A imunidade inata é responsável pela resposta inicial aos microrganismos, uma vez que impede, controla ou elimina a infecção. Esse sistema consiste em barreiras epiteliais, proteínas plasmáticas e células circulantes e teciduais. Dentre esses componentes, os macrófagos possuem grande importância, sendo capazes de controlar e eliminar agentes patogênicos através da fagocitose e produção de espécies reativas de oxigênio e nitrogênio. A ativação de PRRs por constituintes oriundos dos patógenos em macrófagos desencadeia eventos da resposta imune inata, ativados por diversas vias de sinalização intracelular. A via das PI3Ks é conhecida por regular várias funções nas células, como a regulação do ciclo celular, migração e produção de espécies reativas de oxigênio e nitrogênio. O NO é um mediador central na imunidade inata que, após estímulos inflamatórios, é produzido em altas quantidades através da iNOS. Macrófagos deficientes em PI3K produzem menos NO e apresentam prejudicado controle da infecção quando infectados por T. cruzi. O objetivo do presente trabalho foi investigar o papel da via PI3K na produção de NO por macrófagos peritoneais estimulados com LPS. Os macrófagos empregados no estudo, WT e PI3K-/-, possuem o mesmo fenótipo. Observamos que macrófagos PI3K-/- possuem uma menor produção de NO e expressam menos iNOS. A reduzida expressão de iNOS, após estímulo com LPS, é também observada quando macrófagos WT são tratados com inibidores seletivos da PI3K e AKT. Além disso, demonstramos que, concomitantemente à menor expressão da iNOS, ocorre deficiência na fosforilação da AKT e diminuição da ativação do fator de transcrição NF-kB, sugerindo que a PI3K participa da ativação do NF-kB. Foi observado ainda que o tratamento com PTX também diminui a expressão da iNOS. No entanto, macrófagos PAFR-/- expostos ao LPS presentam maior expressão da iNOS, enquanto os macrófagos CCR2-/- apresentam menor expressão dessa enzima nessas condições. Para investigar a implicação da via PI3K in vivo foi administrado LPS i.v., como modelo de choque endotoxemico, no qual observamos maior sobrevida em animais PI3K-/- comparado aos animais WT e menores níveis de nitrito no soro. Nossos dados sugerem que a enzima PI3K é crítica para expressão de iNOS e produção de NO pelos macrófagos, possivelmente através da ativação do receptor CCR2, estando envolvida na fisiopatologia do choque induzido por LPS. / Innate immunity is the initial response to microorganisms, since it prevents, controls and eliminates infection. This system consists in epithelial barriers, plasma proteins and circulating and tissue cells. Among these components, macrophages have great importance, being capable of control and eliminate pathogen agents through phagocytosis and production of reactive oxygen and nitrogen species. Activation of PRRs by pathogens constituents in macrophages triggers events of the innate immune response, activated by various intracellular signaling pathways. PI3Ks pathway is known to regulate several functions in the cell, such as regulation of the cell cycle, migration and production of reactive oxygen and nitrogen species. NO is a central mediator in innate immunity, which after inflammatory stimuli, is produced in high levels by iNOS. PI3K-deficient macrophages produce less NO and exhibit impaired control of infection when infected by T. cruzi. The aim of the present study is to investigate the role of PI3K pathway in NO production by LPS-estimulated peritoneal macrophages. The macrophages used in this study, WT and PI3K- / -, have the same phenotype. We observed that PI3K- / - macrophages have a lower NO production and express less iNOS. The low expression of iNOS after stimulation with LPS was also observed in WT macrophages treated with selective inhibitors of PI3K and AKT. Furthermore, we demonstrate that, along to lower iNOS expression, there is deficiency in AKT phosphorylation and decreased activation of the transcription factor NF-kB, suggesting that PI3K participates of the NF-kB activation. It was also observed that PTX treatment has decreased iNOS expression. However, LPS-exposed PFAR-/- macrophages present greater expression of iNOS, while CCR2-/- macrophage exhibit lower expression of this enzyme under these conditions. To investigate involvement of the PI3K pathway has \"in vivo\",LPS was administered i.v., as an endotoxic model, in which we observed a higher survival in PI3K- / - animals compared to WT animals and lower nitrite levels in serum. Our data suggest that PI3K enzyme is critical to iNOS expression and NO production by macrophages, possibly through activation of the CCR2 receptor, being involved in the LPS-induced shock pathophysiology

Neutrophils form a first line of defence against infections. These short-lived, terminally differentiated cells perform many important functions, including chemotaxis, degranulation, reactive oxygen species (ROS) release and cytokine production. Whilst neutrophils are essential for host immunity, their inappropriate recruitment, activation and/or removal can contribute to excessive inflammation and host damage, as exemplified in autoimmune diseases such as rheumatoid arthritis. It is therefore essential that neutrophil function is tightly regulated. Neutrophils are activated by a range of stimuli, including immune complexes. Neutrophil functions are tightly regulated by intracellular signalling events that are induced by the ligation of cell surface receptors, for example, the binding of immune complexes to Fc receptors. Phosphoinositide 3-kinase (PI3K) and extracellular signal-regulated kinase (Erk) are key signalling intermediates that act downstream of many cell surface receptors. They are involved in the regulation of numerous biological processes in the neutrophil. Using pharmacological interventions, I analysed PI3K signalling in immune complex-stimulated human neutrophils and uncovered a previously uncharacterised, noncanonical signalling pathway, PI3K-Cdc42-Pak-Mek-Erk. This represents an unusual situation where Pak acts as the MAP3K downstream of Cdc42 in a PI3K-dependent fashion. By performing a range of functional experiments, I showed that this unconventional signalling pathway promotes apoptosis in human neutrophils by regulating the ratio between anti- and pro-apoptotic members of the Bcl-2 family proteins. No other immune complex-induced, PI3K-dependent neutrophil function tested depended on PI3K-Cdc42-Pak-Mek-Erk signalling. Mouse knock-outs of all components of this signalling pathway have been described. Immune complex-induced apoptosis was also PI3K-dependent in mouse neutrophils, but experiments performed with inhibitors showed that, in contrast to human neutrophils, this was not dependent on PI3K-Cdc42-Pak-Mek-Erk signalling. The myeloid leukaemia cell line, PLB-985 is amenable to knock-down and can be differentiated to become neutrophil-like. These cells are not notably activated by immune complexes, perhaps because they do not express the major Fcγ receptor, CD16. Since retroviral expression of CD16 in PLB985 cells did not improve their response to activation by immune complexes, I was not able to confirm my observations with human neutrophils genetically. Collectively, I showed that a novel, pro-apoptotic signalling pathway operates downstream of Fcγ receptors in the human neutrophil. The fact that this signalling pathway appears to regulate apoptosis specifically suggests uncoupling pro- and anti-inflammatory effects induced by immune complexes might be possible. This may be helpful in the design of improved therapies of autoimmune diseases such as rheumatoid arthritis, in which immune complex-driven neutrophilic inflammation contributes to disease pathogenesis and where neutrophil apoptosis is disturbed.

Konstitutiv aktivierte Signalwege sind verantwortlich für die malignen Veränderungen in Melanozyten, die zur Entstehung des Melanoms beitragen. Im Mittelpunkt dieser Veränderungen stehen der PI3Kinase-AKT-Signalweg und der MAPK-ERK-Signalweg als wichtige Schlüsselwege in der Zellzykluskontrolle. Daher zielen viele neue Therapieversuche im Kampf gegen das Melanom auf die Kontrolle und Regulation dieser Wege. In dieser vorliegenden Arbeit wurden erstmals die Effekte eines neuen PI3K-Inhibitors, BAY-80-6946, und Wortmannin alleine und in Kombinationsbehandlungen mit den MEK1/2-Inhibitoren PD98059 und U0126 in vitro und in vivo untersucht. Zunächst wurden humane Melanomzellen auf konstitutiv aktivierte Signalwege in vitro per Western Blot untersucht und ihre Wachstumsraten im Mausmodell ermittelt. Die humane Melanomzelllinie LOX zeigte eine hohe konstitutive Expression von aktiviertem AKT und ERK, während A375 nur eine geringe Aktivität dieser beiden Signalwege aufwies. Dennoch besaßen diese beiden Zelllinien ein großes Wachstumspotential im Mausmodell im Vergleich zu anderen getesteten Zelllinien. A375 und LOX wurden in Zellkulturexperimenten mit den PI3Kinase-Inhibitoren Bay-80-6946 und Wortmannin sowie den MEK1/2-Inhibitoren PD98059 und U0126 behandelt und Tumor-relevante Zellfunktionen wie Proliferation und Apoptose gemessen. Die Zelllinien zeigten ein unterschiedliches Ansprechen auf die verschiedenen Inhibitoren und keine der Behandlungen wies eine zufriedenstellendes Ergebnis hinsichtlich der Anti-tumoralen Funktion auf. Bei dualer PI3Kinase- und MEK1/2-Hemmung zeigten sich jedoch deutliche synergistische Effekte, so dass diese Behandlungsform einen vielversprechenden Ansatz im Kampf gegen das Melanom darstellt.

A imunidade inata é responsável pela resposta inicial aos microrganismos, uma vez que impede, controla ou elimina a infecção. Esse sistema consiste em barreiras epiteliais, proteínas plasmáticas e células circulantes e teciduais. Dentre esses componentes, os macrófagos possuem grande importância, sendo capazes de controlar e eliminar agentes patogênicos através da fagocitose e produção de espécies reativas de oxigênio e nitrogênio. A ativação de PRRs por constituintes oriundos dos patógenos em macrófagos desencadeia eventos da resposta imune inata, ativados por diversas vias de sinalização intracelular. A via das PI3Ks é conhecida por regular várias funções nas células, como a regulação do ciclo celular, migração e produção de espécies reativas de oxigênio e nitrogênio. O NO é um mediador central na imunidade inata que, após estímulos inflamatórios, é produzido em altas quantidades através da iNOS. Macrófagos deficientes em PI3K produzem menos NO e apresentam prejudicado controle da infecção quando infectados por T. cruzi. O objetivo do presente trabalho foi investigar o papel da via PI3K na produção de NO por macrófagos peritoneais estimulados com LPS. Os macrófagos empregados no estudo, WT e PI3K-/-, possuem o mesmo fenótipo. Observamos que macrófagos PI3K-/- possuem uma menor produção de NO e expressam menos iNOS. A reduzida expressão de iNOS, após estímulo com LPS, é também observada quando macrófagos WT são tratados com inibidores seletivos da PI3K e AKT. Além disso, demonstramos que, concomitantemente à menor expressão da iNOS, ocorre deficiência na fosforilação da AKT e diminuição da ativação do fator de transcrição NF-kB, sugerindo que a PI3K participa da ativação do NF-kB. Foi observado ainda que o tratamento com PTX também diminui a expressão da iNOS. No entanto, macrófagos PAFR-/- expostos ao LPS presentam maior expressão da iNOS, enquanto os macrófagos CCR2-/- apresentam menor expressão dessa enzima nessas condições. Para investigar a implicação da via PI3K in vivo foi administrado LPS i.v., como modelo de choque endotoxemico, no qual observamos maior sobrevida em animais PI3K-/- comparado aos animais WT e menores níveis de nitrito no soro. Nossos dados sugerem que a enzima PI3K é crítica para expressão de iNOS e produção de NO pelos macrófagos, possivelmente através da ativação do receptor CCR2, estando envolvida na fisiopatologia do choque induzido por LPS. / Innate immunity is the initial response to microorganisms, since it prevents, controls and eliminates infection. This system consists in epithelial barriers, plasma proteins and circulating and tissue cells. Among these components, macrophages have great importance, being capable of control and eliminate pathogen agents through phagocytosis and production of reactive oxygen and nitrogen species. Activation of PRRs by pathogens constituents in macrophages triggers events of the innate immune response, activated by various intracellular signaling pathways. PI3Ks pathway is known to regulate several functions in the cell, such as regulation of the cell cycle, migration and production of reactive oxygen and nitrogen species. NO is a central mediator in innate immunity, which after inflammatory stimuli, is produced in high levels by iNOS. PI3K-deficient macrophages produce less NO and exhibit impaired control of infection when infected by T. cruzi. The aim of the present study is to investigate the role of PI3K pathway in NO production by LPS-estimulated peritoneal macrophages. The macrophages used in this study, WT and PI3K- / -, have the same phenotype. We observed that PI3K- / - macrophages have a lower NO production and express less iNOS. The low expression of iNOS after stimulation with LPS was also observed in WT macrophages treated with selective inhibitors of PI3K and AKT. Furthermore, we demonstrate that, along to lower iNOS expression, there is deficiency in AKT phosphorylation and decreased activation of the transcription factor NF-kB, suggesting that PI3K participates of the NF-kB activation. It was also observed that PTX treatment has decreased iNOS expression. However, LPS-exposed PFAR-/- macrophages present greater expression of iNOS, while CCR2-/- macrophage exhibit lower expression of this enzyme under these conditions. To investigate involvement of the PI3K pathway has \"in vivo\",LPS was administered i.v., as an endotoxic model, in which we observed a higher survival in PI3K- / - animals compared to WT animals and lower nitrite levels in serum. Our data suggest that PI3K enzyme is critical to iNOS expression and NO production by macrophages, possibly through activation of the CCR2 receptor, being involved in the LPS-induced shock pathophysiology

Introduction: Over the past decades, considerable advances in understanding of cell biology at genetic, epigenetic and proteomic levels led to development of new strategies for better outcome of cancer therapy. One of these new strategies is targeting the class I PI3K/Akt/mTOR signaling pathway, in that this pathway plays a key role in regulation of many cellular functions, including proliferation, survival, metabolism, autophagy and motility. Dysregulation of the class I PI3K/Akt/mTOR pathway has been documented in a variety of human tumours and inhibition of this pathway has been observed to hamper tumour proliferation in vitro and prevent tumour progression in vivo and in clinic. More recently, emerging evidence suggests that the class I PI3K/Akt/mTOR pathway is associated with Cancer Stem Cell (CSC) biology, in light of maintenance, viability and conventional therapy resistance of CSCs. The CSC theory conceptualizes that a subset of tumour cells with Stem Cell-like properties, including self-renewal, multipotency, differentiation, and resistance to chemotherapy and radiotherapy, can recapitulate new tumours and resistance to cancer therapy. Materials and Methods: To explore class I PI3K/Akt/mTOR signaling pathway and CSCs as therapeutic targets in canine oncology, in one series of experiments, smallmolecular inhibitors Wortmannin, ZSTK474, KP372-1 and Rapamycin, which selectively target pan-class I PI3K, pan-class I PI3K, Akt and mTOR, respectively, were utilized to treat canine cancer cell lines using inhibitors alone or in combination with conventional therapeutic drugs. The human acute lymphoblastic leukaemia of T-cell origin cell line (Jurkat T cell line) was used as a comparative control. In another, a stem cell culture system was performed to isolate CSCs from canine glioma J3T cell line. Subsequently, microarray analysis of transcriptional expression profiles of J3T spheres (the putative CSCs) versus J3T parental cells was performed. Results: In this study, small molecules ZSTK474 and KP372-1 were found to significantly decrease cell viability at lower micromolar and nanomolar ranges, respectively. Rapamycin decreased cell viability at lower micromolar concentrations. However, the efficacy of Wortmannin varied from one cell line to another. Dissection of the mechanism of these inhibitors using Western Blot analysis and annexin V staining showed that all inhibitors functioned by decreasing phosphorylation of class I PI3K pathway members. Notably, the efficacy of Wortmannin for this pathway inhibition is confined to certain cell lines. In addition, Wortmannin had shorter drug duration than the other three inhibitors. Annexin V staining showed that KP372-1 was a potent inducer of apoptosis, with decreasing potency in hierarchy order, Rapamycin, Wortmannin and ZSTK474. The data obtained from the combination of pan-class I PI3K inhibitor (Wortmannin or ZSTK474) and mTOR inhibitor (Rapamycin) suggested that additive/synergistic effects were, in part, due to inactivation of Akt. The class I PI3K pathway inhibitors enhanced the efficacy of Doxorubicin in SB cells but not in canine REM, 3132 and J3T cells. The CSC colonies of canine glioma J3T cells were successfully isolated and expanded in the neurosphere formation assay. By microarray analysis, several class I PI3K signaling network-associated genes, particularly IGFBP2 (27-fold), FYN (9.3- fold), and DDIT4 (8.5-fold), were found to be highly up-regulated in the J3T CSCs. However, the genes encoding components, such as Akt1 and eIF4E, of class I PI3K/Akt/mTOR axis signaling were either unchanged or down-regulated in the CSCs. The majority of the genes encoding translation initiation factors were also downregulated in the CSCs. Conclusions: This study demonstrates that class I PI3K/Akt/mTOR signaling pathway is critical for proliferation and survival of cell lines derived from human acute lymphoblastic leukemia of T cell origin (Jurkat T cell line) and a variety of canine tumours. However, it appears that this pathway is dispensible for maintainence and viability of the CSCs isolated from canine gloma J3T cell line. This study suggests that the strategy of dual inhibition of class I PI3K and mTOR kinases may have better outcomes than the combination inhibitors of this pathway (such as ZSTK474 and KP372-1) with Doxorubicin in canine oncology.

The phosphoinositide species phosphatidylinositol 3,4,5, trisphosphate (PIP3) is an essential mediator of many vital cellular processes involved in cell growth, survival, and metabolism. The class I PI3Ks are responsible for production of PIP3, and their activity is tightly controlled through interactions with regulatory proteins and activating stimuli. The class IA PI3Ks are composed of three distinct p110 catalytic subunits (p110, p110, p110) and they play different roles in specific tissues due to disparities in both expression and engagement downstream of cell surface receptors. Disruption of PI3K regulation is a frequent driver of numerous human diseases. Growth of all cell types is dependent on PI3K signalling, and development of immune cells relies on a precise balance of PIP3 production. Activating mutations in the genes encoding the catalytic and regulatory subunits of PI3K lead to cancer and immunodeficiencies. The PIK3CA gene encoding the p110 catalytic subunit of class IA PI3K is one of the most frequently mutated genes in cancer, and mutations in the PIK3CD gene encoding the p110 catalytic subunit lead to primary immunodeficiency. All class IA p110 subunits interact with p85 regulatory subunits, and mutations/deletions in different p85 regulatory subunits (PIK3R1, PIK3R2, PIK3R3) have been identified in both cancer and primary immunodeficiencies. By asking how these mutations mediate activation and disease phenotypes, we can identify the natural regulatory molecular mechanisms of class IA PI3Ks. Fundamentally understanding how mutations in PI3K subunits mediate human disease will expand our knowledge of PI3K biology and is essential to the development of novel therapeutics. To identify the molecular mechanisms of class IA PI3K activating mutations, I employed a sophisticated combination of hydrogen-deuterium eXchange mass spectrometry (HDX-MS) with biochemical activity assays to probe the regulatory mechanisms of PI3Ks. HDX-MS measures the exchange rate of amide hydrogens in solution, which in turn can provide information on protein conformation and conformational changes between different states. By comparing PI3K mutants identified in primary immunodeficiency and cancer patients to wild-type enzymes, I have identified dynamic conformational changes induced by activating mutations. Biochemical and biophysical analysis of these mutants led us to generate a panel of engineered mutations to further characterise molecular mechanisms by which class IA PI3Ks are regulated. This thesis will consist of an introduction to class IA PI3K signalling and an introduction to the method of HDX-MS, followed by two data chapters wherein I investigate the mechanisms of activating mutations in PIK3CD followed by an investigation into activating mutations in PIK3R1. A conclusion and discussion of future directions will be presented in the final chapter. This work provides novel insight into the complex regulatory mechanisms of the class IA PI3Ks, which may lead to better understanding of human diseases that activate these enzymes. / Graduate / 2020-04-06

The p85 subunit of phosphatidylinositol 3-kinase (PI3K) has long been thought of as a regulatory subunit that has no other function than the regulation of the p110 catalytic subunit. Our laboratory is studying other roles of the p85 subunit, in particular determining the role of the p85 BH domain. The BH domain has homology to GTPase activating protein (GAP) domains that are involved in the stimulation of monomeric G proteins to hydrolyze their bound GTP to GDP. This converts the G protein from its active conformation to its inactive conformation. We have determined that p85 interacts with Rab proteins, monomeric G proteins that regulate vesicle fusion during the endocytosis of receptors. We have shown that p85 binds to Rab5 regardless of nucleotide-bound state of Rab5. The p85 subunit of PI3K has in vitro GAP activity towards Rab5. It was determined that p85 also has in vitro GAP activity towards Rab4, Rab7, Rab6 as well as the Rho-family G proteins, Rac1 and Cdc42. This GAP activity was localized to the BH domain of p85 and mutation of Arg 274 to Ala abolishes the GAP activity of p85. When this p85R274A mutant was expressed in cells, PDGFR degradation was severely inhibited and there was a corresponding increase in the duration of MAPK and Akt signalling. This increase in cell signalling caused a transformed phenotype in cells expressing the p85 protein with the Arg 274 mutation. These cells have lost contact inhibition for growth, are able to grow independent of attachment as well as in the presence of limited growth factors. They also form tumours in nude mice. These cellular effects seem to be due to an increase in receptor recycling because of the loss of the GAP activity of p85. This increase in receptor recycling may interfere with receptor targeting to the late endosome, which would cause the decrease in receptor degradation that is seen in the p85R274A cells.

The p85 subunit of phosphatidylinositol 3-kinase (PI3K) has long been thought of as a regulatory subunit that has no other function than the regulation of the p110 catalytic subunit. Our laboratory is studying other roles of the p85 subunit, in particular determining the role of the p85 BH domain. The BH domain has homology to GTPase activating protein (GAP) domains that are involved in the stimulation of monomeric G proteins to hydrolyze their bound GTP to GDP. This converts the G protein from its active conformation to its inactive conformation. We have determined that p85 interacts with Rab proteins, monomeric G proteins that regulate vesicle fusion during the endocytosis of receptors. We have shown that p85 binds to Rab5 regardless of nucleotide-bound state of Rab5. The p85 subunit of PI3K has in vitro GAP activity towards Rab5. It was determined that p85 also has in vitro GAP activity towards Rab4, Rab7, Rab6 as well as the Rho-family G proteins, Rac1 and Cdc42. This GAP activity was localized to the BH domain of p85 and mutation of Arg 274 to Ala abolishes the GAP activity of p85. When this p85R274A mutant was expressed in cells, PDGFR degradation was severely inhibited and there was a corresponding increase in the duration of MAPK and Akt signalling. This increase in cell signalling caused a transformed phenotype in cells expressing the p85 protein with the Arg 274 mutation. These cells have lost contact inhibition for growth, are able to grow independent of attachment as well as in the presence of limited growth factors. They also form tumours in nude mice. These cellular effects seem to be due to an increase in receptor recycling because of the loss of the GAP activity of p85. This increase in receptor recycling may interfere with receptor targeting to the late endosome, which would cause the decrease in receptor degradation that is seen in the p85R274A cells.

Gliomblastoma is a highly malignant tumor of the central nervous system that is resistant to radiation and chemotherapy. Evidences accumulated over recent years have indicated the phosphoinositide 3-kinase/Akt signal transduction pathway as one of the major factors implicated in cancer resistance to conventional therapies. In this study we determined whether inhibition of PI3K/Akt signal pathway through pharmacological and/or genetic manipulation could enhance radiation sensitivity in glioma cells. Our results showed 6 of 12 glioma cell lines with activated Akt mostly due to reciprocal down-regulation of PTEN activity (loss-of-function mutations) but not by PIK3CA gain-of-function mutations. U87 and U373 glioma cell lines with PTEN mutation showing strong Akt Ser473 phopshorylation were treated with PI3K inhibitor LY294002 and irradiated with 0, 2.5, 5 and 7.5 Gy of radiation dosages. The results showed LY294002 inhibited Akt actvation in the glioma cells and decreased clonogenic survival in a radiation dose-dependent manner. Expression of dominant-negative Akt and PTEN through adenovirus mediated gene delivery in U87 and U373 glioma cells sensitized tumor cells to radiation treatment. Furthermore, PDK1 and mTOR inhibitors were also used on radiation sensitivity test. But both inhibitors had no radiosensitization in glioma cells. Glioma invasion was linked to advanced tumor stages. Recently, Type 1 insulin-like growth factor regulates tumor invasion have been showed to be mediated through the PI3K/Akt signaling pathway. In this study, we treated glioma cells with LY294002 to analyze its effects on invasion and migration potentials of the tumor cells. The results showed LY294002 inhibited both abilities in most glioma cell lines in vitro. In addition we used adv-PTEN and adv-dnAkt to confirm these results. Adv-PTEN performed dramatic decrease in glioma cell invasion potentials. Furthermore, we investigated whehter PI3K downstream PDK1, and mTOR involved in tumor cell invasion. We used PDK1 and mTOR inhibitors in glioma and determined their effects on invasion by Boyden chamber assay. Unfortunately, both of inhibitors had only limited inhibition on glioma invasion. Take together, our results indicate the feasibility of using PI3K/Akt inhibiting genetic and pharmacological agents to induce glioma cells to become more sensitive to radiation treatment and reduced invasion potentials. However, glioma radiosensitization and invasion may also be regulated by other signaling pathway.

PI3Ks (phosphoinositide 3-kinases), a family of enzymes expressed in immune cells, are activated in response to a wide variety of stimuli by generating second lipid messengers. A subset of singnaling molecules containing lipid-binding pleckstrin homology (PH) domains are downstream molecules of PI3K signaling pathway, essential to mediate the functional outcomes of PI3Ks. Bam32 / DAPP1 is a PH domain-containing adaptor protein, which was discovered from human tonsil germinal centers (GCs); however, its biological function related to GCs, where efficient T-cell-dependent (TD) antibody responses are generated, is unknown. This thesis is focused on the effect of genetic or pharmacological blockade of PI3K p110delta activity on T and B cells, and the role of Bam32 in GC responses. Type 2 cytokine responses are significantly decreased in p110delta-inactivated mice, whereas Type 1 cytokine responses are increased or comparable after primary and secondary immunization. Hallmarks of asthma, airway inflammation and respiratory hyper-responsiveness are dramatically reduced in those mice. Adoptive transfer of OVA-primed splenocytes from normal, but not p110delta-inactivated mice could induce airway eosinophilia in naïve, airway-challenged recipient mice. These data demonstrate a novel functional role for p110delta signaling in induction of Type 2 responses in vivo and may offer a new therapeutic target for Th2-mediated airway disease. Paradoxically, serum IgE levels are markedly increased in OVA-immunized p110delta-inactivated mice despite lower level of swich factor IL-4. In vitro studies showed that p110delta is required to restrain IgE class switch recombination in a B-cell intrinsic manner. Blockade of PI3K activity using broad-spectrum PI3K inhibitors PIK-90 and PI-103 generates similar results. In vivo administration of p110delta-selective inhibitor IC87114 into OVA-immunized mice results in selective elevation of antigen-specific IgE production. Disruption of p110delta signaling leads to increased germline transcription at the epsilon locus (epsilon GLT) and increased induction of activation induced cytidine deaminase (AID) enzyme, suggesting deregulation at the level of the isotype switch process. Moreover, p110delta signaling selectively regulates the expression level of transcription factor Bcl6 and IRF4, which may be responsible for the regulation of AID and epsilon GLT. PI3K signaling regulates multiple steps of GC development, and Bam32 may be involved. GCs dissipate prematurely in Bam32-deficient mice after immunization with OVA/alum. In vitro, Bam32-deficient B cells are functional competent in proliferation, chemotaxis, isotype switching and plasma cell differentiation in response to signals present in GCs. In vivo, Bam32-deficient GC B cells proliferate normally; however, they are more apoptotic. Adoptive transfer studies indicated that intrinsic defect of Bam32-/- B cells leads to premature GC dissolution. Additionally, GCs formed by Bam32-/- B cells contain fewer T cells, implying that Bam32 is required for B cell-dependant T cell accumulation within established GCs. Treatment of Bam32-/- mice with agonistic anti-CD40 fully restored GC persistence and IgG1 isotype switching, demonstrating that Bam32-deficient GC B cells are functionally competent when access to cognate signals is not limiting. Collectively, those data demonstrate that Bam32 is not required for GC initiation, but rather functions in a late checkpoint of GC progression associated with T cell recruitment and GC B cell survival. In general, by focusing on PI3K p110delta and its downstream adaptor protein Bam32, my studies clearly indicate that p110delta is a potential therapeutic target for the treatment of Th2-induced airway inflammation. The unexpected immunomodulatory acitivity on IgE switching associated with multiple PI3K inhibitor compounds is first discovered in this thesis, suggesting that more need to be investigated in this aspect before those inhibitor compounds are widely used in the clinic. Furthermore, the specific regulatory role of Bam32 in GCs represents a unique model for us to study the late GC checkpoint in regarding to in vivo GC B cell and T cell interaction, which is an important issue need to be clarified in order to fully understand GC responses.