This dissertation was intended to identify potential roles for phosphatidylinositol-3 kinase (PI-3 kinase) in the responses of lymphocytes to activation. To understand what functions PI-3 kinase is performing in lymphocytes, experiments were performed to identify proteins that will stably associate with the p85 subunit of PI-3 kinase. Co-precipitation revealed an activation dependent association of p85 with two different phosphotyrosine containing proteins. One protein, pp36-38, is a membrane protein that interacts with PI-3 kinase, PLCγ1, and Grb2/S0S. The other associated protein was identified as the proto-oncogene c-Cbl. The interaction of p85 with cbl was shown to be mediated through the SH2 domains of p85. More importantly, the interactions of p85 with p36-38 and cbl were found to be specific for p85 isoforms. Although the SH2 domains of the α and β isoforms are highly similar in amino acid sequence, they are shown to establish distinct protein interactions in intact cells. Experiments on the cbl/PI-3 kinase complex revealed a stimulation dependent translocation into membrane and insoluble/cytoskeletal fractions of wild type, but not mutant cells. The movement of cbl did not require tyrosine phosphorylation or PI-3 kinase activity. The cbl/PI-3 kinase complex was greatly enhanced in the membrane fraction in contrast to the cytosol, where the largest concentration of cbl can be found. In addition, these complexes were found to form at the membrane in the absence of the tyrosine kinase, p56lck.

INTRODUCTION: Programmed Death Ligand 2 positive B1 cells (L2pB1) cells have a unique immunoglobulin repertoire that is poly-reactive to self-antigens and have previously been shown to have an essential role in autoimmunity. The active accumulation of L2pB1 cells inside tumors grown in vivo led us to hypothesize that this subpopulation of B1a cells may play a role in the immunosurveillance of cancer. Here, we report our investigation of the role of L2pB1 cells in the antitumor response using a three dimensional (3D) murine melanoma and colon cancer models. Our results showed that the depletion of L2pB1 cells rendered the loss of tumor inhibition effects of the syngeneic peritoneal immune cells. METHODS: Lymphocytes were collected from L2pB1 cell depleted and non-depleted peritoneal cavity washout (PCW) from an inducible knockout mouse model. Then tumor spheroids were incubated with PCW cells. Spheroid cross-sectional area (CSA) and volume were measured using a Celigo plate imager and Keyence fluorescence microscope. RESULTS: Tumor spheroid growth was significantly inhibited following incubation with syngeneic PCW but not with splenocytes. Depletion of L2pB1 significantly attenuated the tumor-inhibition effect and showed a negligible difference from the untreated control. This loss of tumor inhibition indicated that L2pB1 cells are essential for the tumor-inhibition effects of autologous peritoneal immune cells. CONCLUSIONS: These findings demonstrate the robust anti-tumor function of L2pB1 cells. In particular, peritoneal L2pB1 cells play an essential role in cancer inhibition. Future studies into the activation and antigen presentation pathways of L2pB1 cells could lead to novel immunotherapy of cancer.

Environ, 60 % des personnes atteintes d’un cancer auront au moins une séance de radiothérapie au cours de la prise en charge thérapeutique de leur maladie. Les doses de radiothérapie sont limitées en raison du risque important de fibrose séquellaire des tissus sains. Les rayonnements ionisants (RI) peuvent induire différents types de mort cellulaire y compris l'apoptose et la sénescence. Les cellules sénescentes ont une sensibilité réduite à l'apoptose et un phénotype sécrétoire inflammatoire. De plus, les RI peuvent induire la production d’espèces réactives de l’oxygène (ERO) qui provoquent des lésions de l'ADN dans les tissus non ciblés, et des effets systémiques associés à l'inflammation. Différentes équipes ont proposé des tests prédictifs de la radiosensibilité individuelle des patients basés sur l’évaluation du taux d'apoptose radio-induite des lymphocytes T CD4+/CD8+ (LT). Cependant, l’impact des différences de sensibilité à l’apoptose/sénescence des sous-populations de LT sur le taux d’apoptose n’a pas été étudié. Notre hypothèse est que la sensibilité à l’apoptose/sénescence radio-induite des LT circulants est associée à la sur/sous-représentation de sous-populations particulières de LT CD4+ dont les fonctions sont en rapport avec la survenue de fibrose. Nos résultats chez le donneur sain montrent que les LT CCR6+Th17 pro-fibrogéniques sont moins sensibles à l’apoptose et plus sensibles à la sénescence que les LT CCR6negTh et les Treg. Cette sénescence peut être préjudiciable car les lymphocytes CCR6+Th17 situés dans les tissus irradiés peuvent sécréter de l'IL-8 et du VEGF-A. La modulation des voies ERO/MAPK ou mTOR pourrait être une cible potentielle pour la prévention de la radiotoxicité induite par les CCR6+Th17 sénescents. Enfin, le ratio de cellules circulantes H2A.J+CCR6+Th17 sénescentes / CCR6+Treg pourrait être utilisé comme marqueur potentiel de la radiosensibilité individuelle. / On average, 60% of cancer patients have at least one radiation session during their care throughout the history of their disease. The doses of radiotherapy are limited because of the high risk of fibrosis-type side effects of healthy tissues. Ionizing radiation can induce a variety of cell death responses including apoptosis, but also senescence. Senescent cells have reduced sensitivity to apoptosis, and a pro-inflammatory secretory phenotype. In addition, ionizing radiations can induce the production of reactive oxygen species (ROS) that cause DNA damage in non-target tissues, and systemic effects associated with inflammation. In order to improve the personalization of radiotherapy, different teams proposed predictive tests of the individual radiosensitivity of patients by establishing a relationship between a low rate of radio-induced apoptosis of CD4+/CD8+ T lymphocytes (LT) and a high risk of secondary fibrosis. However, the impact of the differences in individual cell sensitivity to radiation-induced senescence on the ratio between LT cell subpopulations has not been studied. Our results on healthy donors show that pro-fibrogenic CCR6+ Th17 cells are less sensitive to apoptosis and more susceptible to senescence compared to CCR6neg LT. This senescence can be detrimental as irradiated CCR6+Th17 lymphocytes located in the irradiated tissue can secrete IL-8 and VEGF-A. Modulation of ROS/MAPK or mTOR signaling pathways could be potential targets for the prevention of this CCR6+Th17-induced radiotoxicity. Finally, the ratio of circulating H2A.J+ senescent CCR6+ Th17/CCR6+Treg cells may be used as a potential marker of individual radiosensitivity.

Groundwater contamination is a concern due to the large number of people that can become exposed to the contaminant. The chemicals benzene and phenol are known groundwater contaminants. The main health problem caused by benzene or phenol is bone marrow toxicity. Benzene and phenol are also immunotoxins reported to cause decreased thymic weights, altered lymphocyte mitogenic responses, and lower antibody production. Cytokines are key signaling molecules produced by the cells of the immune system to activate other cells in the immune system, produce antibodies, and recruit other cells to sites of inflammation. The purpose of this study was to determine if exposure to benzene or phenol in drinking water for 30 days could lead to alterations in IL-l, IL-6, and TNFa production in in vitro activated murine macrophages, or in IL-2, IL-3, and IFNy production in in vitro activated murine lymphocytes. Cytokine mRNA and protein production were evaluated to determine if any alteration occurred. Benzene and phenol exposure resulted in significantly decreased thymus weights. Interleukin-2 mRNA production was increased at the medium dose (200 mg/L) but the IL-2 protein secreted from the lymphocytes of benzene-treated mice was unchanged. The macrophages from benzene-treated mice showed a decrease at all dosage levels in both TNFa mRNA and protein production. These macrophages also produced increased JLIa mRNA at the medium benzene concentration, although this increase did not mean an increase of IL-Ia protein secreted. Mice given phenol at the medium (20 mg/L) and high (100 mg/L) dosages had decreased 30-day body weights. The production ofiL-3 mRNA was decreased in the lymphocytes of mice receiving both low and high concentrations of phenol. Lowered TNFa mRNA values were observed in the macrophages from phenoltreated mice. Interleukin-la mRNA production was increased in the macrophages of mice given the low (5 mg/L) dose of phenol. The TNFa cytokine protein was decreased at the low and medium doses, and the IL-l a protein level was decreased at the medium and high doses. The results indicate that benzene and phenol in groundwater should continue to be a concern for public and regulatory agencies.

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第20533号 / 生博第375号 / 新制||生||50(附属図書館) / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 杉田 昌彦, 教授 米原 伸, 教授 清水 章 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21302号 / 医博第4391号 / 新制||医||1030(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 生田 宏一, 教授 椛島 健治, 教授 杉田 昌彦 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21635号 / 医博第4441号 / 新制||医||1034(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 椛島 健治, 教授 岩田 想, 教授 山田 亮 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DGAM

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ABSTRACT: Various recent studies have suggested that regular intake of some non-steroidal anti-inflammatory drugs (NSAIDs) have a preventative effect against several types of tumours including breast cancer. The term nanotechnology refers to technology in which one-billionth of a meter is used as a scale for chemical particle size. This work aims to study the effect of both ibuprofen and aspirin on DNA damage using peripheral blood lymphocytes from breast cancer patients and comparing the results with those from healthy females as a control using the Comet and micronucleus assays. Western blot analysis (WBA) was used to investigate the effect of these drugs on XRCC3 and p53 proteins, whereas QPCR was to evaluate this effect on p53, cox1 and cox2 genes. Two hundred fifty ng/ml of ibuprofen (NP and bulk) and 500 ng/ml of aspirin (NP and bulk) were used to treat the lymphocytes. Both aspirin and ibuprofen caused a reduction in DNA damage and micronucleus formation. Aspirin, both forms, showed a reduction in DNA damage in the Comet and micronucleus assays. Ibuprofen both forms, by contrast, showed a statistically significant reduction in micronucleus frequency in the micronucleus assay, while its preventative effect with the Comet assay was weak or insignificant. NPs of both agents were more effective than bulk sizes. Using the Comet repair assay, aspirin and ibuprofen nano form catalysed DNA repair to a greater extent than their bulk forms. Also, both sizes showed better repair with NSAIDs compared to samples repaired without NSAIDs. In WBA aspirin increased the expression of XRCC3 protein in healthy cells. However, both NSAIDs decreased that expression in cells from BC patients. Furthermore, aspirin increased p53 expression in BC patients lymphocytes. With the QPCR method, results of both aspirin forms increased the expression of the p53 gene in BC patient cells statistically significantly. Both drugs reduced cox1 expression in healthy volunteers and cancer patients lymphocytes. Moreover, cox2 reduction was only in lymphocytes from BC patients. The results of this work are consistent with the view that NSAIDs, particularly aspirin and ibuprofen, could have a promising role in cancer treatment including breast cancer. / Various recent studies have suggested that regular intake of some non-steroidal anti-inflammatory drugs (NSAIDs) have a preventative effect against several types of tumours including breast cancer. The term nanotechnology refers to technology in which one-billionth of a meter is used as a scale for chemical particle size. This work aims to study the effect of both ibuprofen and aspirin on DNA damage using peripheral blood lymphocytes from breast cancer patients and comparing the results with those from healthy females as a control using the Comet and micronucleus assays. Western blot analysis (WBA) was used to investigate the effect of these drugs on XRCC3 and p53 proteins, whereas QPCR was to evaluate this effect on p53, cox1 and cox2 genes. Two hundred fifty ng/ml of ibuprofen (NP and bulk) and 500 ng/ml of aspirin (NP and bulk) were used to treat the lymphocytes. Both aspirin and ibuprofen caused a reduction in DNA damage and micronucleus formation. Aspirin, both forms, showed a reduction in DNA damage in the Comet and micronucleus assays. Ibuprofen both forms, by contrast, showed a statistically significant reduction in micronucleus frequency in the micronucleus assay, while its preventative effect with the Comet assay was weak or insignificant. NPs of both agents were more effective than bulk sizes. Using the Comet repair assay, aspirin and ibuprofen nano form catalysed DNA repair to a greater extent than their bulk forms. Also, both sizes showed better repair with NSAIDs compared to samples repaired without NSAIDs. In WBA aspirin increased the expression of XRCC3 protein in healthy cells. However, both NSAIDs decreased that expression in cells from BC patients. Furthermore, aspirin increased p53 expression in BC patients lymphocytes. With the QPCR method, results of both aspirin forms increased the expression of the p53 gene in BC patient cells statistically significantly. Both drugs reduced cox1 expression in healthy volunteers and cancer patients lymphocytes. Moreover, cox2 reduction was only in lymphocytes from BC patients. The results of this work are consistent with the view that NSAIDs, particularly aspirin and ibuprofen, could have a promising role in cancer treatment including breast cancer. / Libyan Government