Sunlight is the main aetiological agent in the development of non melanoma skin cancer (NMSC), the most commonly diagnosed cancer in the fair skinned population. Mounting epidemiological and molecular data suggest that infection with cutaneous Human papillomavirus (HPV) may act as a co-factor in the early stages of NMSC development. The viral E6 protein can inhibit the apoptotic response to UV induced damage, partly through proteasomal degradation of the pro-apoptotic protein BAK. Upon UV damage, BAK undergoes a series of activating modifications that are linked to changes in phosphorylation status. BAK activation commits a cell to apoptosis through mitochondrial permeabilisation. The aim here was to elucidate the underlying mechanism of HPV5 E6-mediated BAK degradation. Mutation of lysine 113 in BAK and siRNA knockdown of a novel HPV5 E6 associated ubiquitin ligase, HERC1, prevented HPV5 E6 mediated-BAK degradation. A proximity ligation in situ assay showed an interaction between HERC1 and BAK in cells, which was dependent on the presence of the E6 protein and UV irradiation. Probing the BAK conformation and phosphorylation status suggests E6 targets a dephosphorylated BAK monomer before mitochondrial permeabilisation occurs, leading to the proposal of a mechanism for the interaction of BAK with HERC1 which depends on the activation status of BAK and a putative BH3 domain identified in HERC1. Additional work identifies β1-integrin as an interacting protein of a conserved YHDW amino acid motif at the C-terminus of certain β1 HPV E6 proteins. Only expression of E6 proteins with the YHDW motif (HPV5 and 8) in keratinocytes disrupted β1 integrin membrane localisation, altered expression of β1 integrin downstream effectors, such as focal adhesion kinase, and increased cell migration. Mutation of W157A in the HPV5 E6 YHDW motif reduced these effects. Together, this work furthers the understanding of mechanisms by which infection with HPV may promote the early stages of NMSC development.

Transforming-Growth-Factor-beta1 (TGF-b1) ist ein multifunktionelles Zytokin, welches insbesondere Zellwachstum und Zelldifferenzierung koordiniert. TGF-b ist vor allem dafür bekannt, Zellen des Immunsystems zu beeinflussen. TGF-b steuert zum Beispiel die Differenzierung von T-Zellen und und deren Effektorfunktionen. Die Signaltransduktion von TGF-b wird vermittelt durch die Phosphorylierung von Rezeptor-assoziierten Smad-Proteinen (R-Smads). R-Smads werden vom Typ I Rezeptor aktiviert, der seinerseits vom hochaffinen Typ II Rezeptor phosphoryliert wird, sobald der Ligand bindet. Die phosphorylierten RSmads assoziieren darauf mit Co-Smads. Heterooligomere von R-Smads und Co-Smads wandern dann in den Zellkern, wo sie im Zusammenspiel mit Transkriptionsfaktoren wie CBP/p300 oder AP-1 die Transkription TGF-b-spezifischer Zielgene koordinieren. Neue Erkenntnisse lassen vermuten, daß die pleiotropen Effekte von TGF-b durch das Interagieren mit anderen Signalkaskaden entstehen, zum Beispiel mit dem MAP-Kinase-Weg oder der STAT-Kaskade. Wir beschreiben hier den Effekt von TGF-b auf die Effektorfunktionen unterschiedlich stimulierter primärer Maus-Milzzellen und aufgereinigten zytotoxischen CD8+ Maus-TZellen. Langzeitbehandlung mit TGF-b resultierte in der Unfähigkeit der Zellen, Smad2 ligandeninduziert zu phosphorylieren. Entweder wurde überhaupt keine Phosphorylierung beobachtet, oder eine anhaltende Phosphorylierung von Smad2 unabhängig vom Vorhandensein des Liganden. Des weiteren stellten wir einen Zusammenhang zwischen anhaltender Smad2-Phosphorylierung und der Resistenz gegenüber TGF-b induzierter Wachstumshemmung fest. Im Gegensatz dazu zeigen Zellen, die sensitiv sind gegenüber TGF-b vermittelter Wachstumshemmung, keine Smad2-Phosphorylierung mehr. Bezüglich ihrer zytotoxische Aktivtät waren allerdings beide Phänotypen nicht mehr lytisch wirksam, unabhängig von der jeweiligen Smad2-Phosphorylierung. In dieser Arbeit zeigen wir auch die Notwendigkeit eines funktionalen MEK-1-Signalweges auf, der unabdingbar ist, damit TZellen keine Wachstumsinhibierung durch TGF-b mehr erfahren. Das Blockieren dieses Signalweges führt darüberhinaus bei diesen Zellen ebenfalls zu einem veränderten Smad2- Phosphorylierungsmuster. Bezüglich des JNK-Signalweges konnten wir feststellen, daß ein funktional aktiver JNK-Signalweg mit der Resistenz gegenüber TGF-b vermittelter Wachstumsinhibierung einhergeht. Allerdings führt die Zugabe von IFNg und/oder aCD28- Antikörper nicht zu einer veränderten Sensitivität gegenüber TGF-b. Im Gegensatz zuprimären Zellen können die beschriebenen Zusammenhänge in Zellkulturen vom humanen und murinen T Zellen nicht beobachtet werden, und sind somit spezifisch für primare TZellen. Wir beschreiben auch die Klonierung eines chimären dominant-negativen Typ II Rezeptors, der an eine Kinase gekoppelt ist, die bei Aktivierung Zelltod auslöst. Damit soll es in Zukunft möglich sein, T-Zellen gegenüber TGF-b Resistenz zu verleihen. Die hier geschilderten Ergebnisse vertiefen die Kenntnisse über molekulare Mechanismen der Wirkung von TGF-b auf T-Zellen und können vielleicht dazu beitragen, negative Effekte von TGF-b, zum Beispiel in der Tumortherapie, gezielt abzuwenden. / Transforming-Growth-Factor-beta1 (TGF-b1) is a multifunctional cytokine that regulates cell growth and differentiation in many types of cells. TGF-b1 is especially known to exert a variety of regulatory functions in the immune system, such as T cell differentiation and T cell function. Signal transduction of TGF-b1 is mediated by phosphorylation of receptorassociated Smad proteins (R-Smads). R-Smads are phosphorylated by the activated type I receptor, which is itself phosphorylated by the high affinity type II receptor upon ligand binding. The phosphorylated R-Smads then associate with Co-Smads. Heterooligomers of R- and Co-Smads translocate into the nucleus where they regulate transcription of target genes in concert with other transcription factors such as CBP/p300 or AP-1. Recent findings suggest that the pleiotropic effects of TGF-b1 are conferred by crosstalks to other signal transduction pathways such as the MAP-kinases or the STAT-pathway. Here we describe the effect of long-term exposure to TGF-b1 on the effector function of differentially stimulated primary murine splenocytes and purified primary murine CD8+ cytotoxic T cells. Long-term exposure to TGF-b1 results in non-responsiveness to TGF-b1- induced Smad2 phosphorylation. This is seen either by no phosphorylation or sustained phosphorylation of Smad2. Furthermore, we observed a strong correlation between sustained Smad2 phosphorylation and resistance to TGF-b1 mediated growth inhibition. In contrast, splenocyte cultures strongly growth inhibited by TGF-b1 showed no Smad2 phosphorylation. Lytic activity of these cultures, however, was found to be suppressed regardless of proliferation properties and Smad2 phosphorylation pattern. We also describe that a functional MEK-1 pathway is a prerequisite for rendering murine splenocytes unresponsive to TGF-b1 mediated growth inhibition, and that inhibition of the MEK-1 cascade alters the Smad2 phosphorylation pattern. In addition, we show that resistance to TGF-b1 mediated growth inhibition correlates with the activation of the JNK pathway. However, the resistant phenotype was found unable to be reverted upon administration of exogeneous IFNg and/or aCD28 antibody. In human or mouse T cell lines, however, the described correlation between the type of stimulation and TGF-b growth resistance or growth sensitivity is not present. Thus, this correlation is specific for primary T cells. We also cloned a chimeric dominantnegative TGF-b receptor which is coupled to a suicide gene, in order to render T cells resistant to TGF-b mediated effects.These findings shed light on how TGF-b1 mediates its immunosuppressive role, and may help to gain knowledge of averting these TGF-b1 effects in the course of tumor therapy.

Este estudo in vitro teve como objetivo avaliar os efeitos moleculares do TGF-β1, com diferentes períodos de suplementação, sobre a formação do fenótipo osteogênico das células MC3T3-E1, comparando-os com células tratadas com AA+β-GP suplementados com Dex e/ou TGF-β1, sem e com a neutralização dos receptores de TGF-β1. A expressão gênica do próprio TGF-β1 e Smad3 foram analisadas, bem como, a diferenciação das células osteogênicas e a biomineralização. As células tratadas com TGF-β1 sem neutralização de receptores apresentam efeito inibitório nos estágios mais avançados da diferenciação dos osteoblastos e da biomineralização in vitro, mas expressarem alguns marcadores importantes envolvidos na mineralização. Observaram-se nódulos de mineral em todos os tratamentos das células que tiveram os receptores de TGF-β1 neutralizados, mas houve uma diminuição na expressão de alguns genes. Os resultados confirmam a complexidade da via de sinalização do TGF-β1, mostrando que existem lacunas para que seja entendido o mecanismo dessa molécula na biologia osteoblástica. / This in vitro study aimed to evaluate the molecular effects of TGF-β1, with different supplementation time periods on the establishment of MC3T3-E1 cells, comparing with cells treated with AA+β-GP supplemented with Dex and/or TGF-β1, without or with neutralization of TGF-β1 receptors. The gene expression of the TGF-β1 and Smad3 were analyzed, as well as the osteoblast differentiation and biomineralization. The cells treated with TGF-β1 without neutralization of receptors have had inhibitory effect on some important stages of osteoblast differentiation and biomineralization in vitro, but expressed some important mineralization markers. Mineral nodules were observed in all treatments of cells with their TGF-β1 receptors neutralized, but there was a decrease in the expression of some important genes. The results confirm the complexity of the pathway signaling of TGF-β1, showing that there are gaps for understand the mechanisms of this molecule in the biology of osteoblasts.

Functionally active (conformational) autoantibodies directed against the β1-adrenergic receptor (β1-AR) are supposed to have a pathogenic relevance in human heart failure, particularly in idiopathic dilated cardiomyopathy (DCM). Prevalence of anti-β1-autoantibodies (anti-β1-aabs) in the healthy population is almost negligible, whereas it amounts to up to 30% in heart failure patients with idiopathic DCM. As β1-ARs are not restricted to the heart and are also highly expressed in particular segments of the nephron, it is conceivable that such autoantibodies might also affect kidney function to some extent through the activation of renal β1-ARs. In the kidney, β1-ARs are highly abundant in the juxtaglomerular apparatus, the distal convoluted tubules, the collecting duct, and the renal arteries. However, the functional significance of β1-ARs at these particular sites along the nephron is poorly understood, as are the effects of conformational stimulating anti-β1-aabs on renal β1-ARs. From the available literature, it is well known that the β1-adrenergic system is involved in, e.g., the regulation of renin-secretion from juxtaglomerular cells. In addition, the β1-adrenergic system is thought to be involved in the regulation of the urine pH via type B-intercalated cells in the collecting duct. In contrast, the regulation of salt- and fluid-secretion in the medullary collecting duct appears to occur independently from the SNS. As a consequence, the present work aimed to unravel the potential pathophysiological links between renal function, alterations in the cardiovascular system, and circulating agonist-like anti- β1-abs. We analyzed possible renal effects of anti-β1-abs in a human-analogous rat model. After immunization with a GST-fusion protein containing the second extracellular loop (β1-ECII) of the human β1-AR, Lewis-rats develop functionally active, stimulating, conformational anti-β1-ECII-abs. Within the first 6 months, anti-β1-ECII-ab-positive animals develop a hypertensive phenotype, which after 9 months evolves into a DCM phenotype. In n=40 GST/ β1-ECII-immunized Lewis rats and n=40 age-matched, 0.9% NaCl-injected control animals, we sequentially (i.e. at months 1, 2, 3, 6, 9, 12, 15, and 18 after start of immunization) analyzed the changes in renal function on a molecular, functional, and structural level. We could show that the presence of stimulating anti-β1-ECII-abs – even though having detrimental effects on the heart – has only a minor impact on kidney function and structure. Within the first 3 months after induction of anti-β1-ECII-abs, the levels and activity of renin were significantly increased in immunized compared to corresponding control animals, which was confirmed by experiments on isolated perfused kidneys, in which anti-β1-ECII-abs were able to directly induce the liberation of renin. However, within several weeks the initial anti-β1-ECII-ab-mediated RAAS activation was counter-regulated by auto-regulatory mechanisms activated in the kidney. Similarly, glomerular filtration rate (GFR) and renal blood flow (RBF) were initially decreased in the presence of the stimulating anti-β1-ECII-abs, but returned to control values within 3 months after immunization of the animals. Although expression of several pro-fibrotic markers was significantly up-regulated in anti-β1-ECII-ab-positive rats, no significant differences were noted on a histomorphological level with regard to the occurrence of renal fibrosis, glomerular damage, tubular damage, and perivascular fibrosis. Only a mild decrease in glomerular filtration function was observed in the kidneys of anti-β1-ECII-ab-positive animals from immunization-month 12 on, apparent by increased levels of urinary protein. Even though anti-β1-ECII-abs were able to induce mild changes in renal function, their effects were not strong enough to critically damage the kidneys in our rat-model. Differences between immunized anti-β1-ECII-ab-positive and corresponding control rats at later time-points (that is, from immunization-month 12 on) are most likely secondary to the progressive heart failure phenotype that immunized animals develop in the course of the experiment. The present study is the first to focus on the effects of stimulating anti-β1-ECII-abs on the kidney, and on the prevalence of these effects for the heart (referred to as cardio-renal crosstalk). Although our results were obtained in a rat model, they might contribute to better understand the situation in anti-β1-AR-aab-positive human patients. Following the results of our experiments, treatment of such patients should focus on direct and specific neutralization/elimination of stimulating anti-β1-ECII-aab or at least comprise therapeutic strategies that counteract the anti-β1-ECII-aab-effects on the heart by standard treatment for heart failure (i.e. ACE inhibitors, AT1-receptor blockers, and β-blockers) according to current guidelines. / Funktionell aktive, konformationelle Autoantikörper, die gegen den β1-adrenergen Rezeptor (β1-AR) gerichtet sind, sind vermutlich pathologisch relevant bei Herzinsuffizienz, insbesondere bei der idiopathischen Dilativen Kardiomyopathie (DCM). Die Prävalenz solcher Antikörper ist in der gesunden Population vernachlässigbar, wohingegen sie bei der idiopathischen DCM 30% erreicht. Da β1-AR nicht nur im Herzen, sondern auch in der Niere stark exprimiert werden, ist naheliegend, dass solche Antikörper über eine Stimulation renaler β1-AR auch die Nierenfunktion beeinflussen können. In der Niere werden β1-AR überwiegend im juxtaglomerulären Apparat, im distalen Tubulus, in den Sammelrohren und in den renalen Arterien exprimiert. Die Bedeutung der in diesen Bereichen hohen Expression von β1-AR für die Nierenfunktion ist noch nicht vollständig geklärt, ebenso wie die renalen Effekte von stimulierenden β1-AR-Antikörpern. Aus der Literatur ist bekannt, dass das β1-adrenerge System unter anderem an der Renin-Sekretion der juxtaglomerulären Zellen beteiligt ist. Außerdem wird vermutet, dass das β1-adrenerge System in die Regulation des pH-Wertes des Urins über die Typ B-interkalierenden Zellen des Sammelrohrs eingreift, wohingegen die Regulation der Salz- und Wasserexkretion im medullären Sammelrohr wahrscheinlich unabhängig vom sympathischen Nervensystem abläuft. Die vorliegende Arbeit zielt darauf ab, die potentiellen pathophysiologischen Zusammenhänge zwischen renaler Funktion, Änderungen innerhalb des kardiovaskulären Systems und zirkulierenden, agonist-ähnlichen anti-β1-Autoantikörpern darzustellen. Wir haben die möglichen renalen Effekte der anti-β1-AK in einem human-ähnlichen Ratten-Modell untersucht. Nach Immunisierung mit einem GST-Fusionsprotein, welches den zweiten extrazellulären Loop des β1-AR beinhaltet, entwickeln Lewis-Ratten funktionell aktive, stimulierende Antikörper gegen β1-ECII. Anti-β1-ECII-AK-positive Tiere entwickeln nach ca. 6 Monaten einen hypertensiven Phänotyp, welcher nach 9 Monaten in einen DCM Phänotyp übergeht. Wir haben Änderungen der renalen Funktion auf molekularer, funktioneller, und struktureller Ebene in n=40 GST/ β1-ECII-immunisierten Lewis-Ratten und n=40 altersgleichen 0.9% NaCl-injizierten Kontrolltieren sequenziell (d.h. 1, 2, 3, 6, 9, 12, 15 und 18 Monate nach Beginn der Immunisierung) analysiert. Wir konnten zeigen, dass die stimulierenden anti-β1-ECII-Antikörper – obwohl sie eine schädigende Wirkung auf das Herz haben – die Nierenfunktion und -Struktur nur gering beeinflussen. In den ersten Monaten nach Induktion der anti-β1-ECII-AK stieg die Reninkonzentration und -aktivität in den immunisierten Tieren im Vergleich zu den entsprechenden Kontrollen signifikant an. Dieses Ergebnis konnte im Model der isolierten perfundierten Niere bestätigen werden, in dem anti-β1-ECII-AK eine direkte Freisetzung von Renin induzierten. Diese frühe AK-vermittelte Aktivierung des RAAS wurde jedoch innerhalb weniger Wochen durch autoregulatorische Mechanismen der Niere aufgehoben. Ebenso waren anfangs die glomeruläre Filtrationsrate und der renale Blutfluss in anti-β1-ECII-AK-positiven Ratten vermindert, kehrten jedoch nach 3 Monaten zu den Werten der Kontrolltiere zurück. Obwohl die Expression mehrerer pro-fibrotischer Marker signifikant erhöht war, konnten keine signifikanten Veränderungen auf histomorphologischer Ebene hinsichtlich des Auftretens renaler Fibrose, glomerulärer und tubulärer Schädigung, oder perivaskulärer Fibrose festgestellt werden. Lediglich die glomeruläre Filtrationsfunktion war ab dem 12. Immunisierungsmonat zunehmend beeinträchtigt, was sich an einer progredienten Proteinurie zeigte. Obwohl anti-β1-ECII-AK durchaus einen gewissen Effekt auf die Nierenfunktion haben, ist ihr Einfluss nicht stark genug um die Niere kritisch zu schädigen. Unterschiede zwischen immunisierten und Kontrolltieren, welche zu späteren Zeitpunkten (d.h. ab dem 12. Immunisierungsmonat) auftreten, sind wahrscheinlich sekundäre Effekte der progredienten Herzinsuffizienz, die die immunisierten Tiere im Verlauf des Experiments entwickeln. Unsere Studie ist die Erste, die sich mit den Effekten von stimulierenden anti-β1-AK auf die Niere und ihre Zusammenhänge mit der antikörper-induzierten Herzinsuffizienz (dem sogenannten kardio-renalen „Crosstalk“) befasst. Obwohl unsere Ergebnisse in einem Tiermodell erzielt wurden, könnten sie von großem Nutzen sein, um die Krankheitsentwicklung von anti-β1-Autoantikörper-positiven Patienten besser zu verstehen. Unseren Ergebnissen zufolge sollte die Behandlung von Autoimmun-DCM auf eine möglichst direkte und spezifische Neutralisierung/Eliminierung von anti-β1-Autoantikörpern abzielen und gleichzeitig alle kardio- und renal-protektiven Elemente der Standard-Therapie der Herzinsuffizienz (d.h. Gabe von ACE-Hemmern, AT1-Rezeptor-Inhibitoren und β-Blockern) einschließen.

Lübeck, Med. Universität, Habilitation, 2000.

Ulm, Univ. Diss., 2006.

University, Diss., 2002--Köln.

Este estudo in vitro teve como objetivo avaliar os efeitos moleculares do TGF-β1, com diferentes períodos de suplementação, sobre a formação do fenótipo osteogênico das células MC3T3-E1, comparando-os com células tratadas com AA+β-GP suplementados com Dex e/ou TGF-β1, sem e com a neutralização dos receptores de TGF-β1. A expressão gênica do próprio TGF-β1 e Smad3 foram analisadas, bem como, a diferenciação das células osteogênicas e a biomineralização. As células tratadas com TGF-β1 sem neutralização de receptores apresentam efeito inibitório nos estágios mais avançados da diferenciação dos osteoblastos e da biomineralização in vitro, mas expressarem alguns marcadores importantes envolvidos na mineralização. Observaram-se nódulos de mineral em todos os tratamentos das células que tiveram os receptores de TGF-β1 neutralizados, mas houve uma diminuição na expressão de alguns genes. Os resultados confirmam a complexidade da via de sinalização do TGF-β1, mostrando que existem lacunas para que seja entendido o mecanismo dessa molécula na biologia osteoblástica. / This in vitro study aimed to evaluate the molecular effects of TGF-β1, with different supplementation time periods on the establishment of MC3T3-E1 cells, comparing with cells treated with AA+β-GP supplemented with Dex and/or TGF-β1, without or with neutralization of TGF-β1 receptors. The gene expression of the TGF-β1 and Smad3 were analyzed, as well as the osteoblast differentiation and biomineralization. The cells treated with TGF-β1 without neutralization of receptors have had inhibitory effect on some important stages of osteoblast differentiation and biomineralization in vitro, but expressed some important mineralization markers. Mineral nodules were observed in all treatments of cells with their TGF-β1 receptors neutralized, but there was a decrease in the expression of some important genes. The results confirm the complexity of the pathway signaling of TGF-β1, showing that there are gaps for understand the mechanisms of this molecule in the biology of osteoblasts.

Die ETiCS-Studie (Etiology, Titre-Course, and effect on Survival) ist die bisher größte prospektive europäische Studie, die Ursachen und Entstehungsmechanismen kardialer Autoimmunphänomene untersucht. Ziel dieser Dissertation war die umfassende Charakterisierung der beiden prospektiven ETiCS-Kollektive sowie der Vergleich ihrer demographischen, klinischen, laborchemischen und apparativen Charakteristika zum Zeitpunkt des Studieneinschlusses. Die prospektive ETiCS-Studie umfasste im FAMI-Kollektiv (erster akuter Myokardinfarkt) insgesamt n=180 Patienten und im AMitis-Kollektiv (erste akute Myokarditis) n=96 Patienten. Die demographischen Daten, das kardiovaskuläre Risikoprofil sowie die klinische Symptomatik unserer Patienten entsprachen im Wesentlichen den in der Literatur bereits beschriebenen ähnlichen Vergleichskollektiven, mit dem interessanten Unterschied, dass unsere Infarkt-Patienten deutlich jünger waren (57 ± 8 Jahre), als der Durchschnittspatient mit erstmaligem Myokardinfarkt. Als Schlussfolgerung dieser Arbeit für die klinische Praxis lässt sich durch akribische Erhebung der Anamnese und des kardiovaskulären Risikoprofils eines Patienten mit unklaren kardialen Beschwerden mit einer gewissen Wahrscheinlichkeit ein akuter Myokardinfarkt oder eine akute Myokarditis vorhersagen. Das führende klinische Symptom ist mit Thoraxschmerz und Dyspnoe bei beiden Krankheitsbildern recht ähnlich, jedoch sollte bei führender Belastungsdyspnoe und zeitgleich typischen Nebenkriterien (Fieber, Palpitationen, Infektanamnese) primär an eine Myokarditis gedacht werden. Anhand der Ischämiemarker ist der Ausschluss einer akuten Myokardischämie oder einer akuten Herzmuskelentzündung zwar mit großer Sicherheit möglich, bei erhöhten Werten muss jedoch für eine weitere Differenzierung auch die Klinik, die EKG-Diagnostik und die Echokardiographie mit betrachtet werden. Auch bei nicht eindeutigem EKG-Befund sollte die Indikation zur Koronarangiographie nur in Zusammenschau der genannten Befunde gestellt werden. Sobald sich jedoch der Verdacht auf ein akutes Infarktgeschehen erhärtet, sollte ohne Zeitverzögerung eine invasive Diagnostik erfolgen. / The ETiCS study (Etiology, Titre-Course, and effect on Survival) is the largest prospective European study to date to investigate the causes and mechanisms of cardiac autoimmune phenomena. The aim of this dissertation was the comprehensive characterization of the two prospective ETiCS collectives and the comparison of their demographic, clinical, laboratory and instrumental characteristics at the time of study inclusion. The prospective ETiCS study included n=180 patients in the FAMI (first acute myocardial infarction) and n=96 patients in the AMitis (first acute myocarditis) group. The demographic data, cardiovascular risk profile, and clinical symptoms of our patients were broadly similar to those of the other collectives described in the literature, with the interesting difference that our infarction patients were significantly younger (57 ± 8 years) than the average patient with first myocardial infarction. As a conclusion of this work for clinical practice, meticulous evaluation of the medical history and cardiovascular risk profile of a patient with unclear cardiac symptoms allows to predict with a certain probability an acute myocardial infarction or acute myocarditis. The leading clinical symptom is quite similar with chest pain and dyspnea, but in case of leading exercise dyspnea and at the same time typical secondary criteria (fever, palpitations, history of infection) myocarditis should be considered primarily. Although laboratory markers allow the exclusion of acute myocardial ischemia or acute myocarditis with a high degree of certainty, in the case of elevated values, clinical presentation, ECG diagnostic and echocardiography must also be considered for further differentiation. Even if the ECG findings are inconclusive, the indication for coronary angiography should only be made in conjunction with the above-mentioned findings. However, as soon as the suspicion of an acute infarction is confirmed, invasive diagnostics should be performed without delay.

Introdução: Até bem pouco tempo atrás a análise mutacional de regiões gênicas não codificantes raramente eram reportadas em estudos genéticos sobre doenças hereditárias. Geralmente, o enfoque desses estudos recaiam sobre as regiões ditas codificantes, também conhecidas como regiões exônicas. Com o progresso da tecnologia em biologia molecular, especialmente após o desenvolvimento do sequenciamento em larga escala, temos observado um avanço jamais visto antes no entendimento de como opera o genoma humano e consequentemente uma mudança de paradigmas. Estudos mais recentes tem abrangido consistentemente a análise mutacional de regiões não codificantes ou regulatórias. No caso da neuropatia de \"Charcot-Marie-Tooth\" (CMT), pouco ainda se sabe sobre os fenótipos resultantes de mutações localizadas em sequencias regulatórias dos genes relacionados a doença. Por exemplo, na CMT ligada ao cromossomo X do tipo 1 (CMTX1), que é causada por mutações no gene GJB1 que codifica para a proteína canal conexina 32, apenas 0.01% das mutações já detectadas no gene estão presentes na região 5\' UTR (Untranslated Region) (KABZINSKA et al., 2011). Objetivos: Em vista disso, o propósito deste estudo foi a analise mutacional da região não codificante 5\' UTR e região promotora P2 do gene GJB1. Mais precisamente, 600 pares de bases upstream a open Reading frame (ORF) do transcrito do gene GJB1 expresso no sistema nervoso. Métodos: Foram investigados 100 pacientes em andamento no ambulatório de neurologia do Hospital das Clinicas da Faculdade de medicina de Ribeirão preto USP (FMRP - USP) com sintomas sugestivo de CMTX do tipo 1 e que não apresentaram mutação alguma na região codificadora do mesmo gene em triagens posteriores. Para tanto foram realizadas as técnicas de PCR (para amplificação do DNA) e posteriormente o sequenciamento do produto amplificado por meio do sequenciamento de Sanger. Resultados: Três variantes foram detectadas em 4 pacientes não relacionados, duas alterações intrônicas (já descritas) e uma nova alteração, encontrada no exon 1B (não codificante) do gene GJB1. Conclusão: Os resultados obtidos em nosso estudo sugerem que mutações na região 5\' UTR do gene GJB1 na população brasileira são incomuns e aparentemente não são responsáveis por casos sugestivos de CMTX1 sem causa genética atribuída. / Introduction: Until recently mutational analysis of non-coding regions of the DNA were rarely reported on genetic studies about inherited disorders. Usually, the approach of those studies relied primarily on the analysis of the coding regions of the DNA, also known as exonic regions. However, with the progress of science in molecular biology, especially after the development of the high throughput sequencing technology, we have observed an advance never seen before regarding of how the human genome operates and consequently a paradigm shift. New studies have consistently covered these regulatory or non-coding regions of the DNA. In the case of Charcot-Marie-Tooth disease (CMT), little is known about the phenotypes resulting from mutations in the regulatory sequences of the genes related to the disease. For example, in the X linked CMT type 1 (CMTX1), caused by alterations in the GJB1 gene that encodes for the Channel like protein connexin32 (CX32), only 0.01% of the mutations ever found in the gene were actually localized in its 5\' untranslated region (5\'UTR) (KABZINSKA et al., 2011). Objectives: Considering it, the purpose of this study was the mutational analysis of the 5\' non-coding region and promoter region P2 of GJB1 gene. More specifically, 600 base pairs (bp) upstream of the open reading frame (ORF) of the transcript expressed in the nervous system (NS). Methods: 100 patients in progress in the Clinical Hospital of the Medical School at University of São Paulo presenting suggestive clinical diagnoses of CMTX1 were screened for mutations in the region of interest, after mutations in the coding region of GJB1 gene were excluded in previous analysis. Therefore, we performed a Polimerase Chain Reaction (to amplify the DNA) followed by sequencing of the amplified material via Sanger sequencing. Results: We detected 3 alterations in 4 unrelated patients, 2 intronic variants (already described) and a novel mutation in the non-coding exon 1B of the GJB1 gene. Conclusion: The obtained results in our study suggest that mutations in the 5\'UTR of the GJB1 gene in the Brazilian population are uncommon and apparently they are not a major cause of CMTX1 in genetic unassigned patients with a suggestive clinical diagnosis of CMTX1.