Die B-CLL ist eine niedrigmaligne Erkrankung, die im höheren Lebensalter auftritt und für die es weiterhin keine dauerhaft kurative Therapie gibt. Die Erforschung der genetischen Grundlagen dieser Krankheit könnte Erkenntnisse über die Funktionsmechanismen und neue Diagnose- und Therapieansätze erbringen. Ziel dieser Arbeit war es, eine Genbank von CLL-Patienten bzw. gesunden Spendern zu erstellen, um Gene, die für die Entstehung der Erkrankung mitverantwortlich sein könnten, zu identifizieren. Die subtraktive suppressive Hybridisierung (SSH) wurde als Methode eingesetzt, um cDNA-Mischungen zu generieren, in denen differentielle Gene angereichert wurden. In dieser Arbeit wurden drei differentielle Gene identifiziert. Eines dieser Gene kodiert den Oberflächenmarker CD5, der bei CLL-Erkrankten stärker exprimiert wurde. Da CD5 ein für die B-CLL relativ spezifischer Marker ist, bestätigte dieses Ergebnis die Qualität der Genbank. Mit Hilfe der SSH wurde ein Verlust der Expression der cDNA für den Oberflächenmarker CD20 bei CLL-Erkrankten nachgewiesen. CD20 ist möglicherweise ein Kalziumkanal und wird als Ziel des anti-CD20-Antikörpers Rituximab verwendet. Der Verlust von CD20 im Verlauf der Erkrankung konnte in dieser Arbeit angedeutet werden. Weiterhin wurde eine verringerte Expression von cDNA für IkappaBa (Mad-3) bei CLL-Patienten entdeckt. Der Transkriptionsfaktor IkBa spielt im zellulären Ablauf der Apoptose eine wichtige Rolle. Phosphoryliertes IkBa inhibiert die Wirkung von NF-kB. Eine Reduktion von IkBa-cDNA könnte ebenfalls die Funktion von NF-kB beeinflussen. Dieses Ergebnis könnte auch für zukünftige immuntherapeutische Ansätze von Bedeutung sein. Weiterhin sind die nachgewiesenen Gene interessant für die Technik des "DNA-Microarrays". Diese schnelle Methode ermöglicht kostengünstige Diagnosen und könnte daher eine zukunftsweisende Alternative zu herkömmlichen Ansätzen bieten. In der vorliegenden Arbeit konnten differentielle Transkripte auf mRNA-Ebene bei B-CLL-Erkrankten und gesunden Kontrollen festgestellt und untersucht werden. Die Ergebnisse geben neue Einsichten über Onkogene und Tumorsurpressorgene in der B-CLL und eröffnen zukünftige immuntherapeutische Ansätze. / The B-CLL is a malignancy that appears in the higher age and for which it gives further no durably curative therapy. The investigation of the genetic bases of this illness could furnish understandings of the function mechanisms and new diagnosis and therapy extensions. It was the goal of this work of generating a gene bank of CLL-patients and healthy donors, in order to identify genes, that could be responsible for the origin of the disease. The subtractive suppressive hybridisation (SSH) was inserted as a method in order to generate cDNA-mixtures, in which differential genes were enriched. In this work, three differential genes were identified. One of these genes codes for CD5 that in CLL-patients was stronger expressed. Because CD5 is a marker relatively specific for the B-CLL, this result confirmed the quality of the gene bank. By means of the SSH, a loss of the Expression of the cDNA was proved for CD20 in CLL-patients. Possibly CD20 is a calcium canal and is used as a goal of the anti-CD20-antibody Rituximab. The loss of CD20 in the progress of the disease could be indicated in this work. Further a diminished expression was discovered by cDNA for IkBa (Mad-3) in CLL-patients. The transcription factor IkBa plays an important role in the cellular system of apoptosis. Phosphorylated IkBa is inhibiting the effect of NF-kB. A reduction of IkBa-cDNA could influence also the function of NF-kB. This result could be interesting for future immune therapeutic extension. Further the proved genes are interesting for the technology of the "DNA-Microarrays". This fast method enables favorable diagnoses and could offer therefore a leading-edge alternative to conventional extensions. In the existing work, differential transcripts could be assessed on mRNA-levels in B-CLL-patients and healthy donors. The results give new insights over oncogenes and tumor surpressing genes in the B-CLL and open future immune therapeutic extensions.

Cannabinoids have been shown to modulate the immune system in vitro and in animal models. A major area of interest is how cannabinoids impact the brain. A whole variety of neuropathies or brain disorders, such as AIDS dementia, Parkinson’s disease, Multiple Sclerosis and Alzheimer’s disease, are associated with a hyperinflammatory response within the brain. Microglia, the resident macrophages of the brain, are the major cell type responsible for the persistent elicitation of pro-inflammatory cytokines (IL-1a, IL-1b, IL-6, TNFa) and other mediators. In vitro experiments have demonstrated that the partial exogenous cannabinoid agonist delta-9-tetrahydrocannabinol (D9-THC) and the potent synthetic exogenous cannabinoid agonist CP55940 down-regulate the robust production of pro-inflammatory cytokines elicited in response to bacterial lipopolysaccharide (LPS) at the mRNA level. These observations suggest that cannabinoids, devoid of psychotropic properties, have the potential to betherapeutic agents. These highly lipophilic compounds can pass through the blood brain barrier and act through specific cannabinoid receptors, cannabinoid receptor 1 (CB1) and cannabinoid receptor 2 (CB2). CB1 and CB2 are expressed in the brain and the periphery, respectively, and may serve as molecular targets for ablating chronic brain inflammation. Electrophoretic mobility shift assays (EMSA) were used to assess the effects of D9-THC and CP55940 on the LPS-induced binding interactions of the universal transcription factor NFkB to its cognate promoter binding site in BV-2 microglial-like cells. EMSA analyses demonstrated that the D9-THC and CP55940 down-regulated LPS-induced NFkB binding in BV-2 cells in a biphasic manner. Furthermore, reporter activity assays determined that D9-THC and CP55940 attenuated LPS-induced, NFkB transcriptional activity in the same biphasic manner. We then determined the specificity in which cannabinoids inhibit NFkB function. Real-Time RT-PCR analysis demonstrated that BV-2 cells did not express CB1 mRNA, but they do express CB2 mRNA when untreated and stimulated with IFN-g or LPS. We performed specificity studies using CB1 and CB2 selective agonists and antagonists with our reporter activity assays. The CB1-selective agonist ACEA did not affect NFkB transcriptional activity but the CB2-selective agonist O-2137 exerted a significant decrease in activity. Furthermore, the CB1 antagonist SR141716A could not reverse the inhibitory effects of CP55490 but those effects were blocked by the CB2 antagonist SR144528. Lastly, we determined the site of action in which cannabinoids inhibit NFkB function by assessing the effects of D9-THC and CP55940 on NFkB’s inhibitor protein IkBa. IkBa retains NFkB in the cytoplasm until stimulus-induced cell activation. Neither cannabinoid compound was able to inhibit the phosphorylation of IkBa, which initiates its degradation. However both cannabinoids inhibited the complete degradation of IkBa. Western immunoblot analysis also demonstrated that comparable levels of endogenous and phosphorylated p65, the transactivation subunit of the NFkB protein (p65/p50), were detected in the nucleus of LPS-stimulated BV-2 cells pre-treated with or without D9-THC. These results suggest that, in addition to inhibiting the proteolytic degradation of IkBa, there is also a mechanism of action in the nucleus that prevents the proper binding and subsequent transcriptional activity of NFkB. Collectively, these results suggest that cannabinoids suppress pro-inflammatory cytokine gene expression at the transcriptional level, but it is likely that there is more than one signal transduction pathway involved in the cannabinoid-mediated inhibition of NFkB function.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Endometriosis is a gynecological disease in which endometrial glands and stroma are present outside the uterus. Pelvic pain, infertility and decreased quality of life are the main problems caused by this disease carrying epidemiological and social impact. Peritoneal endometriosis which is characterized by the presence of red, black and white pelvic endometriotic lesions is clearly a multifactorial pathology associated with a local inflammatory response in the pelvic cavity. In vitro studies suggest that the transcription factor nuclear factor-kappaB (NF-êB) is implicated in the transduction of proinflammatory signals in endometriosis. The aim of this study was to investigate the involvement and role of the NF-êB pathway in endometriosis in vivo. Firstly, NF-êB activation and intercellular adhesion molecule (ICAM)-1 expression were investigated in thirty-six peritoneal endometriotic lesions from women. Constitutive NF-êB activation, involving p65- and p50-containing dimers, was demonstrated in peritoneal endometriotic lesions by electrophoretic mobility shift assays and supershift analyses, as well as NF-êB (p65) DNA-binding activity immunodetection assays. NF-êB activation and ICAM-1 expression were significantly higher in red lesions than black lesions, while IêBá (NF-êB inhibitory protein) expression was constant, as shown by Western blot analyses. Secondly, endometriosis was induced in nude mice by intraperitoneal injection of fluorescent labeled menstrual endometrium. Two NF-êB inhibitors (BAY 11-7085 and SN-50) were injected intraperitoneally and endometriotic lesions were recovered on day 5. Both NF-êB inhibitors induced a significant reduction in lesion development compared to control mice. NF-êB activation and ICAM-1 expression of endometriotic lesions were significantly reduced in treated mice, and cell proliferation in BAY 11-7085-treated mice. Both inhibitors produced a significant increase in apoptosis of endometriotic lesions, as assessed by active caspase-3 immunostaining and the TUNEL method. In conclusion, this is the first study to show constitutive NF-êB activation in peritoneal endometriotic lesions collected from women and during the initial development of endometriotic lesions in an animal model. Differential levels of NF-êB activation have been established between red and black lesions, providing more evidence on the distinct inflammatory status of these two types of peritoneal endometriotic lesions. In addition, this study offers further insight into the pathways implicated in NF-êB activation in endometriotic lesions, showing the involvement of p50/p65 dimers and suggesting participation of the canonical pathway of NF-êB activation. This study also demonstrates, for the first time, that NF-êB inhibition reduces the initial development of endometriotic lesions by inhibiting the inflammatory response and cell proliferation, and inducing apoptosis of endometriotic lesions. The NF-êB pathway therefore looks to be a promising therapeutic target for endometriosis prevention and treatment.