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Thèse (Ph.D.)--Université Laval, 2004. / Titre de l'écran-titre (visionné le 29 novembre 2004). Bibliogr.

Please read the abstract in the section 00front of this document / Thesis (PhD (Genetics))--University of Pretoria, 2007. / Genetics / unrestricted

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22723号 / 医博第4641号 / 新制||医||1045(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 篠原 隆司, 教授 溝脇 尚志, 教授 増永 慎一郎 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Understanding cancer biomarkers for diagnosis and prognosis leads to improved patient treatments and care. This thesis addresses the relevance of thymidine kinase 1 (TK1) as a cancer biomarker and the role of TK1 in cancer progression. Worldwide, cancer leads to more than 12 million deaths annually. In the United States alone, each year over 1.5 million cases will be diagnosed and over half a million persons will die. The most prevalent cancer types include skin, lung, breast, prostate, and colon. TK1 is known to be present in the serum of patients with multiple cancer types, including lung, breast, colon and prostate. In fact, it is shown to be detectable in cancer patients even before they manifest clinical symptoms. Additionally, the levels of TK1 increase progressively with increasing tumor grade; meaning that levels of TK1 can indicate tumor grade. Cellular proliferation markers such as p53 and Ki-67 have been compared to TK1 in cancer diagnosis and prognosis. TK1 has potential as both a prognostic and diagnostic biomarker in various cancer types including breast. Breast cancer is one of the most aggressive cancer types with 20-30% of diagnosed tumors becoming metastatic. Recent findings have identified additional involvement of TK1 downstream of cellular proliferation in cancer progression, including cellular invasion which is a part of cancer metastasis. These findings while efficacious, fail to identify the individual contribution of TK1 in downstream processes that aid in cancer progression. As mentioned previously, TK1 is upregulated in several different cancer types. We propose that there is an advantage to upregulated levels of TK1 in cancer progression and seek to explore its role specifically in cell invasion and survival. Based on our current understanding of TK1, we first wanted to review the history of TK1 and show the importance of understanding this crucial enzyme. Finally, we report our results from experiments exploring the influence of TK1 in vitro on breast cancer cell invasion and survival.

Despite the progress made in cancer research, cancer remains one of the leading causes of death worldwide. Although the development of new cancer treatments has improved cancer patients' survival rate, a significant number of patients experience refractory and recurrence events with serious side effects. It is known that the immune system actively participates in eliminating cancer. However, cancer cells can develop mechanisms to evade the immune system resulting in immunotolerance. Immunotherapy aids the patient's immune system's ability to recognize and eliminate cancer cells. During the last three decades, immunotherapy has gradually emerged as an effective and more specific approach to treat cancer. Particularly monoclonal antibodies and adoptive cell therapies such as chimeric antigen receptor (CAR) T-cells have proven highly effective. Nevertheless, the success of these novel therapies depends on discovering suitable tumor targets. Recently, we reported localization of Thymidine Kinase 1 (TK1) to the plasma membrane of certain cancer cells but have not found such localization on normal cells. Similarly, another nucleotide salvage pathway enzyme Hypoxanthine Guanine Phosphoribosyltransferase (HPRT), has also been reported to be localized to the plasma membrane of certain cancer cells. Thus, TK1 and HPRT membrane-associated forms can be potential tumor targets for cancer immunotherapy. This dissertation describes the immunotargeting of TK1 for the selective elimination of tumor cells and the surface localization of HPRT on the plasma membrane of cancer cells. Using hybridoma and phage display technologies, we developed monoclonal antibodies (mAb) and isolated human single domain antibodies (sdAb) specific to human TK1. We confirmed that antibodies and sdAbs could target TK1 on the plasma membrane of lung, breast and colon cancer cells, but not on healthy cells. In addition, we demonstrated that cancer cells expressing membrane-associated TK1 (mTK1) co-cultured with human mononuclear cells (MNC) were selectively eliminated through antibody-dependent cell-mediated cytotoxicity (ADCC) when anti-TK1 mAbs were added. Furthermore, we designed novel TK1 specific tumor targeting receptors and expressed them in human T cells and human macrophages. Finally, we proposed using both TK1 and HPRT as biomarkers for the early detection and monitoring of follicular lymphoma (FL), a disease that is usually detected at advanced stages. The knowledge generated from the data presented in this dissertation indicates that TK1 and HPRT may be suitable immunotherapeutic targets for antibody-based and adoptive cell-based therapies against both liquid and solid malignancies. It also proposes the incorporation of TK1 and HPRT as molecular biomarkers for the early detection and monitoring of FL.

La résistance du virus herpes simplex (VHS) aux antiviraux, particulièrement chez des sujets immunosupprimés, et sa capacité d’envahir le système nerveux central soulèvent de nombreuses questions au niveau clinique. Des mutations au sein du gène de la thymidine kinase et de l’ADN polymérase virale sont responsables de la résistance aux traitements antiviraux. Il est donc important d’élucider davantage les différents mécanismes moléculaires à l’origine de ces évènements de résistance et de développer de nouvelles avenues thérapeutiques. De plus, le VHS est la cause la plus fréquente d’encéphalite sporadique et potentiellement fatale dans les pays de l’ouest. Ce type de pathologie est occasionné en partie par une intense réplication virale mais également par une réponse immunologique encore incomprise jusqu’à maintenant. Les travaux présentés dans cette thèse de doctorat ont pour but de développer de nouveaux modèles in vitro et in vivo permettant d’étudier ces deux facettes importantes du VHS. L’impact de certaines mutations et l’effet d’un nouveau traitement dans la résistance du VHS aux antiviraux ont été évalués. La réponse immunitaire innée cérébrale de même qu’une nouvelle approche thérapeutique en rapport avec une encéphalite à VHS ont également été caractérisées. / Herpes simplex virus (HSV) resistance to antiviral treatment is a real concern among immunocompromised population. Mutations localized in the thymidine kinase (TK) and/or the DNA polymerase (pol) genes are mainly responsible for those resistance issues. Thus, it is becoming important to increase knowledge in this area and to develop new therapeutic strategies. Also, HSV has this unique biological property to invade the nervous central system and causes encephalitis. During this type of infection, an important immunological process occurs. However, this inflammatory response is still very controversial and needs to be elucidated. The main objectives of this doctoral thesis consisted of: 1- the characterization of antiviral resistance and 2- the elucidation of the brain inflammatory response to HSV. The first part consisted in the development of an in vitro system allowing the characterization of several mutations in the TK and/or the DNA pol genes responsible for antiviral resistance and the elaboration of a new antiviral strategy. The second part was to characterize the inflammatory response following the induction of HSV-1 encephalitis in a mouse model and to develop an alternative immunomodulatory approache. Mutations localized in conserved and non-conserved regions of the TK are associated with ACV resistance. Hydroxyurea increases the activity of nucleoside (ACV) and nucleotide (CDV) analogues. A delayed glucocorticoid treatment is highly beneficial by decreasing the brain viral load as well as pro-inflammatory cytokine production in the brain of infected mice. TNF- and IL-1permit the initiation of an innate immune response allowing a control of the viral replication and an efficient transition to the adaptive immune response required for viral clearance during HSV-1 encephalitis. A prophylactic treatment with a TLR3 agonist significantly increases the mean life expectancy and survival rate of mice infected with HSV-1 compared to non-treated mice. The experimental models developed during this Ph. D. allow a better understanding of the molecular mechanisms of resistance and of the brain inflammatory response to the HSV.

L'alpha-foetoprotéine (AFP) est une protéine hépatique feotale souvent réexprimée dans les hépatomes. L'utilisation de son promoteur en thérapie génique pourrait donc augmenter la spécificité du traitement. Nous avons tenté de mettre au point un système de thérapie génique utilisant des mutants plus puissants du promoteur de l'AFP associés au gène suicide de la thymidine kinase (TK). La comparaison de l'efficacité du gène TK sauvage et du mutant TK30 à induire la mort cellulaire en présence de la prodrogue ganciclovir (GCV) dans notre lignée cellulaire d'hépatome de rat v7.6 nous a révélé que le mutant n'était pas supérieur au gène sauvage. Nous avons comparé l'efficacité de mutants plus actifs du promoteur de l'AFP, MO1 et M11, à induire l'expression du gène TK placé sous leur contrôle ainsi que leur effet bystander respectif. Le mutant MO1 s'est révélé être le meilleur choix pour la thérapie. Un effet bystander a pu être observé in vivo chez des rats Buffalo injectés avec des v7.6 contenant le plasmide MO1-TK. Nous avons démontré la capacité de l'adénovirus Ad-CMV-LacZ ainsi que nos constructions Ad-MO1-EGFP et Ad-MO1-TK à transduire les hépatomes en culture et chez l'animal (sauf pour Ad-MO1-TK). Finalement, nous avons testé l'efficacité de notre système de thérapie génique in vivo. L'Ad-MO1-TK n'a pas réussi à éliminer les tumeurs. Notre système n'est donc pas encore fonctionnel tel qu'utilisé.

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