In murine and human brown adipose tissue (BAT), mitochondria are powerful generators of heat. Emerging evidence has suggested that the actions of mitochondria extend beyond this conventional biochemical role. In mouse BAT and cultured brown adipocytes, impaired mitochondrial respiratory capacity is accompanied by attenuated expression of Ucp1, a key thermogenic gene, implying a mitochondrial retrograde signaling. However, few have investigated this association in the context of mitochondria-nucleus communication. Using mice with adipose-specific ablation of LRPPRC, a regulator of respiratory capacity, we show that respiration-dependent retrograde signaling from mitochondria to nucleus contributes to transcriptional and metabolic reprogramming of BAT. Impaired respiratory capacity triggers down-regulation of thermogenic and oxidative genes, promoting a storage phenotype in BAT. This retrograde regulation functions by interfering with promoter-specific recruitment of PPARg. In addition, cytosolic calcium may mediate the retrograde signal from mitochondria to nucleus. These data are consistent with a model whereby BAT connects its respiratory capacity to thermogenic gene expression, which in turn contributes to determining its metabolic commitment. Additionally, we find that augmented respiratory capacity activates the thermogenic gene program in inguinal (subcutaneous) white adipose tissue (IWAT) from adipose-specific LRPPRC transgenic mice. When fed a high-fat diet at thermoneutrality, these mice exhibit metabolic improvements as shown by reduced fat mass and improved insulin sensitivity. Furthermore, there is increased recruitment of brown-like adipocytes in IWAT and thus energy expenditure is significantly increased, providing a potential explanation for protection from obesity. These data suggest that augmented respiratory capacity promotes ‘browning’ of IWAT, which has beneficial effects on obesity and diabetes.

L’oxygène singulet ($^1O_2$) est la principale espèce réactive de l’oxygène produite dans le chloroplaste lors d'un stress photo-oxydant. L’$^1O_2$ est hautement cytotoxique s'attaquant aux protéines et lipides membranaires. L’$^1O_2$ est une molécule signal impliquée dans une signalisation rétrograde entre les chloroplastes et le noyau via des médiateurs, menant à la mort cellulaire programmée (MCP), ou à l’acclimatation. Nous avons caractérisé l’implication de la phytohormone salicylate, agissant en aval de la signalisation de la MCP dépendante d’OXI1 (OXIDATIVE SIGNAL INDUCIBLE 1) et du jasmonate. Nous avons mis en évidence une voie de régulation négative faisant intervenir des protéines inhibitrices de la MCP : DAD1 et DAD2. La sur-expression de ces protéines permet à la plante d’éviter de la MCP en inhibant la signalisation contrôlée par OXI1. Dans le Réticulum Endoplasmique (RE) a lieu la conformation d’une grande partie des protéines. Une perturbation dans ce compartiment induit l’activation d’une réponse adaptative appelée l’UPR (Unfolded Protein Response). Nous avons monté que la production d’$^1O_2$ active l’UPR. L’acclimatation à la forte lumière (FL) fait intervenir la branche bZIP28/BiP3 de l’UPR. Un pré-traitement modéré d’un inducteur de stress RE (tunicamycine), induisant l'UPR, déclenche une réponse d’acclimatation à l’$^1O_2$ permettant l’évitement de la MCP lors de l’exposition à la FL. Nous avons réalisé un crible génétique pour rechercher des révertants du mutant \textit{ch1} (un surproducteur d’1$^1O_2$) où l’inhibition de la croissance par l’$^1O_2$ est partiellement levée. Les gènes candidats identifiés feront l’objet d’études complémentaires. / Singlet oxygen ($^1O_2$) is a major reactive oxygen species produced within the chloroplasts during high light (HL) stress. $^1O_2$ has a cytotoxic effect due to its high reactivity towards macromolecules including proteins and membrane lipids. $^1O_2$ also acts as a signal molecule that plays a role in chloroplast-to-nucleus retrograde signaling involving mediators and leading either to programmed cell death (PCD) or to stress acclimation.We have shown the involvement of the phytohormone salicylic acid in HL-induced cell death, acting downstream of the OXI1 kinase and jasmonate. We have also shown a negative regulation of this signaling pathway by PCD inhibitory proteins: DAD1 and DAD2 (DEFENDER AGAINST CELL DEATH 1 and 2). Overexpressing those proteins inhibits OXI1-mediated PCD. Protein folding of most secreted proteins takes place in the Endoplasmic Reticulum (ER). Perturbations in this compartment lead to the activation of an adaptive response called UPR (Unfolded Protein Response). When ER stress is too intense, NRPs-mediated ER stress-induced cell death is activated. We have shown that 1O2 production activates UPR. In particular, the bZIP28/BiP3 UPR branch is activated during acclimation to HL. The induction of UPR by a chemical inducer of ER stress (Tunicamycin) can induce acclimation to $^1O_2$ production and can avoid HL-induced PCD.We performed a genetic screen to search for revertants of the $^1O_2$ overproducing \textit{ch1} mutants in which growth inhibition by$^1O_2$2 is partially released. The candidate genes will have to be confirmed by further phenotypic studies.

Basal forebrain cholinergic neurons (BFCNs) are critical for learning and memory. Profound and early BFCN degeneration is a hallmark of aging and Alzheimer’s disease (AD). BFCNs depend for their survival on the retrograde axonal transport of neurotrophins, proteins critical for neuronal function. Neurotrophins like brain derived neurotrophic factor (BDNF) and pro-nerve growth factor (proNGF) are retrogradely transported to BFCNs from their synaptic targets. In AD, neurotrophin levels are increased within BFCN target areas and reduced in the basal forebrain, implicating dysfunctional neurotrophin transport in AD pathogenesis. However, neurotrophin transport within this highly susceptible neuronal population is currently poorly understood. We began by establishing protocols for the accurate quantification of axonal transport in BFCNs using microfluidic culture. We then determined the effect of age on neurotrophin transport. BFCNs were left in culture for up to 3 weeks to model aging in vitro. BFCNs initially displayed robust neurotrophin transport, which diminished with in vitro age. We observed that the levels of proNGF receptor tropomyosin-related kinase-A (TrkA) were reduced in aged neurons. Additionally, neurotrophin transport in BFCNs derived from 3xTg-AD mice, an AD model, was also impaired. Next, we sought to determine a mechanism for these transport deficits. First, we determined that proNGF transport was solely contingent upon the levels of TrkA. We then found that elevation of oxidative stress, an established AD contributor, significantly reduced both TrkA levels and proNGF retrograde transport. TrkA levels are partially regulated by protein tyrosine phosphatase-1B (PTP1B), an enzyme whose activity is reduced by oxidation. PTP1B antagonism significantly reduced TrkA levels and proNGF retrograde transport in BFCNs. Treatment of BFCNs with PTP1B-activating antioxidants rescued TrkA levels, proNGF transport, and proNGF-mediated axonal degeneration. Our results suggest that oxidative stress contributes to BFCN degeneration in aging and AD by impairing retrograde neurotrophin transport via oxidative PTP1B-mediated TrkA loss. / Thesis / Doctor of Philosophy (PhD) / During aging and Alzheimer’s disease (AD), the connections between neurons, a type of brain cell, break down, causing memory loss. This breakdown begins in a brain area called the basal forebrain. Basal forebrain neurons rely upon the transport of nutrients along their connections with other neurons, called axons, for proper function. This transport process becomes impaired in AD. Our goal was to understand why this happens. First, we determined that axonal transport was impaired with age and in basal forebrain neurons of mice genetically predisposed to develop AD. We recreated these impairments by increasing the levels of harmful molecules called reactive oxidative species (ROS). ROS levels increase with age and become abnormally high during AD. We found that increased ROS impair axonal transport and contribute to the breakdown of basal forebrain neurons. Our work suggests that reducing ROS will help prevent the breakdown of basal forebrain neurons in AD.

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

[pt] Em reservatórios de gás retrógrado com pressões inferiores à pressão de orvalho, a produtividade dos poços pode ser comprometida devido ao aparecimento e acúmulo da fase líquida nas suas imediações. Este fenômeno é conhecido como bloqueio por condensação retrógrada e está associado à uma série de desafios para compreendê-lo. Um deles é a determinação da permeabilidade relativa das fases líquida e gasosa, que comumente advém de curvas de permeabilidade obtidas a partir da extrapolação de poucos dados experimentais. Dessa forma, elas tendem a não representar fielmente efeitos importantes para o escoamento, comprometendo a precisão da modelagem do fluxo no meio poroso. A fim de investigar o efeito das curvas de permeabilidade relativa sobre a formação de bancos de condensado, foi desenvolvido um modelo composicional em escala de reservatório para o estudo do escoamento de gás e condensado. Com o modelo, o uso de curvas de permeabilidade relativa obtidas através de simulação do escoamento de gás retrógrado na escala de poros e de correlações propostas na literatura foi avaliado. Considera-se: sistema isotérmico, escoamento bifásico e incorporação dos efeitos de forças capilares por meio do modelo de permeabilidade relativa. Equações de balanço molar e consistência de volume formam um sistema não linear resolvido pelo método de Newton que fornece pressão e número de mols de cada componente, em todos os volumes de controle do modelo, a cada passo de tempo. Para o equilíbrio de fases, a equação de Peng & Robinson foi implementada com uma rotina de flash a pressão e temperaturas constantes. O modelo foi validado contra a solução analítica para sistema monofásico e por fim, o simulador obteve a evolução temporal das curvas de pressão e saturação em função da distância do poço, a fim de comparar o efeito dos diferentes modelos de curvas de permeabilidade relativa na predição do bloqueio por condensado. Os resultados foram obtidos variando-se a permeabilidade absoluta do meio e a vazão de gás imposta no poço, e o impacto desses parâmetros no acúmulo de condensado foi avaliado. / [en] In gas-condensate reservoirs with pressures below the dew pressure, the productivity of wells can be compromised due to the accumulation of liquid in their surroundings. This phenomenon is known as condensate blockage and there are many challenges to understanding the formation of condensate banks. One of them is the determination of the relative permeability of the liquid and gas phases, which commonly comes from permeability curves obtained from the extrapolation of few experimental data. Thus, they tend not to reliably represent important effects for the flow, compromising the precision of the flow modeling in the porous medium. In order to investigate the effect of relative permeability curves on the formation of condensate banks, a reservoir-scale compositional model was developed for the study of flow of gas and condensate. With the model, the use of relative permeability curves obtained by simulating the gas-condensate flow at the pore-scale and with correlations proposed in the literature was evaluated. It is considered: isothermal system, two-phase flow and incorporation of capillary force effects through the relative permeability model. Molar balance and volume consistency equations form a nonlinear system solved by Newton s method that provides pressure and number of moles of each component, in all control volumes of the model, at each time step. For the phase equilibrium, calculations of the Peng & Robinson equation was implemented in a constant pressure and temperature flash routine. The model was validated against the analytical solution for single-phase flow and, finally, the simulator obtained the temporal evolution of the pressure and saturation as a function of the distance from the well, in order to compare the effect of different models of relative permeability curves in the prediction of condensate blockage. The results were obtained by varying the absolute permeability of the medium and the gas flow imposed in the well, and the impact of these parameters on the accumulation of condensate was evaluated.

<p> The recent discovery of ore grade gold in the Hemlo area of Northern Ontario has provided the initiative for extensive exploration in that region. This study concentrates on rocks from the footwall to the ore zone on the Williams property of Long Lac Mineral Exploration. </p> <p> The footwall rocks consist of fine grained, finely laminated tuffs. The suite has undergone low grade regional metamorphism. Retrograde metamorphic effects are also present but appear to be confined to certain bands. Some of the minerals may have been introduced by the action of metasomatic fluids. </p> <p> The tuffs are quite felsic and many have the composition of a rhyolite. Samples tested for gold have up to 200 times the background concentration of a rhyolite . The gold is associated with high Sio2 and K20 and low Al2O3, CaO, Na2O, Fe2O3 and MgO contents. </p> <p> The ore deposit is similar in many respects to the Bousquet deposit of Northern Quebec. The Bousquet deposit has been interpreted as syngenetic by some authors and therefore it is quite possible that the Hemlo deposit is syngenetic as well. However the metamorphism and possible metasomatism that these rocks have undergone suggests that it may be an epigenetic deposit. Due to the limited number of samples in this study it could not conclusively be determined which hypothesis is correct. </p> / Thesis / Bachelor of Science (BSc)

Among the major phytohormones, the cytokinin exhibits unique features for its ability to positively affect the developmental status of plastids. Even early on in its research, cytokinins were known to promote plastid differentiation and to reduce the loss of chlorophyll in detached leaves. Since the discovery of the components of cytokinin perception and primary signaling, the genes involved in photosynthesis and plastid differentiation have been identified as those directly targeted by type-B response regulators. Furthermore, cytokinins are known to modulate versatile cellular processes such as promoting the division and differentiation of cells and, in concert with auxin, initiating the de novo formation of shoot apical meristem (SAM) in tissue cultures. Yet how cytokinins precisely participate in such diverse cellular phenomena, and how the associated cellular processes are coordinated as a whole, remains unclear. A plausible presumption that would account for the coordinated gene expression is the tight and reciprocal communication between the nucleus and plastid. The fact that cytokinins affect plastid developmental status via gene expression in both the nucleus and plastid is interpreted here to suggest that cytokinin functions as an initiator of anterograde (nucleus-to-plastid) signaling. Based on this viewpoint, we first summarize the physiological relevance of cytokinins to the coordination of plastid differentiation with de novo shoot organogenesis in tissue culture systems. Next, the role of endogenous cytokinins in influencing plastid differentiation within the SAM of intact plants is discussed. Finally, a presumed plastid-derived signal in response to cytokinins for coupled nuclear gene expression is proposed.

No description available.

La progression d’un individu au travers d’un environnement diversifié dépend des informations visuelles qui lui permettent d’évaluer la taille, la forme ou même la distance et le temps de contact avec les obstacles dans son chemin. Il peut ainsi planifier en avance les modifications nécessaires de son patron locomoteur afin d’éviter ou enjamber ces entraves. Ce concept est aussi applicable lorsque le sujet doit atteindre une cible, comme un prédateur tentant d’attraper sa proie en pleine course. Les structures neurales impliquées dans la genèse des modifications volontaires de mouvements locomoteurs ont été largement étudiées, mais relativement peu d’information est présentement disponible sur les processus intégrant l’information visuelle afin de planifier ces mouvements. De nombreux travaux chez le primate suggèrent que le cortex pariétal postérieur (CPP) semble jouer un rôle important dans la préparation et l’exécution de mouvements d’atteinte visuellement guidés. Dans cette thèse, nous avons investigué la proposition que le CPP participe similairement dans la planification et le contrôle de la locomotion sous guidage visuel chez le chat. Dans notre première étude, nous avons examiné l’étendue des connexions cortico-corticales entre le CPP et les aires motrices plus frontales, particulièrement le cortex moteur, à l’aide d’injections de traceurs fluorescents rétrogrades. Nous avons cartographié la surface du cortex moteur de chats anesthésiés afin d’identifier les représentations somatotopiques distales et proximales du membre antérieur dans la partie rostrale du cortex moteur, la représentation du membre antérieur située dans la partie caudale de l’aire motrice, et enfin la représentation du membre postérieur. L’injection de différents traceurs rétrogrades dans deux régions motrices sélectionnées par chat nous a permis de visualiser la densité des projections divergentes et convergentes pariétales, dirigées vers ces sites moteurs. Notre analyse a révélé une organisation topographique distincte de connexions du CPP avec toutes les régions motrices identifiées. En particulier, nous avons noté que la représentation caudale du membre antérieur reçoit majoritairement des projections du côté rostral du sillon pariétal, tandis que la partie caudale du CPP projette fortement vers la représentation rostrale du membre antérieur. Cette dernière observation est particulièrement intéressante, parce que le côté caudal du sillon pariétal reçoit de nombreux inputs visuels et sa cible principale, la région motrice rostrale, est bien connue pour être impliquée dans les fonctions motrices volontaires. Ainsi, cette étude anatomique suggère que le CPP, au travers de connexions étendues avec les différentes régions somatotopiques du cortex moteur, pourrait participer à l’élaboration d’un substrat neural idéal pour des processus tels que la coordination inter-membre, intra-membre et aussi la modulation de mouvements volontaires sous guidage visuel. Notre deuxième étude a testé l’hypothèse que le CPP participe dans la modulation et la planification de la locomotion visuellement guidée chez le chat. En nous référant à la cartographie corticale obtenue dans nos travaux anatomiques, nous avons enregistré l’activité de neurones pariétaux, situés dans les portions des aires 5a et 5b qui ont de fortes connexions avec les régions motrices impliquées dans les mouvements de la patte antérieure. Ces enregistrements ont été effectués pendant une tâche de locomotion qui requiert l’enjambement d’obstacles de différentes tailles. En dissociant la vitesse des obstacles de celle du tapis sur lequel le chat marche, notre protocole expérimental nous a aussi permit de mettre plus d’emphase sur l’importance de l’information visuelle et de la séparer de l’influx proprioceptif généré pendant la locomotion. Nos enregistrements ont révélé deux groupes de cellules pariétales activées en relation avec l’enjambement de l’obstacle: une population, principalement située dans l’aire 5a, qui décharge seulement pendant le passage du membre au dessus del’entrave (cellules spécifiques au mouvement) et une autre, surtout localisée dans l’aire 5b, qui est activée au moins un cycle de marche avant l’enjambement (cellules anticipatrices). De plus, nous avons observé que l’activité de ces groupes neuronaux, particulièrement les cellules anticipatrices, était amplifiée lorsque la vitesse des obstacles était dissociée de celle du tapis roulant, démontrant l’importance grandissante de la vision lorsque la tâche devient plus difficile. Enfin, un grand nombre des cellules activées spécifiquement pendant l’enjambement démontraient une corrélation soutenue de leur activité avec le membre controlatéral, même s’il ne menait pas dans le mouvement (cellules unilatérales). Inversement, nous avons noté que la majorité des cellules anticipatrices avaient plutôt tendance à maintenir leur décharge en phase avec l’activité musculaire du premier membre à enjamber l’obstacle, indépendamment de sa position par rapport au site d’enregistrement (cellules bilatérales). Nous suggérons que cette disparité additionnelle démontre une fonction diversifiée de l’activité du CPP. Par exemple, les cellules unilatérales pourraient moduler le mouvement du membre controlatéral au-dessus de l’obstacle, qu’il mène ou suive dans l’ordre d’enjambement, tandis que les neurones bilatéraux sembleraient plutôt spécifier le type de mouvement volontaire requis pour éviter l’entrave. Ensembles, nos observations indiquent que le CPP a le potentiel de moduler l’activité des centres moteurs au travers de réseaux corticaux étendus et contribue à différents aspects de la locomotion sous guidage visuel, notamment l’initiation et l’ajustement de mouvements volontaires des membres antérieurs, mais aussi la planification de ces actions afin d’adapter la progression de l’individu au travers d’un environnement complexe. / When progressing through a varied environment, an individual will depend on visual information to evaluate the size, shape or the distance and time to contact of objects in his path. This will allow him to plan in advance the gait requirements necessary to avoid or step over these obstacles. This concept is also applicable in situations where the subject must reach a target, as with a predator chasing down its prey. The neural structures involved in generating voluntary gait modifications during locomotion have been extensively studied, but relatively little information is available on the processes that integrate visual information to plan these movements. Numerous studies in the primate suggest that the posterior parietal cortex (PPC) plays an important role in the preparation and execution of visually-guided reaching movements. In this thesis, we investigated the proposition that the PPC is similarly involved in the planning and control of visually-guided locomotion in the cat. Our first study examined the extent of cortico-cortical connections between the PPC and the more frontal motor areas, particularly the motor cortex, using injections of fluorescent retrograde tracers. We mapped the cortical surface of anaesthetized cats to identify the somatotopical representations of the distal and proximal forelimb in the rostral portion of the motor cortex, the forelimb representation in the caudal motor area, and also the hindlimb representation. The injection of different tracers in two selected regions, for every cat, allowed us to visualize the density of divergent and convergent parietal projections to these motor sites. Our analysis revealed a distinct topographical organization of parietal connections with all of the identified motor regions. In particular, the caudal motor representation of the forelimb primarily received projections from the rostral bank of the parietal cortex, while the caudal portion of the PPC strongly projected to the rostral forelimb representation. The latter observation is particularly interesting, since the caudal bank of the PPC receives numerous visual inputs and its target, the rostral motor region, is well-known for its involvement in voluntary motor functions. Therefore, this study suggests that the PPC, through extensive connections with the different somatotopic representations of the motor cortex, could constitute an ideal neural substrate for processes such as inter- and intra-limb coordination, as well as the modulation of visually-guided voluntary movements. Our second study tested the hypothesis that the PPC participates in the modulation and planning of voluntary gait modifications during locomotion in the cat. Using the cortical mapping established in our anatomical study, we recorded the activity of parietal neurons, localized in parts of areas 5a and 5b which are known to project strongly towards motor regions involved in forelimb movements. These recordings were obtained during a locomotion task requiring the cat to step over several obstacles of different sizes. By dissociating the speed of the obstacles from that of the treadmill onto which the cat is walking, our experimental protocol also allows us to increase the importance of visual information from the obstacles and to separate it from the influx of proprioceptive influx generated during locomotion. Our recordings revealed two groups of parietal cells on the basis of their activity in relation with the step over the obstacle: one population, mostly localized in area 5a, discharged solely as the lead forelimb passed over the obstacle (step-related cells), and another group, mainly found in area 5b, that showed significant activity at least one step cycle before the gait modification (step-advanced cells). Additionally, we observed an increase of cell activity in these groups, but particularly in step-advanced cells, when the speed of the obstacles was dissociated from that of the treadmill, demonstrating the growing importance of visual information as the task’s difficulty is increased. Finally, a great number of step-related cells were found to discharge specifically in correlation with muscle activity in the contralateral forelimb, regardless of whether or not it led over the obstacle (limb-specific cells). Inversely, the majority of step-advanced neurons tended to maintain their discharge in phase with the leading limb during the gait modification, independently of its position in relation with the recording site (limb-independent cells). We suggest that this additional disparity indicates diversified functions in PPC activity. For example, limb-specific cells could be involved in modulating the movement of the contralateral forelimb over the obstacle, regardless of its order of passage, while limb-independent neurons could instead specify the type of voluntary movement required to overcome the obstacle. Together, our observations indicate that the PPC can potentially influence the activity of motor centers through extensive cortical networks, and contributes to different aspects of visually-guided locomotion, such as initiation and modulation of voluntary forelimb movements, as well as the planning of these gait modifications to allow an individual to walk through a complex environment.

<p>Акутни панкреатитис је ензиматско инфламаторно оболење панкреаса, са инциденцијом око 17/100000 становника. Најчешћи етиолошки чиниоци који се везују за ово стање су билијарна калкулоза (45%) и конзумација алкохолних пића (35%). Ређи узроци су одређени лекови, хипертриглицеридемија, хиперкалијемија, траума, урођени чиниоци, и идиопатски панкреатитис (20%). По међународној Атланској (Atlanta) класификацији, акутни панкреатитис се може манифестовати у умереној, умерено тешкој и тешкој форми. Умерене форме панкреатитиса се јављају у 80%, карактеришу се едемом органа и имају благ и краткотрајан клинички ток, са стопом морталитета од 1%. Основни циљ лечења болесника са акутним панкреатитисом у прва 24 сата хоспитализације јесте олакшавање тегоба, утврђивање узрока панкреатитиса и процена тежине обољења. Акутни панкреатитис билијарне етиологије узрокован је калкулозом жучне кесе и/ или жучних путева. Препоручени третман билијарне калкулозе, у циљу превенције поновног атака умерене форме билијарног панкреатитиса , подразумева уклањање жучне кесе лапароскопском холецистектомијом са интраоперативном холангиографијом. Уколико се дијагностикује калкулоза жучних канала ради се ендоскопска ретроградна холангиопанкреатографија (ЕРЦП) са ендоскопском папилотомијом (ЕПТ) и уклањем__ калкулуса и детритуса уз жучних водова, са циљем обезбеђивања нормалног протока жучи у дванаестопалачно црево. Циљ овог истраживања је оптимализација редоследа примене ЛХ и ЕРЦП са ЕПТ, идентификацијом предикционих показатеља холедохолитијазе, ради скраћења дужине хоспитализације болесника са умереном формом акутног билијарног панкреатитиса. У спроведеној проспективној анамнестичкој студији, учествовало је 100 болесника лечених од умерене форме акутног билијарног панкреатитиса, у Ургентном центру Клиничког центра Војводине, од 2011. до 2015.године, од којих је код 80 урађена само ЛХ, а код 20 ЛХ и ЕРЦП са ЕПТ. Анализом клиничких, ултразвучних и лабораторијских налаза, идентификовано је 5 статистички значајних предиктора холедохолитијазе; директни и укупни билирубин, алкална фосфатаза (АФ), гама глутирил транспепдидаза (гама ГТ) и це реактивни протеин (ЦРП), на основу којих је омогућено креирање математичког модела за предикцију холедохолитијазе, коришћењем теорије потпорних вектора (СВМ). Установљено је да патолошки налази ових параметара значајно указују на холедохолитијазу, те да је ЛХ препоручена као метода првог избора, код болесника код којих налази предиктора холедохолитијазе нису патолошки. Овако лечени болесници су имали значајно краће време хоспитализације. Поред тога, уколико се интраоперативном холангиографијом (ИОХ) при ЛХ установи холедохолитијаза, ЕРЦП са ЕПТ се може урадити без одлагања.</p> / <p>Akutni pankreatitis je enzimatsko inflamatorno obolenje pankreasa, sa incidencijom oko 17/100000 stanovnika. Najčešći etiološki činioci koji se vezuju za ovo stanje su bilijarna kalkuloza (45%) i konzumacija alkoholnih pića (35%). Ređi uzroci su određeni lekovi, hipertrigliceridemija, hiperkalijemija, trauma, urođeni činioci, i idiopatski pankreatitis (20%). Po međunarodnoj Atlanskoj (Atlanta) klasifikaciji, akutni pankreatitis se može manifestovati u umerenoj, umereno teškoj i teškoj formi. Umerene forme pankreatitisa se javljaju u 80%, karakterišu se edemom organa i imaju blag i kratkotrajan klinički tok, sa stopom mortaliteta od 1%. Osnovni cilj lečenja bolesnika sa akutnim pankreatitisom u prva 24 sata hospitalizacije jeste olakšavanje tegoba, utvrđivanje uzroka pankreatitisa i procena težine oboljenja. Akutni pankreatitis bilijarne etiologije uzrokovan je kalkulozom žučne kese i/ ili žučnih puteva. Preporučeni tretman bilijarne kalkuloze, u cilju prevencije ponovnog ataka umerene forme bilijarnog pankreatitisa , podrazumeva uklanjanje žučne kese laparoskopskom holecistektomijom sa intraoperativnom holangiografijom. Ukoliko se dijagnostikuje kalkuloza žučnih kanala radi se endoskopska retrogradna holangiopankreatografija (ERCP) sa endoskopskom papilotomijom (EPT) i uklanjem__ kalkulusa i detritusa uz žučnih vodova, sa ciljem obezbeđivanja normalnog protoka žuči u dvanaestopalačno crevo. Cilj ovog istraživanja je optimalizacija redosleda primene LH i ERCP sa EPT, identifikacijom predikcionih pokazatelja holedoholitijaze, radi skraćenja dužine hospitalizacije bolesnika sa umerenom formom akutnog bilijarnog pankreatitisa. U sprovedenoj prospektivnoj anamnestičkoj studiji, učestvovalo je 100 bolesnika lečenih od umerene forme akutnog bilijarnog pankreatitisa, u Urgentnom centru Kliničkog centra Vojvodine, od 2011. do 2015.godine, od kojih je kod 80 urađena samo LH, a kod 20 LH i ERCP sa EPT. Analizom kliničkih, ultrazvučnih i laboratorijskih nalaza, identifikovano je 5 statistički značajnih prediktora holedoholitijaze; direktni i ukupni bilirubin, alkalna fosfataza (AF), gama glutiril transpepdidaza (gama GT) i ce reaktivni protein (CRP), na osnovu kojih je omogućeno kreiranje matematičkog modela za predikciju holedoholitijaze, korišćenjem teorije potpornih vektora (SVM). Ustanovljeno je da patološki nalazi ovih parametara značajno ukazuju na holedoholitijazu, te da je LH preporučena kao metoda prvog izbora, kod bolesnika kod kojih nalazi prediktora holedoholitijaze nisu patološki. Ovako lečeni bolesnici su imali značajno kraće vreme hospitalizacije. Pored toga, ukoliko se intraoperativnom holangiografijom (IOH) pri LH ustanovi holedoholitijaza, ERCP sa EPT se može uraditi bez odlaganja.</p> / <p>Acute pancreatitis is an enzymatic inflammatory disease of the pancreas, with an incidence of around 17/100000 inhabitants. The most common etiological factors that are associated with this condition are biliary calculi (45%) and consumption of alcoholic beverages (35%). Less common causes include certain medications, hypertriglyceridemia, hyperkalemia, trauma, congenital factors and idiopathic pancreatitis (20%). According to the Atlanta International classification, acute pancreatitis can be manifested in a moderate, moderately severe and severe forms. Moderate forms of pancreatitis occur in 80%, characterized by pancreatic edema and have mild and short clinical course, with a mortality rate of 1%. The main goal of treatment of patients with acute pancreatitis in the first 24 hours of hospitalization is to facilitate complaints, determining the cause of pancreatitis and assessment of severity of the disease. Acute biliary pancreatitis is caused by calculosis of the gallbladder and / or bile ducts. The recommended treatment of biliary calculi, in order to prevent repeated attacks of moderate biliary pancreatitis, involves the removal of the gallbladder thru laparoscopic cholecystectomy with intraoperative cholangiography. If presence of bile duct calculi is established, an endoscopic retrograde cholangiopancreatography (ERCP) with endoscopic papillotomy (EPT) and removes stones and detritus along the bile ducts is indicated, with the aim of ensuring the normal flow of bile into the duodenum. The aim of this study is the optimization of the order of application LH and ERCP with EPT, the identification of predictable indicators of choledocholithiasis, in order to shorten the length of hospitalization of patients with a moderate form of acute biliary pancreatitis. We conducted prospective case control study, with 100 patients involved, treated for moderate forms of acute biliary pancreatitis in the Emergency Center of the Clinical Center of Vojvodina, from 2011 to 2015, of which 80 made only with LH and 20 with LH at and ERCP with EPT . The analysis of clinical, ultrasound and laboratory findings identified 5 significant predictors of choledocholithiasis; direct and total bilirubin, alkaline phosphatase (AF), gamma glutiril transpepdidase (gamma GT) and C reactive protein (CRP), under which enabled the creation of a mathematical model for predicting choledocholithiasis, using the Support vector machines (SVM). It was found that pathological findings of these parameters indicate a significant choledocholithiasis, and LH is recommended as the first choice in patients in whom there are not present pathological predictors of choledocholithiasis. Thus treated patients had a significantly shorter hospital stay. In addition, if the intraoperative cholangiography (IOH) during LH show choledocholithiasis, ERCP with the EPT can be done without delay.</p>