Cutaneous melanoma is a very aggressive cancer with increasing incidence. It originates from transformed pigmented skin cells (melanocytes). The main risk factor for melanoma development is exposure to UV light and repeated sunburns. In approximately 10 % of cases, melanoma occurs on hereditary basis. Patients with cutaneous melanoma diagnosed in early stages have very good prognosis, with surgical resection of the primary tumour being mostly sufficient for treatment. In contrast, the advanced melanoma stages with metastases are often progressive and refractory to conventional therapies. Cutaneous melanoma is referred to as an immunogenic tumour that is frequently infiltrated by cells of the immune system. Tumours with immune cell infiltration show better prognosis. Spontaneous regression may occur. Over the last few years, progress has been made in the treatment of melanoma using checkpoints molecules (anti-CTLA-4 and anti-PD-1) to activate patients own immune system to recognize tumour lesions. In the tumour microenvironment, cytokines play an important role, enabling communication between cells and regulation of cell proliferation and migration and thus the tumour development. Cytokines (IL-2, IFNα) can be used in adjuvant therapy of melanoma. This work analysed levels of expressed cytokines in...

Extracorporeal membrane oxygenation (ECMO) is a method that allows extracorporeal life support in potentially reversible life-threatening conditions affecting the heart or lungs which are refractory to conventional treatment. Depending on the parameters of its setting, this method affects the haemodynamics of the cardiovascular system and the perfusion of the target organ. From the point of view of its character, the necessity for invasive application, and the function thereof in the conditions of the cardiovascular system, ECMO is regarded as a risky method accompanied by a number of complications. Among the critical complications are thromboembolic complications affecting the central nervous system (CNS) and haemorrhagic complications. The goal of this paper is to present and verify the prerequisites for the formation of periprocedural embolisms affecting the CNS and to evaluate the regional haemodynamics of the CNS. This is done by analysing the presence of embolisms and by analysing the parameters of blood flow rates in the right common carotid artery (arteria carotis communis-ACC) and the corresponding oxygenation of the brain tissue during various flow rate parameters generated by the ECMO support on induced heart failure in a biological porcine model. In the first section of the paper, 8...

The purpose of this dissertation was to study the effects of graft pretension in anterior cruciate ligament (ACL) reconstruction through a series of experiments. First, an in vitro study of 5 human knees was conducted to determine if intact joint kinematics could be restored when using the ideal graft - the intrinsic ACL. The ACL tibial insertion site was freed, and pretensions of 0, 10, 20, 30, and 40 N were applied to the ligament using a custom designed load cell connection. Kinematics during a simulated active extension were compared to those of the intact knee. Intact knee kinematics were not restored. Pretensions that best restored tibial anterior/posterior translation and internal/external rotation ranged from 0-40 N. Furthermore, the pretensions that best restored these kinematic variables were widely disparate in two specimens. Second, the in vitro kinematics during a simulated active extension of human and porcine knees were compared and contrasted both prior to and following transection of the ACL. The ACL limited: (1) tibial anterior translation in both species, (2) tibial internal rotation in humans, and (3) tibial external rotation in pigs. Differences in kinematic patterns for tibial internal/external rotation and abduction/adduction between the species was explained by requirements for biped and quadruped stances. Third, the mechanical characteristics of porcine patellar tendon (PT) were investigated by uniaxial tensile testing at two strain rates. Patella-PT-tibia complexes from freshly sacrificed skeletally immature and mature animals were loaded to failure at elongation rates of 20 and 200 mm/min. Both strain rate and skeletal maturity significantly affected failure mode, tangent modulus, and ultimate stress of the tendons, and hence are important considerations in the mechanical evaluation of porcine PT. Fourth, ACL reconstructions were performed using pretensions of 10 or 20 N in an in vivo porcine model with a specially designed load cell/telemetry system to monitor graft load. Graft pretension was seen to increase during fixation with interference screws. Following sacrifice at 4 weeks, tissues were mechanically, histologically, and biochemically analyzed. A pretension of 20 N resulted in a tissue more similar to the intrinsic ACL. / Ph. D.

L’échoendoscopie thérapeutique occupe une place croissante et incontournable de l’activité médico-chirurgicale en pathologie digestive depuis le développement des sondes sectorielles. Les domaines d’application de cette technique interventionnelle mini-invasive sont de plus en plus nombreux, d’une part en pathologie biliopancréatique et entérale permettant la réalisation d’anastomoses évitant un recours à la chirurgie traditionnelle, et d’autre part en cancérologie digestive car elle permet de délivrer un agent thérapeutique, une source de rayonnement ou des cellules liées au système immunitaire dans une lésion ciblée. L’abord vasculaire est une voie récente et prometteuse de l’échoendoscopie interventionnelle offrant des perspectives thérapeutiques en hépatologie notamment dans le domaine de l’hypertension portale. Ce travail se décompose en trois parties décrivant successivement l’état de l'art des applications de l’échoendoscopie interventionnelle, puis deux travaux originaux expérimentaux : dans un premier temps, le transfert de la technique d’anastomose biliodigestive (réalisée régulièrement dans notre centre) à l’abord vasculaire portosystémique sur animal sain, puis dans un deuxième temps la validité de la technique sur modèle animal de fibrose hépatique. La première série d’expérimentations a permis de mettre au point la technique de création d’un shunt intrahépatique portosystémique par échoendoscopie chez 23 cochons sains, en collaboration avec l’équipe de radiologie vasculaire. Ces procédures ont été réalisées dans une salle de cathétérisme vasculaire expérimentale (laboratoire Caviti) dans le cadre du laboratoire d’accueil ISIT (Image Science for Interventional Techniques, Pr. L. Boyer, Pr. J.-Y. Boire, UMR CNRS/UDA 6284). Les résultats ont été concluants puisque le shunt est apparu faisable dans 91% des cas, fonctionnel dans 81% des cas avec une morbidité de 14,2%. La deuxième série d’expérimentations a permis de valider cette technique d’échoendoscopie chez un modèle porcin de fibrose hépatique par embolisation radiologique, en collaboration avec l’Institut National de Recherche Agroalimentaire de Theix pour la stabulation des animaux. Ce travail multidisciplinaire a permis une collaboration étroite entre endoscopistes, radiologues, chirurgiens viscéraux et anatomopathologistes, s’inscrivant dans une démarche de recherche transversale. Les résultats sont encourageants puisqu’un shunt intrahépatique porto-systémique par échoendoscopie s’est révélé techniquement faisable et fonctionnel sur modèle de fibrose hépatique avec une survie à 7 jours des animaux dans deux tiers des cas. Avant d’envisager une étude princeps chez l’homme, des études complémentaires sont néanmoins nécessaires sur des modèles porcins présentant une fibrose hépatique associée à une hypertension portale en augmentant la durée de stabulation, en homogénéisant la procédure de squelettisation de l’artère hépatique et en adaptant la dose de solution injectée. Cette technique pourrait constituer à terme une alternative en cas d’échec de la technique standard pour complications de l’hypertension portale (shunt intrahépatique portosystémique par voie transjugulaire) chez des patients alors en situation d’impasse thérapeutique. / Therapeutic endoscopic ultrasonography is a growing and essential part of the medical and surgical activity in digestive pathology since the development of sectoral probes. The fields of application of this minimally invasive interventional technique are more and more numerous, on the one hand in biliopancreatic and enteral pathology, allowing the realization of anastomoses without traditional surgery, and on the other hand in digestive oncology, because it makes it possible to deliver a therapeutic agent, a source of radiation or cells linked to the immune system in a targeted lesion. The vascular approach is a recent and promising way of interventional endoscopic ultrasound offering therapeutic perspectives in hepatology, especially in the field of portal hypertension.This work is divided into three parts successively describing the state of the art of the applications of interventional ultrasonoscopy, then two original experimental works: initially, the transfer of the technique of biliodigestive anastomosis (performed regularly in our center ) at the portosystemic vascular approach on healthy animal, then in a second time the validity of the animal model technique of liver fibrosis.The first series of experiments led to the development of the technique for the creation of an intrahepatic shunt portosystemic by echoendoscopy in 23 healthy pigs, in collaboration with the vascular radiology team. These procedures were performed in an experimental vascular catheterization room (Caviti laboratory) as part of the ISIT host laboratory (Image Science for Interventional Techniques, Prof. L. Boyer, Pr. J.-Y.Boire, UMR CNRS / UDA 6284). The results were conclusive since the shunt appeared feasible in 91% of cases, functional in 81% of cases with a morbidity of 14.2%. The second series of experiments validated this endoscopic ultrasound technique in a porcine model of liver fibrosis by X-ray embolization, in collaboration with Theix National Institute for Food Research for Animal Stabling. This multidisciplinary work has allowed close collaboration between endoscopists, radiologists, visceral surgeons and anatomopathologists, as part of a transversal research approach. The results are encouraging since a porto-systemic intrahepatic shunt by endoscopic ultrasonography proved to be technically feasible and functional on a hepatic fibrosis model with a 7-day survival of the animals in two-thirds of the cases. Before considering a primary study in humans, additional studies are nevertheless necessary on porcine models presenting hepatic fibrosis associated with portal hypertension by increasing the duration of stabulation, by homogenizing the procedure of skeletonization of the hepatic artery and by adjusting the dose of injected solution. This technique could eventually be an alternative in case of failure of the standard technique for complications of portal hypertension (intrahepatic portosystemic shunt transjugular) in patients then in a situation of therapeutic impasse.

Doctor of Philosophy / Department of Anatomy and Physiology / Deryl L. Troyer / Many cell types were known to have migratory properties towards tumors and different research groups have shown reliable results regarding cells as delivery vehicles of therapeutics for targeted cancer treatment. Present report discusses proof of concept for 1. Cell mediated delivery of Magnetic nanoparticles (MNPs) and targeted Magnetic hyperthermia (MHT) as a cancer treatment by using in vivo mouse cancer models, 2. Cells surface engineering with chimeric proteins for targeted cancer treatment by using in vitro models. 1. Tumor homing cells can carry MNPs specifically to the tumor site and tumor burden will decrease after alternating magnetic field (AMF) exposure. To test this hypothesis, first we loaded Fe/Fe3O4 bi-magnetic NPs into neural progenitor cells (NPCs), which were previously shown to migrate towards melanoma tumors. We observed that NPCs loaded with MNPs travel to subcutaneous melanoma tumors. After alternating magnetic field (AMF) exposure, the targeted delivery of MNPs by the NPCs resulted in a mild decrease in tumor size (Chapter-2). Monocytes/macrophages (Mo/Ma) are known to infiltrate tumor sites, and also have phagocytic activity which can increase their uptake of MNPs. To test Mo/Ma-mediated MHT we transplanted Mo/Ma loaded with MNPs into a mouse model of pancreatic peritoneal carcinomatosis. We observed that MNP-loaded Mo/Ma infiltrated pancreatic tumors and, after AMF treatment, significantly prolonged the lives of mice bearing disseminated intraperitoneal pancreatic tumors (Chapter-3). 2. Targeted cancer treatment could be achieved by engineering tumor homing cell surfaces with tumor proteases cleavable, cancer cell specific recombinant therapeutic proteins. To test this, Urokinase and Calpain (tumor specific proteases) cleavable; prostate cancer cell (CaP) specific (CaP1 targeting peptide); apoptosis inducible (Caspase3 V266ED3)- rCasp3V266ED3 chimeric protein was designed in silico. Hypothesized membrane anchored chimeric protein (rCasp3V266ED3, rMcherry red) plasmids were constructed. Membrane anchoring and activity of designed proteins were analyzed in RAW264.7 Mo/Ma and HEK293 cells in vitro. Further, Urokinase (uPA) mediated cleavage and release of rCasp3V266ED3 from engineered cells was tested (Chapter-4). Animal models for cancer therapy are invaluable for preclinical testing of potential cancer treatments. Final chapter of present report shows evidence for immune-deficient line of pigs as a model for human cancers (Chapter-5)

La méthode de préservation d’organes la plus utilisée actuellement en transplantation rénale est la conservation statique en hypothermie. Cependant, ce mode de conservation induit des dommages inhérents aux lésions du syndrome d’ischémie/reperfusion (I/R). Cette étude a eu pour objectif d’identifier de nouvelles conditions de préservation des greffons, afin de limiter les lésions d’I/R, en modulant la température de conservation ou par ajout d’un transporteur d’oxygène. Nous avons utilisé deux modèles : in vitro avec des cellules endothéliales et in vivo en autotransplantation rénale chez le porc.Les résultats ont confirmé les effets délétères de la conservation à 4°C contrairement à des conservations à 19°C, 27°C et surtout 32°C, permettant d’obtenir une activité métabolique, une viabilité et une intégrité cellulaire supérieures ainsi qu’une diminution des marqueurs de l’inflammation et du stress oxydant. Nous avons aussi démontré les bénéfices d'un nouveau transporteur d’oxygène, M101, dans deux des solutions de conservation les plus utilisés, UW et HTK. L'utilisation de M101 en conservation statique permet une meilleure reprise de fonction à court terme et une réduction de la fibrose, cause principale de la perte du greffon. Enfin, nous avons montré une conservation des bénéfices de M101 à des doses réduites et déterminé que cette protection était due à une multifonctionnalité de la molécule, combinant un transporteur d’oxygène, une activité superoxyde dismutase et une taille importante (permettant de réguler la pression oncotique). Ce travail a montré de nouvelles pistes de réflexion vers une préservation, et donc une qualité, supérieure des organes à transplanter. / The most used preservation method in renal transplantation is hypothermic cold storage (CS). However, this method induces damages inherent to the ischemia/ reperfusion (I /R) syndrome.My study was aimed at identifying new grafts preservation conditions, to limit I/R damage, by varying storage temperature or by adding an oxygen carrier.We used two models: in vitro with endothelial cells and in vivo in pig renal autotransplantation. The results confirmed the deleterious effects of 4°C storage in contrast to conservations at 19°C, 27°C and above 32°C, resulting in improved metabolic activity, cellular viability and integrity as well as a significant reduction in markers of inflammation and oxidative stress. Then we demonstrated the benefits of a new oxygen carrier, M101, in the two most used preservation solutions, UW and HTK. Indeed, use of M101 in CS protocols improved short-term function recovery and reduced fibrosis development, main cause of graft loss. Finally, we have shown that the benefits of M101 were preserved at lower doses and we determined that this protection was due to a multifunctionality of the molecule, combining oxygen transport, superoxyde dismutase activity and a large size (regulating oncotic pressure). This work permitted the uncovering of new concepts towards improved organ preservation and quality for transplantation.

The causative role of the huntingtin (HTT) gene in Huntington's disease (HD) has been identified more than 25 years ago. The extension of CAG repeat stretch over 39 repeats in exon 1 of one HTT allele results in full penetrance of this neurodegenerative disorder. While the identification of the causative mutation raised hopes that development of the therapeutic compound will be easily achievable, the patients and their families are still waiting for treatment until now. The main reason for that might be the complex cellular function HTT that makes the determination of the pathologic mechanism difficult and the development of treatments even more challenging. Although a lot of different animal models have been generated until now, establishing a suitable model has still not been achieved yet. Due to its anatomy, physiology, and genetics, the minipig seems to be a suitable candidate for neurodegenerative disease models. Indeed, the existing Transgenic (Tg) Libechov minipig model manifests signs typical for HD in patients, but on the other hand significant inconsistencies have also been observed. The finding of malformation that partially shows the situation in human patients is true for both, the male reproductive tract as well as for the brain. The reason for this might be the fact the genetic...

Huntington's disease (HD) is an autosomal dominant hereditary neurodegenerative disease characterized by motor, cognitive and behavioral disorders. HD is caused by expansion of CAG triplet (cytosine-adenosine-guanine) located in a gene on the short arm of the fourth chromosome. This expansion encodes an aberrant polyglutamine chain in the protein huntingtin. Physiological and mutated huntingtin (in case of HD) are expressed in almost all tissues and influences many cellular functions. The prevalence of HD in population is about 1 per 10.000. The disease is currently incurable and its mechanisms are not sufficiently understood. Besides affecting the central nervous system HD also affects peripheral tissues, including skeletal muscles. HD disrupts mitochondrial function and damages oxidative phosphorylation system, which has the task of producing energy in the form of ATP in cells. Research of transgenic minipig model for HD could help elucidate the mechanisms of disease's pathogenesis and potential therapeutic strategy. In this diploma thesis, immunodetection with help of specific antibodies to detect changes in amount of 14 selected mitochondrial proteins in skeletal muscle tissue of three age groups of transgenic HD minipigs - 24, 36 and 48 months old was used. Gradual progression in reduced...

Huntingtons's disease (HD) is devastating neurodegenerative disorder manifesting by motor disturbances, cognitive decline and personal changes. The huge effort to find a cure for HD has brought several promising therapeutic treatments on the scene. Each of the prospective approaches needs to be investigated for safety, tolerability and efficacy. Mouse and rat models were a lot helpful in examination of pathological mechanisms of HD, but they are not sufficient for completion of pre-clinical testing. Therefore, we aimed to generate transgenic HD minipig to overcome the gap between rodents and humans. Minipig transgenic for the first 548 aminoacids of human mutant huntingtin gene (TgHD) under the control of human HD promotor was manipulated by lentiviral transduction of porcine one-cell stage embryos. Currently, six generations of minipigs expressing single copy of N-truncated human mutant huntingtin protein (mtHtt) with a repetition of 124 glutamines are at disposal. The more the model simulates the disease symptoms the better it is for translational research as the efficacy of the cure can be finer evaluated. Hence, the second aim was to demonstrate HD-like phenotype in our model. Testicular degeneration that preceded the clinical symptoms onset was observed as a consequence of expression of mtHtt....

The causative role of the huntingtin (HTT) gene in Huntington's disease (HD) has been identified more than 25 years ago. The extension of CAG repeat stretch over 39 repeats in exon 1 of one HTT allele results in full penetrance of this neurodegenerative disorder. While the identification of the causative mutation raised hopes that development of the therapeutic compound will be easily achievable, the patients and their families are still waiting for treatment until now. The main reason for that might be the complex cellular function HTT that makes the determination of the pathologic mechanism difficult and the development of treatments even more challenging. Although a lot of different animal models have been generated until now, establishing a suitable model has still not been achieved yet. Due to its anatomy, physiology, and genetics, the minipig seems to be a suitable candidate for neurodegenerative disease models. Indeed, the existing Transgenic (Tg) Libechov minipig model manifests signs typical for HD in patients, but on the other hand significant inconsistencies have also been observed. The finding of malformation that partially shows the situation in human patients is true for both, the male reproductive tract as well as for the brain. The reason for this might be the fact the genetic...