Einleitung: Kardiovaskuläre Erkrankungen stellen weltweit die häufigste Todesursache dar. Bis heute gibt es keine ursächliche Therapie für die Herzinsuffizienz, die die gemeinsame Endstrecke einer Vielzahl von unterschiedlichen kardialen Erkrankungen bildet. Auch die Gentherapie hat in der Kardiologie, anders als in anderen Bereichen, noch keinen großen Fortschritt erzielen können. Das Kalziumsensorprotein S100A1 stellt aber einen vielversprechenden Kandidaten für die kardiale Gentherapie dar, da es als zentraler Regulator der Herzfunktion und des Kalziumsignalwegs innerhalb der Kardiomyozyten identifiziert werden konnte. Ziele der Untersuchungen: Diese translationale Studie sollte dazu beitragen, dem Ziel einer kardialen Gentherapie der kardialen Dysfunktion einen Schritt näher zu kommen. Zum einen sollte hierzu ein auf adeno-assoziierten Viren (AAVs) basierender Vektor (rAAV) des Serotyps 5 in Verbindung mit einem kardiospezifischen Promotor (CMVenh/0,26 kb-MLC) auf seine Anwendbarkeit und Sicherheit für die kardiale Gentherapie untersucht werden. Durch eine umfangreiche Testung auf präexistierende neutralisierende Serumfaktoren (NSF) sollte zudem untersucht werden, ob das Schwein generell ein geeignetes Modelltier für AAV5-basierte präklinische Studien darstellt. Zum anderen sollte in einem endpunktbasierten Studiendesign der Effekt der hS100A1-Gentherapie nach Myokardinfarkt (MI) im humanrelevanten Großtiermodell weiter charakterisiert werden. Tiere, Material und Methoden: Insgesamt wurden 83 juvenile Schweine aus einem kommerziellen Herkunftsbetrieb verwendet. Vor Versuchsbeginn wurde das Serum von 40 Tieren mittels eines auf Durchflusszytometrie basierenden Zellreporter-Assays auf präexistierende NSF gegen AAV5 untersucht. Für die Hauptstudie wurde bei 8 Tieren eine 2 stündige perkutane Okklusion des Ramus circumflexus der linken Koronararterie (LCX) durchgeführt und so ein ischämischer Myokardinfarkt mit anschließender Reperfusion und resultierender kardialer Dysfunktion induziert. Nach 2 Wochen wurden Infarktgröße und Herzfunktion mittels Kardio-Magnetresonanztomographie (MRT) evaluiert. Die Tiere wurden in die Therapiegruppe (AAV5-hS100A1, 5 Tiere) oder Kontrollgruppe (AAV5-hRluc, 3 Tiere) aufgeteilt. Der Gentransfer (1x1013 virale Genomkopien (vgc)/Tier)) erfolgte per retrograder koronarvenöser Infusion. 12 Wochen nach Gentransfer wurde eine erneute Kardio-MRT Untersuchung durchgeführt. Zur Überprüfung der pharmakologischen Sicherheit wurden während des Versuchs serielle Blut und Elektrokardiogramm (EKG) Untersuchungen durchgeführt. Am Versuchsende wurden die Tiere schmerzfrei getötet und ihre Organe für weitere molekularbiologische Untersuchungen entnommen. Zur Untersuchung der Verteilung und Transkriptionseffizienz der vgc wurde aus den Gewebeproben DNA und RNA isoliert und anschließend eine quantitative Echtzeit-Polymerase-Kettenreaktion (qPCR) durchgeführt. Zusätzlich erfolgte eine Next-Generation Sequenzierung der myokardialen RNA, die mit einer gewichteten Gen Co-Expressionsanalyse (WGCNA) und anschließender Anreicherungsanalyse untersucht wurde. Zur Reduktion der Tierzahl wurde die Studie Endpunkt orientiert durchgeführt: sobald die beiden Gruppen signifikant unterschiedliche Ergebnisse in den Endpunkten (EF und Infarktausweitung) erzielten, wurde die Studie beendet. Ergebnisse: Die Ergebnisse zeigen eine niedrige anti-AAV5-Seroprävalenz in der Hausschweinpopulation. Die AAV5-hS100A1-Gentherapie führte nach 12 Wochen zu einer signifikanten Verringerung der Infarktausweitung und zu einer signifikant höheren EF im Vergleich zur Kontrollgruppe (ungepaarter zweiseitiger Student t-Test, p < 0,05). Die EKG- und Blutuntersuchungen ergaben keine Hinweise auf Toxizität. Die Transkriptomanalyse der Myokardproben lieferte mit der EF und Infarktausweitung signifikant und stark negativ korrelierende pathophysiologisch relevante Signalwege. Dabei scheint u.a. eine antiinflammatorische Wirkung von AAV5-hS100A1 von großer Bedeutung zu sein. Erstmals konnte auch eine Interaktion zwischen S100A1 und der kardioprotektiven Retinsäure gezeigt werden. In einer aufgrund der hohen Mortalität während der MI-Induktion eingeschobene Versuchsreihe mit 72 Tieren konnte durch den Wechsel des Narkosegases von Isofluran auf Sevofluran die Mortalität signifikant gesenkt werden (einseitiger Fisher’s exact test, p < 0,05). Schlussfolgerungen: Das Schwein stellt ein geeignetes Modelltier für AAV5-basierte Versuchsvorhaben dar. Das zu testende Konstrukt AAV5-hS100A1 mit CMVenh/0,26 kb-MLC Promotor zeigte bei einer hohen pharmakologischen Sicherheit eine robuste und weitestgehend kardiospezifische Expression des Transgens 12 Wochen nach Gentransfer. Es verfügt somit über ein großes therapeutisches Potenzial. Die Studie konnte dazu beigetragen, neue Signalwege zu identifizieren, die für den Wirkmechanismus von S100A1 relevant sein könnten. Durch die Änderung des Narkosegases konnte die Mortalität bei der MI-Induktion gesenkt werden. In zukünftigen MI-Studien sollte daher die Aufrechterhaltung der Inhalationsanästhesie bevorzugt mittels Sevofluran erfolgen.:Inhaltsverzeichnis Abkürzungsverzeichnis 1 Einleitung 2 Literaturübersicht 2.1 Koronare Herzkrankheit, Myokardinfarkt und Herzinsuffizienz 2.1.1 Definition und Epidemiologie 2.1.2 Infarktheilung 2.1.3 Therapiemöglichkeiten 2.2 (Kardiale) Gentherapie 2.2.1 Vektoren 2.2.1.1 AAV5 2.2.2 Promotoren 2.2.3 Applikationsmethoden 2.3 S100A1 als therapeutisches Protein in der kardialen Gentherapie 2.3.1 Die Struktur von S100A1 2.3.2 Die Funktion von S100A1 2.3.3 Die kardiale S100A1-Gentherapie 2.4 Das Schwein in der translationalen Forschung 3 Material und Methoden 3.1 Material für die Aufarbeitung der Proben im Labor 3.1.1 Geräte und Verbrauchsmaterialien 3.1.2 Reagenzien und Chemikalien 3.1.3 Kits 3.1.4 Medien und Puffer 3.2 Material für den Großtier-OP und das Kardio-MRT 3.2.1 Geräte 3.2.2 Katheter und Schleusen 3.2.3 Medikamente und Medizinprodukte 3.3 Allgemeiner Versuchsaufbau 3.3.1 Versuchstiere 3.3.2 Versuchsaufbau 3.3.3 Vorbereitung, Narkose, perioperative Überwachung und Versorgung 3.3.4 Blutentnahme 3.3.5 Gefäßzugänge 3.3.6 Induktion des Myokardinfarkts 3.3.6.1 Änderung der Narkoseaufrechterhaltung während der MI-Induktion 3.3.7 Kardio-MRT – Durchführung 3.3.8 Gentransfer 3.3.9 Virale Vektoren 3.3.10 Organentnahme 3.4 Untersuchung auf neutralisierende Antikörper und Serumfaktoren 3.5 Bestimmung der Genexpression mittels qPCR 3.5.1 Homogenisierung und RNA-/DNA-Isolierung 3.5.2 cDNA-Synthese 3.5.3 Primer und Probes 3.5.4 qPCR im multiplex-Ansatz 3.5.5 Quantifizierung der Vektorgenomkopien mittels SYBR-qPCR 3.6 Kardio-MRT – Auswertung 3.7 Transkriptomanalyse 3.7.1 Next-Generation RNA-Sequenzierung – Durchführung 3.7.2 NGS – Auswertung 3.7.3 Hauptkomponentenanalyse 3.7.4 Gewichtete Gen Korrelation Netzwerk Analyse 3.7.5 Anreicherungsanalyse 3.8 Blutanalyse 3.9 Elektrokardiogramm – Auswertung 3.10 Statistische Auswertung 4 Ergebnisse 4.1 Erfüllung der Einschlusskriterien zur Aufnahme in die Studie 4.1.1 Seroprävalenz von neutralisierenden Antikörpern und Serumfaktoren gegen AAV5 4.1.2 Infarktgröße vor Gentherapie 4.2 Mortalität beim perkutanen LCX-Ischämie-/Reperfusionsmodell 4.3 Gentransfer 4.3.1 Überprüfung der Spezifität der Primer und Probes 4.3.2 Distribution der viralen Vektoren im linken Ventrikel 4.3.3 Transgenexpression im linken Ventrikel 4.3.4 Trankriptionseffizienz im linken Ventrikel 4.3.5 Systemische Verteilung 4.4 Einfluss der hS100A1-Gentherapie auf die kontraktile Funktion und Infarktgrößenentwicklung nach Myokardinfarkt 4.4.1 Effekte auf die Ejektionsfraktion 4.4.2 Effekte auf die Infarktausweitung 4.4.3 Ergebnisse der Transkriptomanalyse 4.5 Einfluss der hS100A1-Gentherapie auf die pharmakologische Sicherheit 4.5.1 Blutanalyse 4.5.2 Elektrokardiogramm 5 Diskussion 5.1 Eignung des Tiermodells 5.1.1 Seroprävalenz von neutralisierenden Antikörpern und Serumfaktoren in der Versuchstierart Schwein 5.1.2 Das perkutane LCX-Ischämie-/Reperfusionsmodell als experimentelles Modell zur Untersuchung der Infarktausweitung 5.1.3 Einfluss des Narkosegases auf die Mortalitätsrate beim perkutanen LCX-Ischämie-/ Reperfusionsmodell 5.2 Eignung des Vektorkonstruktes für die kardiale Gentherapie 5.2.1 Kardiale und systemische Biodistribution, Expression und Transkriptionseffizienz 5.2.2 Pharmakologische Sicherheit der AAV5-hS100A1-Gentherapie 5.2.2.1 Blutanalyse 5.2.2.2 Elektrokardiogramm 5.3 Effekte der hS100A1-Gentherapie 5.3.1 Einfluss auf die Ejektionsfraktion 5.3.2 Einfluss auf die Infarktausweitung 5.4 Limitationen des Studiendesigns und des tierexperimentellen Modells 5.5 Fazit und Ausblick 6 Zusammenfassung 7 Summary 8 Literaturverzeichnis 9 Anhang 10 Danksagung / Introduction: Cardiovascular diseases are the prevailing cause of death worldwide. To date, there is no causal therapy for heart failure, which represents the common endpoint of a large number of different cardiac diseases. Even the promising gene therapy approach has not yet been able to achieve relevant progress in this field. Identified as a central regulator of cardiac activity and calcium signaling in cardiomyocytes, the calcium-sensor protein S100A1 represents a highly suitable candidate for cardiac gene therapy. Objective: The overall goal of this translational study is to advance the field of gene therapy for cardiac dysfunction. On the one hand, the on adeno-associated viruses (AAVs) based vector (rAAV) of serotype 5 was tested in combination with a cardiac-specific promotor (cmvenh/0,26 kb-mlc) for its applicability and safety for cardiac gene therapy. Beforehand, extensive testing for preexisting neutralizing serum factors (nsf) was performed to decipher whether the pig is a suitable model for AAV5-based preclinical studies. On the other hand, the effect of the hS100A1 gene therapy after myocardial infarction (MI) was further characterized in a clinically relevant large animal model with an endpoint-based study design. Animals, materials and methods: A total of 83 juvenile farm pigs were used. Before starting the experiment, we analyzed serum from 40 animals for preexisting nsf against AAV5 using a flow cytometry-based cell reporter assay. For the main study, we used 8 animals in which we induced an ischemic myocardial infarction with subsequent reperfusion by occluding percutaneously the ramus circumflexus of the left coronary artery (LCX) for 2 hours to generate cardiac dysfunction. After 2 weeks, we evaluated infarct size and cardiac function with cardiac magnetic resonance imaging (MRI). Animals were divided into the treatment group (AAV5-hS100A1, 5 animals) and the control group (AAV5-hRluc, 3 animals). We applied gene transfer (1x1013 viral genome copies (vgc)/animal) using retrograde coronary venous infusion. We repeated the cardiac MRI 12 weeks after gene transfer. Serial blood and electrocardiogram (ECG) tests were performed during the experiment to verify pharmacological safety. At the end of the study, the animals were euthanized and their organs were collected for further molecular analyses. To investigate the distribution and transcriptional efficiency of the vectors, we isolated DNA and RNA from the tissue samples and performed real-time quantitative polymerase chain reaction (qPCR). In addition, a next-generation sequencing of myocardial RNA was conducted and analyzed with weighted gene co-expression network analysis (WGCNA) and subsequent enrichment analysis. To reduce the number of animals, the study was end point oriented: When reaching significant differences in the primary end points (ejection fraction (EF) and infarct extension), the study was terminated. Results: The results demonstrate a low anti-AAV5 seroprevalence in the farm pig population. After 12 weeks, the AAV5-hS100A1 gene therapy resulted in a significant reduction of infarct extension and a significantly higher EF compared to the control group (unpaired two-sided Student t-Test, p < 0.05). ECG and blood tests did not show any indications of toxicity. The transcriptome analysis of the myocardial samples provided a significant negative correlation between relevant pathological signaling pathways and EF/infarct extension, thus giving clues to underlying mechanisms. Among these, an anti-inflammatory effect of AAV5-hS100A1 appears to be of major importance. For the first time, we could also demonstrate an interaction between S100A1 and the cardioprotective retinoic acid. Due to the high mortality during MI-induction, we incorporated a test series with 72 animals. By changing the anesthetic gas from isoflurane to sevoflurane, we could significantly reduce the mortality (one-sided Fisher's exact test, p < 0.05). Conclusions: The pig represents a suitable model for AAV5-based studies. 12 weeks after gene transfer, the construct AAV5-hS100A1 with cmvenh/0.26 kb-mlc promoter showed a robust and mostly cardiospecific expression of the transgene accompanied by high pharmacological safety. Thus, it provides great therapeutical potential. The study contributed to identify novel signaling pathways that may be relevant for S100A1’s therapeutic actions. By changing the anesthetic gas, we could reduce the mortality during infarct induction. Therefore, in future MI studies, sevoflurane should be used preferably to maintain inhalation anesthesia.:Inhaltsverzeichnis Abkürzungsverzeichnis 1 Einleitung 2 Literaturübersicht 2.1 Koronare Herzkrankheit, Myokardinfarkt und Herzinsuffizienz 2.1.1 Definition und Epidemiologie 2.1.2 Infarktheilung 2.1.3 Therapiemöglichkeiten 2.2 (Kardiale) Gentherapie 2.2.1 Vektoren 2.2.1.1 AAV5 2.2.2 Promotoren 2.2.3 Applikationsmethoden 2.3 S100A1 als therapeutisches Protein in der kardialen Gentherapie 2.3.1 Die Struktur von S100A1 2.3.2 Die Funktion von S100A1 2.3.3 Die kardiale S100A1-Gentherapie 2.4 Das Schwein in der translationalen Forschung 3 Material und Methoden 3.1 Material für die Aufarbeitung der Proben im Labor 3.1.1 Geräte und Verbrauchsmaterialien 3.1.2 Reagenzien und Chemikalien 3.1.3 Kits 3.1.4 Medien und Puffer 3.2 Material für den Großtier-OP und das Kardio-MRT 3.2.1 Geräte 3.2.2 Katheter und Schleusen 3.2.3 Medikamente und Medizinprodukte 3.3 Allgemeiner Versuchsaufbau 3.3.1 Versuchstiere 3.3.2 Versuchsaufbau 3.3.3 Vorbereitung, Narkose, perioperative Überwachung und Versorgung 3.3.4 Blutentnahme 3.3.5 Gefäßzugänge 3.3.6 Induktion des Myokardinfarkts 3.3.6.1 Änderung der Narkoseaufrechterhaltung während der MI-Induktion 3.3.7 Kardio-MRT – Durchführung 3.3.8 Gentransfer 3.3.9 Virale Vektoren 3.3.10 Organentnahme 3.4 Untersuchung auf neutralisierende Antikörper und Serumfaktoren 3.5 Bestimmung der Genexpression mittels qPCR 3.5.1 Homogenisierung und RNA-/DNA-Isolierung 3.5.2 cDNA-Synthese 3.5.3 Primer und Probes 3.5.4 qPCR im multiplex-Ansatz 3.5.5 Quantifizierung der Vektorgenomkopien mittels SYBR-qPCR 3.6 Kardio-MRT – Auswertung 3.7 Transkriptomanalyse 3.7.1 Next-Generation RNA-Sequenzierung – Durchführung 3.7.2 NGS – Auswertung 3.7.3 Hauptkomponentenanalyse 3.7.4 Gewichtete Gen Korrelation Netzwerk Analyse 3.7.5 Anreicherungsanalyse 3.8 Blutanalyse 3.9 Elektrokardiogramm – Auswertung 3.10 Statistische Auswertung 4 Ergebnisse 4.1 Erfüllung der Einschlusskriterien zur Aufnahme in die Studie 4.1.1 Seroprävalenz von neutralisierenden Antikörpern und Serumfaktoren gegen AAV5 4.1.2 Infarktgröße vor Gentherapie 4.2 Mortalität beim perkutanen LCX-Ischämie-/Reperfusionsmodell 4.3 Gentransfer 4.3.1 Überprüfung der Spezifität der Primer und Probes 4.3.2 Distribution der viralen Vektoren im linken Ventrikel 4.3.3 Transgenexpression im linken Ventrikel 4.3.4 Trankriptionseffizienz im linken Ventrikel 4.3.5 Systemische Verteilung 4.4 Einfluss der hS100A1-Gentherapie auf die kontraktile Funktion und Infarktgrößenentwicklung nach Myokardinfarkt 4.4.1 Effekte auf die Ejektionsfraktion 4.4.2 Effekte auf die Infarktausweitung 4.4.3 Ergebnisse der Transkriptomanalyse 4.5 Einfluss der hS100A1-Gentherapie auf die pharmakologische Sicherheit 4.5.1 Blutanalyse 4.5.2 Elektrokardiogramm 5 Diskussion 5.1 Eignung des Tiermodells 5.1.1 Seroprävalenz von neutralisierenden Antikörpern und Serumfaktoren in der Versuchstierart Schwein 5.1.2 Das perkutane LCX-Ischämie-/Reperfusionsmodell als experimentelles Modell zur Untersuchung der Infarktausweitung 5.1.3 Einfluss des Narkosegases auf die Mortalitätsrate beim perkutanen LCX-Ischämie-/ Reperfusionsmodell 5.2 Eignung des Vektorkonstruktes für die kardiale Gentherapie 5.2.1 Kardiale und systemische Biodistribution, Expression und Transkriptionseffizienz 5.2.2 Pharmakologische Sicherheit der AAV5-hS100A1-Gentherapie 5.2.2.1 Blutanalyse 5.2.2.2 Elektrokardiogramm 5.3 Effekte der hS100A1-Gentherapie 5.3.1 Einfluss auf die Ejektionsfraktion 5.3.2 Einfluss auf die Infarktausweitung 5.4 Limitationen des Studiendesigns und des tierexperimentellen Modells 5.5 Fazit und Ausblick 6 Zusammenfassung 7 Summary 8 Literaturverzeichnis 9 Anhang 10 Danksagung

The identification of known asteroids on existing CCD pictures would allow us to obtain accurate astrometric and photometric asteroid properties. Some asteroids might have ambiguous orbital elements, thus their identification along with their exact positions on multiple picture frames could significantly improve their orbital elements. Furthermore, the possibility of identifying known asteroids on older pictures, sometimes preceding their discovery date, might allow the study of non-gravitational effects like the Yarkovsky effect. Identifying a potential Yarkovsky effect on asteroids is challenging because it is extremely weak. However, this effect cumulates with time, therefore, it is necessary to find astronomical pictures that are as old as possible. In addition, we need to collect high quality CCD pictures and use a methodology that would allow obtaining a statistically significant sample of asteroids. To accomplish this, we decided to use the online archive of the Subaru telescope at Mauna Kea Hawaii because it has a prime-focus camera with a very high resolution of 80 millions pixels very well suited to capture serendipitous asteroids. In addition, the Subaru online archive has pictures from the last 10 years. iv The methodology used in this thesis is to build a database that contains the orbital elements of all the known asteroids, allowing us to write a program that calculates the approximate position of all the asteroids at the date and time of each CCD picture we collect. To obtain a more precise position, the program also interfaces the JPL NASA Horizons on-line computation service. Every time an asteroid is found on a picture, Horizons sends its theoretical location back to the program. A later visual identification of this asteroid at this theoretical location on the picture triggers its input into our sample for further study. This method allowed us to visually confirm 508 distinct asteroids on 692 frames with an average diameter of 3.6 km. Finally, we use the theory (given in appendix A) to calculate the theoretical drift of these asteroids that we compare with the one we measured on the CCD pictures.

Mouse polyomavirus (MPyV) is a model virus of the Polyomaviridae family. Polyomaviruses are small non-enveloped DNA viruses. They cause severe problems to immunocompromised patients. Their oncogenic potential is known in animals and humans. Trafficking of MPyV within the cell is not clear yet. The virus enters via smooth monopinocytic vesicles and continues to early and late endosomes. From there, the virus is transported to the ER by unknown mechanism. It bypasses Golgi aparatus (GA). One possible pathway is from late endosomes to trans-Golgi network (TGN) facilitated by Rab9 GTPase and then in COPI vesicles to the ER. In this thesis, the effect of inhibitors of retrograde transport (Brefeldin A, Golgicide A) on MPyV infection was evaluated. Brefeldin A is not completely specific; it has effect on whole endosomal system. Golgicide A causes specific disruption of transport via TGN and GA. Both inhibitors suppressed infection of MPyV. Confocal microscopy revealed colocalization of some MPyV virions with markers of TGN and COPI vesicles. MPyV didn't colocalize with cis-Golgi marker. Unfortunately, the effect of overexpression of Rab9 dominant negative mutant couldn't been evaluated due to its high cytotoxicity. However, overexpression of wild type Rab9 slightly increased infectivity. The results...

A Esclerose Lateral Amiotrófica (ELA) é uma doença neurodegenerativa progressiva de evolução rápida, caracterizada pela perda seletiva dos neurônios motores (NM) superiores e inferiores. Recentemente, as células gliais centrais (astrócito, microglia e oligodendrócito) mostraram-se tóxicas aos NM, porém os detalhes moleculares não estão completamente elucidados. Em relação às células gliais periféricas, alterações eletrofisiológicas no nervo ciático do modelo animal da ELA na idade pré-sintomática foram reportadas pelo nosso grupo e os achados de denervação precoce tanto no modelo animal quanto em pacientes sugerem a participação das células de Schwann (CS) na morte neuronal retrógrada na ELA, teoria conhecida como dying back. Nesse contexto, as CS mostraram-se capazes de induzir a retração axonal e a denervação das junções neuromusculares, eventos precoces na doença, ocorrendo possivelmente na fase présintomática. O objetivo deste trabalho foi verificar a influência das CS do modelo experimental na fase pré-sintomática e do paciente com evolução recente da forma esporádica da ELA, na sobrevida e no tamanho dos prolongamentos dos NM in vitro e entender a natureza molecular do fenômeno. Culturas de CS altamente purificadas foram obtidas a partir do nervo ciático do camundongo modelo animal e do nervo periférico de pacientes com ELA. Os NM da medula espinal de camundongos neonatos foram co-cultivados com as CS. A neurodegeneração foi avaliada pela presença do marcador Fluoro-Jade C (FJC). Os NM também foram tratados com o meio condicionado das culturas de CS do modelo animal ou dos pacientes com ELA. Os motoneurônios tiveram os seus prolongamentos contados e a morte neuronal foi identificada pela presença do FJC. Diversos fatores neurotróficos foram quantificados no meio condicionado das culturas de CS pela técnica de ELISA. A reação em cadeia da polimerase quantitativa (do inglês, quantitative polymerase chain reaction - qPCR) foi realizada para detectar alterações nas CS e no nervo periférico que pudessem estar relacionadas com disfunção na unidade CS/NM. Os resultados mostraram que os NM cultivados na ausência das CS mostraram-se mais susceptíveis à morte. Os NM cocultivados com as CS ELA mostraram maior número de perfis neurodegenerativos em comparação com os NM co-cultivados com as CS controle. Após o tratamento com o meio condicionado das CS ELA, os NM mostraram redução no tamanho dos prolongamentos e aumento do número de células em neurodegeneração em comparação com o grupo controle. Quantidades reduzidas dos fatores neurotróficos foram encontradas no meio condicionado das culturas de CS ELA. Alterações na expressão gênica das CS e no nervo periférico evidenciaram disfunções na unidade CS/NM que podem estar contribuindo para o processo neurodegenerativo visto na ELA. Conclui-se que a falência nos mecanismos de neuroproteção pelas CS ELA é um importante mecanismo implicado na morte neuronal, com grande potencial terapêutico / Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by the selective loss of upper and lower motor neurons (MN). Recently, central glia (astrocytes, microglias and olygodendrocytes) were toxic to the MN, but the molecular aspects have not fully described. In relation to the peripheral glia, electrophysiological changes in the sciatic nerve of ALS animal model in the presymptomatic stage have been reported by our group and early denervation findings in both animal models and patients suggests the participation of Schwann cells (SC) in the retrograde neuronal death of ALS , theory known as dying back. In this context, the SC proved to be able to induce axonal retraction and denervation of the neuromuscular junctions, early events in the disease, possibly occurring in the pre-symptomatic phase. The aim of this thesis was to investigate the influence of SC of pre-symptomatic experimental model and from patient with recent evolution of ALS sporadic form, in the survival and axonal length of MN in vitro and understand the molecular nature of the phenomenon. Highly purified SC cultures were obtained from the sciatic nerve of the animal model and from ALS patient\'s peripheral nerve. MN from the newborn mouse spinal cord were co-cultured with SC and the neurodegeneration was assessed by the presence of the marker Fluoro-Jade C (FJC). MN were also treated with conditioned medium from cultures of SC of the animal model or ALS patients. MN had their neuronal length measured and neuronal degeneration was identified by the presence of the FJC. Several neurotrophic factors were measured in conditioned medium of mice and ALS patient\'s SC cultures by ELISA. The chain reaction quantitative polymerase (qPCR) was performed to detect changes in the SC and peripheral nerve that could be related with dysfunction in the functional unit SC/MN. The MN co-cultured with ALS SC showed a greater number of neurodegenerative profiles compared with MN cocultured with control SC. After treatment with ALS SC conditioned medium, MN showed a reduction in the neuronal length and increased number of cells in neurodegeneration compared with the control group. Lower levels of neurotrophic factors were found in the conditioned medium of ALS SC cultures. Changes in the gene expression of SC and peripheral nerve showed dysfunctions in SC/MN unit, which may be contributing to the neurodegenerative process seen in ALS. In conclusion, the failure of neuroprotection by ALS SC is an important mechanism implicated in the MN cell death, with great therapeutic potential

Introdução: O diagnóstico histológico das estenoses biliares é fundamental na definição da terapêutica a ser empregada, devido à heterogeneidade dos resultados dos estudos comparando o uso do escovado citológico e da biópsia transpapilar na colangiopancreatografia retrógada endoscópica (CPRE) com a punção aspirativa ecoguiada com agulha fina (ECO-PAAF) no diagnóstico histológico da estenose biliar maligna, e o fato de não existirem revisões sistemáticas e metanálises comparando esses métodos, este estudo propõe comparar esses dois métodos no diagnóstico histológico da estenose biliar maligna, através de revisão sistemática e metanálise da literatura. Métodos: Utilizando as bases de dados eletrônicas Medline, Embase, Cochrane, LILACS, CINAHL, e Scopus foram pesquisados estudos datados anteriormente a novembro de 2014. De um total de 1009 estudos publicados, foram selecionados três estudos prospectivos comparando ECO-PAAF e CPRE no diagnóstico histológico da estenose biliar maligna e cinco estudos transversais comparando ECO-PAAF com o mesmo padrão-ouro dos outros três estudos comparativos. Todos os pacientes foram submetidos ao mesmo padrão-ouro. Foram calculadas as variáveis do estudo (prevalência, sensibilidade, especificidade, valores preditivos positivos e negativos e acurácia) e realizada a metanálise utilizando os softwares Rev Man 5 e Meta-DiSc 1.4. Resultados: Um total de 294 pacientes foi incluído na análise. A probabilidade pré-teste para estenose biliar maligna foi de 76,66%. As sensibilidades médias da CPRE e da ECO-PAAF para o diagnóstico histológico da estenose biliar maligna foram de 49% e 76,5%, respectivamente; especificidades foram de 96,33% e 100%, respectivamente. As probabilidades pós-teste também foram determinadas: valores preditivos positivos de 98,33% e 100%, respectivamente, e valores preditivos negativos de 34% e 58,87%. As acurácias foram 60,66% e 82,25%, respectivamente. Conclusão: A ECO-PAAF é superior a CPRE com escovado citológico e/ou biópsia transpapilar no diagnóstico histológico da estenose biliar maligna. No entanto, um teste de ECO-PAAF ou CPRE com amostra histológica negativa não pode excluir a estenose biliar maligna, pois ambos os testes apresentam baixo valor preditivo negativo / Background and Aims: Due the heterogeneity of the results of studies comparing the use of ERCP-based brush cytology and forceps biopsy and EUS-guided fine-needle aspiration for the diagnosis of malignant biliary stricture, and the fact that there are no systematic reviews and meta-analysis comparing these methods, in this review, we will compare ERCP against EUS-FNA for tissue diagnosis of malignant biliary stricture. Design: A systematic review of comparative studies (prospective or retrospective) was conducted analyzing EUS and ERCP for tissue diagnosis of malignant biliary stricture. Methods: The databases Medline, EMBASE, Cochrane, LILACS, CINAHL, and Scopus were searched for studies dated previous to November 2014. We identified three prospective studies comparing EUS-FNA and ERCP for the diagnosis of malignant biliary stricture and five cross sectional studies comparing EUS-FNA with the same gold standard of the other three studies. All patients were submitted to the same gold standard method. We calculated study variables (prevalence, sensitivity, specificity, positive and negative predictive values, and accuracy) and performed a meta-analysis using the Rev Man 5 and Meta-DiSc 1.4 softwares. Results: A total of 294 patients were included in the analysis. The pretest probability for malignant biliary stricture was 76.66%. The mean sensitivities of ERCP and EUS-FNA for tissue diagnosis of malignant biliary stricture were 49% and 76.5%, respectively; specificities were 96.33% and 100%, respectively. The post-test probabilities, positive predictive value (98.33% and 100%, respectively) and negative predictive value (34% and 58.87%, respectively) were determined. The accuracies were 60.66% and 82.25%, respectively. Conclusion: EUS- FNA is superior to ERCP with brush cytology and forceps biopsy for diagnosing malignant biliary strictures. However, a negative EUS-FNA or ERCP test may not exclude malignant biliary stricture because both have low negative post-test probabilities

Introdução: O acesso biliar ecoguiado é um método de drenagem alternativo à drenagem percutânea transhepática (DPTH) e à cirurgia em pacientes com obstrução biliar distal incurável que falharam drenagem por Colangiopancreatografia Endoscópica Retrógrada (CPRE). Nos casos em que a drenagem ecoguiada anterógrada transpapilar (ou transanastomótica) e o rendez-vous ecoguiado não podem ser realizados como primeira opção, a coledocoduodenostomia (CDT) e a hepaticogastrostomia (HPG) ainda podem ser realizadas em pacientes selecionados. Estas duas vias de drenagem não anatômicas criam uma fístula entra a via biliar e o estômago ou duodeno. Não há dados na literatura que determinem superioridade de uma ou outra técnica. Objetivo: Comparar o sucesso técnico, sucesso clínico e fatores associados entre as duas vias de drenagem em pacientes com obstrução da via biliar distal maligna incurável que não lograram sucesso na drenagem por CPRE ou rendez-vous ecoguiado. Métodos: Entre abril de 2010 e dezembro de 2013, 49 pacientes com obstrução biliar distal maligna incurável que falharam CPRE e rendez-vous ecoguiado foram randomizados para CDT ou HPG. Dados referentes ao sucesso técnico, sucesso clínico, tempo de procedimento, complicações, qualidade de vida e sobrevida foram coletados até três meses após o procedimento. Todos os procedimentos foram realizados em um centro terciário de endoscopia pelo mesmo endoscopista. Próteses biliares parcialmente recobertas (Boston Scientific, Wallflex, 10 mm, 8 cm ou 6 cm) foram utilizadas em todos os pacientes com sucesso técnico. Nos casos de HPG a punção ecoguiada foi intra-hepática no ducto hepático esquerdo. Nos casos de CDT a punção foi extra-hepática no segmento distal não obstruído do colédoco. Após a punção foi realizada colangiografia com introdução de um fio guia hidrofílico de 0,035 polegada. Dilatação com cateter e um dispositivo de needle knife foi realizada para permitir introdução do sistema de disparo da prótese biliar com 8,5 Fr. Resultados: Quarenta e nove procedimentos foram realizados (25 HPG e 24 CDT). Todos os pacientes tinham dilatação da via biliar intra e extra-hepática. A taxa de sucesso técnico foi de 96 % para HPG e de 91% para CDT (p = 0,609). A taxa de sucesso clínico foi de 91% para o grupo HPG e de 77% para o grupo CDT (p = 0,234). No grupo da HPG 5 pacientes (20%) tiveram complicações (3 sangramentos, 2 biliomas e uma bacteremia). No grupo da CDT 3 pacientes (12,5%) tiveram complicações (1 bilioma, 1 sangramento e 1 perfuração). Somente o caso da perfuração necessitou tratamento cirúrgico. As outras complicações foram tratadas clinicamente. O tempo de procedimento médio foi de 47,83 min para a HPG e de 48,88 min para a CDT (p = 0,843). Conclusão: O presente estudo não demonstrou diferença estatisticamente significante em relação ao sucesso técnico, sucesso clínico, complicações e tempo de procedimento entre os dois grupos estudados. Mais estudos são necessários para elucidar o papel de cada via de drenagem / Background: EUS-guided biliary access is an alternative for percutaneous access or surgery in patients with malignant unresectable distal biliary obstruction and failed ERCP. When rendezvous or anterograde transpapillary/transanastomotic intervention fails as primary drainage options, a Choledochoduodenostomy (CDT) or a Hepaticogastrostomy (HGT) can still be performed in selected patients. This procedure creates a new \" \" y I w one route or the other should be recommended. Aim: To compare technical and clinical success and possible associated factors between the two different drainage routes CDT and HGT in patients with distal unresectable malignant biliary obstruction that failed standard ERCP and EUS-guided rendez vouz (RV) maneuver. Methods: Between April/2010 and December/2013 49 consecutive jaundiced patients with distal unresectable malignant biliary obstruction that failed previous ERCP and EUS-guided RV maneuver were elected randomly to undergo either EUS-guided CDT or HGT. Data including indications, clinical and technical success, procedural times and complications with a three-month follow-up were prospectively collected in a database. All procedures were performed in a tertiary center by the same endoscopist. A partially covered SEMS (Boston Scientific, Wallflex, 10 mm, 8 cm or 6 cm) was used in all technically successful procedures. After puncture of left hepatic duct in case of HGT or the distal unobstructed segment of common bile duct in case of CDT a cholangiogram was obtained followed by advancement of a 0,035-inch guide wire into the biliary system. Bougies and wire-guided needle-knife were used to perform track dilation to allow passage of an 8.5 Fr stent delivery system. Results: Forty-nine cases (25 HGT and 24 CDT) were performed. All patients had intra and extra hepatic biliary dilation. Technical success rate was 96 % for HGT and 91% for CDT (p = 0.609). Clinical success rate was 91% for HPG and 77% for CDT (p = 0.234). In the HGT group five patients (20%) had complications (3 bleeding, 2 bilomas and 1 bacteremia). In the CDT group 3 patients (12.5%) had complications (1 biloma, 1 bleeding and 1 perforation). Only the perforation patient required surgery. All other complications were managed clinically. The median procedural time was 47.83 min for HGT and 48.88 min for CDT (p = 0.843). Conclusion: No significant difference was found in regards to technical or clinical success, complications and procedure time between the two drainage routes. More studies are needed to clarify situations in which the CDT or the HGT should be advocated

O MCH é um neuropeptídeo sintetizado preferencialmente no hipotálamo que exibe projeções para todo o neuroeixo, podendo modular vários processos fisiológicos, como a ingestão aguda de alimento. Um alvo de suas projeções é o núcleo accumbens (Acb), componente do estriado ventral envolvido na recompensa de estímulos hedônicos. Nossa contribuição visa mapear as células imunorreativas (ir) ao MCH responsáveis pelas aferências do Acb, onde também investigamos aposições entre os sistemas MCH, GABA e ChAT. Utilizaram-se os métodos de simples e duplas imunoperoxidases e combinação de imunofluorescência e captação de traçadores retrógrados. A maior densidade de fibras ir-MCH ocupa a parte shell do Acb (AcbSh); as aferências ir-MCH partem, por exemplo, da área hipotalâmica lateral e área perifornicial. Controles anterógrados foram obtidos a partir da LHA. Observaram-se aposições entre terminais ir-MCH e células ir-GABA e ir-ChAT, no AcbSh. Assim, sugere-se que os territórios hipotalâmicos que contém MCH inervem o AcbSh e contatem outros sistemas neuroquímicos no Acb. / MCH is a neuropeptide synthesized preferentially in the hypothalamus that shows projections throughout neuraxis, and may modulate various physiological processes such as food intake. One target of its projections is the nucleus accumbens (Acb), a component of the striatum involved in reward of hedonic stimuli. Our contribution aims to map immunoreactive (ir) cells responsible for the MCH inputs of the Acb, and to investigate appositions between MCH fibers, GABA and ChAT cells. We used the single and double immunoperoxidase methods and immunofluorescence combined with retrograde tracer. The highest density of fiber MCH-ir occupies the shell part of the Acb (AcbSh); MCH-ir inputs are mainly from lateral hypothalamic and perifornical areas. Anterograde controls were obtained from the LHA. We have founded appositions between MCH-ir terminals and GABA- and ChAT-ir cells at AcbSh. Thus, is suggested that hypothalamic areas containing MCH innervate the AcbSh and contact other neurochemical systems systems in the Acb.

Les toxines Shiga (Stx) sont produites par Shigella dysenteriae et certaines espèces d’E. coli transmisent aux humains par la consommation d'aliments contaminés et causant des maladies graves. La toxine Stx est libérée par les bactéries dans l'intestin et par la suite, traverse les vaisseaux sanguins en aval pour atteindre leurs principaux organes cibles, notamment les reins. Les dommages causés aux reins peuvent entraîner des complications graves notamment Le syndrome hémolytique urémique (SHU). A ce jour, il n’existe aucun traitement disponible contre le SHU. Les toxines Stx usent du transport rétrograde intracellulaire pour infester les cellules endothéliales rénales et atteindre leur cible cytosolique, l'ARN ribosomal 28S. Via un screening à haut débit, il a été démontré que le composé Rétro-2 bloque le trafic rétrograde de Stx à l'interface Endosome-TGN, sans affecter la morphologie des organites cellulaires et le trafic des protéines endogènes. Au cours de cette thèse, une analyse des relations structure fonction du composé Retro-2 nous a permis d’identifier les régions de l'inhibiteur qui sont critiques pour l'activité de protection. Nous avons identifié un dérivé dihydroquinazolinone nommé Rétro-2.1 qui est à ce jour l'inhibiteur le plus puissant contre les toxines Stx. Afin d’identifier la cible moléculaire de Retro-2.1, nous avons développé des sondes photo-activables bio-actives. En outre, les données de diffraction des rayons X ont révélé que de l'activité antitoxine réside principalement dans l’énantiomère S. (S) -Retro-2.1 est 500 fois plus puissant contre Stx (50 nM) que la molécule initiale. Cette étude peut donner lieu à un nouveau concept thérapeutique ciblant la voie de transport rétrograde de la toxine à l'intérieur de la cellule hôte. Une telle stratégie thérapeutique pourrait donc être étendue à d'autres agents pathogènes qui usent également du trafic rétrograde pour une intoxication des cellules hôtes. Ce nouveau concept thérapeutique qui permet de cibler les cellules hôtes et non l'agent pathogène représente une véritable percée dans la découverte de médicaments à large spectre et réduit le risque de développement d’une résistance chez l’agent pathogène. / Shiga toxins (Stx) are produced by Shigella dysenteriae and certain species of E. coli that can be transmitted to humans primarily through consumption of contaminated foods and may cause severe disease. Stx is released by the bacteria in the intestine and subsequently, could cross the downstream blood vessels to reach their main target organs such as kidney. Damage to the kidney can result in serious life-threatening complication hemolytic uremic syndrome, for which there is no proven safe treatment available other than supportive care. Stx invades renal endothelial cells in a retrograde manner from cell surface to the endoplasmic reticulum in order to gain access to its cytosolic target, 28S rRNA. By using HTS, it was previously demonstrated that the compound Retro-2 blocks retrograde trafficking of Stx at the early endosome-TGN interface, without affecting the morphology of cellular organelles and trafficking of other endogenous proteins. In this work, different regions of the lead inhibitor Retro-2 that are critical for the protective activity have been determined by systematic structure-activity relationship studies. It allowed us to identify a dihydroquinazolinone derivative, named Retro-2.1 that is the most potent inhibitor of Stx to date and also to develop bio-active photo-activatable probes with the aim of identifying the molecular target of Retro-2 derivatives. Further, crystal X-ray diffraction data revealed that the antitoxin activity resides mainly in the S-enantiomer. (S)-Retro-2.1 has displayed 500 fold more potency (50 nM) than parent molecule against Stx cytotoxicity. This study may result in a new therapeutic concept - targeting the retrograde transport route of toxin inside host cell - for the treatment of Stx-producing E. coli infections and could therefore be extended to other pathogens that also traffic via the retrograde transport. Such a new therapeutic concept that target the host cells and not the pathogen itself would represent a real breakthrough in drug discovery leading to broad spectrum drugs.

O objetivo desde estudo foi avaliar a eficiência de remoção de smear layer e a influência da resistência de união de cimentos de silicato de cálcio de um novo inserto ultrassônico para agitação do irrigante em preparos retrógrados. Metodologia Foram selecionados 112 caninos unirradiculados, que foram formatados e obturados de forma convencional com a subsequente ressecção apical. Seguidamente, foram conformados 4 grupos de avaliação, ultra-som/solução salina; convencional/solução salina; ultra-som/EDTA17% e convencional/EDTA17%. Para analisar a remoção do smear layer, 72 espécimes foram divididos em 4 grupos (n=18), e retroprepararados utilizando uma ponta ultrassônica. Os espécimes foram clivados, obtendo-se dois segmentos iguais (mesial e distal). Os segmentos retrocavitários foram observados através da microscopia eletrônica de varredura (MEV), a partir de três regiões representativas a 250x, obtendo-se imagens correspondentes à pré-irrigação. Posteriormente, foram executados os protocolos de irrigação e novamente obtidas imagens representativas da pós-irrigação. Para o teste de push-out, 40 dentes alocados em 4 grupos (n=10) foram empregados, conforme os diferentes protocolos de irrigação previamente mencionados. Os espécimes foram retrobturados e a obtenção dos corpos-de-prova realizada a partir de um novo seccionamento apical de 2mm±0.1 de cada espécime. A resistência de união foi determinada por meio de uma máquina de ensaios mecânicos e o tipo de falha foi determinado por meio de MEV. Os dados foram comparados por meio dos testes de Kruskal-Wallis e Dunn para a remoção de smear layer e ANOVA e Tukey no teste de push-out. Resultados O grupo ultrassom/EDTA17% removeu significativamente uma maior quantidade do smear layer, observando-se uma maior porcentagem de túbulos dentinários abertos em comparação com os outros grupos (P < 0.05). Na resistência de união, o grupo ultrasom/ EDTA17% mostrou os maiores valores na resistência de união (6.42±0.69) seguido pelos grupos convencional/EDTA17%, ultra-som/solução salina e convencional/solução salina respectivamente (P < 0.05). O padrão de falha adesivo foi o mais predominante, enquanto que no grupo ultrassom/EDTA17% foi preponderantemente mista (8/10). Conclusões O novo inserto ultrassônico para ativação do irrigante promoveu uma maior remoção de smear layer além de favorecer maior resistência de união do material retrobturador às paredes dentinárias em preparos retrógrados, principalmente se associado ao EDTA17%. / To evaluate the smear layer removal capacity and its influence on the bond strength of a calcium silicate-based cement of a new ultrasonic tip for irrigant agitation in rootend cavity preparations. Methods: One hundred and twelve maxillary and mandibular canines were chemomechanically instrumented, root filled and the root apexes resected. Specimens were randomly distributed into 4 experimental groups: ultrasonic/saline solution; conventional/ saline solution; ultrasonic/EDTA 17% and conventional/EDTA solution 17%. The analysis of the smear layer removal employed seventy-four specimens divided into 4 groups (n=18), and the retrograde cavities were prepared using an ultrasonic tip. Next, the specimens were split longitudinally and obtained two halves (mesial and distal). Scanning electron microscopy (SEM) was used to examine and score the internal surfaces for dentinal tubule opening before and after to perform the irrigation protocols. Images were taken at three representative areas with a magnification of 250x. The push-out test employed forty specimens allocated in four groups (n=10), according to the different irrigation protocols previously described. The specimens were retrofilled, sectioned perpendicularly and obtained apical slices at 2mm±0.1 from the apex. Each disc/dentin/retrofilling material was placed in a testing machine for the bond strength measurement and the failure mode was observed by SEM. The smear layer removal data were analysed and compared using Kruskal-Wallis and Dunn. Push-out data were analysed using ANOVA and Tukey\'s test. Results The ultrasonic/EDTA 17% group removed the smear later more effectively, and showed the best tubule opening condition than the others three groups. (P < 0.05). Ultrasonic/EDTA 17% group demonstrated the highest mean bond strength values (6.42±0.69) compared with conventional/EDTA, ultrasonic/saline solution and conventional/saline solution, respectively (P < 0.05). Most failure modes were predominantly adhesive (55%), whereas in the ultrasonic/EDTA 17% group was mixed (80%). Conclusion The new ultrasonic tip for irrigant agitation showed higher percentage of smear layer removal and improved the push-out bond strength of the retrograde root filling to the root-end dentine surfaces, mainly when associated to EDTA 17%.

ν Octantis is a spectroscopic binary with a semi-major axis and period of 2.55 AU and 2.9 years, respectively. Ramm et al. (2009) discovered a 52 ms^(-1) radial-velocity (RV) perturbation with a period of 417 days in this system. All evidence, both photometric and spectroscopic, suggests the perturbation is the result of a 2.5 MJ planet orbiting the primary star. However, when assuming a “normal” prograde coplanar orbit, celestial mechanics predicts this orbit is unstable, contradicting the observed stability. Simulations by Eberle and Cuntz (2010) showed a retrograde orbit for the planet to be stable for at least 10^7 years. In this thesis, we performed a 10^8 -yr simulation of the retrograde orbit, and found it remained stable. Simulations over a range of planetary semi-major axes, eccentricities, and primary/secondary masses showed that stable retrograde orbits are not possible past a semi-major axis of 1.315 +/- 0.092 AU . Therefore, planetary retrograde orbits are most likely inherently more stable than prograde orbits owing to the absence of stability at known mean-motion resonances. Eccentricity simulations showed that the period of the planet's dominant eccentricity variation is related to the planet's semi-major axis by a second order exponential. However, retrograde orbits tend to have longer eccentricity periods than prograde orbits at the same semi-major axis. There is also evidence that this eccentricity period is connected to the orbital stability. By fitting a keplerian to both Ramm et al. (2009) and current radial velocities, the period of the ν Octantis binary was determined to be 1050.04 +/- 0.02 days with an eccentricity of 0.2359 +/- 0.001 . The planetary orbital solution for just the data reduced in this thesis gave a period of 416.9 +/- 2.1 days and an eccentricity of 0.099 +/- 0.015 , with an RMS scatter of 9.6 ms^(-1). Therefore, the orbital elements are within 1σ of the Ramm et al. (2009) elements. Assuming a retrograde coplanar orbit about the primary star then the planet has a mass of M_pl = 2.3 M_J and a semi-major axis of a_pl = 1.21 +/- 0.09 AU.