The cellular inhibitor of apoptosis protein (cIAP)-2 plays an important role in the protection against apoptosis by inhibiting the endogenous IAP inhibitor Smac, thus allowing other members of the IAP family, such as XIAP to block caspases. Additionally, cIAP2 functions as a ubiquitin ligase and mediates survival/proliferative signaling through NF-κB. cIAP2 is overexpressed in many human cancers and is believed to play an oncogenic role. This led to the development of small molecule IAP antagonists aimed at eliciting apoptosis in cancer cells. However, the loss of cIAP2 is also associated with multiple myeloma, in which constitutively active NF-κB signaling contributes to pathogenesis of the disease and suggests that cIAP2 may also perform a tumour suppressive function. We demonstrate a novel role for cIAP2 in maintaining p53 levels in mammary epithelial cells that express wildtype p53. Downregulation of cIAP2 resulted in activation of IKKs, which led to increased Mdm2-mediated degradation of p53. cIAP2 depletion also led to increased phosphorylation of ERK1/2. Reduction of p53 levels, in combination with survival signaling provided by NF-κB and MEK-ERK pathways were associated with increased colony formation in vitro and increased DMBA-induced adenocarcinomas in cIAP2-null mice. Treatment of cells with IAP antagonists resulted in significant cytotoxicity only in p53-mutant MDA-MB-231 cells, which was associated with autocrine production of TNF-α. We propose that the transcription of TNF-α is potentiated by gain-of-function mutation in p53 since downregulation of mutant p53 in MDA-MB-231 cells decreased TNF-α mRNA. Downregulation of cIAPs in p53-mutant cells resulted in a decrease in nuclear IKK-α, which may result in decreased IKK-α-mediated survival signaling. In contrast, cIAP downregulation in p53-wildtype cells resulted in no change in nuclear IKK-α, degradation of the corepressor SMRT and cell survival. We show that the effects of cIAP2 downregulation are context-dependent. Downregulation of cIAP2 in p53-wildtype cells results in a decrease in p53 and an increase in survival and proliferative signaling. These results suggest a tumour suppressor function for cIAPs that may account for cIAP mutation-associated cancers such as multiple myeloma. Moreover, our data also defines gain-of-function p53 mutation as a possible contributor to IAP antagonist sensitivity.

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There are approximately 10^14 bacteria belonging to nearly 1000 different species in the human gastrointestinal (GI) tract that co-exist with host cells. Within the GI tract, signaling molecules secreted by both eukaryotic and prokaryotic cells are abundant. Recent studies have shown that both bacteria and human cells recognize and respond to the signals from each other, presumably to gain a competitive advantage. The cross-recognition of signals is known as Inter-kingdom (IK) signaling and this phenomenon is considered to be important in the onset of infections in the GI tract. Of the eukaryotic signaling molecules present in the GI tract, the neuroendocrine hormone norepinephrine (NE) is considered to be important in the context of infections as NE is produced at very high concentration in the intestine under post traumatic stress, is known to increase bacterial virulence and infection, and has also been shown to be a potent chemoattractant for GI tract pathogens such as enterohemorrhagic E. coli (EHEC). The focus of this study is on elucidating the mechanisms underlying the recognition and chemotaxis of bacteria towards NE. While chemotaxis has been typically investigated in the context of bacteria moving towards a metabolizable source (e.g., amino acids), chemotaxis is potentially important in the onset of infections in the human GI tract. In this study we use a microfluidic plug assay to investigate the receptor and mechanism utilized by a model bacterium Escherichia coli in its chemotactic response to NE. A series mutant of E. coli RP437 strains of knockouts for four MCP-encoding genes was used in this study. The results from the microfluidic plug assay were then confirmed quantitatively by capillary assay. We have shown that Tsr receptor is necessary for chemotaxis of NE for E. coli RP437, and attraction of E. coli towards NE may require an additional receptor. Results from the priming experiments suggest that exposure to NE may result in the de novo expression of co-receptor(s) that are crucial to chemotaxis towards NE. The requirement for high cell density also suggests the possibility that NE per se may not be an attractant for E. coli, but could be a precursor that is modified into a chemoattractant by cells. These results are expected to further our understanding of bacterial chemotaxis and its role in bacterial colonization and infection of the human GI tract.

Glucose is a primary and essential energy source for humans. It is broken down from complex carbohydrates in the diet and absorbed across the gut epithelium into the blood stream. Glucose homeostasis is important as hyperglycermia causes damage of pancreatic and peripheral cells. In response to a meal glucose is principally taken up by fat and muscle tissues and this response is activated by insulin release from pancreatic beta cells. Insulin stimulates the translocation of GLUT4 from the intracellular storage vesicles to the plasma membrane in fat and muscle cells. Although many proteins have been implicated in this process, the key insulin-regulated substrate has not been determined yet. In the present study, the phosphoserine/threonine binding protein 14-3-3 was used as a tool to affinity-purify insulin-stimulated phosphoproteins from 3T3-L1 adipocytes. By using mass spectrometry 38 proteins were identified, reflecting the important role of 14-3-3 in mediating many insulin-regulated processes. Among the potential phosphoproteins was Myosin 1C (Myo1c), an actin-associated molecular motor, which has previously been implicated in insulin-stimulated GLUT4 trafficking in adipocytes. I showed that insulin stimulates the activation of CaMKII which phosphorylates Myo1c at S701 in a Ca2+/PI3K-dependent manner. Myo1c phosphorylation induced its interaction with 14-3-3-proteins, reduced calmodulin-binding and stimulated its in vitro ATPase activity. Insulin-dependent stimulation of Myo1c phosphorylation and its ATPase activity were both required for GLUT4 translocation. By using yeast two-hybrid techniques, I identified a candidate ligand of the Myo1c tail, Armcx5, and demonstrated the in vivo interaction in 3T3-L1 adipocytes. The siRNA-mediated knockdown of Armcx5 inhibited insulin-stimulated glucose uptake and GLUT4 translocation. These results suggest that the regulation of Myo1c and its ligand Armcx5 are essential in insulin-regulated GLUT4 trafficking, possibly playing a key role in vesicle fusion.

B lymphocytes constitutes an important arm of the immune system, and their response to antigen is largely dependent upon signal transduction through the B cell receptor (BCR). Such a potent receptor, however, needs to be further balanced by positive and negative regulators to prevent harmful effects that may arise from inappropriate stimulation. Src family protein tyrosine kinase Lyn is involved in both positive and negative regulation, since the both gain-of-function Lyn and loss-of-function Lyn mutations caused autoimmunity in mice. The exact signalling pathway(s) regulated by Lyn in B cells, however, are still not clear. Work presented in this thesis attempts to elucidate the biochemical mechanisms that underline the double-edged nature of Lyn in BCR signalling. (For complete abstract open document)

Diss. (sammanfattning) Linköping : Univ., 2003. / Härtill 5 uppsatser.

Thesis (Ph. D.)--Cornell University, August, 2008. / Vita. Includes bibliographical references (leaves 126-141).

Thesis (Ph.D.)--University of Alabama at Birmingham, 2010. / Title from PDF title page (viewed on June 28, 2010). Includes bibliographical references (p.179-204).

Made available in DSpace on 2014-06-11T19:22:49Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-01-27Bitstream added on 2014-06-13T20:27:33Z : No. of bitstreams: 1 pintojunior_dac_me_prud_prot.pdf: 1186583 bytes, checksum: e83d66af1d0fd4767ad1c9b69a60014b (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A obesidade é uma condição que afeta muitos indivíduos e está relacionada a vários tipos de disfunções,como a resistência insulínica.Esta patologiase dá devido a uma falha na sinalização entre o hormônio, proteínas intracelularese o GLUT4 em células musculares e adiposas. Alguns fatores contribuem para aumentar esta falha, como o aumento da fosforilação em serinado IRS –1 e atividade pró -inflamatória. Uma maneira indireta de se avaliar o grau de inflamação é analisar a expressão do SOCS3,que estámais expressoquando há maior atividade inflamatória. Aprática de exercício físico aparece como uma importante ferramenta, pois melhora a sensibilidade insulínica e pode aumentara expressão do transportador de glicose até mesmo em animais obesos. Oobjetivo do trabalho foiavaliar o efeito dos exercícios aeróbio contínuo e com pesoscombinadossobre a via de sinalização da insulina e expressão de transportador... / Obesity is a condition that affects manypeople and is related with some kindof diseases, like insulin resistance.This pathology occurs because of animpairmenton activation of GLUT4 machinery (insulin signaling, activation of intracellular proteins) in skeletal muscle and adipose tissue. Some factors contributedirectly with this illness, like increase IRS –1 serinephosphorylationand pro –inflammatoryactivity. An indirect way to measure inflammation istoanalyze expression of SOCS3, because when it is overexpressed means thatthere are more pro –inflammatory activity.Literature shows that physical exercise can improve insulin sensitivity by increases of GLUT4 expression even in obese rats. So we aimed... (Complete abstract click electronic access below)

Abstract The mammalian metanephric kidney develops through ureteric bud branching morphogenesis and tubule formation and involves secreted inductive signals and possibly their antagonists to regulate the process. Sprouty (spry) genes encode antagonists of FGFs and the EGF signalling pathways. To get an insight to potential developmental roles of the spry genes, the expression of spry1, 2 and 4 was analyzed in developing kidney. Spry1 is expressed in the ureteric bud, and spry2 and 4 in the ureteric bud, the kidney mesenchyme and the nephrons deriving from it suggesting developmental roles for the sprys in kidney development. Spry function was addressed in vivo in the kidney by targeting hspry2 expression to the ureteric bud with a Pax2 promoter. Hspry2 expression led to development of small, ectopic and cystic kidneys. Ureter branching was reduced and there was less glomeruli in a smaller kidney compared to the wild type controls. Spry2 may antagonize signalling of FGF2 and lead to changes in FGFR1 and FGFR3 expression. In organ culture ectopic FGFs restored ureteric branching of the hSpry2 transgenic kidneys suggesting that hSpry2 may antagonize FGF signalling in embryonic kidney. In addition to changes in FGFs, hspry2 expression also lead to downregulation of GDNF and BMP4. We conclude that the Sprouty-FGFs-FGFR signaling is important for kidney development. Wnt2b is a recently identified member of the Wnt family of secreted growth factors, but its function in organogenesis is unknown. In the kidney Wnt2b is localized to the perinephric mesenchymal cells at the initiation of organogenesis. Wnt2b signalling supported ureteric bud growth and branching in vitro. Ureteric bud that was co-cultured with Wnt2b expressive cells or incubated with a known Wnt pathway regulator lithium, and then recombined with isolated kidney mesenchyme led to recovery of the expression of some ureteric epithelial marker genes and reconstitution of early kidney development. Hence, Wnt2b signalling is critical for induction of ureteric branching in vitro. Type XVIII collagen is a matrix molecule and may be involved in Wnt signalling. Roles of type XVIII collagen in kidney and lung organogenesis was analysed. Type XVIII collagen expression correlated with the differences in epithelial branching in both of these organs and its expression in the epithelial tissue was mutually exclusive. In recombinants of ureteric bud and lung mesenchyme, type XVIII collagen expression pattern shifted from kidney to lung type and was accompanied by a shift in epithelial Sonic Hedgehog (Shh) expression and by ectopic lung Surfactant Protein C in the ureteric bud. Blocking of type XVIII collagen function prevented ureteric development with lung mesenchyme and associated with reduction in the expression of Wnt2. Taken together, the findings suggest critical roles for Sprouty2, Wnt2b and type XVIII collagen in controlling pattern formation and the mode of ureteric bud branching in the embryonic kidney.