O receptor P2X4 (canal iônico controlado por adenosina-5\'-trifosfato-ATP) está amplamente distribuído no sistema nervoso central e, após sua ativação, pode regular os níveis de cálcio intracelulares via permeação direta e por ativação de canais de cálcio voltagem-dependentes. Tem sido proposto que a atividade do receptor pode ser importante na plasticidade sináptica. Tendo em vista a importância do receptor P2X4, sobretudo na fisiologia do sistema nervoso central, é útil caracterizá-lo farmacologicamente e entender os mecanismos moleculares que regulam sua atividade. Examinamos o papel que resíduos específicos N- e C-terminais desempenham na atividade do receptor P2X4 humano, combinando técnicas de biologia molecular, bioquímica e patch-clamp em células de rim de embrião humano (células HEK-293T). Células HEK-293T expressando o receptor P2X4 wild-type apresentaram correntes iônicas, cujas amplitudes dependeram da concentração de ATP, fornecendo um valor de EC50 de 1,37 ± 0,21 µM. Os receptores mutantes E14A e D16A exibiram respostas ao ATP equiparáveis àquelas do receptor selvagem, ao passo que os mutantes Y15A e T17A não foram funcionais, apesar de serem expressos na membrana plasmática das células. A inibição de tirosina fosfatases por pervanadato diminuiu fortemente correntes induzidas por ATP. Subsequente análise de citometria de fluxo na presença de um anticorpo contra resíduos de fosfotirosina indicaram que, entre as células que expressam o receptor P2X4, a percentagem de células fosfo-tirosina-positivas é a mesma para os mutantes Y372A (86 ± 10%) e Y378A (79 ± 6.9%), mas substancialmente menor para os mutantes Y15A (35 ± 12%), Y367A (48 ± 6.4%) e Y372F (31 ± 1.7%), quando comparados com células que expressam o receptor wild-type (76 ± 5.6%). Resultados semelhantes foram obtidos quando quantificamos a expressão relativa de proteínas fosforiladas em resíduos de tirosina e expressamos através dos valores de intensidade de fluorescência média. Ensaios de western-blot revelaram que mesmo o mutante T17A é fosforilado em resíduos de treonina, sugerindo que o receptor P2X4 contém outros sítios de fosforilação. Entretanto, nenhum sinal de fosfotirosina foi detectado no receptor wild-type e nos mutantes, em que resíduos de tirosina foram substituídos por alanina ou fenilalanina. Não parece ser o resíduo Y15 o alvo de tal fosforilação, cabendo a ele um papel estrutural mais importante. Nossos dados também sugerem que a fosforilação em resíduos de tirosina de proteínas intermediárias regula a atividade do receptor P2X4. / The human P2X4 receptor (ATP-gated ion channel) is widely distributed in the CNS and, after activation, participates in regulation of levels of intracellular calcium through direct permeation and activation of voltage-dependent calcium channels with well-defined functions including synaptic plasticity. Given the importance of the P2X4 receptor, especially in CNS physiology, we investigated the role that specific N- and C-termini residues play in human P2X4 receptor activity, by combining techniques of molecular biology, biochemistry and patch-clamping in human embryonic kidney cells (HEK-293T cells). HEK-293T cells expressing the wild-type P2X4 receptor showed ionic currents whose amplitudes depended on the ATP concentration, providing an EC50 value of 1.37 ± 0.21 mM. E14A and D16A receptor mutants exhibited responses to ATP comparable to those ones of wild-type receptor, whereas Y15A and T17A mutants were not functional, despite being expressed in the plasma membrane of cells. The inhibition of tyrosine phosphatases by pervanadate decreased strongly ATP-induced currents. Subsequent flow cytometry analysis in the presence of an antibody against phosphotyrosine residues indicated that, among the cells that express the P2X4 receptor, the percentage of phosphotyrosine-positive cells was the same for Y372A (86 ± 10%) and Y378A (79 ± 6.9%) mutants, however, substantially lower for Y15A (35 ± 12%), Y367A (48 ± 6.4%) and Y372F (31 ± 1.7%) mutants when compared with cells expressing the wild-type receptor (76 ± 5.6%). Similar results were obtained by quantifying the relative expression of phosphotyrosine proteins. Western blot assays revealed that even the T17A mutant was phosphorylated at threonine residues, suggesting that the human P2X4 receptor also contains further phosphorylation sites. However, no phosphotyrosine-antibody signal was detected in the wild-type receptor and mutants in which tyrosine residues were replaced by alanine or phenylalanine. The residue Y15 is supposedly not the target of such phosphorylation, despite its important structural role. However, the present work indicates that tyrosine phosphorylation of intermediate signaling proteins regulates P2X4 receptor activity.

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and the third most common cause of cancer-related deaths. Systemic therapies are the main treatment options for HCC patients with advanced disease (∼ 80% of all cases). However, only very moderate clinical responses are achieved with most of the conventional therapies. Thus, more effective therapeutic strategies are much needed. The PI3K/Akt/mTOR signaling pathway, which plays a critical role in controlling cell proliferation and survival, is aberrantly activated in ∼ 45% HCC, suggesting it to be a potential target for HCC treatment. Moreover, emerging evidences indicate that activation of the PI3K/Akt/mTOR pathway may be associated with resistance to many cytotoxic chemotherapies, including microtubule targeting agents. In this study, by gene expression profiling and gene ontology analysis, "microtubule-related cellular assembly" was identified to be the major biological/functional process involved in HCC development, suggesting that microtubule is also an important therapeutic target for HCC. With these understandings, it is hypothesize in this thesis that combined targeting of a key component ofthe PI3K/Akt/mTOR pathway, namely the mammalian target of rapamycin (mTOR) and the microtubule would be an effective therapeutic strategy for HCC. The objectives of the thesis are to examine the therapeutic potential of microtubule targeting, mTOR targeting, and combined targeting of the microtubule and mTOR in both in vitro and in vivo models of HCC. / In summary, the PI3K/Akt/mTOR pathway and the microtubule represent promising therapeutic targets for HCC treatment. The findings from this thesis offer a rationale for combining mTOR inhibitors with microtubule targeting agents for effective HCC treatment. / In the second part, the effect of mTOR inhibition, either alone or in combination with an additional microtubule targeting agent (vinblastine) was investigated in HCC. Temsirolimus, an mTOR inhibitor, suppressed HCC cell proliferation in as early as 24 hrs with an IC50 of 1.27+/-0.06muM (Huh7), 8.77+/-0.76muM (HepG2), and 52.95+/-17.14muM (Hep3B). Vinblastine (1nM) alone caused 30--50% growth inhibition in 3 HCC cell lines. In these HCC cell lines, it was found that temsirolimus/vinblastine combination resulted in an additive to synergistic effect (when compared to single agents alone) with maximum growth inhibition of 80--90% as early as 24 hrs upon treatment. This marked growth inhibition was accompanied with cell cycle arrest at both G1 and G2/M phases, and PARP cleavage (a hallmark for apoptosis). Moreover, the combination specifically caused concerted down-regulation of several important anti-apoptotic and survival proteins (survivin, Bcl-2 and Mcl-1), which was not observed in single agent treatments. It was hypothesized that inhibition of these key anti-apoptotic/survival proteins may represent a novel mechanistic action of this highly effective combination approach of dual targeting of mTOR and microtubule by temsirolimus/vinblastine in HCC cells. Indeed, transient over-expression of each of these genes (survivin, Bcl-2 or Mcl-1) in HCC cells did partially rescue the growth inhibitory effect of the temsirolimus/vinblastine combination. More importantly, this novel combination significantly suppressed the growth of HCC xenografts in nude mice (when compared with single agents alone). / In the third part, the anti-tumor effect of another mTOR inhibitor everolimus in combination with microtubule targeting agents, vinblastine and patupilone (a microtubule-stabilizing agent), was investigated in HCC cells. Everolimus/vinblastine combination resulted in an additive to synergistic effect accompanied with cell cycle arrest at both G1 and G2/M phases, and PARP cleavage. The combination also caused concerted down-regulation of anti-apoptotic and survival proteins (survivin, Bel-2 and Mel-1) as observed with the temsirolimus/vinblastine combination. However, everolimus only moderately enhanced the sensitivity of patupilone for reasons unknown. / Taxanes are the major chemotherapeutic agents that target the microtubule. In the first part of the thesis, the anti-tumor activity of two taxanes, paclitaxel and docetaxel (which are known to stabilize microtubules) was examined and compared with doxorubicin (a DNA intercalating agent). Across all three HCC cell lines tested, it was found that the microtubule targeting agents, taxanes, were more efficacious than doxorubicin. This supports the initial finding that microtubule assembly process is functionally important in HCC. Recent studies demonstrated that using nanoparticles for drug delivery can greatly enhance therapeutic efficacy and reduce side-effects. Therefore, the nanoparticle albumin-bound (nab)-paclitaxel was employed to further evaluate the therapeutic efficacy of such a delivery strategy in HCC models. In all three HCC cell lines tested, nab-paclitaxel was found to be the most effective agent, with an average IC50 value of 0.16--10.42nM, when compared to non-conjugated taxanes (paclitaxel, docetaxel) and doxorubicin. In vitro analysis showed that nab-paclitaxel was able to induce cell cycle arrest at G2/M phase and apoptosis in HCC cells. In vivo study demonstrated that nab-paclitaxel readily inhibited the growth of HCC xenografts with lower toxicity when compared to paclitaxel, docetaxel and doxorubicin. Moreover, specific silencing of a key regulatory protein for microtubule dynamics, Stathmin 1, by siRNA significantly enhanced the effect of nab-paclitaxel in HCC cells, resulting in synergistic growth inhibition in vitro. / Zhou, Qian. / Advisers: Winnie Yeo; Vivian Lui; Nathalie Wong. / Source: Dissertation Abstracts International, Volume: 73-06, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 148-164). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Cronobacter species cause serious infections such as meningitis and enteritis in newborns and neonates, with the major vehicle being contaminated powdered infant formula. The main objectives of this study were i) to identify potential virulence factors, such as enterotoxin production; ii) characterize the gene(s) involved in adhesion and invasion of the human brain microvascular endothelial cells (HBMEC); and iii) determine whether strains from clinical, food, and environmental sources differ in their ability to produce surface-attached bacterial aggregates, known as biofilms. Random transposon mutagenesis was used on strains demonstrating the best adherence and invasion to blood- brain barrier cell lines (BBB). Isogenic mutants were then screened for increased or decreased adherence and invasion. Screening of the transposon library identified one isogenic mutant of a clinical strain which lost the ability to adhere to BBB cells. The transposon rescue revealed the insertion site to be within a diguanylate cyclase (DGC) gene. The major function of DGC in many Gram-negative bacteria is to synthesize cyclic diguanylate (c-di-GMP), a secondary bacterial metabolite known for regulating biofilm formation, motility, and virulence or aspects of microbial pathogenicity. Based on the findings of this study, DGC appears to play an important role in Cronobacter species’ ability to produce biofilms and may also have a role of the pathogenicity in the microorganism.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 107-130).

Dissertation (Ph.D.)--University of South Florida, 2008. / Includes vita. Includes bibliographical references.

The intracellular human pathogen Salmonella enterica serovar Typhi (S. typhi) causes the systemic disease known as typhoid fever. This disease afflicts approximately 17,000,000 people every year, of which over 600,000 cases are fatal. / Sequencing of the S. typhi genome has allowed a better understanding of the pathogenesis caused by this bacterium. In silico research on the genome sequence identified three open reading frames, termed prpZ gene cluster, present in the Ty2 and multi-drug resistant CT18 strains of S. typhi but absent in all other sequenced serovars of S. enterica. Further analysis of this gene cluster revealed that the three genes are transcribed as an operon that encodes two eukaryotic-like Ser/Thr kinases (PrkX and PrkY) and a protein phosphatase 2C (PP2C) (PrpZ). / A previous study has shown that the recombinant His-PrpZ protein has all the hallmarks of a PP2C. Typically, PP2Cs hydrolyze phosphoserine and phosphothreonine residues. In addition, His-PrpZ was found to hydrolyze phosphotyrosine residues, making it a dual specificity phosphatase. A subsequent investigation implicates the prpZ gene cluster in S. typhi virulence as the survival of a prpZ operon deletion mutant is compromised after 48 hours of macrophage infection when compared to wild type bacteria. / It is clear from these results that the prpZ operon plays a role in the pathogenesis of S. typhi. To determine the role of these three genes in virulence, an in vitro characterization of PrkY was carried out as well as an examination of the possible physiological roles of PrpZ. / We have demonstrated that PrkY is an active protein kinase capable of phosphorylating artificial substrates in the presence of Mg2+ and/or Mn2+. Optimal phosphorylation of substrates is achieved in the presence of 5mM Mg2+ at pH 8.0. In addition, we have identified a putative interaction between PrkY and PrpZ, leading to an inhibition of the kinase activity of PrkY. While exploring the possible physiological functions of PrpZ, we have found that this protein is secreted by Ty2 S. typhi in both LB and in the low pH, low phosphate and low Mg 2+ LPM medium. / These findings suggest that PrkY and PrpZ may have antagonistic effects in a S. typhi specific virulence pathway involved in the modulation of host cell signaling by secreted bacterial virulence factors.

Macrophages are active participants in many important biological processes, including antimicrobial activity, tumour surveillance, apoptotic cell clearance, homeostasis and wound healing. The activity of all cells is under the direct influence of their genetic makeup and macrophages are no exception. Natural resistance-associated macrophage protein 1 (Nramp1, also known as SLC11A1) is a macrophage-restricted gene that confers resistance to intracellular pathogens in mice. Mitogen activated protein kinase activated protein kinase 2 (MAPKAPK-2 or MK2), a substrate of p38 MAPK, is known to influence the activation of macrophages in response to stressors, including the Toll-like receptor (TLR)-4 ligand LPS. Like NRAMP1, MK2 has also been shown to influence the efficiency of the antibacterial response. The present study evaluates the role of NRAMP1 and MK2 in TLR-mediated cytokine induction and their role in cutaneous wound healing. Mice lacking NRAMP1 are severely impaired in their rate of cutaneous wound healing. Nramp1 gene ablation has been associated with lower levels of SLPI, a protein previously demonstrated to influence the rate of wound healing in a non-redundant fashion. Macrophages derived from Nramp1-null mice are less efficient in activating p38 MAPK signaling, which results in lower levels of MK2 phosphorylation. The reduced level of p38 MAPK and MK2 activation in Nramp1-null macrophages also correlates with decreased cytokine induction in response to TLR7 ligand stimulation of these cells. Using p38 MAPK inhibitor and MK2-deficient macrophages, we demonstrate that TLR7- and TLR9-mediated cytokine induction is directly under the control of this signaling pathway. Furthermore, cytokine induction is regulated by MK2 at the post-transcriptional level. Macrophage-induced cytokines play an important role in cutaneous wound healing. Since MK2-deficient macrophages are severely impaired in their ability to induce cytokines following activation, we next evaluated the role of MK2 in cutaneous wound healing. Our results demonstrate that the rate of wound healing is significantly delayed in the absence of MK2. The level of cytokine expression in the wounds is impaired and macrophages are major players in cutaneous wound healing. Our data also show that intradermal transfer of macrophages with intact MK2 significantly improved wound healing kinetics. Overall, the studies presented in this dissertation demonstrate the importance of NRAMP1 and MK2 in the modulation of macrophage gene expression, and their important role in the control of cutaneous wound healing.

The objectives of these experiments were to evaluate the main effects and interactive effects between dietary supplementation of a mannan oligosaccharide based product, Actigen® (ACT), and different levels of threonine in monogastrics, as evidenced by growth parameters, immune function, and intestinal health characteristics. In nursery piglets, ACT supplementation decreased average daily feed intake (P=0.04), but had no effect on body weight or feed conversion ratio. There were no noted differences between threonine levels on performance traits. There were no consistent differences in complete blood count or cytokine gene expression profiles in the blood. The highest level of threonine, 77% true ileal digestible threonine:lysine (Thr:Lys), increased villus height (P=0.007) and villus height:crypt depth (P=0.01). The lowest level, 57% Thr:Lys, decreased villus surface area (P=0.04) and goblet cell density (P=0.04). Supplementation with ACT increased total goblet cell area (P=0.02) and density (P=0.05). There were no interactions observed between ACT and Thr:Lys levels. In broiler chicks, feeding a diet containing 0.56% threonine decreased body weight (PPPP=0.07). On d 7, birds supplemented with ACT had heavier spleens as a percentage of body weight (P=0.01) compared to no ACT. When adjusted for body weight, the 0.56% threonine fed birds had smaller spleens (P=0.05) on d 7 when compared to the other threonine levels. Humerus (PPP=0.04) and birds fed 0.56% threonine had a higher concentration of potassium (PP=0.03) and few goblet cells (P=0.04) on d 7 when compared to the other threonine levels. In d 21 jejunum, supplementation with ACT reduced apical width (P=0.03) and surface area (P=0.02). An interaction was observed between ACT and threonine level in the jejunum on d 21 on basal width (P=0.03) and surface area (P=0.02), indicating that in diets lacking ACT, excess threonine increased villus size. Overall, ACT and threonine acted primarily independently to modulate the intestinal architecture of both nursery piglets and broiler chicks. However, in broiler chicks ACT and threonine interacted to alter villus size. These results indicate that ACT and threonine have direct effects on the intestines of monogastrics.

Us3, a serine/threonine kinase encoded by all alphaherpesviruses, plays diverse roles during virus infection. Recently, work done in our laboratory determined that Us3 orthologues from herpes simplex type 2 (HSV-2) and pseudorabies virus (PRV) are capable of disrupting promyelocytic leukaemia (PML) protein nuclear bodies (-NBs). PML-NBs are discrete, dynamic nuclear bodies named for PML, their essential structural component and one that plays a key role in diverse cellular processes, including transcriptional regulation, apoptosis, and cellular antiviral defense. In infected cells, PML-NBs exert transcriptional silencing on the viral genome to prevent viral gene expression and virus replication. Based on this finding, my studies were aimed to understand the mechanism and physiological function of Us3-mediated PML-NB disruption. The degradation of one or more cellular proteins seems necessary for this Us3 activity, as the proteasome inhibitor, MG132, dramatically reduced Us3-mediated PML-NB disruption. The target of this proteasome activity is not likely PML protein, as Us3 expression did not lead to detectable PML protein degradation. Nonetheless, the involvement of proteasome activity suggests that Us3 may utilize the host ubiquitylation pathway to disrupt PML-NBs. Supporting this hypothesis, PRV and HSV-2 Us3 orthologues were shown to interact with KLHL21, a substrate adaptor protein for cullin-3 ubiquitin ligase. PRV and HSV-2 Us3 were re-localized to PML-NBs when co-expressed with KLHL21, and knock-down of KLHL21 prevented Us3-mediated PML-NB disruption. Taken together, these findings suggest that Us3-KLHL21 complex recruits the cullin-3 ubiquitin ligase to PML-NBs, where subsequent ubiquitylation of unknown target(s) leads to PML-NB disassembly. Since it is well established that PML is an important antiviral effector induced by interferon (IFN), Us3 may contribute to viral resistance to IFN by disrupting PML-NBs. Favoring this hypothesis, virus yield and viral gene transcription were dramatically reduced in IFN-exposed cells in the absence of Us3. These reductions were associated with an increased number of PML-NBs in the absence of Us3, and were partially recovered in cells knocked down for PML. Therefore, by disrupting PML-NBs, Us3 may alleviate IFN-induced, host-mediated transcriptional silencing of the viral genome, allowing efficient viral gene transcription and replication in cells exposed to IFN. / Thesis (Ph.D, Microbiology & Immunology) -- Queen's University, 2014-04-28 16:36:54.079

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 4 uppsatser.