion studies key enzymes of the degradative metabolism and their genetic background were clearly identified. Hence, the results of this work contribute to verify so far just hypothesized metabolic steps by detailed enzymatic and genetic studies. Based on detected metabolites, first studies on MTBE biodegradation already postulated an oxidative pathway via TBA, 2-methyl-1,2-propane-diol (MPD) and 2-HIBA. In case of a monoxygenatic hydroxylation of the methoxy group of MTBE a hemiacetale results as reaction product, from which the tertiary alcohol TBA can be formed easily in subsequent reactions. By comparing wild type strain L108 with the spontaneous mutant strain L10, we were now able to clearly show that the cytochrome P-450 monoxygenase system EthABCD accounts solely for this MTBE-oxidizing activity. It is also the only enzyme catalyzing the corresponding hydroxylation of ETBE, TAME and TAEE. In strain L108 this enzyme complex is expressed constitutively. TBA, which is also generated from hydroxylation of ETBE, is, as postulated and verified by this study, degraded by a different monoxygenase resulting in MPD. Via Tn5-mediated mutations this enzyme was confirmed as Rieske non-heme mononuclear iron monooxygenase MdpJ. MPD is further altered to the corresponding branched acid 2-HIBA, presumably by two dehydrogenation reactions. For the degradation of 2-HIBA, diverse hypotheses exist on the basis of known enzymatic reactions. Another Tn5 mutation now gave evidence, that in the mentioned beta-proteobacteria the novel mutase HcmAB linearizes 2-HIBA to 3-hydroxybutyric acid (3-HB) dependent on cobalamin and coenzyme A (CoA). Sequence comparison revealed, that strain L108 acquired all three key enzyme complexes, EthABCD, MdpJ and HcmAB via horizontal gene transfer (HGT). For TAME and TAEE a completely new degradation pathway was found. In strain L108, the resulting degradation product tert-amyl alcohol (TAA) of these ethers is, like TBA, also specifically oxidized by MdpJ. In Tn5-deletion studies and metabolite analyzes, however, no hydroxylation could be detected. Instead, TAA is rather desaturated. Therefore within the metabolism no diols or acids analogue to MPD or 2-HIBA were formed. Instead, via MdpJ TAA is initially degraded to the unsaturated tertiary alcohol and hemiterpene 2-methyl-3-butene-2-ol. Prenol (3-methyl-2-buten-2-ol), prenal (3-methyl-2-buten-2-al) and 3-methyl crotonic acid were detected as additional metabolites. Hence, an isomerization of the branched acid by HcmAB is apparently irrelevant in TAA biodegradation. Accordingly it could be shown, that deletion mutants for HcmAB indeed could not grow on TBA, but are still able to grow on TAA, just as fast as the wild type, in fact. The tertiary alcohol 2-methyl-3-butene-2-ol is presumably transformed via another isomerase resulting in the primary alcohol prenol. Prenol is further oxidized by postulated dehydrogenases to 3-methyl crotonic acid. This would also be in correlation to the already observed degradation pathway of the monoterpene linalool in other bacteria. However, the responsible enzymes in strain L108 are not yet identified. Besides this principal gain of knowledge in the degradation of xenobiotic ether structures and the evolution of degradative microorganism, the now confirmed key enzymes EthB, MdpJ and HcmAB, respectively their coding genes, can be used as specific markers to monitor natural degradation processes in in situ studies. On this basis, the presence of active microorganisms and additionally - derived from the confirmed single key enzymes - a potentially complete degradation can be concluded. In the long run, it might be possible to stimulate the natural microbiological activity, e.g. via bioaugmentation with degradation specialists. Furthermore, regarding the potential progress of remediation procedures, potentially limiting steps can be distinguished via respective markers and narrowed down as possible cause of deficient degradation activity. However, such function-based monitoring requires specific verification. Therefore, subsequent studies have to analyze, if there is a sequence diversity among these three key enzymes. Previous sequence comparisons hypothesize that up to 60% accordance in the protein sequence in homologues of MdpJ and HcmA they can still be assumed to possess the same enzymatic function. This diversity has to be considered in the development of specific probes.:Bibliographische Darstellung Eidesstattliche Erklärung Danksagung Abstract Kurzfassung Abkürzungsverzeichnis 1. Einleitung 1.1. Tertiäre Ether als Benzin-Oxygenate - Hintergrund und Umweltproblematik 1.2. Mikrobiologischer Abbau tertiärer Ether 1.3. Postulierter Abbauweg 1.4. Monitoring-Tools für biologischen Abbau 1.5. Ziel dieser Arbeit 1.6. Referenzen der Einleitung 2. Die initiale Etherspaltung des Stammes L108 2.1. Die Ethermonooxygenase EthB 2.2. Supplemental Material 3. Die spezifische Alkoholmonooxygenase MdpJ 3.1. Die Alkoholmonooxygenase MdpJ als Hydroxylase und Reduktase 3.2. Supplemental Material 4. Die 2-HIBA-Mutase HcmAB des Stammes L108 4.1. Die 2-HIBA-Mutase HcmAB 4.2. Supplemental Material 5. Der TAA-Abbau des Stammes L108 5.1. Der TAA-Abbau des Stammes L108 5.2. Supplemental Material 6. Diskussion 6.1. Nachweis der Schlüsselenzyme in Stamm L108 durch Mutation 6.2. Nutzen für den Nachweis natürlichen Abbaus 6.3. Der TAA-Metabolismus als neuartiger Abbauweg 6.4. Mikrobiologische Anpassung an Xenobiotika am Beispiel MTBE 6.5. Ausblick 6.6. Referenzen der Diskussion Anhang Curriculum Vitae Publikationsverzeichnis Tagungsbeiträge Nachweis über Anteile der Co-Autoren / Die Einführung bleifreien Benzins in den 1970er-Jahren und die hohe Emissionsbelastung von Ballungszentren durch den Straßenverkehr insbesondere seit den 1990er-Jahren erforderte den Einsatz alternativer Benzinadditive, um eine Verbesserung der Verbrennung zu erreichen. Die Nutzung sauerstoffhaltiger Kohlenwasserstoffe als Antiklopfmittel und als sogenannte Oxygenate bot sich an, da diese eine effizientere Verbrennung mit gleichzeitig niedrigeren gesundheits- und umweltschädigenden Emissionen fördern. Zu den Oxygenaten gehören die synthetischen Ether Methyl-tert-butylether (MTBE), Ethyl-tert-butylether (ETBE), tert-Amylmethylether (TAME) und tert-Amylethylether (TAEE). Eine herausragende Stellung nimmt MTBE ein. Innerhalb weniger Jahre wurde es zum hauptsächlich verwendeten Oxygenat weltweit. Seitdem führten jedoch über 100.000 Leckagen, zumeist in Tankstellennähe, innerhalb weniger Jahre zu ebenso zahlreichen Kontaminationen des Grundwassers mit Oxygenaten. Aufgrund der hohen Wasserlöslichkeit kommt es dabei zu einer besonders schnellen und großflächigen Ausbreitung der Ether. Derart belastetes Grundwasser weist schon bei geringsten Etherkonzentrationen einen als terpentinartig wahrgenommenen Geruch und Geschmack auf und kann daher nicht mehr als Trinkwasserzufuhr genutzt werden. Es bedarf einer Lösung dieses Problems. Die chemischen Parameter der Ether senken allerdings die Effizienz anderweitig erfolgreich genutzter technischer Sanierungsverfahren auf Basis von z. B. Adsorption oder Aerisierung. Auch gegenüber mikrobiellen Abbau erweisen sie sich als rekalzitrant. Die Suche nach oxygenatabbauenden Mikroorganismen führte zwar zur Anreicherung vieler Isolate, welche die Oxygenate cometabolisch partiell oder sogar komplett oxidieren, nur sehr wenige Kulturen sind aber zu autarkem Wachstum auf diesen Ethern fähig. Dazu gehören die Beta-Proteobacteria Methylibium petroleiphilum PM1 und der dieser Arbeit zugrunde liegende Aquincola tertiaricarbonis L108. Der Stamm L108 zeichnet sich durch ein vergleichsweise gutes Wachstum auf MTBE aus und ist als bisher einzig bekanntes Isolat in der Lage, auch ETBE, TAME und TAEE ähnlich schnell zu mineralisieren. Die vorliegende Arbeit handelt von dem scheinbar besonders gut an den Oxygenatabbau adaptierten Stoffwechsel des ursprünglich aus MTBE-kontaminiertem Grundwasser angereicherten Stammes L108. Durch verschiedene Deletionsstudien wurden Schlüsselenzyme des Abbaus und deren genetischer Hintergrund eindeutig identifiziert. Die Ergebnisse der genetischen, enzymatischen und physiologischen Studien des Wildtyps im Vergleich zu den erzeugten Deletionsstämmen tragen dazu bei, bisher nur postulierte Reaktionsschritte zu verifizieren. Schon seit den ersten Studien zum MTBE-Abbau wird anhand markanter Metabolite ein oxidativer Abbau via TBA, 2-Methyl-1,2-propandiol (MPD) und 2-Hydroxyisobuttersäure (2-HIBA) vermutet. Im Fall einer Hydroxylierung der Methoxygruppe von MTBE wird ein Hemiacetal als Reaktionsprodukt erzeugt, aus dem nachfolgend leicht der tertiäre Alkohol TBA entstehen kann. Durch den Vergleich des Wildtyps mit der Spontanmutante Stamm L10 konnte jetzt gezeigt werden, dass hierfür allein das Cytochrom-P450-Monooxygenasesystem EthABCD verantwortlich ist. Dieses katalysiert auch exklusiv die entsprechende Hydroxylierung von ETBE, TAME und TAEE. In Stamm L108 wird das Enzym konstitutiv exprimiert. TBA, das auch aus der Hydroxylierung von ETBE resultiert, wird, wie postuliert und in dieser Arbeit verifiziert, durch eine weitere Monooxygenase zu MPD abgebaut. Durch eine Tn5-Transposon-vermittelte Mutation konnte verifiziert werden, dass es sich bei diesem Enzym um die Rieske-nicht-Häm-Monooxygenase MdpJ handelt. MPD wird im weiteren Verlauf voraussichtlich durch zwei Dehydrogenierungen zur korrespondierenden, verzweigten Säure 2-HIBA gewandelt. Zum 2-HIBA-Abbau gibt es, basierend auf bekannten Enzymreaktionen, diverse Hypothesen. Anhand einer weiteren Tn5-Mutation konnte jetzt bestätigt werden, dass in den genannten beta-Proteobacteria die neuartige Mutase HcmAB wirksam ist, welche 2-HIBA abhängig von Cobalamin und Coenzym A (CoA) zu 3-Hydroxybuttersäure (3-HB) linearisiert. Sequenzvergleiche ergaben, dass Stamm L108 die Schlüsselenzyme des Etherabbaus, EthABCD, MdpJ und HcmAB, durch horizontalen Gentransfer erworben hat. Für TAME und TAEE wurde ein völlig neuer Abbauweg gefunden. In Stamm L108 wird der beim Abbau dieser Ether entstehende tert-Amylalkohol (TAA) wie TBA ebenfalls exklusiv durch MdpJ oxidiert. Durch die Tn5-Deletionsstudien und durch Analyse der Metabolite konnte allerdings keine Hydroxylierung nachgewiesen werden. TAA wird durch MdpJ vielmehr desaturiert. Somit entstehen im Abbauweg keine zu MPD und 2-HIBA analogen Diole und Säuren, sondern TAA wird zunächst durch MdpJ zu einem ungesättigten tertiären Alkohol, dem Hemiterpen 2-Methyl-3-buten-2-ol, abgebaut. Prenol (3-Methyl-2-buten-2-ol), Prenal (3-Methyl-2-buten-2-al) und 3-Methylcrotonsäure wurden als weitere Metabolite des TAA-Stoffwechsels detektiert. Somit spielt eine Isomerisierung einer tertiär verzweigten Säure durch HcmAB im TAA-Abbauweg offensichtlich keine Rolle. Entsprechend konnte gezeigt werden, dass Deletionsmutanten für hcmAB zwar nicht mehr auf TBA, aber immer noch auf TAA wachsen können, und das genauso schnell, wie der Wildtyp. Der tertiäre Alkohol 2-Methyl-3-buten-2-ol wird wahrscheinlich durch eine andere Isomerase zum primären Alkohol Prenol umgewandelt und dieser dann durch Dehydrogenasen zur Methylcrotonsäure oxidiert. Dies würde dem bereits in anderen Bakterien beobachteten Abbauweg des Monoterpens Linalool entsprechen. Die in Stamm L108 dafür verantwortlichen Enzyme wurden aber noch nicht identifiziert. Neben diesem grundsätzlichen Erkenntnisgewinn zum Abbau der xenobiotischen Etherverbindungen und der Evolution degradativer Mikroorganismen, können die hier bestätigten Schlüssel-enzyme EthABCD, MdpJ und HcmAB bzw. deren codierende Gene als spezifische Marker zum Monitoring natürlicher Abbauprozesse für in-situ-Untersuchungen genutzt werden. Auf dieser Basis kann auf die Anwesenheit aktiver Mikroorganismen und zudem noch - abgeleitet aus der Präsenz der einzelnen Schlüsselenzyme - auf einen potenziell kompletten Abbau geschlossen werden. Darauf aufbauend kann die natürliche mikrobiologische Aktivität durch nachfolgende biotechnologische Maßnahmen stimuliert werden, zum Beispiel durch eine Bioaugmentation mit Abbauspezialisten. Des weiteren können mögliche limitierende Schritte hinsichtlich des potenziellen Verlaufs der Sanierungsmaßnahme über Präsenztiter der betreffenden Marker gezielter verfolgt und als etwaige Ursachen defizitärer Abbauleistungen eingegrenzt werden. Voraussetzung für dieses funktionsbasierte Monitoring ist allerdings der spezifische Nachweis. Somit sollte in nachfolgenden Studien analysiert werden, ob es bei den drei Schlüsselenzymen eine Sequenzdiversität gibt. Die bisherigen Sequenzvergleiche lassen zumindest vermuten, dass bis etwa 60% Übereinstimmung der Proteinsequenzen bei Homologen von MdpJ und HcmA noch mit der gleichen Enzymfunktion zu rechnen ist. Diese Diversität sollte bei der Entwicklung von spezifischen Sonden berücksichtigt werden.:Bibliographische Darstellung Eidesstattliche Erklärung Danksagung Abstract Kurzfassung Abkürzungsverzeichnis 1. Einleitung 1.1. Tertiäre Ether als Benzin-Oxygenate - Hintergrund und Umweltproblematik 1.2. Mikrobiologischer Abbau tertiärer Ether 1.3. Postulierter Abbauweg 1.4. Monitoring-Tools für biologischen Abbau 1.5. Ziel dieser Arbeit 1.6. Referenzen der Einleitung 2. Die initiale Etherspaltung des Stammes L108 2.1. Die Ethermonooxygenase EthB 2.2. Supplemental Material 3. Die spezifische Alkoholmonooxygenase MdpJ 3.1. Die Alkoholmonooxygenase MdpJ als Hydroxylase und Reduktase 3.2. Supplemental Material 4. Die 2-HIBA-Mutase HcmAB des Stammes L108 4.1. Die 2-HIBA-Mutase HcmAB 4.2. Supplemental Material 5. Der TAA-Abbau des Stammes L108 5.1. Der TAA-Abbau des Stammes L108 5.2. Supplemental Material 6. Diskussion 6.1. Nachweis der Schlüsselenzyme in Stamm L108 durch Mutation 6.2. Nutzen für den Nachweis natürlichen Abbaus 6.3. Der TAA-Metabolismus als neuartiger Abbauweg 6.4. Mikrobiologische Anpassung an Xenobiotika am Beispiel MTBE 6.5. Ausblick 6.6. Referenzen der Diskussion Anhang Curriculum Vitae Publikationsverzeichnis Tagungsbeiträge Nachweis über Anteile der Co-Autoren

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Oxygen dependent prolyl-4-hydroxylation of the alpha-subunit of the hypoxia inducible transcription factor (HIF-alpha) plays an essential role in the hypoxic response. Hydroxylation of proline residues in the N- or C-terminal oxygen dependent degradation domains (NODD or CODD) increases the affinity of HIF-alpha to the von Hippel-Lindau protein (pVHL) by approx. 1000 fold so signalling for HIF-alpha degradation. With limiting oxygen, HIF-alpha hydroxylation slows, it dimerises with HIF-beta and activates the transcription of a gene array. Prolyl-4-hydroxylation also stabilises the triple helix structure of collagen, the most abundant human protein. Both the collagen and the HIF prolyl hydroxylases (PHDs) are Fe(II) and 2-oxoglutarate (2OG) dependent oxygenases. Crystal structures of PHD2 in complex with CODD were determined in the current study. Together with biochemical analyses, the results demonstrate that catalysis involves a mobile region of PHD2 that encloses the hydroxylation site and stabilises the PHD2.Fe(II).2OG complex. When bound to PHD2 the pyrrolidine ring of the non-hydroxylated proline-residue adopts a C⁴-endo conformation. Evidence is provided that 4R-hydroxylation enables a stereoelectronic effect that changes the proline conformation to the C⁴-exo state, as observed when hydroxylated HIF-alpha is bound to pVHL and in collagen. The results help to rationalise NODD/CODD selectivity data for PHD isoforms and the effects of clinically observed mutations on PHD2 catalysis. Analyses on the interaction of nitric oxide with PHD2 are described and discussed with respect to regulation of the hypoxic response by nitric oxide.

Hypoxia-inducible factor (HIF) is the master regulator of genes involved in adaptation to hypoxia. The stability and transcriptional activity of HIF are regulated by post-translational hydroxylations: prolyl hydroxylation by the prolyl hydroxylase domain-containing enzymes PHD1 – 3 earmarks HIF for proteasomal degradation, whilst asparaginyl hydroxylation by factor inhibiting HIF (FIH) blocks the interaction of HIF with the transcriptional coactivators p300/CBP. The PHDs and FIH hydroxylate HIF directly from molecular oxygen and are therefore oxygen sensors. Recent literature shows that FIH also hydroxylates a number of proteins containing an ankyrin-repeat domain (ARD). Together with reports suggesting that the PHDs are involved in HIF-independent pathways, this suggests that the HIF hydroxylases may have a wide range of non-HIF targets. This thesis describes my investigations into novel substrates of the HIF hydroxylases. This work has characterized the FIH-dependent hydroxylation of the ARD-containing protein Notch1, and defined a consensus sequence for hydroxylation that corresponds to the ankyrin-repeat consensus. Using this consensus potential sites of hydroxylation in a novel ARD FIH substrate, myosin phosphatase targeting subunit 1 (MYPT1), were identified then subsequently confirmed and characterized. Notch1 competes with HIF for FIH hydroxylation. My experiments show that this occurs because Notch1 is a more efficient substrate than HIF, whilst studies on MYPT1 and other proteins indicate that competitive inhibition of FIH may be a general property of ARDs. There are more than 300 ARD proteins in the human genome, and this thesis demonstrates that FIH may hydroxylate a significant percentage of these. In addition to the analysis of ARD hydroxylation a proteomic investigation into novel PHD3 substrates has identified two candidate proteins, suggesting that the PHDs may also have multiple targets. These results have important implications for oxygen sensing, and indicate that post-translational hydroxylation is likely to be a widespread modification in cell biology.

A deficiência da 21-hidroxilase é uma doença genética comum, causada por mutações no gene CYP21A2, que codifica a enzima 21-hidroxilase (P450c21). A deficiência da 21-hidroxilase afeta a síntese de cortisol e aldosterona e promove acúmulo de precursores, que são desviados para a síntese de andrógenos. Observa-se três principais fenótipos: a forma clássica virilizante simples (VS), na qual as meninas nascem com virilização da genitália externa e ambos os sexos apresentam virilização pós-natal; a forma perdedora de sal (PS), na qual além da virilização, ambos os sexos apresentam crise de perda de sal no período neonatal; e a forma não clássica (NC), na qual os sintomas de hiperandrogenismo iniciam-se mais tardiamente, na infância, adolescência ou idade adulta. Os estudos in vitro das mutações do CYP21A2 demonstram que existe uma boa correlação do grau de comprometimento da atividade enzimática conferido pelo genótipo com o fenótipo. Entretanto, existem algumas divergências como: pacientes que apresentam quadro clínico e hormonal de forma não clássica, nos quais mutações não são identificadas em um ou em ambos os alelos do CYP21A2, e pacientes que apresentam o fenótipo mais leve do que o predito pelo genótipo. Essas divergências sugerem a presença de fatores moduladores do fenótipo na deficiência da 21-hidroxilase. A primeira hipótese foi de que houvesse mutações no gene P450 óxido-redutase (POR), que codifica uma flavoproteína que doa elétrons para as enzimas microssomais P450, inclusive a P450c21, passo fundamental para a atividade enzimática das mesmas. A segunda hipótese foi de que outras enzimas P450, que não a P450c21, tivessem a capacidade de realizar a 21-hidroxilação extra-adrenal da progesterona e 17OH-progesterona (17OHP), sendo então capazes de modular o fenótipo da perda de sal e/ou virilização. Os citocromos P450 hepáticos CYP2C19 e CYP3A4, responsáveis pelo metabolismo de drogas, são capazes de realizar 21-hidroxilação da progesterona, porém essa atividade nunca foi comparada com a atividade de 21-hidroxilação da P450c21, a fim de que se possa extrapolar a importância dessa atividade extra-adrenal in vivo. A casuística desse estudo constou de 11 pacientes com a forma NC e genótipo incompleto, e 6 pacientes com fenótipo discordante do genótipo (5 com genótipo predizendo forma PS e manifestando forma VS, e 1 com genótipo predizendo VS e apresentando forma NC). O gene POR foi sequenciado em todos 17 pacientes e 10/30 alelos não relacionados apresentavam o polimorfismo A503V. O estudo funcional foi realizado através da expressão em bactéria do P450c21, do POR selvagem (PORwt) e da variante do POR A503V, e de estudo enzimático in vitro comparando a 21-hidroxilação da P450c21 promovida pela PORwt e POR A503V. Essa comparação foi realizada através de cálculos dos parâmetros que avaliam cinética enzimática (Km, Vmax e Vmax/Km). Apesar da variante POR A503V diminuir significativamente a atividade da P450c17 in vitro, nosso estudo demonstrou que ela não altera a capacidade de 21-hidroxilase da P450c21. Portanto, não existem substituições no POR que justifiquem as discordâncias de fenótipo/genótipo na deficiência da 21-hidroxilase nesta casuística. Para avaliarmos o papel da 21-hidroxilação extra-adrenal, as enzimas P450c21, as enzimas P450 hepáticas CYP2C19 e CYP3A4 e o POR foram expressos em bactéria. A capacidade das enzimas P450c21, CYP2C19 e CYP3A4 de 21- hidroxilar progesterona e 17OHP foram avaliadas in vitro e medidas através dos mesmos parâmetros de cinética enzimática. Comparado com a P450c21, o Vmax/Km da 21-hidroxilação da progesterona pelos CYP2C19 e CYP3A4 foram 17% e 10%, respectivamente. Os citocromos hepáticos não apresentaram atividade de 21-hidroxilação da 17OHP. Considerando que uma atividade residual mínima de 21-hidroxilação da progesterona é satisfatória para produzir quantidades suficientes de aldosterona para se evitar a perda de sal, este estudo sugere que a atividade de 21-hidroxilação extra-adrenal do CYP2C19 e CYP3A4 é capaz de melhorar o fenótipo de perda de sal, mas não o da deficiência de glicocorticóide. O gene CYP2C19, ao contrário do CYP3A4, é extremamente polimórfico, e são descritos vários haplótipos com diferentes atividades enzimáticas, os quais explicam as diferenças no metabolismo de várias drogas utilizadas na prática clínica. O único polimorfismo ultra-metabolizador é o CYP2C19*17, representado por duas substituições na região promotora que aumentam a transcrição do gene em 2-4 vezes. Os indivíduos que apresentam o haplótipo ultra-metabolizador podem ter maior atividade de 21-hidroxilação extraadrenal da progesterona e, dessa forma, não apresentarem a crise de perda de sal. O gene CYP2C19 foi sequenciado nos cinco pacientes com genótipo de forma perdedora de sal e que não desidrataram como predito, e encontramos o haplótipo ultra-metabolizador em homozigose em 1/5 pacientes e em heterozigose em 1/5 pacientes. Heterozigose para o CYP2C19*17 também foi encontrada no grupo controle (indivíduos perdedores de sal com genótipo/fenótipo concordantes). Portanto, o haplótipo CYP2C19*17 em heterozigose é insuficiente para modular a perda de sal e, provavelmente, em homozigose pode evitar a perda de sal. Em conclusão, pela primeira vez descrevemos o efeito modulador de uma variante alélica dos citocromos hepáticos P450 melhorando o fenótipo da forma perdedora de sal; no entanto, os demais casos permanecem com indefinição do genótipo. Estes resultados sugerem que não apenas uma única enzima, mas múltiplas enzimas extra-adrenais estão possivelmente envolvidas na modulação do fenótipo da deficiência da 21-hidroxilase. / Adrenal 21-hydroxylase deficiency is a common genetic disorder, caused by mutations in the CYP21A2 gene, which encodes the 21-hydroxylase P450c21. The 21-hydroxylase deficiency disrupts cortisol and aldosterone biosynthesis and leads to accumulation of androgen precursors. There are 3 main phenotypes: the simple virilizing (SV) form, in which girls present with virilized external genitalia at birth and both sexes present with precocious pseudopuberty; the salt wasting (SW) form, characterized by additional salt-wasting crisis in the neonatal period in both sexes; and the nonclassic (NC) form, in which the hyperandrogenic signs occur later in life, during childhood, adolescence or adulthood. In vitro studies show a good correlation between the degree of enzymatic impairment determined by genotype and phenotype. However, there are some discrepancies as: patients with clinical and hormonal profiles of nonclassic form in whom mutations are not found in one or both alleles, and patients with milder phenotype than the ones predicted by genotyping. These discrepancies suggest the existence of modulatory factors in 21- hydroxylase deficiency phenotype. The first hypothesis was that there were mutations in P450 oxidoreductase (POR), a gene which encodes a flavoprotein that donates electrons for all microsomal P450s, including P450c21, an essential step for P450s activity. The second hypothesis was that other enzymes that not P450c21 could perform extra-adrenal 21-hydroxylation of progesterone and 17OHprogesterone (17OHP), modulating salt balance and virilization. Hepatic drugmetabolizing P450 enzymes CYP2C19 and CYP3A4 can 21-hydroxylate progesterone; however, this activity was never compared to 21-hydroxylation performed by P450c21, in order to determine the importance of this extra-adrenal activity in vivo. The present cohort consisted of 11 patients with nonclassic form and incomplete genotype and 6 patients with genotype/phenotype discrepancies (genotype-predicted SW form and manifested SV form, n=5; genotyped-predicted SV form and manifested NC form, n=1). The POR gene was sequenced in these 17 patients, and 10/30 alleles presented the polymorphism A503V. P450c21, PORwt and POR A503V were expressed in bacteria, assayed in vitro, and the kinetics parameters Km, Vmax and Vmax/Km were calculated. Although POR A503V variant decreases the activity of P450c17, our study showed that it does not alter the 21-hydroxylation by P450c21. Therefore, there are no POR variants in this cohort that could explain discrepancies between genotype and phenotype in 21- hydroxylase deficiency. To evaluate the hypothesis of extra-adrenal 21- hydroxylation, P450c21, CYP2C19, CYP3A4, and POR were expressed in bacteria, assayed in vitro, and kinetic parameters assessed. Compared to P450c21, the Vmax/Km of 21-hydroxylation of progesterone by CYP2C19 and CYP3A4 was 17% and 10%, respectively. Neither CYP2C19 nor CYP3A4 were able to 21-hydroxylate 17OHP. Considering that little 21-hydroxylation activity is enough to produce sufficient amount of aldosterone to avoid the dehydration, this study suggests that extra-adrenal 21-hydroxylation by CYP2C19 and CYP3A4 may be able to ameliorate the mineralocorticoid, but not the glucocorticoid deficiency. CYP2C19 is very polymorphic, and some haplotypes can modify enzymatic activity. For example, the unique ultrametabolizer allele CYP2C19*17 has two polymorphisms in the promoter region that increase gene transcription in 2-4 times. Thus, patients harboring the CYP2C19 ultra-metabolizer allele could present an increased extraadrenal 21-hydroxylation of progesterone, and hence be able to avoid salt wasting crisis. CYP2C19 gene was sequenced in the 5 patients who did not present salt wasting crisis as expected, and 1/5 patients was homozygous and 1/5 patients was heterozygous for the CYP2C19*17 allele. Heterozygosity was also present in the control group (patients with salt wasting form and genotype/phenotype concordance). Therefore, heterozygosity for CYP2C19*17 seems to be insufficient to modulate salt balance but homozygosity might be able to avoid salt wasting crisis. In conclusion, we described for the first time the modulatory effect of an allelic variant of an extra-adrenal P450 enzyme ameliorating the salt wasting phenotype in a patient with 21-hydroxylase deficiency. Taken together with the remaining undefined genotypes, these results suggest that multiple extra-adrenal enzymes, rather than a single one, modulate the phenotypic expression of defects in 21-hydroxylase.

Introdução: A Hiperplasia Adrenal Congênita por deficiência da 21-hidroxilase (HAC) é uma doença com mortalidade neonatal elevada sendo elegível para programas públicos de Triagem Neonatal (TN). A HAC é causada por mutações no gene CYP21A2, as quais acarretam diferentes comprometimentos da atividade enzimática e resultam em espectro amplo de manifestações clínicas. Apesar da eficiência da TN para diagnosticar os casos graves, a taxa elevada de resultados falso-positivos (RFP), principalmente relacionados à prematuridade, é um dos maiores problemas. Porém, resultados falso-negativos também podem ocorrer em coletas antes de 24 horas de vida. No Brasil, a coleta da amostra neonatal difere entre os municípios, podendo ser no terceiro dia de vida como após. Objetivo: Avaliar se os valores da 17OH-progesterona neonatal (N17OHP) das coletas no terceiro dia de vida diferem significativamente das coletas a partir do quarto dia. Determinar qual percentil (99,5 ou 99,8) pode ser utilizado como valor de corte para a N17OHP, de acordo com o peso ao nascimento e tempo de vida na coleta, a fim de que proporcione taxa menor de RFP. Métodos: Foi avaliada, retrospectivamente, a N17OHP de 271.810 recém-nascidos (Rns) de acordo com o tempo de vida na coleta (G1: 48 - = 72h) e peso ao nascimento (P1: <= 1.500g, P2: 1.501-2.000g, P3: 2.001-2.500g e P4: >= 2.500g), pelo método imunofluorimétrico. Testes com resultados alterados foram confirmados no soro por Espectrometria de Massas em Tandem - LC-MS/MS. Rns afetados e/ou assintomáticos e com valores persistentemente elevados de 17OHP sérica foram submetidos ao estudo molécular, sequenciamento do gene CYP21A2. Resultados: os valores da N17OHP no grupo G1 foram significativamente menores do que em G2 em todos os grupos de peso (p < 0.001). A taxa de RFP em G1 e G2 foi de 0,2% para o percentil 99,8 e de 0,5% para o percentil 99,5 em ambos os grupos. O percentil 99,8 da N17OHP foi o melhor valor de corte para distinguir os Rns não afetados dos afetados, cujos valores são: G1 (P1: 120; P2: 71; P3: 39 e P4: 20 ng /mL) e em G2 (P1: 173; P2: 90; P3: 66 e P4: 25 ng/mL). Vinte e seis Rns do grupo G1 apresentaram a forma perdedora de sal (PS) (13H e 13M), nestes a N17OHP variou de 31 a 524 ng/mL e vinte Rns no grupo G2 (8H e 12M), nestes a N17OHP variou de 53 a 736 ng/mL. Para ambos os grupos foram encontrados três Rns com a forma virilizante simples (1H e 2M) e os valores da N17OHP variaram de 36 a 51 ng/mL. Resultados falso-negativos não foram relatados. O valor preditivo positivo (VPP) no teste do papel filtro foi de 5,6% e 14,1% nos grupos G1 e G2, respectivamente, ao se utilizar o percentil 99,8, e de 2,3% e 7% nos grupos G1 e G2 ao se utilizar o percentil 99,5. Dentre os casos com TN alterada (RFP), 29 deles também apresentaram 17OHP sérica elevada quando dosada por LC-MS/MS. Os casos assintomáticos foram acompanhados até normalização da 17OHP sérica e/ou submetidos ao estudo molecular, que identificou dois Rns com genótipo que prediz a forma não clássica. Conclusão: a melhor estratégia para otimização do diagnóstico da HAC na triagem neonatal é se padronizar valores de corte da N17OHP em dois grupos de acordo com o tempo de vida na coleta (antes e depois de 72 horas), subdivididos em quatro grupos de peso. A utilização dos valores de corte do percentil 99,8 se mantém eficaz no diagnóstico da HAC-21OH na triagem neonatal, reduzindo de forma significativa a taxa de RFP, sem perda do diagnóstico da forma PS / Introduction: Congenital Adrenal Hyperplasia (CAH) due to 21-hydroxylase is disorder with high mortality rate, being eligible for Public Programs of Newborn Screening (NBS). It is caused by mutations in the CYP21A2 gene leading to different impairments of enzyme activity and consequently to a spectrum of clinical manifestations. Despite the efficacy of NBS in diagnosing the severe clinical forms, the main concern is the high rate of false-positive results (FPR), mainly related to prematurity. False-negative results can also occur due to precocious sample collections, especially before 24 hs of life. Neonatal samples are collected at different times across our country, at third day of life or after. Objective: To determine if the values of neonatal 17OH-progesterone (N17OHP) collected on the third day of life differ significantly to the ones collected after the fourth day. To evaluate the best percentile of N17OHP values, according to birth-weight and age at the sample collection, to be used as a cut-off in NBS, in order to result in lower rate of FPR. Methods: N17OHP of 271,810 newborns (NB) according to sample collection time (G1: 48 - = 72 hs) and birth-weight (P1: <= 1,500g, P2: 1,501-2,000g, P3: 2,001-2,500 and P4: >2,500g) were retrospectively evaluated. N17OHP was measured by immunofluorimetric assay. N17OHP tests with altered values were confirmed through serum 17OHP by Tandem Mass Spectrometry - LC-MS/MS. Affected newborns (NB) and those asymptomatic with persistently increased serum 17OHP levels were submitted to molecular analysis, entire CYP21A2 gene sequencing. Results: N17OHP values of G1 on the third day of life were significantly lower than those of G2, in all birth-weight groups (p < 0.001). The FPR rate in G1 and G2 was 0.2% using the 99.8th and 0.5% in G1 and G2 using the 99.5th. The 99.8th percentile of N17OHP values was the best cut-off for distinguishing unaffected from affected NBs: G1 (P1: 120 ng/mL; P2: 71; P3: 39 and P4: 20 ng/mL) and G2 (P1: 173 ng/mL; P2: 90; P3: 66 and P4: 25 ng/mL). Twenty six (13M and 13F) NB were diagnosed with the salt wasting (SW) form, their N17OHP values ranged from 31 to 524 ng/mL. Twenty (8M, 12F) NB were diagnosed with the SW form in G2 group, N17OHP values ranged from 53 to 736 ng/mL. Three NB were diagnosed with the simple virilizing form (1M and 2F) in G1 and G2, their N17OHP values ranged from 36 to 51 ng/mL. False-negative results were not reported. The positive predictive value (PPV) of an altered neonatal test using the 99.8 percentile was 5.6% and 14.1% in G1 and G2, respectively, and using the percentile 99.5 was 2.3% and 7% in G1 and G2, respectively. Among cases with abnormal N17OHP result (RFP), 29 of them still presented with increased serum 17OHP levels through LC-MS/MS. Asymptomatic cases were followed up until normalization of serum 17OHP and/or submitted to molecular analysis, which identified two Rns with genotype that predicts the nonclassical form. Conclusions: the best strategy to optimize the CAH-NBS is to adjust N17OHP levels into two groups according to age at sample collection (before and after 72 hs), subdivided into four birth-weight groups. The use of N17OHP values according to the 99.8th remains effective to perform the CAH diagnosis CAH and significantly reduces the RFP rate without loss of diagnosis of the severe clinical forms

<p>The developing nervous system depends largely on extracellular cues to shape its complex network of neurons. Classically, neurotrophins are known to be important mediators in this process. More recently, Bone Morphogenetic Proteins (BMPs), belonging to the Transforming Growth Factor beta (TGFβ) superfamily of secreted cytokines, have been shown to exert a wide range of effects, such as cellular growth, differentiation, survival and apoptosis, both in the developing and adult nervous system. They signal via serine/threonine kinase receptor essentially to the Smad pathway, which carries the signal to the nucleus where the transcription of target genes is regulated.</p><p>This thesis investigates the functions of BMPs in the nervous system, using a set of different models. Firstly, a targeted deletion of GDF10 (BMP3b) in the mouse was established to evaluate the role of this growth/differentiation factor in the hippocampal formation, a brain area known to be involved in memory processing. Other members of the TGFβ superfamily likely compensate for the lack of GDF10, since no detectable alterations in hippocampal function or gene transcription profile have been found. Secondly, a mouse model was set up, with the aim to study impaired BMP-signalling in dopaminergic neurons. The tyrosine hydroxylase (TH) locus was used to drive the expression of dominant negative BMP receptors by means of bicistronic mRNAs. TH is the rate-limiting enzyme in the biosynthesis of catecholamine and the mice described, show a graded decrease of TH-activity resulting in severe to mild dopamine deficiency. The contribution of the dominant negative BMP receptors to the phenotype is however secondary to the apparent TH hypomorphism. The final theme of this thesis is the potentiating effects of BMPs on neurotrophin-induced neurite outgrowth as studied in explanted ganglia from chick embryos and in the rat phaeochromocytoma cell line PC12. A number of pharmacological inhibitors of intracellular signalling kinases were applied to the cultures in order to reveal the contribution of different pathways to the enhanced neurite outgrowth. We made the unexpected finding that inhibition of MEK signalling mimicked the potentiating effects of BMP stimulation in the chick system. The underlying mechanisms for the synergistic effects, however, are still an enigma.</p>

The developing nervous system depends largely on extracellular cues to shape its complex network of neurons. Classically, neurotrophins are known to be important mediators in this process. More recently, Bone Morphogenetic Proteins (BMPs), belonging to the Transforming Growth Factor beta (TGFβ) superfamily of secreted cytokines, have been shown to exert a wide range of effects, such as cellular growth, differentiation, survival and apoptosis, both in the developing and adult nervous system. They signal via serine/threonine kinase receptor essentially to the Smad pathway, which carries the signal to the nucleus where the transcription of target genes is regulated. This thesis investigates the functions of BMPs in the nervous system, using a set of different models. Firstly, a targeted deletion of GDF10 (BMP3b) in the mouse was established to evaluate the role of this growth/differentiation factor in the hippocampal formation, a brain area known to be involved in memory processing. Other members of the TGFβ superfamily likely compensate for the lack of GDF10, since no detectable alterations in hippocampal function or gene transcription profile have been found. Secondly, a mouse model was set up, with the aim to study impaired BMP-signalling in dopaminergic neurons. The tyrosine hydroxylase (TH) locus was used to drive the expression of dominant negative BMP receptors by means of bicistronic mRNAs. TH is the rate-limiting enzyme in the biosynthesis of catecholamine and the mice described, show a graded decrease of TH-activity resulting in severe to mild dopamine deficiency. The contribution of the dominant negative BMP receptors to the phenotype is however secondary to the apparent TH hypomorphism. The final theme of this thesis is the potentiating effects of BMPs on neurotrophin-induced neurite outgrowth as studied in explanted ganglia from chick embryos and in the rat phaeochromocytoma cell line PC12. A number of pharmacological inhibitors of intracellular signalling kinases were applied to the cultures in order to reveal the contribution of different pathways to the enhanced neurite outgrowth. We made the unexpected finding that inhibition of MEK signalling mimicked the potentiating effects of BMP stimulation in the chick system. The underlying mechanisms for the synergistic effects, however, are still an enigma.

El objetivo general de este trabajo ha sido estudiar la posible implicación de diferentes cascadas de proteín kinasas en las modificaciones cardiacas que se producen tras la administración de naloxona a ratas dependientes de morfina. Los datos obtenidos indican que durante la abstinencia a morfina se produce un aumento del turnover de NA, de la actividad TH y de su fosforilación en serina 40 y 31, lo que sugiere la puesta en marcha de mecanismos post-transcripcionales. Por otra parte, la vía de la PKA estaría implicada en el incremento del turnover de NA, en el aumento de TH total y en la fosforilación y activación de TH en serina 40 durante dicho síndrome. Por último, la vía de la PKC sería una de las vías implicadas en la expresión de c-fos, así como la de las ERK, que estaría también implicada en la activación de TH en serina 31. / The main aim of this work was to study the posible involvement of different protein kinases in the cardiac adaptive changes induced during morphine withdrawal. Our results show an increase of NA turnover, TH activity and TH phosphorylation at serine 31 and 40, suggesting starting post-trascriptional mechanisms. On the other hand, PKA transduction system could be implicated in the enhanced NA turnover, in the total TH increase and in the phosphorylation and activation of TH at serine 40 during this syndrome. Finally, PKC pathway would be involved in c-Fos expression as well as ERK system which would also be responsible for TH phosphorylation at serine 31.

The capacity of the olfactory neuraxis to undergo neuronal replacement and axon targeting following injury, has led to scrutiny concerning the molecular and physical determinants of this growth capacity. This is because injury to the central nervous system, in contrast, leads to permanent disconnection of neurons with targets. Olfactory ensheathing cells (OECs), a specialized glial cell, may contribute to olfactory repair, and have been used to promote recovery from spinal cord injury. However, there mechanisms underlying OEC-induced regeneration are poorly appreciated. To understand these mechanisms, OECs from the lamina propria (LP OECs) or olfactory bulb (OB OECs) were transplanted into a lesion of the dorsolateral funiculus. While both cells demonstrated reparative capacities, LP and OB OECs differentially promoted spinal fibre growth; large-diameter neurofilament-positive, CGRP-positive, and serotonergic fibres sprouted in response to both LP and OB OEC transplantation, whereas substance-P and tyrosine hydroxylase-positive neurons grew more extensively following OB or LP OEC transplantation, respectively. To further understand the growth of spinal cord neurons in response to OECs, a proteomic analysis of OEC secreted factors was performed, identifying secreted protein acidic and rich in cysteines (SPARC) as a mediator of OEC-induced outgrowth in vitro. To test the contributions of SPARC to spinal cord repair after OEC transplantation, cultures of LP OECs from SPARC null and wildtype (WT) mice were transplanted into a crush of the dorsolateral funiculus. Substance P and tyrosine hydroxylase positive axon sprouting was significantly reduced in SPARC null OEC-treated animals, suggesting that individual factors may contribute to OEC-promoted regeneration. To investigate the effect of OECs on corticospinal (CST) neurons, an in vitro assay was developed using postnatal day 8 CST neurons. Coculture of CST neurons with OB OECs produced extensive axon elongation. Application of OB OEC secreted factors increased CST neurite branching, but did not increase axon elongation. In contrast, plating of CST neurons on OB OEC plasma membrane resulted in extensive axon elongation. Furthermore, the OB OEC plasma membrane could overcome CST neurite outgrowth inhibition induced by an outgrowth inhibitor. Together these findings provide insight into OEC mechanisms of neurite outgrowth and axon regeneration.