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By employing a comprehensive exploration of the molecular mechanisms of human platelet activation, this thesis investigates the mechanism of platelet activation and their function, thereby contributing to advancements in platelet biology and pharmacology. The findings in this thesis will help to develop future kinase-related antiplatelet drugs for cardiovascular diseases, inflammation and cancer.

Cancer is a major global health issue, causing millions of deaths per year. Cancer or resistance against anti-cancer drugs can be favored by amino acid mutations in proteins, such as kinases. These so-called missense mutations can likely hap- pen in a living organism and can therefore alter the proteins properties, like stability, specificity or activity. Modern methods of drug discovery could help to develop new anti-cancer drugs with less side effects, higher specificity or a completely new mechanism of action. By predicting kinase mutations, that are likely to be either drug resistant or oncogenic, scientists could develop new kinase inhibitors for future use when such a predicted mutation is found in a cancer patient. Such a workflow was developed and is capable of predicting drug resistant or oncogenic mutations, that have a single or double point codon mutation as a cause. The foundation of good results is the selection of the right protein structures – each calculation needs an active conformation and an inactive/inhibited conformation of the protein. The workflow further uses the calculated stability of the protein to estimate the effect of an amino acid mutation on the protein where a stabilized active conformation and a destabilized inactive/inhibited confor- mation are good indicators for an oncogenic or drug resistant mutation. For a rough classification, three cutoffs are applied to the raw calculation results. Additional refinement is achieved by including mutational signatures, which express the probability of a certain amino acid mutation according to empirical studies. The final step is a pareto ranking, that uses the stability values in combination with the frequency and the mutational signature to rank the mutations according to their probability to be drug resistant or oncogenic. The workflow was validated with two protein kinases – extracellular-signal regulated kinase 2 (ERK2) and Bruton’s tyrosine kinase (BTK). ERK2 is an important part of a s, submitted by Lukas Forster, BSc., Masterarbeit University Innsbruck 2023

Here, we describe the expression of Bruton’s Tyrosine Kinase (BTK) in head and neck squamous cell carcinoma (HNSCC) cell lines as well as in primary HNSCC samples. BTK is a kinase initially thought to be expressed exclusively in cells of hematopoietic origin. Apart from the 77 kDa BTK isoform expressed in immune cells, particularly in B cells, we identified the 80 kDa and 65 kDa BTK isoforms in HNSCC, recently described as oncogenic. Importantly, we revealed that both isoforms are products of the same mRNA. By investigating the mechanism regulating oncogenic BTK-p80/p65 expression in HNSSC versus healthy or benign tissues, our data suggests that the epigenetic process of methylation might be responsible for the initiation of BTK-p80/p65 expression in HNSCC. Our findings demonstrate that chemical or genetic abrogation of BTK activity leads to inhibition of tumor progression in terms of proliferation and vascularization in vitro and in vivo. These observations were associated with cell cycle arrest and increased apoptosis and autophagy. Together, these data indicate BTK-p80 and BTK-p65 as novel HNSCC-associated oncogenes. Owing to the fact that abundant BTK expression is a characteristic feature of primary and metastatic HNSCC, targeting BTK activity appears as a promising therapeutic option for HNSCC patients.

Brutons tyrosine kinase (BTK) is a critical downstream signaling element from the B-cell receptor (BCR) that has been effectively inhibited in B-cell cancers by irreversible, covalent inhibitors including ibrutinib and acalabrutinib. All FDA-approved covalent BTK inhibitors rely on binding to the cysteine 481 (C481) amino acid within the active site of BTK, thus rendering it inert. While covalent BTK inhibitors have been very successful in multiple B-cell malignancies, improving both overall survival and progression-free survival relative to chemoimmunotherapy in phase 3 trials, they can be limited by intolerance and disease progression. Pirtobrutinib is a novel, highly selective, and non-covalent BTK inhibitor that binds independently of C481, and in a recent, first-in-human phase 1/2 clinical trial was shown to be extremely well tolerated and lead to remissions in relapsed/refractory patients with multiple B-cell malignancies. Here, we review the pharmacologic rationale for pursuing non-covalent BTK inhibitors, the clinical need for such inhibitors, existing safety, and resistance mechanism data for pirtobrutinib, and the forthcoming clinical trials that seek to define the clinical utility of pirtobrutinib, which has the potential to fulfill multiple areas of unmet clinical need for patients with B-cell malignancies.

In the last decade data piled up indicating that BTK – for twenty years considered as a “private matter” of bone marrow-derived cells – it is expressed and plays important and different roles also outside of the hematopoietic compartment and, most notably, in tumor cells. Initial evidence that BTK plays a critical role in B cell-derived malignancies prompted the chase for specific inhibitors, the forefather of which entered the clinic in a record time and paved the way for an ever increasing number of new molecules to be trialed. The growing interests in BTK also led to the discovery that, in solid tumors, two novel isoforms are mainly expressed and actionable liabilities for target therapy. Remarkably, the different isoforms appear to be involved in different signaling pathways which will have to be attentively specified in order to define the area of therapeutic intervention. In this perspective we briefly summarize the progress made in the last decade in studying BTK and its isoforms in cancer cells and define the open questions to be addressed in order to get the most benefits from its targeting for therapeutic purposes.

Bruton's tyrosine kinase is a tyrosine kinase expressed in the entire hematopoietic lineage, except for T cell progenitors, and plays an essential role in the BCR pathway. Inhibition of BTK has proven to be an effective strategy in some lymphomas and chronic lymphocytic leukemia. Ibrutinib and acalabrutinib are BTK inhibitors approved in Argentina.Zanubrutinib is a more specific iBTK, which would explain some differences with those previously developed. Many studies with zanubrutinib in different B-lymphoproliferative syndromes are in development or with different follow-up and it demonstrate a more selective inhibition with more complete and sustained occupation of BTK, which would result in greater benefits in efficacy and safety., La tirosina kinasa de Bruton es una tirosina kinasa expresada en todo el linaje hematopoyético, excepto en los progenitores de células T, y cumple un papel esencial en la vía del BCR. La inhibición de la BTK ha probado ser una estrategia efectiva en algunos linfomas y en leucemia linfática crónica. Ibrutinib y acalabrutinib son inhibidores de BTK aprobados en Argentina. Zanubrutinib es un iBTK más específico, lo que explicaría algunas diferencias con los previamente desarrollados. Muchos estudios con zanubrutinib en diferentes síndromes linfoproliferativos B se encuentran en desarrollo o con diferente tiempo de seguimiento y demuestran una inhibición más selectiva por parte de zanubrutinib con ocupación más completa y sostenida de la BTK, lo que resultaría en mayores beneficios en eficacia y seguridad. ia y seguridad.

The B cell receptor (BCR) pathway has been identified as a potential therapeutic target in a number of common B cell malignancies, including chronic lymphocytic leukemia, diffuse large B cell lymphoma, Burkitt lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone B cell lymphoma, and Waldenstromʹs macroglobulinemia. This finding has resulted in the development of numerous drugs that target this pathway, including various inhibitors of the kinases BTK, PI3K, and SYK. Several of these drugs have been approved in recent years for clinical use, resulting in a profound change in the way these diseases are currently being treated. However, the response rates and durability of responses vary largely across the different disease entities, suggesting a different proportion of patients with an activated BCR pathway and different mechanisms of BCR pathway activation. Indeed, several antigen-dependent and antigen-independent mechanisms have recently been described and shown to result in the activation of distinct downstream signaling pathways. The purpose of this review is to provide an overview of the mechanisms responsible for the activation of the BCR pathway in different B cell malignancies and to correlate these mechanisms with clinical responses to treatment with BCR inhibitors.

Background: Bruton’s tyrosine kinase (BTK) is involved in the immune response and its deficiency impairs B cell maturation. We evaluated the expression of a novel BTK isoform, p65BTK, in colorectal cancer (CRC), to identify its impact on survival. Materials and Methods: This retrospective study evaluated 87 consecutive stage III CRC patients treated at the National Cancer Institute of Aviano (1999-2017). Multiple specimens were collected and analyzed for staining intensity and percentage of tumor cells positive for p65BTK. Prognostic impact was tested by univariate Cox regression analysis. Results: After a median follow-up of 82.59 months, median disease-free survival (DFS) and overall survival (OS) were 11.67 months and 31.33 months, respectively. Interestingly, 10% of patients did not express p65BTK. For the immunohistochemistry IHC intensity 1, the best cutoff point was 1% of p65BTK positivity; for IHC intensity 2, it was 50%; and for IHC intensity 3, it was 80%. Through univariate analysis, patients with highly expressed p65BTK (IHC intensity 3 and ≥80%) were shown to have the worst prognosis in terms of DFS (HR: 6.23; p = 0.005; 95% C.I. 1.75-22.79) and OS (HR: 2.54; p = 0.025; 95% C.I. 1.12-5.76). Conclusions: p65BTK is frequently expressed in CRC and, if highly expressed, is an unfavourable prognostic factor. However, further confirmation is needed and its potential targeting needs to be studied

Chikungunya virus (CHIKV) is transmitted by Aedes mosquitoes and causes prolonged arthralgia in patients. After crossing the mosquito midgut barrier, the virus disseminates to tissues including the head and salivary glands. To better understand the interaction between Aedes albopictus and CHIKV, we performed RNASeq analysis on pools of mosquito heads and parts of the thorax 8 days post infection, which identified 159 differentially expressed transcripts in infected mosquitos compared to uninfected controls. After validation using RT-qPCR (reverse transcriptase-quantitative polymerase chain reaction), inhibitor of Bruton’s tyrosine kinase (BTKi), which has previously been shown to be anti-inflammatory in mammals after viral infection, was further evaluated for its functional significance. Knockdown of BTKi using double-stranded RNA in a mosquito cell line showed no significant difference in viral RNA or infectivity titer. However, BTKi gene knocked-down cells showed increased apoptosis 24 hours post-infection compared with control cells, suggesting involvement of BTKi in the mosquito response to viral infection. Since BTK in mammals promotes an inflammatory response and has been shown to be involved in osteoclastogenesis, a hallmark of CHIKV pathogenesis, our results suggest a possible conserved mechanism at play between mosquitoes and mammals. Taken together, these results will add to our understanding of Aedes Albopictus interactions with CHIKV.

La leucemia linfocítica crónica (LLC) es la leucemia más frecuente en nuestro país afectando mayoritariamente a mayores de 65 años. En los últimos años el tratamiento de la leucemia linfocítica crónica se ha visto modificado debido al descubrimiento de mo léculas cuyo mecanismo de acción es la inhibición del receptor de las células B. Concretamente Ibrutinib ha sido autorizado por la EMA para usarse en monoterapia como primera línea en pacientes adultos con LLC y en monoterapia o asociación con Bendamustina y Rituximab (BR) en pacientes adultos con LLC en recaída que ya hayan tenido un tratamiento previo. El objetivo de este trabajo es evaluar la efectividad y seguridad de Ibrutinib en práctica clínica habitual en pacientes con LLC del Hospital Universitario Virgen Macarena. Estudio observacional retrospectivo de 15 pacientes con LLC tratados con Ibrutinib. La mediana de tiempo en tratamiento fue 12´5 meses (rango 0,5-29). Se incluyeron 9 hombres (60%) y 6 mujeres (40%) con una media de edad de 70 años. La deleción 17p fue positiva en 3 pacientes y negativa en 9. La mutación p53 estaba presente en 2 pacientes y negativa en 4. Ibrutinib se usó como primera línea en 13,33%, en segunda 73,33% y en tercera 13,33% de pacientes. Los efectos adversos causaron en 5 casos la interrupción temporal del tratamiento y en uno la suspensión definitiva. Actualmente continúan con el tratamiento 9 pacientes (60%). Los motivos de suspensión del fármaco fueron exitus (3 pacientes), progresión de la enfermedad (1), falta de respuesta (1) y toxicidad (1).

La línea celular murina MEL (Murine Erythroleukemia cell line) deriva de progenitores eritroides transformados con el complejo vírico Friend, formado por el “spleen focus forming virus” (SFFV) y el “Friend murine leukemia virus” (F-MuLV). Un atributo extremadamente útil de las células MEL radica en su capacidad para retomar el programa de diferenciación mediante la utilización de inductores químicos tales como el hexametilen-bisacetamida (HMBA). En un intento por identificar posibles dianas del HMBA se establecieron líneas celulares eritroleucémicas, derivadas de MEL, que son resistentes a la acción del agente inductor (MEL-R). En un estudio reciente, se han identificado los genes Was y Btk con mayor expresión diferencial en MEL respecto a MEL-R3. Estos genes comparten dos características comunes importantes: están implicados en la organización del citoesqueleto de actina y se activan específicamente en el linaje hematopoyético. Las mutaciones producidas en Was y Btk provocan el desarrollo de enfermedades hematológicas severas. La pérdida de función de Was desencadena el desarrollo del síndrome de Wiskott-Aldrich, asociado a defectos en una gran cantidad de procesos celulares que inducen inmunodeficiencias, trombocitopenia y alteraciones autoinmunes. Asimismo, la inhibición de Btk afecta a las vías de señalización TLR2 y TLR4 de células mieloides, la función de la interleuquina 10 y STAT3 de células dendríticas.

La línea celular murina MEL (Murine Erythroleukemia cell line) deriva de progenitores eritroides transformados con el complejo vírico Friend, formado por el “spleen focus forming virus” (SFFV) y el “Friend murine leukemia virus” (F-MuLV). Un atributo extremadamente útil de las células MEL radica en su capacidad para retomar el programa de diferenciación mediante la utilización de inductores químicos tales como el hexametilen-bisacetamida (HMBA). En un intento por identificar posibles dianas del HMBA se establecieron líneas celulares eritroleucémicas, derivadas de MEL, que son resistentes a la acción del agente inductor (MEL-R). En un estudio reciente, se han identificado los genes Was y Btk con mayor expresión diferencial en MEL respecto a MEL-R3. Estos genes comparten dos características comunes importantes: están implicados en la organización del citoesqueleto de actina y se activan específicamente en el linaje hematopoyético. Las mutaciones producidas en Was y Btk provocan el desarrollo de enfermedades hematológicas severas. La pérdida de función de Was desencadena el desarrollo del síndrome de Wiskott-Aldrich, asociado a defectos en una gran cantidad de procesos celulares que inducen inmunodeficiencias, trombocitopenia y alteraciones autoinmunes. Asimismo, la inhibición de Btk afecta a las vías de señalización TLR2 y TLR4 de células mieloides, la función de la interleuquina 10 y STAT3 de células dendríticas.

Background: Bruton’s tyrosine kinase (BTK) is involved in the immune response and its deficiency impairs B cell maturation. We evaluated the expression of a novel BTK isoform, p65BTK, in colorectal cancer (CRC), to identify its impact on survival. Materials and Methods: This retrospective study evaluated 87 consecutive stage III CRC patients treated at the National Cancer Institute of Aviano (1999-2017). Multiple specimens were collected and analyzed for staining intensity and percentage of tumor cells positive for p65BTK. Prognostic impact was tested by univariate Cox regression analysis. Results: After a median follow-up of 82.59 months, median disease-free survival (DFS) and overall survival (OS) were 11.67 months and 31.33 months, respectively. Interestingly, 10% of patients did not express p65BTK. For the immunohistochemistry IHC intensity 1, the best cutoff point was 1% of p65BTK positivity; for IHC intensity 2, it was 50%; and for IHC intensity 3, it was 80%. Through univariate analysis, patients with highly expressed p65BTK (IHC intensity 3 and ≥80%) were shown to have the worst prognosis in terms of DFS (HR: 6.23; p = 0.005; 95% C.I. 1.75-22.79) and OS (HR: 2.54; p = 0.025; 95% C.I. 1.12-5.76). Conclusions: p65BTK is frequently expressed in CRC and, if highly expressed, is an unfavourable prognostic factor. However, further confirmation is needed and its potential targeting needs to be studied

The 5th Scientific Conference of the International Pemphigus and Pemphigoid Foundation (IPPF), "Pemphigus and Pemphigoid: A New Era of Clinical and Translational Science" was held in Orlando, Florida, on May 15-16, 2018. Scientific sessions covered recent, ongoing, and future clinical trials in pemphigus and bullous pemphigoid, disease activity and quality of life instruments, and the IPPF Natural History Study. Furthermore, the meeting provided an opportunity to hear firsthand from patients, investigators, and industry about their experience enrolling for clinical trials.

The 5th Scientific Conference of the International Pemphigus and Pemphigoid Foundation (IPPF), "Pemphigus and Pemphigoid: A New Era of Clinical and Translational Science" was held in Orlando, Florida, on May 15-16, 2018. Scientific sessions covered recent, ongoing, and future clinical trials in pemphigus and bullous pemphigoid, disease activity and quality of life instruments, and the IPPF Natural History Study. Furthermore, the meeting provided an opportunity to hear firsthand from patients, investigators, and industry about their experience enrolling for clinical trials.

La transcription des symboles et des caractères spéciaux utilisés dans la version originale de ce résumé n’a pas été possible en raison de limitations techniques. La version correcte de ce résumé peut être lue dans le document original. Ce projet de recherche vise à développer un biopesticide à base de Bacillus thuringiensis var kurstaki, Btk, efficace contre la tordeuse des bourgeons de l’épinette et compétitif vis-à-vis des biopesticides commerciaux. Ce projet est né dans un contexte de lutte contre cette tortricidae qui est un des ravageurs le plus destructeur des forêts du Québec et en Amérique du Nord en général. Cet insecte endémique provoque une épidémie environ tous les 30 ans, chacune durant en moyenne 20 ans, ce qui affecte grandement l’industrie forestière, qui est très importante dans l’activité économique de la province. Le coût de la matière première pour l’obtention de biopesticide à base de Btk représente jusqu’à 40% du coût global de production. En effet, ces coûts qui sont inhérents au biopesticide à base de substrat semi-synthétique réduisent son accessibilité. Afin de s’affranchir de cette contrainte, la production de biopesticide à base de Btk, grâce à des matières alternatives suscite beaucoup d’intérêt. L’usage de substrats alternatifs tels que les eaux usées d’industries d’amidon ou des boues secondaires issues du traitement des résidus agroalimentaires permettrait de réduire le coût sur l’obtention du biopesticide ainsi que la valorisation de résidus qui posent un problème de traitement et de leur élimination finale dans l’environnement. Ces matières contiennent une forte concentration de la demande chimique en oxygène (DCO), sous la forme de carbone et de sources d'azote (amidon, gluten, protéines, fibres et autres minéraux). La production du biopesticide est caractérisée par la production de Btk à partir d’un substrat dans un bioréacteur, suivie d’une étape de centrifugation pour concentrer les matières actives de ce bacille (cellules tota

Most terrorist attacks in recent years have resulted in the quick death or capture of the suspect. This thesis examines the hypothesis that terrorism in the United States, from groups such as Al-Qaeda and ISIS, changes in tone, scope, and scale to obtain multiple attacks from each individual adherent. While historically most serial killers try to conceal their crimes, some have taunted the government and the populace with their acts and, in so doing, engendered tremendous fear in large groups of people over significant periods. This thesis examines three cases of well-known serial killer events—the Zodiac killer, BTK, and the D.C. Beltway snipers—and compares them to three recent cases of terrorism—in Boston, San Bernardino, and Orlando. It employs a comprehensive comparison of these six incidents to study the congruency, differences, discourse, patterns, and effects of each to examine the possible impacts and implications of terrorists who use tactics similar to serial killers. The output provides key takeaways pertaining to possible policy implications for the law enforcement community and its situational awareness., http://archive.org/details/imaginingashiftt1094560446, Civilian, San Francisco Police Department, Approved for public release; distribution is unlimited.

Honeybee populations around the world are experiencing a decrease in colony numbers probably due to a combination of different causes, such as diseases, poor nutrition and frequent applications of insecticides to control pests. Previous studies about the effect of pesticide Bacillus thuringiensis subsp. kurstaki (Btk) on Apis mellifera L. report different results. The aim of this study was to analyze the effect of field aerial applications of Btk on bee colony performance, specifically on the brood cell percentage evolution, which can be used as an indicator of queen health and brood development breeding rates. To achieve it, the brood cell surface was photographed in every sampling, and data were analyzed using a method based on image treatment software. A total of 480 pictures were examined from two groups of four nucleus hives in two areas, one receiving aerial spraying with Btk and the other without treatment. A mixed factorial design was realized to analyse the data showing no differences in colony performance between the two groups of colonies either before the treatment, during and at the end of the assay. Furthermore, the brood surface ratio of Btktreated/ untreated increased along the experiment. Therefore, the results of the present study suggest that Btk aerial applications did not affect the brood development of honeybees under natural conditions. Nevertheless further field studies are required to ascertain a safe use of Btk in forest pest management

Honeybee populations around the world are experiencing a decrease in colony numbers probably due to a combination of different causes, such as diseases, poor nutrition and frequent applications of insecticides to control pests. Previous studies about the effect of pesticide Bacillus thuringiensis subsp. kurstaki (Btk) on Apis mellifera L. report different results. The aim of this study was to analyze the effect of field aerial applications of Btk on bee colony performance, specifically on the brood cell percentage evolution, which can be used as an indicator of queen health and brood development breeding rates. To achieve it, the brood cell surface was photographed in every sampling, and data were analyzed using a method based on image treatment software. A total of 480 pictures were examined from two groups of four nucleus hives in two areas, one receiving aerial spraying with Btk and the other without treatment. A mixed factorial design was realized to analyse the data showing no differences in colony performance between the two groups of colonies either before the treatment, during and at the end of the assay. Furthermore, the brood surface ratio of Btktreated/ untreated increased along the experiment. Therefore, the results of the present study suggest that Btk aerial applications did not affect the brood development of honeybees under natural conditions. Nevertheless further field studies are required to ascertain a safe use of Btk in forest pest management

The binding of antigen (Ag) to the B cell receptor (BCR) induces the activation of intracellular signaling and the reorganization of the actin cytoskeleton. However, the function of actin reorganization and the mechanisms by which BCR signaling and actin reorganization is coupled have not been well studied. This thesis has investigated how BCR signaling regulates actin reorganization and how actin remodeling in turn influences BCR signalig. My studies show that the key stimulatory signaling molecule of the BCR, Bruton's tyrosine kinase (Btk), is critical for actin polymerization at the activation surface and BCR clustering and B cell spreading, events that are essential for signaling initiation and amplification. The key inhibitory signaling molecule, SH2-containing phosphatidylinositol-5 phasphatase (SHIP-1), is important for removal of F-actin from the activation surface, and actin-mediated B cell contraction and the formation of BCR central clusters. SHIP-1 suppresses actin polymerization by inhibiting Btk-dependent activation of Wiskott-Aldrich syndrome protein (WASP). These results suggest that BCR signaling can regulate B cell morphology and surface BCR clustering via modulationg actin dynamics. To understand the roles of actin reorganization in BCR signaling, I investigated the effects of gene knockout of the two actin regulators, WASP and its homolog, neuronal (N)-WASP. My results show that both WASP and N-WASP are required for optimal BCR clustering, B cell spreading, and BCR signaling, but they play distinct roles. WASP promotes actin polymerization, B cell spreading, BCR clustering, and signaling amplification, and N-WASP inhibits actin polymerization at the activation surface and promotes B cell contraction, BCR central cluster formation, and signaling attenuation. Importantly, B cell-specific N-WASP knockout causes increases in the levels of autoantibody. In addition, WASP and N-WASP negatively regulate each other, compete for Arp2/3, and are inversely

Cancer is a multistep process in which mutation of 4 to 6 genes has to occur for the cell to become fully oncogenic. Very often, genes affected by such mutations encode for molecules belonging to the pathways allowing DNA repair or controlling the apoptotic process. Many anticancer drugs act by inducing DNA damage, which in turn triggers apoptosis. Inability of tumour cells to undergo chemotherapy-induced apoptosis is a main mechanism of drug resistance. Therefore, in order to find rational targets to tailor therapy is necessary to identify novel genes involved in modulating apoptosis induced by chemotherapeutics. Recently in our lab a loss-of-function RNAi-library-based phenotypic screen has been performed which identified 49 novel genes whose silencing reverts 5-fluoraouracil (FU) resistance in a model colon cancer cell line (HCT116p5KO). The aim of the project was to study the role of one of the targets identified in the abovementioned screen, the Bruton’s tyrosine kinase (BTK), so far assumed to be expressed only in hematopoietic lineages. Our findings suggest that BTK could be an interesting candidate to be targeted in the therapy of drug-resistant colon cancers and a marker of drug-resistance., GRASSILLI, EMANUELA, 1692, open, open, Narloch, Narloch, R

The Signalosome is a term used to define a putative signalling complex, which assembles near the plasma membrane in response to external signals received at cell surface receptors and then migrates towards downstream effectors. It is proposed to regulate the level of intracellular Ca2+ and subsequent downstream signalling events. To date it has been defined to consist of BTK, BLNK, BCAP, VAV, PLCγ2 and PI3K1-4 in B-Cells. This work entailed initiating a new proteomic approach to investigate the nature and extent of Bruton’s tyrosine kinase, Btk, involvement in the signalosome – inherently, the aim was to study multiple interactions of Btk with other molecules. By transfecting host cells with a Btk gene-transfer plasmid, virus particles were produced that were used to up-regulate and analyse the expression of Btk in three haematopoietic cell lines: B-cells, Pre-B-cells and a myeloid cancer cell. The construction of a new gene-transfer vector was successfully carried out by plasmid sub-cloning and it was subsequently found to effectively transfect the host cells and produce virus particles. The recombinant virus particles were employed with success in transducing three haematopoietic cell lines and with immunopurification and subsequent gel separation protein signalosome complexes were obtained ready for analysis by mass spectrometrical fingerprinting (to be carried out as a joint effort in Mount Sinai Hospital in Toronto, Canada).

Macrophages play an essential role in the defense against infection by phagocytosis and by production of inflammatory mediators. Such mediators are TNF? and eicosanoids. Many microbial agents elicit arachidonate release in macrophages that lead to the formation of eicosanoids. This thesis is based on studies performed with the use of microbial constituents which induce arachidonate release or/and TNF? production. Whole gram-positive bacteria S.aureus, the bacterial products LPS and peptidoglycan, the yeast preparation zymosan and ?-glucan were used to stimulate the cells. The main focus has been to elucidate the potential role of the non-receptor tyrosine kinases from the Src family (SFK) and the Tec kinase Btk. in signaling pathways induced by microbial constituents. SFK are important in bacteria and yeast induced arachidonate release and these studies bring forward that SFK has a pivotal role in acting proximally of several known members of the signaling like, ERK, p38 and PLC?2. Btk is also observed to be a part signaling pathway downstream of SFK. Differences were detected between bacteria and zymosan elicted responses, probably due to use of different receptors. Zymosan can bind to several receptors and our results suggest that zymosan-induced arachidonate release is mediated by the ?-glucan receptor dectin-1. Furthermore we show that the adaptor protein gab-2 is affected by ?-glucan and zymosan stimulation, indicating a role in dectin-1 signaling. SFK are also seen to be involved in TNF? production induced by microbial constituents, but their mode of action is still unknown. In summery this thesis has contributed to an increased understanding of the role of SFK in eicosanoid and TNF? production in macrophages. Furthermore has we contributed to knowledge about the signaling pathway resulting in arachidonate release.