Dissertationen zum Thema „Cytokinesis Genetic aspects“

Errata pasted onto back page. Bibliography: p. 133-149. The RhoGEF activity of PBL is shown to be acting predominantly by the activation of Rho1 and downstream signaling pathways required for contractile ring function during cytokinesis. Genetic evidence suggests this could be through the activation of Diaphanous (an FH protein) to reorganize the actin cytoskeleton, as well as through the activation of Rho-kinase which results in the phosphorylation, and activation of myosin. Highlights a possible role for PBL during contractile ring function at a later stage that previously thought. Genetic interaction screens were employed to identify regulators of PBL activity during cytokinesis. CDK1 was identified genetically as a candidate for regulating PFB activity, but functional studies in vivo showed that this regulation was not by direct phophorylation of the PBK consensus CDK1 suites tested. Further screening has identified other possible components pf PBL signaling pathways, but a role during cytokinesis for these interactors remains to be confirmed. The eye phenotypes described provide ideal systems for the identification of components of PBL signaling pathways in Drosophila. The high level of conservation in the mechanism of cytokinesis from yeast to mammals would also suggest that the identified interactors would most likely represent components of cytokinesis pathways in all eukaryotes.

The Best M.Phil Thesis in the Faculties of Dentistry, Engineering, Medicine and Science (University of Hong Kong), Li Ka Shing Prize, 2001-2003.
published_or_final_version
abstract
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Medicine
Master
Master of Philosophy

Citocinas são moléculas que controlam e modulam a atividade de numerosas células por se ligarem a seus receptores específicos. As diferenças observadas na produção de citocinas entre indivíduos podem ser, pelo menos em parte, explicadas pelos polimorfismos genéticos como o polimorfismo de um único nucleotídeo (SNP). Em 181 indivíduos saudáveis não-aparentados da cidade do Rio de Janeiro (região Sudeste - Brasil), nós analisamos os polimorfismos de citocinas em genes que codificam para Fator de Necrose Tumoral-alfa (TNF-a), Fator de Crescimento Transformante-beta (TGF-b), Interleucina-10, Interleucina-6 e Interferon-gama (IFN-g). Reação em cadeia da polimerase utilizando-se iniciadores sequencia-específicos foi realizada com auxílio do kit comercial CytGen (One Lambda Inc. Canoga Park, CA, USA). Ao todo, 8 polimorfismos foram analisados: TNF-a (-308G/A); TGF-b (códon 10C/T, códon 25C/G); IL-10 (-1082A/G, -819T/C, -592A/C); IL-6 (-174C/G) e IFN-g (+874T/A). Os dados observados foram comparados a três grupos de população de diferentes regiões do Brasil (São Paulo, Paraná e Bahia) e a três populações de outros continentes (Itália, Eslováquia e Negros Norte-Americanos). O teste qui-quadrado foi utilizado para as comparações. Nossa análise da população do Rio de Janeiro mostrou que os as freqüências alélicas em IL-10, IL-6 e IFN-g são desigualmente distribuídos entre Brancos, Mulatos e Negros (p<0,05). A comparação com populações de outras regiões do Brasil revelou que Rio de Janeiro e Bahia possuem freqüências alélicas e genotípicas de TGF-b (códon 25) estatisticamente diferentes (p=0,004 e p=0,002, respectivamente). Ainda, a freqüência alélica na população do Rio de Janeiro é significativamente diferente quando comparada à população da Itália [IL-6 (-174), p=0,0092; e IFN-g (+874) p=0,0418)]; Eslováquia [IL-10(-1082), p=0,006; IL-6(-174), p=0,0002; e IFN-g(+874), p=0,0335]; e Afro-Americanos [IL-10(-819), p=0,0446; IL-6(-174), p<0,0001; e IFN-g(+874), p<0,0001]. Adicionalmente, observamos que a diferença na distribuição dos haplótipos em IL-10 (-1082/-819/-592) na população do Rio de Janeiro em comparação com a da Itália (p=0,0293) e Afro-Americanos (p=0,0025) é significativa. Portanto, concluímos que os polimorfismos em IL-10, IL-6 e IFN-g estão distribuídos de acordo com a etnia na população do Rio de Janeiro. A população do Rio de Janeiro possui freqüências de polimorfismos diferentes das populações de Bahia, Itália, Eslováquia e Afro-Americanos, mas semelhantes à população de São Paulo/Paraná. Nossas observações poderão ser úteis para futuros estudos e associação entre polimorfismos genéticos de citocinas e doenças na população do Rio de Janeiro.
Cytokines are molecules that control and modulate the activities of numerous target cells via binding to specific receptors. The observed differences in the cytokine production among individuals can be, at least, explained by genetic polymorphisms like single nucleotide polymorphism (SNP). In 181 unrelated healthy Brazilian individuals from Rio de Janeiro City, we investigated the polymorphisms of cytokine genes encoding Tumor Necrosis Factor-alpha (TNFA), Transforming Growth Factorbeta (TGFB), Interleukin (IL)-10, IL-6, and Interferon-gamma (IFNG). Polymerase chain reaction using sequence-specific primers genotyping was performed for these gene cytokines using the avaiable comercial kit CytGen (One Lambda Inc., CA, USA). Eight polymorphisms were tested: TNF-a (-308G/A); TGF-b (códon 10C/T, códon 25C/G); IL-10 (-1082A/G, -819T/C, -592A/C); IL-6 (-174G/C) and IFN-g (+874T/A). Chi-square test was used for comparisons. Observed data were compared to three other populations from different regions of Brazil (São Paulo, Paraná and Bahia) and three populations of other countries (Italy, Slovakia and North American Blacks). Our analysis from Rio de Janeiro population showed that the alleles frequencies in IL-10, IL-6 and IFN-g are unevenly distributed among Whites, Blacks and Mulatos (p<0.05). The comparison with populations from other regions of Brazil showed that Rio de Janeiro and Bahia have genotypic and allelic frequencies of TGF-b (códon 25) statistically different (p=0,004 and p=0,002, respectively). Also, the allelic frequency in the population of Rio de Janeiro is significantly different when compared to the population of Italy [IL-6 (-174), p=0.0092, and IFN-g (+874), p=0.0418] ; Slovak [IL-10 (-1082), p=0006; IL-6 (-174), p=0.0002, and IFN-g (+874), p=0.0335] and Afro-Americans [IL-10 (-819), p=0.0446, IL-6 (-174), p<0.0001, and IFN-g (+874), p<0.0001]. Additionally, we observed that the distribution of haplotypes in IL-10 (-1082/-819/-592) in the population of Rio de Janeiro in comparison with that of Italy (p = 0.0293) and Afro-Americans (p =0,0025) is statistically different. Therefore, we conclude that the polymorphisms in IL-10, IL-6 and IFN-g are distributed according to ethnicity in the population of Rio de Janeiro. The population of Rio de Janeiro have different frequencies of polymorphisms of the population of Bahia, Italy, Slovak and Afro-American, but similar to the population of São Paulo/Paraná. Our observations may be useful for future studies in association between genetic polymorphisms of cytokines and disease in the Rio de Janeiro population.

[Truncated abstract] Bone remodeling is intricately regulated by osteoclast-mediated bone resorption and osteoblast-mediated bone formation. The elevation in osteoclast number and/or activity is a major hallmark of several common pathological bone disorders including post-menopausal osteoporosis, osteoarthritis, Paget's disease, and tumour-mediated osteolysis. Receptor activator of nuclear factor kappa B ligand (RANKL) is a key cytokine for osteoclast differentiation and activation. The association of RANKL to its cognate receptor, RANK, which is expressed on osteoclast precursors and mature osteoclasts, is essential for osteoclast formation and activation. The intimate interaction between RANKL and RANK triggers the activation of a cascade of signal transduction pathways including NF-κB, NFAT, MAPK and PI3 kinase. Although osteoclast signaling pathways have been intensively studied, the precise molecules and signaling events which underlie osteoclast differentiation and function remain unclear. In order to dissect the molecular mechanism(s) regulating osteoclast differentiation and activity, this thesis herein explores the key RANKL/RANK-mediated signaling pathways. Four truncation mutants within the TNF-like domain of RANKL [(aa160-302), (aa160-268), (aa239-318) and (aa246-318)] were generated to investigate their potential binding to RANK and the activation to RANK-signal transduction pathways. All were found to differentially impair osteoclastogenesis and bone resorption as compared to the wild-type RANKL. The impaired function of the truncation mutants of RANKL on osteoclast formation and function correlates with their reduced ability to activate crucial RANK signaling including NF-κB, IκBα, ERK and JNK. Further analysis revealed that the truncation mutants of RANKL exhibited differentially affinity to RANK as observed by in vitro pull-down assays. ... It is possible that Bryostatin 1 acts via upregulation of a fusion mechanism as the RANKL-induced OCLs are morphologically enlarged, exhibiting increased nuclei number expressing high level of DC-Stamp. Furthermore, Rottlerin was shown to inhibit NF-κB activity, whereas Bryostatin 1 showed the opposing effects. Both inhibitor and activator were also found to modulate other key osteoclastic signaling pathways including NFAT and total c-SRC. These findings implicate, for the first time, Protein Kinase C delta signaling pathways in the modulation of RANKL-induced osteoclast differentiation and activity. Taken together, the studies presented in this thesis provide compelling molecular, biochemical and morphological evidence to suggest that: (1) RANKL mutants might potentially serve as peptide mimic to inhibit RANKL-induced signaling, osteoclastogenesis and bone resorption. (2) A cross talk mechanism between extracellular Ca2+ and RANKL exist to regulate on osteoclast survival. (3) TPA suppressed RANKL-induced osteoclastogenesis predominantly during the early stage of osteoclast differentiation via modulation of NF-κB. (4) Selective inhibition of Protein Kinase C signaling pathways involved in osteoclastogenesis might be a potential treatment method for osteoclast-related bone diseases. (5) Protein Kinase C delta signaling pathways play a key role in regulating osteoclast formation and function.

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O câncer gástrico é uma doença caracterizada como multifatorial associada a fatores ambientais e genéticos. A carcinogênese do estômago pode progredir de uma inflamação crônica da mucosa gástrica, resultante da infecção pela bactéria Helicobacter pylori que ativa a resposta inflamatória do hospedeiro. Portanto, selecionamos um grupo de polimorfismos presentes em genes de citocinas pró-inflamatórias (IL8, TNFA e TNFB), anti-inflamatórias (IL10 e IL-1RN) e receptores de reconhecimento de padrões moleculares associados aos microorganismos, denominados toll like receptor (TLR). Considerando a escassez e controvérsia de estudos de polimorfismos desses genes em câncer gástrico e seu envolvimento na carcinogênese de estômago, propôs-se avaliar, pelas técnicas de PCR- alelo específico ou PCR-RFLP, a associação dos polimorfismos TLR2 -196 a –174 del, TLR4 (+896 A/G rs4986790 e +1196 C/T rs4986791), IL-1RN VNTR, TNFB 252A/G (rs909253), TNFA (-308 G/A rs1800629 e –857 C/T rs1799724), IL8 (-251 T/A rs4073 e –845 T/C rs2227532) e IL10 (-592 C/A rs1800872) com risco de câncer gástrico, gastrite crônica e fatores de risco ambientais. Também, procurou-se associar, pela técnica de qPCR em tempo real, os polimorfismos com os níveis de expressão dos genes das citocinas, cujas variantes ocorrem em regiões promotoras (TNFA, IL8 e IL10). De modo geral, a genotipagem dos referidos genes foi realizada em amostras de DNA de 723 indivíduos (207 de câncer gástrico-CG; 276 de gastrite crônica-GC e 246 controles saudáveis-C), enquanto que nas análises de expressão gênica foi utilizado o cDNA de tecido gástrico proveniente de 45 pacientes com CG e 47 com GC. Observou-se que, os SNPs TLR4+1196C/T, TNFB+252A/G, TNFA-308G/A e IL8-251T/A não foram associados com o risco de gastrite crônica e câncer gástrico. Contudo, as freqüências dos genótipos TLR2 ins/del +del/...
Gastric cancer is a multifactorial disease characterized as associated with environmental and genetic factors. The carcinogenesis of the stomach can progress to a chronic inflammation of the gastric mucosa, resulting from infection by the bacterium Helicobacter pylori that activates the inflammatory response of the host. Therefore, we selected a group of polymorphisms in genes of pro-inflammatory cytokines (IL8, TNFA and TNFB), anti-inflammatory (IL10 and IL-1RN) and receptor recognition of molecular patterns associated with microorganisms, the toll like receptors (TLR). Considering the scarcity and controversy of these polymorphisms studies in gastric cancer and their involvement in carcinogenesis of the stomach, this study proposed to evaluate, by PCR allele-specific or PCR-RFLP the association of polymorphisms TLR2 -196 to -174 del , TLR4 (rs4986790 and rs4986791), IL-1RN VNTR, TNFB 252A/G (rs909253), TNFA (rs1800629 and rs1799724), IL8 (rs4073 and rs2227532) and IL10 (rs1800872) with risk of gastric cancer, chronic gastritis and environmental risk factors. Also, we tried to associate, for the technique of real-time qPCR, polymorphisms with levels of expression of cytokine genes whose variations occur in the promoter region (TNFA, IL8 and IL10). Overall, the genotyping of these genes was performed on DNA samples from 723 individuals (207 gastric cancer-GC, 276 chronic gastritis-CG, and 246 healthy controls-C), whereas in the analysis of gene expression was used cDNA of gastric tissue from 45 patients with GC and 47 CG. It was observed that TLR4 SNPs +1196C/T, TNFB +252A/G, TNFA-308G/A and IL8-251 T/A were not associated with risk of chronic gastritis and gastric cancer. In the analysis of polymorphisms of toll like receptor, the frequencies of genotypes TLR2 ins / del + del / del and TLR4 +896 AG were significantly higher (p <0.01) in GC group (33.5% and 13% respectively)... (Complete abstract click electronic access below)

Orientador: Ana Elizabete Silva
Banca: Dertia Villalba Freire-Maia
Banca: Spencer Luiz Marques Payao
Banca: Débora Aparecida Pires de Campos Zuccari
Banca: Mariangela Torreglosa Ruiz
Resumo: O câncer gástrico é uma doença caracterizada como multifatorial associada a fatores ambientais e genéticos. A carcinogênese do estômago pode progredir de uma inflamação crônica da mucosa gástrica, resultante da infecção pela bactéria Helicobacter pylori que ativa a resposta inflamatória do hospedeiro. Portanto, selecionamos um grupo de polimorfismos presentes em genes de citocinas pró-inflamatórias (IL8, TNFA e TNFB), anti-inflamatórias (IL10 e IL-1RN) e receptores de reconhecimento de padrões moleculares associados aos microorganismos, denominados toll like receptor (TLR). Considerando a escassez e controvérsia de estudos de polimorfismos desses genes em câncer gástrico e seu envolvimento na carcinogênese de estômago, propôs-se avaliar, pelas técnicas de PCR- alelo específico ou PCR-RFLP, a associação dos polimorfismos TLR2 -196 a -174 del, TLR4 (+896 A/G rs4986790 e +1196 C/T rs4986791), IL-1RN VNTR, TNFB 252A/G (rs909253), TNFA (-308 G/A rs1800629 e -857 C/T rs1799724), IL8 (-251 T/A rs4073 e -845 T/C rs2227532) e IL10 (-592 C/A rs1800872) com risco de câncer gástrico, gastrite crônica e fatores de risco ambientais. Também, procurou-se associar, pela técnica de qPCR em tempo real, os polimorfismos com os níveis de expressão dos genes das citocinas, cujas variantes ocorrem em regiões promotoras (TNFA, IL8 e IL10). De modo geral, a genotipagem dos referidos genes foi realizada em amostras de DNA de 723 indivíduos (207 de câncer gástrico-CG; 276 de gastrite crônica-GC e 246 controles saudáveis-C), enquanto que nas análises de expressão gênica foi utilizado o cDNA de tecido gástrico proveniente de 45 pacientes com CG e 47 com GC. Observou-se que, os SNPs TLR4+1196C/T, TNFB+252A/G, TNFA-308G/A e IL8-251T/A não foram associados com o risco de gastrite crônica e câncer gástrico. Contudo, as freqüências dos genótipos TLR2 ins/del +del/...(Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Gastric cancer is a multifactorial disease characterized as associated with environmental and genetic factors. The carcinogenesis of the stomach can progress to a chronic inflammation of the gastric mucosa, resulting from infection by the bacterium Helicobacter pylori that activates the inflammatory response of the host. Therefore, we selected a group of polymorphisms in genes of pro-inflammatory cytokines (IL8, TNFA and TNFB), anti-inflammatory (IL10 and IL-1RN) and receptor recognition of molecular patterns associated with microorganisms, the toll like receptors (TLR). Considering the scarcity and controversy of these polymorphisms studies in gastric cancer and their involvement in carcinogenesis of the stomach, this study proposed to evaluate, by PCR allele-specific or PCR-RFLP the association of polymorphisms TLR2 -196 to -174 del , TLR4 (rs4986790 and rs4986791), IL-1RN VNTR, TNFB 252A/G (rs909253), TNFA (rs1800629 and rs1799724), IL8 (rs4073 and rs2227532) and IL10 (rs1800872) with risk of gastric cancer, chronic gastritis and environmental risk factors. Also, we tried to associate, for the technique of real-time qPCR, polymorphisms with levels of expression of cytokine genes whose variations occur in the promoter region (TNFA, IL8 and IL10). Overall, the genotyping of these genes was performed on DNA samples from 723 individuals (207 gastric cancer-GC, 276 chronic gastritis-CG, and 246 healthy controls-C), whereas in the analysis of gene expression was used cDNA of gastric tissue from 45 patients with GC and 47 CG. It was observed that TLR4 SNPs +1196C/T, TNFB +252A/G, TNFA-308G/A and IL8-251 T/A were not associated with risk of chronic gastritis and gastric cancer. In the analysis of polymorphisms of toll like receptor, the frequencies of genotypes TLR2 ins / del + del / del and TLR4 +896 AG were significantly higher (p <0.01) in GC group (33.5% and 13% respectively)... (Complete abstract click electronic access below)
Doutor

Errata pasted onto back page.
Bibliography: p. 133-149.
149 p., [29] leaves of plates : ill. (chiefly col.) ; 30 cm.
Title page, contents and abstract only. The complete thesis in print form is available from the University Library.
The RhoGEF activity of PBL is shown to be acting predominantly by the activation of Rho1 and downstream signaling pathways required for contractile ring function during cytokinesis. Genetic evidence suggests this could be through the activation of Diaphanous (an FH protein) to reorganize the actin cytoskeleton, as well as through the activation of Rho-kinase which results in the phosphorylation, and activation of myosin. Highlights a possible role for PBL during contractile ring function at a later stage that previously thought. Genetic interaction screens were employed to identify regulators of PBL activity during cytokinesis. CDK1 was identified genetically as a candidate for regulating PFB activity, but functional studies in vivo showed that this regulation was not by direct phophorylation of the PBK consensus CDK1 suites tested. Further screening has identified other possible components pf PBL signaling pathways, but a role during cytokinesis for these interactors remains to be confirmed. The eye phenotypes described provide ideal systems for the identification of components of PBL signaling pathways in Drosophila. The high level of conservation in the mechanism of cytokinesis from yeast to mammals would also suggest that the identified interactors would most likely represent components of cytokinesis pathways in all eukaryotes.
Thesis (Ph.D.)--University of Adelaide, Dept. of Molecular Biosciences, 2002?

Errata pasted onto back page.
Bibliography: p. 133-149.
149 p., [29] leaves of plates : ill. (chiefly col.) ; 30 cm.
The RhoGEF activity of PBL is shown to be acting predominantly by the activation of Rho1 and downstream signaling pathways required for contractile ring function during cytokinesis. Genetic evidence suggests this could be through the activation of Diaphanous (an FH protein) to reorganize the actin cytoskeleton, as well as through the activation of Rho-kinase which results in the phosphorylation, and activation of myosin. Highlights a possible role for PBL during contractile ring function at a later stage that previously thought. Genetic interaction screens were employed to identify regulators of PBL activity during cytokinesis. CDK1 was identified genetically as a candidate for regulating PFB activity, but functional studies in vivo showed that this regulation was not by direct phophorylation of the PBK consensus CDK1 suites tested. Further screening has identified other possible components pf PBL signaling pathways, but a role during cytokinesis for these interactors remains to be confirmed. The eye phenotypes described provide ideal systems for the identification of components of PBL signaling pathways in Drosophila. The high level of conservation in the mechanism of cytokinesis from yeast to mammals would also suggest that the identified interactors would most likely represent components of cytokinesis pathways in all eukaryotes.
Thesis (Ph.D.)--University of Adelaide, Dept. of Molecular Biosciences, 2002?

Errata is pasted onto back end paper.
Includes bibliographical references (26 leaves).
115, [68] leaves, [8] leaves of plates : ill. (some col.) ; 30 cm.
This study entailed work towards the isolation of the pbl gene and preliminary characterisation of a candidate pbl transcript. Plasmid rescue of the genomic DNA flanking the inserted P element led to the isolation of a third candidate p61 cDNA, the 1A cDNA. This data suggests that the IA cDNA is encoded by the p61 gene.
Thesis (Ph.D.)--University of Adelaide, Dept. of Genetics, 1998

"November 2002"
Bibliography: p. 177-194.
194 p. : ill., plates (some col.) ; 30 cm.
Title page, contents and abstract only. The complete thesis in print form is available from the University Library.
Thesis (Ph.D.)--University of Adelaide, Dept. of Molecular Biosciences, 2003

Interleukin (IL)-3, IL-5 and granulocyte-macrophage-colony-stimulating factor (GM-CSF) are cytokines that affect the growth, survival and differentiation of many cells within the haematopoietic system. The functions of these factors are mediated by membrane bound receptor complexes that are composed of specific ligand binding subunits (α)and a common signal transducing subunit(hβc). Constitutively activated mutants of hβc have been previously identified that are able to confer factor-independent signalling in a number of haematopoietic cell lines (including FDC-P1 and FDB-1). These activated mutants fall into two classes defined by the location of the mutation and their biochemical and leukaemogenic properties. In particular, the transmembrane mutant, V449E, causes an acute myeloid leukaemia in vivo, whereas the extracellular mutants (FI∆ or I374N) cause chronic myeloproliferative disorders. The work described in this thesis used the activated hβc mutants to uncover novel transcriptional events induced by the GM-CSF/IL-3/IL-5 receptor complex and to define pathways associated with proliferation and differentiation. Large-scale gene expression profiling techniques were used to investigate the genes involved in these biological processes in the murine myelomonocytic cell line FDC-P1, and the bi-potent FDB-1 myeloid cell line, which are responsive to IL-3 and GM-CSF. Membrane arrays were used to identify differences in gene expression between I374N and V449E expressing FDC-P1 cells. This technique revealed that the gene Ptpmt1 was differentially expressed between V449E and I374N, which was subsequently confirmed by Northern blotting. This finding suggested that the phosphatase encoded by Ptpmt1 may be involved in the different outcomes induced by these two hβc mutants. Northern analysis also revealed Ptpmt1, Nab1 and Ddx26b to be regulated in response to human GM-CSF in FDC-P1 cells expressing human GM CSFα and hβc. A large-scale cDNA microarray experiment was also performed to identify genes that are selectively expressed during differentiation of FI∆ expressing FDB-1 cells, compared to proliferating V449E expressing FDB-1 cells over 24 hours. A comprehensive analysis approach was adopted to examine the microarray data and identify differentially expressed genes. Among the genes displaying differential expression were Btg1, S100a9, Cd24, and Ltf found to be differentiation-associated and Bnip3, Cd34, Myc, Nucleophosmin, and Nucleostemin found to be proliferation-associated. Hipk1, Klf6, Sp100, and Sfrs3 were also identified as potential transcriptional regulators during growth and differentiation. Northern analysis was used to confirm differences in expression for these 13 genes between FI∆ and V449E expressing FDB-1 cells. Eleven of the 13 genes examined were confirmed to be differentially expressed between FI∆ and V449E expressing FDB-1 cells over 24 hours. Furthermore, six genes (Btg1, Hipk1, Cd24, Cd34, Klf6 and Nucleostemin) examined over 72 hours revealed differences in gene expression at early (6-12 hours) and late (48-72 hours) time points. Cell surface expression of CD24 protein was also shown to be induced upon FI∆ expression or GM-CSF induced differentiation of FDB-1 cells, consistent with elevated levels of Cd24 mRNA in FI∆ cells over time. Based on their confirmed gene expression differences seen on the microarrays and Northern analysis, four genes (Btg1, Cd24, Klf6 and Nucleostemin) were selected for over-expression analysis in FDC-P1 or FDB-1 cells, in order to gain insights into the function of these genes. Optimisation of the retroviral infection process was performed so that the role of these genes in proliferation and differentiation could be investigated in the FDB-1 model. Such preliminary functional experiments in FDB-1 cells will enable prioritisation of the genes for further analysis of their function in primary cells. Thus, the work in this thesis describes the first use of microarrays to identify gene expression differences between hβc mutants with differential activities affecting myeloid growth and differentiation.
http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1284103
Thesis (PhD)-- School of Medicine, 2005

Interleukin (IL)-3, IL-5 and granulocyte-macrophage-colony-stimulating factor (GM-CSF) are cytokines that affect the growth, survival and differentiation of many cells within the haematopoietic system. The functions of these factors are mediated by membrane bound receptor complexes that are composed of specific ligand binding subunits (α)and a common signal transducing subunit(hβc). Constitutively activated mutants of hβc have been previously identified that are able to confer factor-independent signalling in a number of haematopoietic cell lines (including FDC-P1 and FDB-1). These activated mutants fall into two classes defined by the location of the mutation and their biochemical and leukaemogenic properties. In particular, the transmembrane mutant, V449E, causes an acute myeloid leukaemia in vivo, whereas the extracellular mutants (FI∆ or I374N) cause chronic myeloproliferative disorders. The work described in this thesis used the activated hβc mutants to uncover novel transcriptional events induced by the GM-CSF/IL-3/IL-5 receptor complex and to define pathways associated with proliferation and differentiation. Large-scale gene expression profiling techniques were used to investigate the genes involved in these biological processes in the murine myelomonocytic cell line FDC-P1, and the bi-potent FDB-1 myeloid cell line, which are responsive to IL-3 and GM-CSF. Membrane arrays were used to identify differences in gene expression between I374N and V449E expressing FDC-P1 cells. This technique revealed that the gene Ptpmt1 was differentially expressed between V449E and I374N, which was subsequently confirmed by Northern blotting. This finding suggested that the phosphatase encoded by Ptpmt1 may be involved in the different outcomes induced by these two hβc mutants. Northern analysis also revealed Ptpmt1, Nab1 and Ddx26b to be regulated in response to human GM-CSF in FDC-P1 cells expressing human GM CSFα and hβc. A large-scale cDNA microarray experiment was also performed to identify genes that are selectively expressed during differentiation of FI∆ expressing FDB-1 cells, compared to proliferating V449E expressing FDB-1 cells over 24 hours. A comprehensive analysis approach was adopted to examine the microarray data and identify differentially expressed genes. Among the genes displaying differential expression were Btg1, S100a9, Cd24, and Ltf found to be differentiation-associated and Bnip3, Cd34, Myc, Nucleophosmin, and Nucleostemin found to be proliferation-associated. Hipk1, Klf6, Sp100, and Sfrs3 were also identified as potential transcriptional regulators during growth and differentiation. Northern analysis was used to confirm differences in expression for these 13 genes between FI∆ and V449E expressing FDB-1 cells. Eleven of the 13 genes examined were confirmed to be differentially expressed between FI∆ and V449E expressing FDB-1 cells over 24 hours. Furthermore, six genes (Btg1, Hipk1, Cd24, Cd34, Klf6 and Nucleostemin) examined over 72 hours revealed differences in gene expression at early (6-12 hours) and late (48-72 hours) time points. Cell surface expression of CD24 protein was also shown to be induced upon FI∆ expression or GM-CSF induced differentiation of FDB-1 cells, consistent with elevated levels of Cd24 mRNA in FI∆ cells over time. Based on their confirmed gene expression differences seen on the microarrays and Northern analysis, four genes (Btg1, Cd24, Klf6 and Nucleostemin) were selected for over-expression analysis in FDC-P1 or FDB-1 cells, in order to gain insights into the function of these genes. Optimisation of the retroviral infection process was performed so that the role of these genes in proliferation and differentiation could be investigated in the FDB-1 model. Such preliminary functional experiments in FDB-1 cells will enable prioritisation of the genes for further analysis of their function in primary cells. Thus, the work in this thesis describes the first use of microarrays to identify gene expression differences between hβc mutants with differential activities affecting myeloid growth and differentiation.
Thesis (PhD)-- School of Medicine, 2005

研究背景及實驗目的: 非酒精性脂肪性肝炎(NASH)使得肥胖和2 型糖尿病變得複雜，肝臟炎症的持續產生是其主要的發病機理。CXCL10 是一種促進炎症的細胞因數，其在肥胖和2 型糖尿病中的表達顯著升高。CXCL10 以及其受體CXCR3 是否在NASH 的發生發展中起作用尚不清楚。在本研究中，我們探索了CXCL10 以及其受體CXCR3 在脂肪性肝炎中的功能， 並評估了CXCL10 在NASH 中的臨床價值。
實驗方法：CXCL10 基因敲除鼠，CXCR3 敲除鼠以及野生型C57BL/6 小鼠給予蛋氨酸膽鹼缺乏食(MCD)4 周或者8 周。CXCL10 的信號通路以及下游靶點通過細胞因數分析，cDNA array, 蛋白DNA 結合實驗，自噬溶酶體系統分析進行檢測。為了闡明CXCL10 抑制對NASH 的預防治療作用，我們給MCD 餵養的小鼠注射抗CXCL10 抗體。用不同濃度的CXCL10 抗體以及CXCR3 抑制劑NIBR2130 幹預MCD 培養的肝細胞株AML-12。臨床研究中，我們收集了147個非酒精性脂肪肝患者以及73 個健康對照的血清，用酶聯免疫吸附試驗檢測血清中CXCL10 的水準。
結果：野生型小鼠給予MCD 餵養後，CXCL10 以及CXCR3 的表達升高，並出現脂肪性肝炎的表現。然而，MCD 飼養的CXCL10 以及CXCR3 基因敲除鼠中，脂肪性肝炎明顯減輕。CXCL10 通過促炎細胞因數的產生以及NK-κB 信號通路促進MCD 飼養的小鼠NASH 的發生。CXCL10 通過促進脂質合成的基因SREBP-1c, ChREBP 和 SCD-1 引起脂肪變性，並通過CYP2E1 以及 C/EBPβ 的上調引起氧化應激。值得注意的是，自噬的損傷在CXCL10 以及CXCR3 導致的脂肪性肝炎的進展中起重要作用。 MCD 飼養的野生型小鼠中p62 以及LC3-II 表達明顯高於CXCL10 以及CXCR3 基因敲除鼠。通過抗CXCL10 抗體中和CXCL10 可以減輕MCD 食引起的小鼠脂肪性肝炎以及MCD 培養液引起的AML-12 細胞損傷。高選擇性的CXCR3 抑制劑NIBR2130 也可以抑制MCD 引起的肝細胞損傷。我們進一步研究了CXCL10 的臨床應用價值，發現NASH 患者血清以及肝臟中CXCL10 的水準明顯升高。更重要的是，血液中CXCL10 的水準與肝小葉炎症程度有關，是NASH 的獨立危險因素。
結論：我們的研究首次發現CXCL10 以及其受體CXCR3 通過促進炎症，脂質聚集，氧化應激以及自噬缺乏在NASH 的發病中起重要作用。抑制CXCL10 或者CXCR3 為NASH 患者的治療提供了新的方法。CXCL10 可作為NASH 患者非侵入性診斷的標誌物。
Background and aims: Non-alcoholic steatoheaptitis (NASH) complicates obesity and type 2 diabetes, while recruitment and perpetuation of liver inflammation is central to its pathogenesis. Expression of C-X-C motif chemokine 10 (CXCL10), a proinflammatory cytokine, correlates positively with obesity and type 2 diabetes. Whether CXCL10 and its receptor CXCR3 play a role in NASH is unknown. In this study, we investigated the functional significance of CXCL10 and its receptor CXCR3 in steatoheaptitis. Moreover, the clinical impact of CXCL10 in NASH was examined.
Methods: Gene-deleted CXCL10 (CXCL10-/-), CXCL10 receptor CXCR3 (CXCR3-/-) and C57BL/6 wildtype (WT) mice were fed methionine and choline-deficient (MCD) diet for 4 or 8 weeks. Cytokine profiling assay, cDNA array, protein-DNA binding activity assay and autophagosome-lysosome system analysis of CXCL10 signaling and downstream targets were performed. In other experiments, we injected neutralizing anti-CXCL10 monoclonal antibodies (mAb) into MCD diet-fed WT mice, while AML-12 cells were cultured in MCD medium in the presence of anti-CXCL10 mAb or CXCR3 inhibitor (NIBR2130) for 24 hours. Human serum was obtained from 147 patients with biopsy-proven non-alcoholic fatty liver disease and 73 controls. Circulating CXCL10 levels were determined by enzyme-linked immunosorbent assay.
Results: MCD-fed WT mice developed steatohepatitis with higher hepatic CXCL10 and CXCR3 expression. CXCL10-/- and CXCR3-/- mice were refractory to MCDinduced steatohepatitis. In WT mice with steatohepatitis, but not in CXCL10-/- mice, CXCL10 was associated with the induction of pro-inflammatory chemokines and cytokines, as well as activation of nuclear factor-κB (NF-κB) signaling. CXCL10 expression was linked to steatosis through lipogenic factors, including liver X receptors and its downstream targets (SREBP-1c, ChREBP and SCD-1), and also to oxidative stress (up-regulation of CYP2E1 and C/EBPβ). In particular, autophagy deficiency was involved in CXCL10- and CXCR3-induced steatohepatitis as indicated by p62 and LC3-I/II protein accumulation in MCD-fed WT mice than in CXCL10-/- and CXCR3-/- mice. Moreover, the impaired autophagic function was related to the reduction of lysosomal function in CXCL10- or CXCR3-induced NASH. Blockade of CXCL10 by anti-CXCL10 mAb protected against MCD-induced steatohepatitis in vivo and against MCD-mediated injury to AML-12 cells in vitro. The highly selective CXCR3 antagonist NIBR2130 also inhibited MCD-induced injury in AML-12 hepatocytes. We further investigated the clinical impact of CXCL10 and found circulating and hepatic CXCL10 levels were significantly higher in human NASH. Importantly, circulating CXCL10 level was correlated with the degree of lobular inflammation and was an independent risk factor for NASH patients.
Conclusions: We demonstrate for the first time that CXCL10 and its receptor CXCR3 plays a pivotal role in the pathogenesis of NASH by promoting inflammation, fatty acid accumulation, oxidative stress and autophagy deficiency. Blockade of CXCL10 or CXCR3 is a potential novel approach for NASH intervention. CXCL10 is a noninvasive biomarker for NASH patients.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Zhang, Xiang.
Thesis (Ph.D.) Chinese University of Hong Kong, 2014.
Includes bibliographical references (leaves 145-167).
Abstracts also in Chinese.

Indiana University-Purdue University Indianapolis (IUPUI)
Neurofibromatosis type 1 is a genetic disease that results from either heritable or spontaneous autosomal dominant mutations in the NF1 gene, which encodes a protein serving, at least in part, to accelerate the intrinsic hydrolysis of active Ras-GTP to inactive Ras-GDP. A second-hit NF1 mutation precedes predominant NF1 neoplasms, including juvenile myelomoncytic leukemia (JMML) and plexiform neurofibroma formation, potentially fatal conditions with no medical therapy. While NF1 loss of heterozygosity (LOH) in myeloid progenitor cells sufficiently engenders leukemogenesis, plexiform neurofibroma formation depends on LOH in Schwann cells and Nf1 heterozygosity in the hematopoietic system. Specifically, recruited Nf1+/- mast cells accelerate tumorigenesis through secreted cytokines and growth factors. Nf1+/- mast cells depend upon deregulated signaling in c-kit pathways, a receptor system conserved in hematopoietic stem cells (HSCs). Accordingly, Nf1-/- myeloid progenitor cells, which can induce a JMML-like disease in mice, also demonstrate deregulated c-kit receptor signaling. C-kit-activated Nf1+/- mast cells and Nf1-/- myeloid progenitors both show increased latency and potency of active Erk1 and Erk2, the principal cytosolic-to-nuclear effectors of canonical Ras-Raf-Mek signaling. Thus, Erk represents a potential regulator of leukemogenesis and tumor-associated inflammation. However, single and combined Erk1 and Erk2 roles in HSC function, myelopoiesis, and mature mast cell physiology remain unknown, and recent hematopoietic studies relying on chemical Mek-Erk inhibitors have produced conflicting results. Here, we show that hematopoietic stability, myelopoiesis, and mast cell generation require Erk1 or Erk2, but individual isoforms are largely dispensable. Principally, Erk-disrupted hematopoietic stem cells incorporate BrdU but are incapable of dividing, a novel and cell type-specific Erk function. Similarly, mast cell proliferation requires Erk but cytokine production proceeds through other pathways, elucidating molecule-specific functions within the c-kit cascade. Based on these findings, we have reduced tumor mast cell infiltration by treating genetically-engineered tumor model mice with PD0325901, a preclinical Mek-Erk inhibitor. Moreover, we have devised a quadruple transgenic HSC transplantation model to examine dual Erk disruption in the context of Nf1 nullizygosity, testing whether diseased hematopoiesis requires Erk. These insights illuminate cell-specific Erk functions in normal and Nf1-deficient hematopoiesis, informing the feasibility of targeting Mek-Erk in NF1-associated disease.

Indiana University-Purdue University Indianapolis (IUPUI)
CD4 T helper cells control immunity to pathogens and the development of inflammatory disease by acquiring the ability to secrete effector cytokines. Cytokine responsiveness is a critical component of the ability of cells to respond to the extracellular milieu by activating Signal Transducer and Activator of Transcription factors that induce the expression of other transcription factors important for cytokine production. STAT4 is a critical regulator of Th1 differentiation and inflammatory disease that attenuates the gene-repressing activity of Dnmt3a. In the absence of STAT4, genetic loss of Dnmt3a results in de-repression of a subset of Th1 genes, and a partial increase in expression that is sufficient to observe a modest recovery of STAT4-dependent inflammatory disease. STAT4 also induces expression of the transcription factors Twist1 and Etv5. We demonstrate that Twist1 negatively regulates Th1 cell differentiation through several mechanisms including physical interaction with Runx3 and impairing STAT4 activation. Following induction by STAT3-activating cytokines including IL-6, Twist1 represses Th17 and Tfh differentiation by directly binding to, and suppressing expression of, the Il6ra locus, subsequently reducing STAT3 activation. In contrast, Etv5 contributes only modestly to Th1 development but promotes Th differentiation by directly activating cytokine production in Th9 and Th17 cells, and Bcl6 expression in Tfh cells. Thus, the transcription factors Twist1 and Etv5 provide unique regulation of T helper cell identity, ultimately impacting the development of cell-mediated and humoral immunity.

Indiana University-Purdue University Indianapolis (IUPUI)
The patients with Cystic fibrosis (CF), an inherent genetic disorder, suffer from chronic bacterial infection in the lung. In CF, modification of epithelial cells leads to alteration of the lung environment, such as inhibition of ciliary bacterial clearance and accumulation of thickened mucus in the airways. Exploiting these conditions, opportunistic pathogens like Pseudomonas aeruginosa cause lifelong persistent infection in the CF lung by forming into antibiotic-resistant aggregated communities called biofilms. Airway infections as well as inflammation are the two major presentations of CF lung disease. P. aeruginosa strains isolated from CF lungs often contain mutations in the mucA gene, and this mutation results in higher level expression of bacterial polysaccharides and toxic lipoproteins. In a previous work, we have found a putative lipoprotein gene (PA4326) which is overexpressed in antibiotic-induced biofilm formed on cultured CF-derived airway cells. In the current work, we speculated that this particular putative lipoprotein affects cellular cytotoxicity and immune-stimulation in the epithelial cells. We found that mutation of this gene (ΔPA4326) results in reduced airway cell killing without affecting other common virulence factors.Moreover, we observed that this gene was able to stimulate secretion of the proinflammatory cytokine IL-8 from host cells. Interestingly, we also found that ΔPA4326 mutant strains produced less pyocyanin exotoxin compared to the wild type. Furthermore, our results suggest that PA4326 regulates expression of the pyocyanin biosynthesis gene phzM, leading to the reduced pyocyanin phenotype. Overall, these findings implicate PA4326 as a virulence factor in Pseudomonas aeruginosa. In the future, understating the molecular interplay between the epithelial cells and putative lipoproteins like PA4326 may lead to development of novel anti-inflammatory therapies that would lessen the suffering of CF patients.