Diss. (sammanfattning) Umeå : Umeå universitet, 2008. / Härtill 4 uppsatser.

De nos jours, la cryoconservation est une technique très largement utilisée dans les protocoles d’assistance à la reproduction ou comme outil pour la sauvegarde des ressources génétiques. Toutefois, la chienne est un modèle animal complexe pour l’application des biotechnologies de la reproduction du fait de ses nombreuses singularités anatomiques et physiologiques. L’objectif de notre travail était d’étudier et de développer une méthode de cryoconservation des ressources génétiques chez la chienne par le biais de deux types de ressources : les embryons et le tissu ovarien. Après avoir mis au point une méthode de collecte d’embryons, nous nous sommes appliqués à la constitution d’un stock d’embryons cryoconservés en prévision d’un transfert embryonnaire. L’étude et le développement d’un protocole de cryoconservation du tissu ovarien ont été abordés après avoir adapté et validé nos méthodes d’analyses in vitro. L’utilisation de plans d’expériences factoriels fractionnaires a permis de mettre en évidence les facteurs les plus influents sur la qualité de la réserve folliculaire (nature du cryoprotecteur pénétrant, cinétique de congélation, étapes d’équilibration) et de proposer un protocole de cryoconservation. La combinaison du DMSO incorporé en un seul bain d’équilibration avec une vitesse de congélation de 0,3°C/min est apparue comme la combinaison la plus appropriée à la cryoconservation de tissu ovarien chez la chienne et a permis d’observer, après xénogreffe de tissu ovarien cryoconservé, une reprise de la croissance folliculaire et de l’activité hormonale du tissu greffé / Nowadays, cryopreservation is widely used in animal assisted reproduction or safeguarding of genetic resources. Nevertheless, the bitch is a complex animal model concerning the use of this biotechnology, due to numerous anatomical and physiological peculiarities. The aim of our research work was to investigate and develop a method of cryopreservation of genetic resources in the bitch by exploring two kinds of resources: embryos and ovarian tissue. After the setting up of a method for embryo collection, we have built up a stock of cryopreserved embryo for subsequent embryo transfer. After a preliminary validation of our in vitro assessment methods, the investigation and development of a cryopreservation protocol has been conducted. The use of fractional experimental design allowed us to highlight the main factors affecting the follicular pool quality (CPA nature, freezing rate and equilibration steps). The combination of DMSO incorporated in a unique equilibration bath with a freezing rate of 0.3°C/min appeared to be suitable for the cryopreservation of bitch ovarian tissue. Finally, Follicular growth and hormonal activity resumption have been observed after xenotransplantation of cryopreserved bitch ovarian tissue

Les cellules souches cancéreuses (CSC) représentent une sous-population de cellules tumorales à l’origine de l’hétérogénéité et de la croissance tumorale. Les CSC sont plus résistantes aux traitements, et à l’origine de la rechute et des métastases. L’identification des CSC constitue actuellement un enjeu majeur dans le développement de nouvelles thérapies ciblées pour inhiber la croissance tumorale et éradiquer le cancer. Dans ce travail, nous avons cherché à identifier, caractériser, et cibler les CSC dans l’adénocarcinome gastrique. Des modèles murins de xénogreffe de tumeurs primaires de patients atteints d'adénocarcinome gastrique hors cardia de types intestinal et diffus ont été développés, ainsi qu’un modèle de tumorsphere in vitro afin d’évaluer les capacités tumorigéniques de sous-populations tumorales. Nous avons identifié CD44 et l'aldéhyde déshydrogénase (ALDH) comme marqueurs d’enrichissement des CSC dans les 2 types d’adénocarcinomes gastriques, l’ALDH représentant un marqueur plus spécifique que CD44. Nous avons ensuite étudié l'effet de l’acide rétinoïque tout trans (ATRA), et nous avons montré que l'ATRA inhibe la formation et la croissance des tumorspheres in vitro ainsi que la croissance tumorale in vivo. Cet effet de l’ATRA passe par l’inhibition de l’expression des marqueurs souches et des capacités d'auto-renouvèlement des CSC. En conclusion, CD44 et ALDH sont des marqueurs de CSC dans les adénocarcinomes gastriques hors cardia de types intestinal et diffus, et le traitement par l’ATRA constituerait une stratégie commune de traitement pour cibler spécifiquement les CSC et inhiber la croissance tumorale dans ces deux types de cancer gastrique. / Cancer stem cells (CSCs) are a subpopulation of tumor cells at the origin of the heterogeneity and growth of tumors. CSCs are more resistant to treatment, and are responsible for relapse and metastasis. The identification of CSCs is a major challenge for the development of new targeted therapies to inhibit tumor growth and eradicate cancer. In this work, we aimed to identify, characterize, and target CSCs in gastric adenocarcinoma. Mouse models of primary tumor xenografts from intestinal and diffuse type non-cardia gastric adenocarcinomas from patients were developed, as well as an in vitro tumorsphere assay, to assess the tumorigenic capacity of subpopulations of tumor cells. We identified CD44 and aldehyde dehydrogenase (ALDH) as CSC enrichment markers in the two types of gastric adenocarcinoma, ALDH representing a more specific marker than CD44. We then studied the effect of All-trans retinoic acid (ATRA), and showed that it inhibited the formation and growth of tumorspheres in vitro and tumor growth in vivo. This effect of ATRA is due to the inhibition of stem marker expression and the self-renewal capacity of CSCs. In conclusion, CD44 and ALDH are effective CSC markers in intestinal and diffuse type non-cardia gastric adenocarcinomas, and treatment with ATRA provides a common treatment strategy to specifically target CSCs and inhibit tumor growth in both subtypes of this gastric cancer.

L’infection chronique par Helicobacter pylori touche plus de la moitié de la population mondiale et est la principale cause connue de l’adénocarcinome gastrique. La metformine, un antidiabétique oral, est de plus en plus étudiée pour ses propriétés antitumorales dans de nombreux types de cancers. Cependant, ses effets potentiels ont très peu été étudiés dans le cancer gastrique. Des expériences réalisées sur des lignées cellulaires cancéreuses gastriques et des tumeurs de patients amplifiées par xénogreffe chez la souris (PDX), ont permis de confirmer les propriétés antitumorales de la metformine. La metformine, en traitement préventif et curatif, diminue la capacité de formation des tumorsphères, l’expression des marqueurs de CSC gastriques, et la capacité d’auto-renouvellement propre aux CSC. Dans un second temps, nous avons montré que la metformine est capable d’inhiber la croissance bactérienne de H. pylori invitro et in vivo. Enfin, les effets de l’infection par H. pylori ont été étudiés sur la voie de signalisation mTOR par la réalisation d’une analyse transcriptomique et de western-blots sur des lignées cellulaires gastriques. Ceci a permis de démontrer que H. pylori inhibe le complexe mTORC1.Pour conclure, ce travail de thèse a permis i) de démontrer la capacité de la metformine à cibler les CSC gastriques, ii) de découvrir une nouvelle propriété antibactérienne de la metformine vis-à-vis de H. pylori, iii) de démontrer que H. pylori inhibe la voie de signalisation mTOR. / Chronic infection with Helicobacter pylori affects more than half of the world's population and is the main known cause of gastric adenocarcinoma. Metformin is an oral antidiabetic drug used to treat type 2 diabetes patients, and is being increasingly studied for its antitumoral properties in several cancer types. However, its potential effects in gastric cancer have not been thoroughly studied. Experiments performed on gastric cancer cell lines and patient-derived gastric carcinoma xenografts (PDX), have confirmed the antitumoral properties of metformin. Metformin, in preventive and curative treatment, decreases the tumorsphere formation, the expression of gastric CSC markers and the self-renewal capacity of CSC. In a second time, we have shown that metformin is able to inhibit the bacterial growth of H. pylori in vitro and in vivo. Finally, the effects of the H. pylori infection have been studied on the mTOR signaling pathway using a transcriptomic analysis and western blots, performed on gastric cancer cell lines. These show the ability of H. pylori to inhibit mTORC1.To conclude, this thesis work allowed i) to demonstrate the ability of metformin to target gastric CSCs, ii) to discover a new antimicrobial property of metformin against H. pylori, iii) to demonstrate that H. pylori inhibits the mTOR signaling pathway.

Lung cancer leads in mortality among all types of cancer in the US and Non-small cell lung cancer (NSCLC) is the major type of lung cancer. Immuno-compromised mice bearing xenografts of human lung cancer cells represent the most common animal models for studying lung cancer biology and for evaluating potential anticancer agents. However, orthotopic lung cancer models based on intrapulmonary injection of suspended cancer cells feature premature leakage of the cancer cells to both sides of the lung within five days, which generates a quick artifact of metastasis and thus belies the development and progression of lung cancer as seen in the clinic. Based on intrapulmonary inoculation of multicellular spheroids (MCS), we have developed the first orthotopic xenograft model of lung cancer that simulates all four clinical stages of NSCLC progression in mice over one month: Stage 1 localized tumor at the inoculation site; Stage 2 multiple tumor nodules or larger tumor nodule on the same side of the lung; Stage 3 cancer growth on heart surface; and Stage 4 metastatic cancer on both sides of the lung. The cancer development was monitored conveniently by in vivo fluorescent imaging and validated by open-chest anatomy, ex vivo fluorescent imaging, and histological studies. The model enjoys high rates of postoperative survival (100%) and parenchymal tumor establishment (88.9%). The roughness of the inoculated MCS is associated negatively with the time needed to develop metastatic cancer (p=0.0299). In addition, we have constructed a co-culture MCS that consisted of A549-iRFP lung cancer cells and WI38 normal human fibroblast cells. The pro-proliferation effect and the high expression of α-smooth muscle actin (α-SMA) by the co-cultured WI38 cells indicated their transformation from normal fibroblasts to cancer-associated fibroblasts (CAFs). The morphology of the co-culture MCS features a round shape, a tight internal structure, and quicker development of roughness. The large roughness value of co-culture MCS suggests that small co-culture MCS could be inoculated into mice lung with a small needle to reduce the surgical trauma. Taken together, a new orthotopic model of NSCLC has been developed, which would facilitate future development of medications against lung cancer.

Cancer is the second leading cause of death in the US and worldwide, accounting for 16% of deaths worldwide in 2015. Of more than 100 types of cancers affecting humans, breast cancer is the most common cancer among women and is the second leading cause of death in women. Triple negative breast cancer (TNBC) is a subtype of breast carcinomas defined by the lack of the expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor (HER2 /neu). The prognosis and survival of TNBC patients remains the poor due to the lack of effective targeted therapy. Nanotechnology-based drug delivery systems, such as liposomes, are widely investigated to enhance anticancer efficacy by concentrating the drug molecules in the tissues of interest and by altering the pharmacokinetic profile. Taking advantage of the pH gradient in the tumor microenvironment, pH-triggered release is a promising strategy to enhance the anticancer efficacy of drug delivery systems against TNBC. Previously, a strategy in our lab has been developed to render saturated and pegylated liposomes pH-sensitive: protonation-induced conformational switch of lipid tails, using trans-2-aminocyclohexanol lipids (TACH, flipids) as a molecular trigger. Based on previous work in our lab, pH-sensitive liposomes (fliposomes) composed of C-16 flipids with amine group of morpholine (MOR) and azetidine (AZE) demonstrated optimized triggered release in response to the tumor’s low pH microenvironment. In this study, different preparation methods were developed and optimized to produce viable fliposomes with high doxorubicin (DOX) encapsulation efficiency. In vitro release assays were established and validated to accurately reflect pH-triggered release of fliposomes. The physicochemical properties of DOX-loaded fliposomes were characterized and their pH-dependent release were investigated. Factors influencing the desirable attributes of liposomes, such as size, pH-sensitivity, stability and drug-loading capacity were explored. Based on these characterizations, central composite design (CCD) was utilized to optimize the formulation of fliposome with two critical factors, flipids and cholesterol. Cell viability assays on traditional monolayer and innovative three-dimensional multicellular spheroids (3D MCS) of TNBC cell lines were conducted to evaluate the anticancer efficacy of the resultant fliposomes in vitro. The constructed 3D MCS carried heterogeneously distributed live and apoptotic cells, as well as acidity inside the 3D MCS based on confocal microscopic imaging studies. The distribution and penetration of DOX-loaded fliposomes into 3D MCS was imaged by confocal microscopy in comparison to DOX-loaded non pH-sensitive liposomes and free DOX. As a result, fliposome manifested superior anticancer activity against TNBC 3D MCS by efficient penetration into 3D MCS, followed by tuning up the release rate of the anticancer agent DOX. A TNBC orthotopic xenograft model was established by transplanting TNBC into the murine mammalian fat pad, which maintains the organ-specific tumor microenvironment of the original organ . A pilot pharmacokinetic study was conducted in order to correlate the pH response and stability properties with the in vivo stability of the optimized AZE-C16 fliposome. The antitumor efficacy was comparable between free DOX and DOX-loaded stealth liposome with tumor volumes of ~ 80-90% of the control treatment 32 days post first dose. In contrast, the DOX-loaded fliposome, especially MOR-C16 fliposome, exhibited a significantly higher antitumor efficacy and delayed progression compared to free DOX and stealth liposome treatments. Taken together, DOX-loaded fliposomes were successfully prepared and optimized for in vivo application. They were able to achieve superior activity against TNBC in vitro and in vivo, facilitated by enhanced release of the anticancer drug DOX after penetration inside TNBC tumor.

No description available.

Osteoarthritis is one of the most common causes of disability in adults in America. It is a progressive and degenerative disease where the articular cartilage is broken down and lost from the surfaces of bones causing chronic pain and swelling in the joints, and currently has no cure. The most commonly osteoarthritis starts from a focal lesion on the cartilage surface, which will expand on the surface and downwards through the thickness of the tissue. The current gold standard for correcting cartilage focal lesions is the osteochondral autograft/allograft transplantation (OAT), which replaces the defect with a fresh osteochondral graft. The main limiting factor for using the OAT comes from the limited number of autograft and allografts that are available for implantation. To address the concern of graft availability, this study will look at the development of a porcine osteochondral xenograft (OCXG). The first aim of this research is to establish a decellularization protocol that will remove the antigens and cellular debris, which are the leading causes of graft rejection when implanting animal tissue in humans. The second aim of this study is restoring the mechanical strength of the OCXG that was lost during the decellularization process through crosslinking the tissue using genipin and epigallocatechin gallate (EGCG). The third aim is comparing the performance of the complete crosslinked OCXG at different degrees of crosslinking in a long-term goat animal model. The final aim is an alternative way to correct focal lesions through the development of an injectable collagen stabilizing treatment with genipin and punicalagin that will slow or stop the growth of a lesion and prevent osteoarthritis.

Neuartige Vakzinierungsstrategien zur Aktivierung einer Tumor-spezifischen zellulären Immunantwort sind vielversprechende Ansätze zur Therapie von Tumoren, insbesondere Human Papillomvirus (HPV)-assoziierte Tumore. Bisherige HPV-Impfstudien zeigen zwar die Aktivierung einer spezifischen zellulären Immunantwort, eine Tumorreduktion bleibt jedoch aus. Um diesen Effekt auf Immunzellebene zu definieren, wurde die Wirkung der HPV-Vakzine Ad p14 im Mausmodell und an Untersuchungsmaterial humaner Tumore analysiert. In Mäusen bildeten sich HPV+ TC1-Tumore einer frühen Entwicklungsphase nach Vakzinierung zurück. Tumore einer späten Entwicklungsphase wuchsen dagegen in zwei Intervallen aus. Immunologische Eigenschaften der Tumorzellen blieben dabei unverändert. Unterschiede zeigten sich in den Frequenzen Tumor-infiltrierender Lymphozyten; in progressiven Phasen wurden nur CD4+ T Zellen nachgewiesen, in Regressionsphasen zusätzlich zytotoxische CD8+ T Zellen. Immunmodulatoren, wie Interferon alpha oder DTA-1, einem Antikörper für den Glucocorticoid-induzierten Tumornekrosefaktor-Rezeptor, unterstützten die Wirkung der Vakzine; letzterer erhöhte die Anzahl zytotoxischer CD8+ T Zellen und führte zur Abstoßung der TC1-Tumore. HPV+ Tumorgewebe des Menschen, wie auch ihre Vorstufen, zeigten im Vergleich zu anderen Tumoren, wie Bronchial oder Kolonkarzinomen einen signifikant höheren Anteil an CD4+ und CD8+ T Zellen und an Forkhead Box P3+ regulatorischen T Zellen. Die Ergebnisse deuten darauf hin, dass die immunologischen Abläufe bei der Entwicklung HPV-assoziierter Tumore mit denen vorangeschrittener chronischer Erkrankungen vergleichbar sind, in denen sich CD4+ und CD8+ T Zellantworten erschöpfen während sich gleichzeitig immunsuppressive Mechanismen verstärken. Um die Entwicklung von Impfstoffen zur Therapie HPV-assoziierter Tumore zu verbessern sollten diese Mechanismen ausführlicher betrachtet werden. / Novel vaccination strategies, activating cellular tumour specific immune responses represent a promising approach for the treatment of cancer. Especially featured for these treatments are tumours evolving from chronic human papillomavirus (HPV) infections. But current strategies have not yet proved efficacious for complete tumour regression. Addressing cellular immunological aspects of tumour vaccination, this work focused on effects of HPV vaccine Ad p14 in mice and in samples of human tumours. In mice vaccination resulted in complete regression of early stage murine HPV+ TC1 tumours. Late stage TC1 tumours increased discontinuously. During that process, TC1 cells preserved their immunological characteristics. But frequencies of tumour-infiltrating lymphocytes varied; in progressing tumours only CD4+ T cells occurred, in temporary regressing tumours also CD8+ T cells were detected. Immune modulators, like interferon alpha or glucocorticoid-induced tumour necrosis factor receptor targeting antibody DTA-1 aggravated the effects of vaccination; latter raised cytotoxic CD8+ T cell numbers and resulted in complete tumour regression. Human HPV+ tumours as well as HPV+ precancerous stages revealed numbers of CD4+ and CD8+ T cells and especially of forkhead box P3+ regulatory T cells that were significantly increased compared to melanoma, bronchial or colon carcinoma. To assist further analysis of human HPV-associated cervical cancer and facilitate studies on therapeutic approaches, a humanized mouse model was established. The present work points to immunological exhaustion in the development of HPV-related tumours comparable to chronic diseases where CD4+ and CD8+ T cells exhaust and immunosuppression by regulatory T cells increases at the same time. For the development of appropriate strategies to enhance efficacy in HPV-associated tumour therapy, further knowledge of mechanisms involved in specific T cell activation, T cell exhaustion and immunosuppression is necessary.

No description available.