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1
Knight, Deborah. "Novel schizophrenia risk genes and gene expression." Thesis, Cardiff University, 2012. http://orca.cf.ac.uk/47378/.
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ZNF804A was (at the time this work started) one of only a few robustly implicated schizophrenia susceptibility genes, due to replicated genome-wide significant evidence for association between a polymorphism in the gene and schizophrenia. Determining the function of the ZNF804A protein, which is currently unknown, may provide a way of elucidating the pathophysiology of this relatively common, complex disorder. Based on the hypothesis that the ZNF804A protein regulates gene expression or splicing, the aim of this thesis was to identify genes that exhibit altered expression or splicing in brain tissue from mice in which the orthologue Zfp804a carries a nonsense mutation. No robust evidence was obtained that showed the effects of the mutation on differential expression in individual genes. Although this finding does not support the hypothesis that ZNF804A acts directly to regulate gene expression, the results may reflect the possibility that effects on gene expression may be too subtle to be detected using the methods applied. Evidence was obtained to show the mutation affected the alternative splicing of a number of individual genes, which could suggest a role for ZNF804A in the direct or indirect regulation of alternative splicing. Through RNA sequencing, I identified a novel transcript in Zfp804a with an alternative exon upstream of the Refseq exon 1. I also showed that a proportion of the significant splicing differences identified in mutants were artefacts of strain differences in gene sequences that are likely to affect the efficiency of hybridisation on the exon array. Genes identified as differentially spliced between mutants and wildtypes were enriched in axon guidance and cell adhesion pathways, both thought to be important during development. The findings of this thesis suggest the novel hypothesis that ZNF804A effects risk for schizophrenia via aberrant splicing in the above pathways that are critical to normal brain development. Further studies with increased power are required to understand the effects on gene expression.
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Preuten, Tobias. "Organellar gene expression." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2010. http://dx.doi.org/10.18452/16142.
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Zusätzlich zu der eubakteriellen RNA-Polymerase (RNAP) der Plastiden sind im Zellkern von Arabidopsis thaliana drei weitere, phagentypische RNAP kodiert, die jeweils aus nur einer Einheit aufgebaut sind. Die Enzyme RpoTp und RpoTm werden in die Plastiden, bzw. die Mitochondrien transportiert, während RpoTmp in beiden Organellen zu finden ist. Um die Lichtabhängigkeit der RpoT-Gene zu untersuchen, wurde die lichtinduzierte Akkumulation ihrer Transkripte in 7-Tage alten Keimlingen, sowie 3- bzw. 9-Wochen alten Rosettenblättern mittels quantitativer real-time PCR ermittelt. Die entwicklungsabhängige Regulation der RpoT-Transkript-Akkumulation wurde außerdem während der Blattentwicklung analysiert. Zusätzlich wurde der Einfluss des circadianen Rhythmus untersucht. Es stellte sich heraus, dass die Transkriptakkumulation aller drei RpoT-Gene stark lichtinduziert war und nur marginalen circadianen Schwankungen unterlag. In weiteren Versuchen mit verschiedenen Lichtrezeptor-Mutanten und unterschiedlichen Lichtqualitäten wurde der Einfluss multipler Rezeptoren auf den Prozess der Lichtinduktion gezeigt. In den Zellen höherer Pflanzen finden sich drei Genome. Die Biogenese von Chloroplasten und Mitochondrien, sowie lebenswichtige Prozesse, wie Atmung und Photosynthese setzen oftmals die Aktivität von Genen auf mindestens zwei dieser Genome voraus. Eine intrazelluläre Kommunikation zwischen den verschiedenen Genomen ist daher unumgänglich für einen funktionierenden Stoffwechsel der Pflanze. In dieser Arbeit wurde herausgestellt, dass die Zahl mitochondrialer Genkopien in photosynthetisch inaktiven Arabidopsis-Keimlingen drastisch erhöht ist. Bei der Untersuchung des DNA-Gehaltes in Proben, die Altersstufen von 2-Tage alten Keimblättern bis hin zu 37-Tage alten, seneszenten Rosettenblättern umfassten, fand sich ein deutlicher Anstieg der Kopienzahlen in älteren Rosettenblättern. Außerdem unterschieden sich die Kopienzahlen der untersuchten Gene zum Teil erheblich voneinander.In addition to eubacterial-like multi-subunit RNA polymerases (RNAP) localized in plastids and the nucleus, Arabidopsis thaliana contains three phage-like single-unit, nuclear-encoded, organellar RNAPs. The enzymes RpoTp and RpoTm are imported into plastids and mitochondria, respectively, whereas RpoTmp shows dual targeting properties into both organelles. To investigate if expression of the RpoT genes is light-dependent, light-induced transcript accumulation of RpoTm, RpoTp and RpoTmp was analyzed using quantitative real-time-PCR in 7-day-old seedlings as well as in 3- and 9-week-old rosette leaves. To address the question whether RpoT transcript accumulation is regulated differentially during plant development transcript abundance was measured during leaf development. Additionally, effects of the plants circadian rhythm on RpoT transcript accumulation were analyzed. Transcripts of all three RpoT genes were found to be strongly light-induced even in senescent leaves and only marginally influenced by the circadian clock. Further analyses employing different photoreceptor mutants and light qualities revealed the involvement of multiple receptors in the light-induction process. The biogenesis of mitochondria and chloroplasts as well as processes like respiration and photosynthesis require the activity of genes residing in at least two distinct genomes. There have to be ways of intracellular communication between different genomes to control gene activities in response to developmental and metabolic needs of the plant. In this study, it was shown that gene copy numbers drastically increased in photosynthetically inactive Arabidopsis seedlings. Mitochondrial DNA contents in cotyledons and leaves ranging in age from 2-day-old cotyledons to 37-day-old senescent rosette leaves were examined. A common increase in senescing rosette leaves and drastic differences between individual genes were found, revealing the importance of an integrative chondriome in higher plant cells.
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Jia, Yizhen, and 贾亦真. "Bioinformatics study of the lineage and tissue specificity of genes and gene expression." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2010. http://hub.hku.hk/bib/B45540652.
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Berggren, Bremdal Karin. "Evolution of MHC Genes and MHC Gene Expression." Doctoral thesis, Uppsala universitet, Evolutionsbiologi, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-122011.
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Polymorphism in coding regions and regions controlling gene expression is the major determinant of adaptive differences in natural populations. Genes of the major histocompatibility complex (MHC) possess a high level of genetic variation, which is maintained by selection over long coalescence times. MHC genes encode antigen-presenting molecules in the adaptive immune system, which protects the host from infectious diseases. However, MHC molecules may also present self-peptides and for most autoimmune diseases there is a genetic factor associated with the MHC. MHC genes have been used to learn about the interplay of selection and historical population events. In domestic dogs and their progenitor, the wolf, I explored factors associated with domestication and breed formation and their influence not only on MHC coding regions but also on the haplotypic structure of the class II region. Polymorphism and strong selection was demonstrated in the proximal promoters of MHC genes in dogs and wolves. Hence, genetic variation associated with MHC gene expression may have at least equal importance for a well functioning immune system. Associations between promoter sequences and particular coding alleles suggested allele-specific expression patterns. SNP haplotypes of the MHC class II region revealed ancestral as well as convergent haplotypes, in which combinations of alleles are kept by selection. Interestingly, weaker allelic associations were found between different genes and between coding regions and promoters in dogs compared to wolves. Potentially, this could cause insufficient defense against infections and predispose dogs to autoimmune diseases. For example, I identified a site in the promoter region that showed a consistent difference between haplotypes conferring susceptibility and protection to diabetes in dogs, which should be investigated further. Furthermore, I investigated how selection and demographic changes associated with glacial and inter-glacial periods have affected MHC variation in European hedgehogs and extended the prevailing knowledge concerning their population history.
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Bashiardes, Evy. "Gene polymorphisms, gene expression and atherosclerotic plaques." Thesis, Imperial College London, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.420882.
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Smith, Erin N. "Gene-environment interaction in yeast gene expression /." Thesis, Connect to this title online; UW restricted, 2008. http://hdl.handle.net/1773/5025.
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Lemons, Derek Scott. "Gene expression and evolution." Diss., [La Jolla] : University of California, San Diego, 2010. http://wwwlib.umi.com/cr/ucsd/fullcit?p3397172.
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Thesis (Ph. D.)--University of California, San Diego, 2010.Title from first page of PDF file (viewed March 23, 2010). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references (p. 98-111).
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Humphries, Clare Ruth. "Gene expression in schizophrenia." Thesis, Imperial College London, 1997. http://hdl.handle.net/10044/1/7770.
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Fischer, Heléne. "Gene expression in carcinogenesis /." Stockholm, 2001. http://diss.kib.ki.se/2001/91-628-4961-1/.
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Hornan, Daniel Mark. "Human macular gene expression." Thesis, University College London (University of London), 2005. http://discovery.ucl.ac.uk/1444745/.
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The human macula is essential for precise vision. It contains many more cone photoreceptors than the peripheral retina, especially in the fovea. Cones are known to express specific opsins and other proteins that form part of the phototransduction cascade. However, relatively little is known about retinal macular gene expression compared with the rod-rich peripheral retina. I obtained human donor eyes and used foveo-macular and macular punches and sections of peripheral retina to study differential gene expression. I combined multiple microarray experiments with quantitative PCR, statistical, and bioinformatic analyses. I identified several known and previously unidentified retinal genes that are more abundant in the macula. I went on to characterize proteins encoded by histone deacetylase 9 and the morpheus gene family. Both were expressed in the human macula, especially in the photoreceptors. Several other genes also provided insight into the mechanisms of precise vision and its maintenance. Genes identified by this approach are excellent candidates for macular disease.
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Tebbutt, Scott James. "Pollen-specific gene expression." Thesis, University of East Anglia, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.334705.
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Al-Nbaheen, May Salem. "Liver specific gene expression." Thesis, University of Bath, 2002. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.760790.
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Herbert, John Matthew Jeff. "Endothelial cell gene expression." Thesis, University of Birmingham, 2013. http://etheses.bham.ac.uk//id/eprint/3803/.
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Tumour angiogenesis is a vital process in the pathology of tumour development and metastasis. Targeting markers of tumour endothelium provide a means of targeted destruction of a tumours oxygen and nutrient supply via destruction of tumour vasculature, which in turn ultimately leads to beneficial consequences to patients. Although current anti-angiogenic and vascular targeting strategies help patients, more potently in combination with chemo therapy, there is still a need for more tumour endothelial marker discoveries as current treatments have cardiovascular and other side effects. For the first time, the analyses of in-vivo biotinylation of an embryonic system is performed to obtain putative vascular targets. Also for the first time, deep sequencing is applied to freshly isolated tumour and normal endothelial cells from lung, colon and bladder tissues for the identification of pan-vascular-targets. Integration of the proteomic, deep sequencing, public cDNA libraries and microarrays, delivers 5,892 putative vascular targets to the science community. These analyses identify Endothelial Specific Molecule 1 as a pan vascular target and lysyl oxidase-like 2 as putative novel vascular target. It is envisioned vascular targets and angiogenesis genes in this data will destroy or inhibit tumour vessel growth without the side effects manifest with current clinical regimens.
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Li, Yan 1978 July 15. "Gene expression array simulator." Thesis, Massachusetts Institute of Technology, 2002. http://hdl.handle.net/1721.1/87263.
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Thesis (M.Eng.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, June 2002."May 10, 2002.
Includes bibliographical references (leaf 141).
by Yan Li.
M.Eng.
15
Johansson, Karin. "Analysis of immunoglobulin gene expression focus on Oct2 /." Lund : Dept. of Cell and Molecular Biology, Lund University, 1995. http://catalog.hathitrust.org/api/volumes/oclc/39776663.html.
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Gould, Barbara. "The genomics of labour : global gene expression profiling and oxytocin receptor gene expression." Thesis, McGill University, 2003. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=84251.
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Premature labour and subsequent premature birth is a leading cause of neonatal morbidity and mortality. We studied marine labour at term and preterm with models of intrauterine infection and ovariectomy using Affymetrix microarray U74Av2 (containing 12,488 probe sets) and real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) to identify novel candidate genes involved in normal and preterm labour. Strict statistical analysis revealed 320, 188, and 74 genes to be significantly induced or suppressed during normal, infection-induced, and ovariectomy-induced labour, respectively. Novel genes identified include: associated with normal labour: alpha fetoprotein, apolipoprotein A1, fibrinogen polypeptides, cytochrome P45011a, Purkinje cell protein 4 (PCP4), and chloride channel calcium activated 3; associated with infection: numerous inflammatory mediators, alpha fetoprotein, apolipoprotein Al, and fibrinogen polypeptides. Small proline-rich protein 2 family genes were induced by ovariectomy and infection. PCP4 gene was induced after ovariectomy, but suppressed at normal labour. Only seven genes were significantly regulated (each induced) at labour in all models implying that unique gene networks are involved in normal and preterm labour induced by various stimuli. These included genes for plasminogen activator inhibitor 1 and for contraction associated proteins (CAPS) required for uterine activation and uterotonin stimulation of contractions.The oxytocin receptor (OTR) gene encodes one such CAP. Northern blot and real-time RT-PCR demonstrated its up-regulation prior to labour in each model, preferentially in normal labour. Uterine contraction promotes increased central and peripheral oxytocin release and synaptic plasticity. To further examine the role of the OTR, we developed an OTR-lacZ reporter mouse. We mapped, by X-gal histochemistry, the distribution of OTR gene expression in the early postparturient mouse brain and identified novel regions of expression. These included the piriform cortex, entorhinal cortices, and parasubiculum, which support memory function. Dorsal tegmental, vestibular, and lateral reticular nuclei expression suggests the transmission of locomotor inputs. Hypoglossal, facial, and spinal trigeminal nuclei support maternal behaviours. We also more accurately demarcated OTR gene expression in the solitary tract nucleus responsible for relaying contraction stimulation of oxytocin release.
These studies provide a more accurate knowledge base for the development of successful therapies to decrease the incidence of premature labour.
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Herrig, Danielle Kay. "Evaluating gene flow, gene expression divergence, and hybrid expression in Drosophila sister species." Diss., University of Iowa, 2016. https://ir.uiowa.edu/etd/2222.
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A primary goal of evolutionary biology is to elucidate the factors necessary for a single interbreeding species to become two independent species. Observations and data collected and recorded since the 6th century B.C. have added to our comprehension of the “the origin of species—that mystery of mysteries” (DARWIN 1859). To continue to add to our knowledge of how speciation occurs and how species interact, it is crucial to determine 1) how different categories of genes evolve as species diverge, 2) what happens to hybrids of two species, and 3) if genetic exchange is allowed between species, where it is located.
In the first research aim of my dissertation, I look for population genetic trends and signatures of gene flow in a minimally studied set of Drosophila sister species using sequences of 26 nuclear and mitochondrial regions in 29 isofemale lines of D. subobscura and D. madierensis. Standard population genetic tests revealed that the X chromosome evolves faster than the autosomes in these species. We also find evidence of genetic exchange for some autosomal genes while both the sex chromosomes and mitochondrial genomes remain distinct between species.
In the second research aim of my dissertation, I assess the rates of gene expression evolution for sex-biased genes located on the X chromosome and autosomes. We find that gene expression evolves faster in males than females and find evidence of faster-X evolution that is exclusive to genes expressed at higher levels in males. The X chromosome has previously been shown to have a disproportionately large influence on hybrid male sterility compared to autosomes. I investigate this trend and find that the sex chromosomes have a large influence on autosomal expression levels in hybrid males and hybrid females. Specifically, uniparental inheritance of the X chromosome results in greater differences between reciprocal hybrids and higher levels of hybrid misexpression.
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Nascimento, Helvia. "Caracterização da expressão genica de celulas tumorais de pacientes com adenocarcinoma esporadico do colon." [s.n.], 2007. http://repositorio.unicamp.br/jspui/handle/REPOSIP/310956.
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Orientador: Carmen Silvia Passos Lima, Fernando Ferreira CostaTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
Made available in DSpace on 2018-08-12T11:53:50Z (GMT). No. of bitstreams: 1 Nascimento_Helvia_D.pdf: 1845231 bytes, checksum: 16f227072d706aacb01f06165bd8a553 (MD5) Previous issue date: 2007
Resumo: Os mecanismos moleculares envolvidos na origem do adenocarcinoma de cólon esporádico (ACE) ainda não estão completamente elucidados. Recentemente, o método da análise seriada da expressão gênica (SAGE) foi descrito como eficaz para identificar a expressão total de genes de tipos celulares diversos, mas esta análise não foi realizada em células epiteliais purificadas do ACE moderadamente diferenciado. Nós caracterizamos pelo método SAGE a expressão gênica total de células epiteliais neoplásicas do cólon de um paciente com ACE moderadamente diferenciado (SAGE CC) e de células epiteliais normais do cólon de um paciente com megacólon chagásico (SAGE CN). Foram geradas, após o seqüenciamento automático, 44.004 e 43.570 tags totais das bibliotecas SAGE CC e SAGE CN, representando 16.484 e 13.479 tags únicas, respectivamente. Na comparação entre as bibliotecas, 171 transcritos diferencialmente expressos foram identificados (P< 0,001; expressão diferencial = 5), incluindo 10% de transcritos que podem representar genes não descritos. As expressões de 10 genes diferencialmente expressos foram quantificadas pela reação em cadeia da polimerase em tempo real (qPCR) na amostra de células epiteliais neoplásicas (SAGE CC), com o intuito de validar os resultados obtidos pelo SAGE, e, posteriormente, em amostras de células epiteliais de outros cinco pacientes com o mesmo tipo de doença. As expressões foram concordantes em 80% dos genes (CEACAM6, KLK6, LYZ, PFN1, S100A8, S100A9, VIL2 e ZFHX1B) e discordantes nos demais 20% (PLA1A e ZNF277). As expressões dos genes de interesse, quantificadas pelos dois métodos, foram similares na amostra SAGE CC e nas amostras dos demais pacientes com a doença. Foram observadas expressões anormais de genes envolvidos com a proliferação e diferenciação celular e com a resposta ao stress em células epiteliais neoplásicas. Foram também visualizadas expressões anormais de genes não relacionados com a doença e de genes ainda não identificados. Em conjunto, os nossos resultados podem contribuir para a identificação de genes relacionados com a origem ou a progressão do ACE moderadamente diferenciado e, ainda, para a descoberta de agentes terapêuticos específicos que controlem a proliferação anormal das células neoplásicas.
Abstract: The molecular mechanisms involved in sporadic colon adenocarcinoma (SCA) are still not completely elucidated. Recently, the serial analysis of gene expression (SAGE) method has allowed the global analysis of genes expressed in diverse cellular types but there are no studies in purified epithelial cells of SCA moderately differenciated. We have characterized through SAGE the global gene expression of neoplastic epithelial cells from a SCA moderately differenciated patient (SAGE CC) and normal epithelial cells from a megacolon patient (SAGE CN). After automatic sequencing, a total of 44.004 tags from SAGE CC and 43.570 tags from SAGE CN profiles were generated, representing 16.484 and 13.479 unique tags, respectively. Comparing both profiles, 171 differentially expressed transcripts were identified (P< 0.001; fold = 5), including 10.0% that may represent novel transcripts. The expression of 10 selected genes was further investigated by realtime polymerase chain reaction (qPCR) in the SCA moderately differenciated epithelial cells sample (SAGE CC), with the purpose of to validate the results obtained by the SAGE method, and also in five epithelial cells samples from the same type of SCA patients. Similar expressions were seen in 80% (CEACAM6, KLK6, LYZ, PFN1, S100A8, S100A9, VIL2 e ZFHX1B) and discordant expressions were seen in 20% (PLA1A e ZNF277) of analysed genes. On SAGE CC sample and samples of the SCA patients, all genes presented similar expressions measured by both methods. We observed abnormal expression of genes involved with cell proliferation and differentiation, and with response to stress in neoplastic epithelial cells. Also, were found abnormal expressions of genes not related with the disease and not identified genes. Together, our results may contribute for the identification of genes involved in the origin or progression of SCA moderately differenciated, as well as for the discovery of new therapeutical agents, with specific action on abnormal proliferation of the neoplastic cells.
Doutorado
Ciencias Basicas
Doutor em Clínica Médica
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McGillivray, Shauna Marie. "Regulation of gonadotrope gene expression /." Diss., Connect to a 24 p. preview or request complete full text in PDF formate. Access restricted to UC campuses, 2006. http://wwwlib.umi.com/cr/ucsd/fullcit?p3208634.
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Marciniak, Jennifer Yuko. "Variability in eukaryotic gene expression /." Diss., Connect to a 24 p. preview or request complete full text in PDF formate. Access restricted to UC campuses, 2005. http://wwwlib.umi.com/cr/ucsd/fullcit?p3208639.
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Ma, Pinchao. "Gene expression and splicing efficiency." Thesis, University of Ottawa (Canada), 2008. http://hdl.handle.net/10393/27801.
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Most eukaryotic protein-coding genes are split into exons and introns, and introns need to be spliced for the production of mature mRNA by pre-mRNA splicing. Pre-mRNA splicing is very important for eukaryotic gene expression, because it is not only a key step in producing mature mRNA, but can also affect transcription and translation. The purpose of this study is to investigate the relationship between gene expression and splicing efficiency since the relationship has not been studied systematically from a bioinformatic approach. In this thesis research, we focus on the question of how gene expression would constrain the evolution of three principal splicing signals: the donor splice site, the acceptor splice site, and the branchpoint sequence (BPS).
We chose yeast, Saccharomyces cerevisiae, as the model organism in this study due to its many research advantages such as relatively simple splicing mechanism and extensive genome-wide characterization of gene expression at both transcript and protein levels. We first studied the relationship between gene expression and the strength of the donor and acceptor splice sites in the yeast, with the latter being characterized by position weight matrix scores. We found that donor and acceptor splice sites in highly expressed genes have significantly higher mean, but smaller variance, of splicing strength than that in lowly expressed genes. In addition, genes with extremely low splice site strength tend to be spliced by non-spliceosomal mechanisms, and the last nucleotide on the exon side in the donor splice site (immediately upstream of the 5'GU dinucleotide) is important for splicing. We further studied the relationship between gene expression and BPS in the yeast. The results revealed that the BPS strength of highly expressed yeast intron-containing genes (ICGs) is significantly higher than that of lowly expressed yeast ICGs. Moreover, highly expressed yeast ICGs have significantly longer distance between the donor splice site and BPS (Si distance) and slightly longer distance between BPS and the acceptor splice site (S2 distance) than lowly expressed yeast ICGs. The long Si distance of highly expressed yeast ICG does not indicate the potential of enhancing splicing efficiency through forming secondary structure in the region between the donor splice site and BPS.
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Molloy, Timothy John St George Clinical School UNSW. "Gene expression in healing tendon." Awarded by:University of New South Wales. St George Clinical School, 2006. http://handle.unsw.edu.au/1959.4/23939.
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Tendon injury is painful and often debilitating, and is a one of the most prevalent soft tissue injuries encountered in the clinic. While common, the underlying molecular and genetic processes of tendon damage and repair remain poorly understood. The work described herein used genome-wide expression analyses to investigate tendon injury and healing from three perspectives. The first identified novel gene expression in tendon fibroblasts following their stimulation with nitric oxide (NO). Of particular relevance to tendon healing was the observation that stimulated fibroblasts express a number of extracellular matrix (ECM) genes in response to NO in a dose-dependent manner, and that NO significantly affects cellular adhesion, a critical process during tendon repair. These findings will be of use when optimising dosages of NO delivery in future work investigating NO as potential treatment for tendon injuries. The second study examined gene expression in an acute tendon injury model in the rat at 1, 7, and 21 days post injury, roughly representing the inflammation, proliferation, and remodelling phase of wound repair. Several novel genes and pathways were found to be differentially expressed at each stage of healing. Of particular interest were the presence of a significant number of genes related to glutamate signaling, a method of cellular communication that has not previously been shown to exist in tendon. Also upregulated were a number of genes which have previously only been associated with embryonic development. Finally, gene expression in a supraspinatus tendinopathy model in the rat was investigated. Several genetic pathways were identified in tendinopathic tendons which have not previously been associated with the disease, and, analogous to the acute injury model study, glutamate signaling gene overexpression was also prevalent. Further in vitro studies showed that the expression of these genes in tendon fibroblasts were stimulated by glutamate treatment, which in turn upregulated pro-apoptotic pathways causing cell death. This may prove to be an important causative factor in the tendon degeneration seen in tendinopathy, in which apoptosis has been identified as playing a significant role. The results of these studies contribute to a better understanding of the aetiology of several extremely common pathologies of this soft tissue, and may help to develop more targeted therapies for increasing the efficacy of tendon healing in future.
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Palm, Kaia. "Regulation of neuronal gene expression /." Stockholm, 1998. http://diss.kib.ki.se/search/diss.se.cfm?19980612palm.
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Fritz, Georg. "Strategies of bacterial gene expression." Diss., lmu, 2012. http://nbn-resolving.de/urn:nbn:de:bvb:19-155355.
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Romanuik, Tammy Lee. "Gene expression in prostate cancer." Thesis, University of British Columbia, 2008. http://hdl.handle.net/2429/5313.
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Development and maintenance of the prostate is dependent on androgens and the androgen
receptor. The androgen pathway continues to be important in prostate cancer. Here, we evaluated
the transcriptome of prostate cancer cells in response to androgen using Long Serial Analysis of
Gene Expression (L0ngSAGE) libraries. We identified 35 genes with novel associations to
androgen signalling and validated 24 of these genes using quantitative real time-polymerase
chain reaction (qRT-PCR). These genes were: ARL6IF5, BL VRB, C]9orf48, C]orfJ22,
C6orf66, CAMK2NJ, CCNI, DERA, ERRFI], GLUL, GOLFH3, HMJ3, HSP9OB], MANEA,
NANS, NIPSNAP3A, SLC4JA], SOD], SVIF, TAOK3, TCP], TMEM66, USP33, and
VTAJ. The physiological relevance of these expression trends was evaluated in vivo using the
LNCaP Hollow Fibre model.
There is no cure for castration-recurrent prostate cancer (CRPC), and the mechanisms underlying
the disease are not known. To address this problem, we assayed the transcriptome of LNCaP
human prostate cancer cells as they progress to castration-recurrence in vivo using replicate
L0ngSAGE libraries. We identified 96 novel genes consistently differentially expressed in
CRPC. The expression profiles support a role for the transcriptional activity of the androgen
receptor genes (CCNH, CUEDC2, FLNA, and FSMA 7), steroid synthesis and metabolism genes
(DHCR24, DHRS7, ELOVL5, HSDJ 7B4, and OPRKJ), neuroendocrine cell genes (ENO2,
MAOA, OPRK], SJOOA]O, and TRPM8), and proliferation genes (GAS5, GNB2L], MT-ND3,
NKX3-], PCGEM], PTGFR, STEAFJ, and TMEM3OA) in castration-recurrence.
Screening for prostate cancer using serum levels of prostate-specific antigen has resulted in the
over-treatment of indolent disease. Novel diagnostic and prognostic markers for prostate cancer
are required. To address this need, the levels of 27 transcripts were investigated with qRT-PCR.
Expression of POP3 (100 kb from EST CF140309) was prostate-specific, with restricted
expression ofADAM2, POP1 (50 kb from AK000023), POP4 (truncated TMEFF2), POP 10
(intron ofADAM2), ELOVL5, RAMP], and SPON2. ELO VL5, NGFRAP1, POP5 (intron of
NCAM2), POP8 (intron of EFNA5), RAMP], SPON2, and TMEM66 were differentially
expressed between laser microdissected tumour and normal clinical samples of prostatic tissue.
These studies suggest that ADAM2, ELOVL5, POP 1, POP3, POP4, POP 10, RAMP], and SPON2
may be good candidates for biomarkers of prostate cancer.
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Khan, Mahmood Ali 1962. "Peroxidoxin gene expression in Leishmania." Thesis, McGill University, 2001. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=33792.
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Leishmania protozoans are the etiologic agents of the disease leishmaniasis. The parasite exists in two morphological forms: promastigote and amastigote. Promastigotes are found in the gut of the sandfly vector while amastigotes reside inside the vertebrate macrophage. Leishmania, an obligate intracellular parasite, resists toxic reactive oxygen species (ROS) from both endogenous and exogenous sources. Like other protozoa, Leishmania lacks some of the antioxidant defence enzymes such as catalase and glutathione peroxidase (Gpx) that are usually found in aerobic cells. Instead they possess the antioxidant thiol compound trypanothione, in association with specific trypanothione linked antioxidant enzymes such as peroxidoxins. The transformation from promastigote to amastigote is a crucial step for parasite infection and survival. The molecular basis for this transformation is not clearly understood. Recently it was shown that the peroxidoxin gene is present in multiple copies in Leishmania. In the present study we examined the potential of antisense RNA and double stranded RNA (dsRNA) to perform functional knockout of the peroxidoxin gene. Towards that end antisense RNA and dsRNA expressing plasmids, targeting the peroxidoxin gene, were constructed. Leishmania promastigotes were subsequently transfected with these plasmids and the levels of peroxidoxin gene expression were studied. The results from this study suggest that there is no apparent reduction in either the levels of peroxidoxin mRNA or the protein in the transfected promastigotes as compared to the non-transfected cells.
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Mason, J. O. "Regulation of immunoglobulin gene expression." Thesis, University of Cambridge, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.384506.
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Naik, Sandhia. "Nutrition and intestinal gene expression." Thesis, Queen Mary, University of London, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.406381.
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Tam, Wai Keung. "Cytokine gene expression in glomerulonephritis." Thesis, Imperial College London, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.363081.
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Al-Lawati, Sabah Ali Redha. "Differential gene expression in schizophrenia." Thesis, Imperial College London, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.420028.
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Ebert, Benjamin L. "Oxygen regulation of gene expression." Thesis, University of Oxford, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.296895.
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Muszynska, Dorota. "Gene expression profiling in Keratoconus." Thesis, Queen's University Belfast, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.602693.
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Keratoconus (MIM#148300), a common bilateral, progressive corneal thinning disorder, is the leading indication for corneal transplantation in the developed world. Keratoconus usually arises in the teenage years and presents a significant health burden in work-age adults. Despite the visual and social impact of keratoconus, our lack of understanding of the molecular pathology of keratoconus is a major obstacle to the development of new therapeutic approaches. This study represents the first reported application of massively parallel sequencing of mRNA (RNA-Seq) to perform whole genome transcriptome analysis of keratoconic keratocytes. Genes-enrichment analysis identified several pathway maps that are disrupted in keratoconic keratocytes and associated with the pathogenesis of keratoconus. Microarray gene expression was used to validate differentially expressed genes identified by RNA-Seq in a global manner, whereas quantitative reverse transcriptase polymerase chain reaction was performed on selected candidate genes. Wnt signalling, TGF-beta signalling. ECM-matrix interactions, oxidative stress and inflammatory- related genes were specifically identified in keratoconic keratocytes and implicated in the pathogenesis of keratoconus. Analysis of individual target genes identified altered expression of both known and novel keratoconus-related genes, in particular, SFRP1, BMP4, CBS, POSTN, C0L11Al, COL4Al, SOD1, IL6, and SP3. Functional analyses and expression profiling of keratoconic keratocytes harbouring a novel heterozygous missense mutation (c.l920G>T; p.Gln640His) in the zinc finger E-box binding gene 1 (ZEB1) was also performed. The mutant ZEB1 protein was stable and localised to the nucleus resulting in an enhanced transcriptional repressor of known ZEB1 targets, involved in epithelialmesenchymal transition and collagen synthesis. This ZEB1 mutation results in a gain-In-function with enhanced transcriptional repression of a number of gene targets associated with keratoconus, corneal thickness and Fuchs endothelial corneal dystrophy. This study has identified a number of molecular targets for keratoconus and provides a significant insight into the gene pathways involved in keratoconus pathogenesis. Further functional studies can build on this evidence to interrogate disease pathogenesis, identify novel genes and develop molecular therapies for keratoconus.
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Ng, W. C. "Studies on vitellogenin gene expression." Thesis, Open University, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.484402.
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Curtis, R. K. "Control analysis of gene expression." Thesis, University of Cambridge, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.598230.
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This thesis describes the development of the application of modular regulation analysis, a subset of metabolic control analysis to microarray data. Microarray experiments measure complex changes in the abundance of many mRNAs under different conditions. Current analysis methods, such as clustering, cannot distinguish between direct and indirect effects on expression, or calculate the relative importance of mRNAs in effecting responses. Modular regulation analysis of microarray data reveals and quantifies which mRNA changes are important for cellular responses. The mRNAs are clustered, then how perturbations alter each cluster (integrated response co-efficients) and how strongly those clusters affect an output response is calculated (elasticity co-efficients). The product of these values quantifies how an input changes a response through each cluster (partial response co-efficients). Once identified, important clusters that mediate a large proportion of the response may suggest targets for investigation of, for example, disease mechanisms, and way of modifying that response, such as potential knockout, overexpression or drug targets. Two published datasets were used throughout the development of the method. This determined the requirements of a suitable dataset, and involved the creation of a test to exclude problematic experiments from the dataset. Analyses of the two datasets using the final method reveal that two mRNA clusters transmit most of the response of yeast doubling time to galactose; one contains mainly galactose metabolic genes, and the other a regulatory gene. Analysis of the response of yeast relative fitness to 2-deocy-D-glucose reveals that control is distributed between several mRNA clusters. Monte Carlo analysis revealed that the co-efficients were not statistically significant, due to the large amount of experimental error in the dataset. However, modular regulation analysis should become more applicable in practice as microarray technology is improving rapidly.
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Vijayan, Vikram. "Circadian Gene Expression in Cyanobacteria." Thesis, Harvard University, 2012. http://dissertations.umi.com/gsas.harvard:10665.
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Cyanobacteria are photosynthetic prokaryotes that live in aquatic environments. The cyanobacterium Synechococcus elongatus PCC 7942, (hereafter S. elongatus) coordinates its day and night behaviors via a circadian clock. The clock is entrained by light/dark cycles but continues to run in constant light conditions. The core circadian clock in S. elongatus is encoded by post-translational modifications of three Kai proteins, but the extent and mechanism of circadian gene expression are unknown. We provide the first unbiased characterization of circadian gene expression in S. elongatus, demonstrating that \(\sim 65\%\) of genes display oscillation in continuous light conditions, with some genes peaking in expression at subjective dawn and others at subjective dusk. We next sought to identify the mechanism by which such a large fraction of the genome could be rhythmically controlled. Through bioinformatic, correlative, and perturbation experiments, we find that circadian changes in chromosome topology/supercoiling are sufficient to drive rhythmic expression (Chapter 2). To further investigate how chromosome topology can control gene expression we performed a high resolution characterization of transcripts and RNA polymerase across the S. elongatus genome (Chapter 3). Bioinformatic analysis of transcription start sites suggests that the AT/GC content a particular region of the promoter is informative in defining the phase at which a transcript is maximally expressed. We find that these sequences are sufficient to drive circadian gene expression at a particular phase and that mutation of single nucleotides in this region can reverse the expression phase of a transcript (Chapter 4). To understand the role of chromosome dynamics in circadian gene expression and cyanobacterial physiology, we tagged and followed chromosomes over multiple cell divisions. We find that S. elongatus cells harbor multiple ordered copies of a single chromosome, and the organization of chromosomes in the cytoplasm facilitates equal segregation of chromosomes to daughter cells (Chapter 5).
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Allan, G. "Gene expression during keratinocyte differentiation." Thesis, University of Newcastle Upon Tyne, 1988. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.233424.
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MacLeod, Ronald. "Gene expression in human neutrophils." Thesis, University of Liverpool, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.317206.
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Rennie, Sarah. "Regulatory complexity in gene expression." Thesis, University of Edinburgh, 2017. http://hdl.handle.net/1842/29503.
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The regulation of gene expression is the driver of cellular differentiation in multicellular organisms; the result is a diverse range of cell types each with their own unique profile of expression. Within these cell types the transcriptional product of a gene is up or down regulated in response to intrinsic and extrinsic stimuli according to its own regulatory programme encoded within the cell. The complexity of this regulatory programme depends on the requirements of the gene to change expression states in different cell lineages or temporally in response to a range of conditions. In the case of many housekeeping genes integral to the survival of the cell, this programme is simple - switch on the gene and leave it on, whereas often the required level and precision of regulatory control is much more involved and lends to subtle changes in expression. This raises many questions of precisely where and how that regulatory information is encoded and whether different biological systems encode it in the same way. This project attempts to answer these questions through the development of novel approaches in quantifying the output of this regulatory programme according to the state changes as observed from the expression profile of a given gene. Measures of complexity in gene expression are calculated over a wide range of cell types and conditions collected using CAGE, which provides a quantitative estimate of gene expression that precisely defines the promoter utilised to initiate that expression. As expected, housekeeping genes were found to be amongst the least complex, as a result of their uniform expression profiles, as well as those genes highly restricted in their expression. The genes most complex in their expression output were those associated with the presence of H3K27me3 repressive marks; genes poised for activation in a specific set of cell types, as well as those enriched in DNAse I hypersensitive sites in their upstream region but not necessarily conserved in that region. Evidence also suggests that different promoters associated with a gene contribute in different ways to its resultant regulatory complexity, suggesting that certain promoters may be more crucial in driving the regulation of some genes. This allows for the targeting of such promoters in the analysis of certain diseases implicated by changes in regulatory regions. Indeed, genes known to be associated with diseases such as leukaemia and Alzheimer’s are found to be highly complex in their expression.
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Warnefors, Anna Maria Linne´a. "Evolution of human gene expression." Thesis, University of Sussex, 2011. http://sro.sussex.ac.uk/id/eprint/6979/.
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During evolution, biological differences between species can arise not only due to structural differences between genes, but also following changes in how, where and when genes are active. However, we know much less about this second aspect, because large-scale comparative transcriptomics only became feasible relatively recently. In this thesis, I will therefore investigate several aspects of gene expression evolution, with emphasis on our own species. A first step to understanding regulatory evolution is to determine how variation in gene expression is created. Transposable elements (TEs) are genomic parasites that can affect their host genome in a number of ways, including gene expression. In Chapter 2, I investigate to what extent transposable elements (TEs) have contributed to expression differences between humans and chimpanzees. Once expression variation has been established, a combination of selection and drift will decide which variants are passed on to future generations. It is of particular interest to identify changes that were established through positive selection, as these are adaptive. In Chapter 3, I describe a new method to detect positive selection acting on gene expression and apply it to data from humans and chimpanzees. Human gene expression is regulated through several mechanisms associated with transcription and post-transcriptional processing. In Chapter 4, I consider the long-term evolution of the human genome and investigate whether genes have reached their maximum capacity in terms of regulatory complexity. Finally, in Chapter 5, I explore the relationship between gene regulation and sequence conservation by identifying and analysing extremely conserved elements in the genome of the fruit fly Drosophila melanogaster.
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Sweeney, Glen E. "Differential gene expression in Physarum." Thesis, University of Leicester, 1987. http://hdl.handle.net/2381/35166.
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Physarum polycephalum has a life-cycle that encompasses two very distinct vegetatively growing cell-types; uninucleate microscopic amoebae, and multinucleate macroscopic plasmodia. cDNA libraries have been prepared from both amoebae and plasmodia using the M13 vector mp8. Clones from the two libraries were screened using a differential hybridisation procedure that identifies clones derived from mRNAs much more abundant in one cell-type than in the other. For both the amoebal library and the plasmodial library it was found that about 5% of the clones represented genes preferentially expressed in the cell-type from which the library was prepared. Some of the cell-type-specific clones obtained were used to probe northern blots of amoebal and plasmodial RNA. Two of the plasmodial-specific clones were found to be derived from highly abundant mRNAs, constituting between 0.5% and 2% of total plasmodial mRNA. Selected clones were then used to look at changes in mRNA concentrations during development by probing northern blots of RNA from amoebae, plasmodia and intermediate cell-types. It was found that the plasmodial-specific mRNAs examined fell into two classes; those expressed early in development, and those expressed late in development. The amoebal-specific clones analysed constituted a single group, with each of the probes used detecting an mRNA whose concentration declined markedly in early development. Some of the changes in gene expression were examined more quantitatively by dot blotting. It was found that the difference in concentration of cell-type-specific mRNAs between amoebae and plasmodia varied from between 10 fold to greater than 100 fold. Analysis of the pattern of gene expression was begun in two mutant strains of Physarum which are unable to complete development. Results obtained from a strain which is arrested late in development (RA612) suggest that the developmentally arrested cells express the plasmodial-specific genes which are activated early in development, but not those that become active late in development. A few of the clones have been sequenced, but no homologies with genes sequenced in other systems were detected.
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Collier, William. "Gene expression regulation in Pneumoviruses." Thesis, University of Warwick, 2017. http://wrap.warwick.ac.uk/102038/.
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Members of the Pneumoviridae virus family are responsible for severe respiratory tract disease in their hosts. Human respiratory syncytial virus (hRSV) is responsible for over 200,000 deaths worldwide each year and bovine respiratory syncytial virus (bRSV) causes major economic loss to the cattle industry worldwide. The current model for all nonsegmented negative-sense single stranded RNA virus gene expression, is that mRNA is generated in a polar gradient, with decreasing levels of mRNA transcribed from genes further along the genome from the 3 ́ end. With the exception of translation of ORF-2 located on the bicistronic M2 mRNA, translation of Pneumoviridae mRNAs is thought to be regulated through the levels of mRNA abundance. Translation of M2 ORF-2 has been characterised as being regulated by the non-canonical mechanism of coupled translation termination/initiation in pneumonia virus of mice (PVM), hRSV and avian metapneumovirus (APV). This mechanism is reliant on a proportion of the elongating ribosome translating the upstream M2 ORF-1, terminating and reinitiating translation of M2 ORF-2. Although the initiation site for M2 ORF-2 is similar in bRSV to other members of this family that use the mechanism of coupled translation, the mechanism has not been characterised. Using the technique of ribosomal profiling to analyse steady state viral mRNA abundance and viral translation in both hRSV and bRSV-infected cells, it was observed that for certain viral mRNAs, levels of mRNA abundance did not follow the standard polar transcription model. This was characterised by an increase in the levels of mRNA abundance between the mRNA’s respective gene and its upstream neighbour. The increase was observed in the same group of mRNAs in both viruses suggesting that factors other than the transcription polar gradient influence levels of viral mRNA abundance. It was also observed that levels of proportional translation did not match the respective proportional levels of mRNA abundance for certain viral mRNAs in both viruses. This would suggest that translation of viral genomes is not primarily controlled by mRNA abundance and instead other translational regulatory factors influence levels of translation. The mechanism of bRSV M2 ORF-2 translation was also characterised using reporter plasmids assays. It was identified that the mechanism of initiation of translation of M2 ORF2 used, was not that of coupled translation termination/initiation used by other members of this family. Instead it was observed that translation of M2 ORF-2 used an internal initiation mechanism located inside M2 ORF-1 to initiate translation. The mechanism of coupled translation termination/initiation used for translation of PVM M2 ORF-2 was also further characterised. It was observed that translation of M2 ORF-2 was reliant on upstream sequence in the M2 ORF-1 sequence. A predicted mRNA secondary structure was identified in this region and when disrupted, inhibited translation of M2 ORF2. This was similar to the mechanism of coupled translation used in hRSV, suggesting that the mechanism used by this family is reliant on a mRNA secondary structure located upstream of the initiation site.
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Fioroni, Orietta Maria. "Gene expression in cultured cells." Thesis, University of Leicester, 1989. http://hdl.handle.net/2381/35455.
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Dedifferentiation is the process by which specialised quiescent cells give rise to heterotrophic, dividing cells. This process may be initiated in vivo as a response to wounding, or in vitro during culture initiation. This thesis is concerned with evaluating whether the process of dedifferentiation and maintenance of the fast-dividing, dedifferentiated state by culture, is dependant upon major changes in gene expression. In particular, the role of transcription, as mirrored by changes in steady state mRNA levels, in these putative changes in gene expression has been investigated. Mechanically isolated Asparagus officinalis mesophyll cells were used to study dedifferentiating cells, and suspension cultures of Petunia hybrida to investigate the established dedifferentiated state. This thesis shows that dedifferentiation in Asparagus officinalis is accompanied by major changes in the steady state mRNA profiles of the cells. A group of novel transcripts appearing in dedifferentiating asparagus cells were termed DDl, and targeted for further study. Two cDNA clones coding for DDl transcripts were isolated and characterised, and antibodies to DDl raised for serological work. Only minor differences were found between the steady state mRNA populations of Petunia hybrida cultured cells and seedlings, and these were mainly caused by transcripts disappearing in culture; no transcripts specific to the suspension culture system were detected. The results presented in this thesis are used to foward the hypothesis that changes in gene expression involving de novo transcription may only occur in response to major changes in environmental conditions. It is suggested that the basal transcription pattern for cells in established state is probably common to all cell types with regards to primary cell functions such as growth, division and catabolism. In such established states, the control of metabolism probably resides within the biochemical pathways utilised by the cell at any moment in time.
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Heur, J. Martin. "Lysosomal Regulation of Gene Expression." University of Cincinnati / OhioLINK, 2002. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1026512116.
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Gaspar, Paulo Miguel da Silva. "Gene optimization for heterologous expression." Master's thesis, Universidade de Aveiro, 2010. http://hdl.handle.net/10773/7238.
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Mestrado em Engenharia de Computadores e TelemáticaCom o uso de computadores para assistir investigadores na área da biologia na resolução de tarefas complexas, o seu potencial surgiu como uma ajuda preciosa para alcançar o que está para além das capacidades humanas. Para um biólogo, nos tempos que correm, lidar com um computador é uma tarefa tão trivial como realizar experiencias em laboratório. Assim, a capacidade fornecida pela tecnologia computacional, juntamente com as centenas de aplicações e ferramentas de software que já existem, concedem à Biologia um apoio significativo para a investigação e desenvolvimento. O ramo da Biologia Molecular tem testemunhado um uso crescente destas capacidades tecnológicas, sobretudo nos programas de sequenciação de genomas, que traduzem a informação genética de seres vivos para formatos digitais. Como fruto destes projectos, são gerados grandes volumes de dados de várias espécies, que são disponibilizados. Em consequência, muitos sistemas de bioinformática tem como objectivo analisar estes dados. Novas descobertas e avanços requerem novas ferramentas e técnicas. Esta tese debruça-se sobre o problema das metodologias de redesenho de genes, estudando e reunindo várias características conhecidas dos genes e o seu impacto na criação de proteínas, na perspectiva das estratégias de manipulação de sequências de genes. Estas características e algoritmos de redesenho devem ser encaixados numa só ferramenta que permita aos investigadores estudar mais apropriadamente os genes e os factores que influenciam as suas sequências. Também objecto de estudo nesta tese é a capacidade de combinar esses factores de forma óptima, num só processo de redesenho.
As computers started assisting biology researchers in complex tasks, their potential arose as a precious aid to achieve what was beyond human capacity. In modern times, for a biologist, dealing with a computer is as trivial as working with test tubes in the laboratory. Thus, the power provided by computational technology along with hundreds of software applications and tools that already exist, grant biology a signi_cant support for research and development. Molecular biology has witnessed an increased use of these technological capabilities, especially with the genome sequencing projects that translate the genetic information from living beings into digital formats. Large volumes of data from various species are, thus, generated and made available. Analyzing that data is now the goal of many bioinformatics systems. Consequently, new discoveries and advancements demand new tools and techniques. This thesis lays on the problem of gene redesign methodologies, by studying and gathering the available known gene characteristics and its impact on protein production, from the perspective of their sequence manipulation strategies. These characteristics and redesign algorithms should be assembled into a single package tool, to allow researchers to better study genes and all factors that inuence their sequence. Also a subject of study is the capacity to correctly and optimally combine those factors into a single redesign process.
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Lyons, Scott K. "Studies on conditional gene expression." Thesis, University of Edinburgh, 1999. http://hdl.handle.net/1842/21371.
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Using a double replacement targeting approach, I attempted to replace the p53 promoter region with inducible promoter sequence. Targeting constructs were designed to produce a mutant p53 locus, which would then subsequently allow the rapid and simple introduction of a variety of transcriptional regulatory sequences. The first stage vector was introduced into ES cells, and of 508 HAT resistant clones screened, 1 clone possessed a recombinant p53 locus. Time constraints did not permit full characterisation of this clone, either in vitro or in vivo. In a parallel approach, I used one of the candidate promoter systems to generate an interferon α/β inducible p53 transgene. Sarcoma cells, derived from an engineered homozygous p53 deficient mouse, were established as lines in vitro and stably transfected with this construct. Analysis of resultant inducible clones demonstrated little significant transgene mediated effect upon tumour cell cycle. However, a clear transgene dependent apoptotic phenotype was observed following UV irradiation. To test the possible extent of therapeutic benefit resulting from the introduction of p53 gene expression into p53 null tumour cells in vivo, 5 x 106 inducible sarcoma cells were injected sub-cutaneously into the flanks of SCID animals. Tumours grew at the site of injection within 3 weeks, at which time the animals were injected intra-peritoneally with synthetic double stranded RNA (pI:pC) to induce transgene expression. No p53 protein could be detected in these tumour samples by either western or immuno-histochemical analyses. Subsequent re-establishment and analysis of cell lines derived from these tumours indicate that, during the clonal expansion of the original inoculate in the SCID animal, selection had occurred for a final presenting tumour cell with lost or silenced p53 transgene expression. Following pI:pC injection of SCID mice, an acute apoptotic phenotype was observed in the crypt compartments of the small intestine which resulted in the death of approximately 20 % of all injected animals.
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Mizumoto, Hiroyuki. "Gene Expression Mechanisms of Dianthovirus." Kyoto University, 2004. http://hdl.handle.net/2433/147733.
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Kyoto University (京都大学)0048
新制・課程博士
博士(農学)
甲第10883号
農博第1389号
新制||農||887(附属図書館)
学位論文||H16||N3894(農学部図書室)
UT51-2004-G730
京都大学大学院農学研究科応用生物科学専攻
(主査)教授 奥野 哲郎, 教授 遠藤 隆, 教授 泉井 桂
学位規則第4条第1項該当
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Leonard, Pauline Catherine. "Gene expression profiling of osteosarcoma." Thesis, University College London (University of London), 2006. http://discovery.ucl.ac.uk/1445814/.
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Purpose: The aim of the work described in this thesis was to determine whether needle core and open biopsies from osteosarcoma (OS) provide sufficient quality of mRNA for cDNA array analyses which might then provide insights into the expression profile of OS. Experimental Design: Sixteen samples collected from OS and two established cell lines were used for array analyses. A primary cell culture was also established from one of the OS biopsies. Total RNA was extracted and probes generated for cDNA arrays. A reference probe was included for computational analyses. Results: cDNA probes were made for twenty five samples. Two of these samples were needle core bone biopsies. Twenty two cDNA probes were used for the generation of microarray data. Previous established statistical analysis confirmed the reliability of array data obtained in sixteen of the twenty two samples. Known genes involved in bone metabolism, osteoblast differentiation and cancer cell growth, were identified as up- or down-regulated in OS. Conclusions: Without amplification of RNA, OS tissue including small core bone biopsies, are amenable to cDNA array analysis. Known and novel putative markers for OS, that could have prognostic value, were identified.
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Kim, Michael S. "Gene Expression in Bone Cells." Thesis, Griffith University, 2006. http://hdl.handle.net/10072/366180.
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Osteoclast formation is a complex process that is yet to be clearly defined. Osteoclasts differentiate from monocytic precursors to large multinuclear cells via the actions of two crucial cytokines: macrophage colony stimulating factor (M-CSF) and receptor activator of NFKB ligand (RANKL). These two cytokines bind to the osteoclast precursor cells, activating various down stream signalling pathways, inducing genes required for differentiation and for activation of osteoclasts. Exposure of monocytic precursors to M-CSF alone leads to differentiation into macrophages. Osteoclast differentiation was suppressed by granulocyte macrophage colony-stimulating factor (GM-CSF), resulting in mononuclear cells, lacking tartrate-resistant acid phosphatase (TRAP) and a bone resorptive phenotype. Further analysis determined GM-CSF dosage and temporal effects on osteoclast formation, where higher doses and earlier treatments of GM-CSF result in greater suppression of osteoclast formation. To understand the TRAP negative mononuclear cell phenotype, various osteoclast related markers and nuclear factors were tested using quantitative real-time PCR. GM-CSF suppressed the mRNA expression of osteoclast markers, including TRAP and cathepsin K (CTSK). CTSK is a cysteine protease, involved in osteoclast activity of bone resorption. Furthermore, GM-CSF down regulated the expression of critical osteoclast-related nuclear factors, including nuclear factor of activated T-cells, cytoplasmic (NFATcI), which has been identified as playing a critical role in osteoclast differentiation and ftinction in mice and to some extent in humans. The suppression of crucial osteoclast markers and transcription factors by GM-CSF indicated an overriding of the RANKL signal and possible switching of the cellular phenotype away from osteoclasts.
To determine the cellular phenotype of GM-CSF driven cell differentiation, flow cytometry analysis was employed. As the cells visualised as dendritic cell like, CDIa, a dendritic cell surface marker, was selected for investigation. CDIa was highly expressed in GM-CSF, M-CSF and RANKL (GMR) treated cells and was absent in osteoclasts (M-CSF and RANKL treatment). The CDI a observations were indicative of GM-CSF overcoming the RANKL signal for osteoclastogenesis and directing differentiation to dendritic-like cells. To ftirther understand the osteoclastogenesis suppressive effect of GM-CSF, a 19,000 gene cDNA microarray assay was examined. The microarray experiment showed that the CC chemokine, monocyte chemotactic protein I (MCP-l), was profoundly repressed by GM-CSF. CC chemokines are chemoattractants that are induced during inflammation and recruit monocytes to the site of inflammation. MCP-l and other CC chemokines, RANTES (regulated on activation normal T cell expressed and secreted) and macrophage inflammatory protein I alpha (MIP I a) permitted formation of TRAP positive multinuclear cells in the absence of RANKL. However, these cells were negative for bone resorption. In the presence of RANKL, MCP-1 significantly increased the number of TRAP positive multinuclear bone resorbing osteoclasts (p= 5.7x 105, while RANTES and MIPI a mildly increased the number of bone resorbing TRAP positive multinuclear cells. Furthermore, CC chemokines, MCP-1, RANTES and MIP I a are all induced when authentic bone resorbing human osteoclasts differentiate from monocyte precursors in vitro following M-CSF-RANKL treatment. The addition of MCP- 1, RANTES or MIP I a appeared to reverse GM-CSF suppression of osteoclast formation, resulting in TRAP positive multinuclear cells. However, only MCP- I recovered the bone resorption phenotype, while other
chemokines, RANTES or MTPIa did not. The cognate receptors for MCP-1, in particular, CCR2b and CCR4, were potently induced by RANKL (12.6 and 49-fold, p= 4.0x107 and 4.0x108, respectively), whereas the chemokine receptors for RANTES and MTP I a (CCR I and CCR5) were not regulated by RANKL. Chemokine treatment in the absence of RANKL also induced MCP- 1, RANTES and MIP I a. Unexpectedly, treatment with MCP-I in the absence of RANKL resulted in 458-fold induction of CCR4 (p I.0xI010), while RANTES treatment resulted in two fold repression (p= I .Ox ioj. Since CCR2b and CCR4 are cognate MCP-I receptors, these data support the existence of an MCP-I autocrine loop in human osteoclasts differentiated using RANKL. All three chemokines in the absence of RANKL can induce TRAP positive multinuclear cells that are negative for bone resorption. However, as MCP-I can significantly increase the number of osteoclast formation and recover the bone resorbing osteoclast phenotype from GM-CSF suppression, MCP-1 is the most potent chemokine involved in osteoclast formation. MCP-1 induced TRAP positive multinuclear cells were characterised and found to be positive for calcitonin receptor (CTR) and a number of other osteoclast markers, including NFATcI. As NFATcI is associated with osteoclast maturity in mice and has even been referred to as a master regulator of osteoclast differentiation and ftinction, a strong induction of NFATcI should theoretically allow bone resorption of MCP-l mediated TRAP positive multinuclear cells. Although great NFATcI mRNA induction and activated nuclear NFAT were observed, MCP-1 did not result in the formation of bone resorbing osteoclasts in the absence of RANKL. Despite the similar visual phenotype and expression of mature osteoclast markers TRAP and CTR when compared to osteoclasts, RANKL treatment was required for the MCP- I induced TRAP positive, CTR positive, multinuclear cells to possess bone resorption activity. This suggested that MCP-1 mediated TRAP positive multinuclear cells were primed for RANKL signal, to ftirther differentiate into authentic osteoclasts. The lack of bone resorption was ftirther correlated with a deficiency in expression of certain genes related to bone resorption, such as CTSK and matrix metalloproteinase 9 (MMP9) and integrin aV. Another observation with implications for absence of the bone resorptive activity in MCP- I cell was the absence or disruption of the F-actin ring structure, correlating with the lack of integrin aV mRNA expression. It was hypothesised that as MCP-1 mediated TRAP positive multinuclear cells possessed a high induction of CTR, the addition of calcitonin would block multinucleation. Indeed, the exogenous calcitonin blocked the MCP-I induced formation of TRAP positive, CTR positive, multinuclear cells as well as bone resorption activity in the osteoclast controls, indicating that calcitonin acts at two stages of osteoclast differentiation in the human PBMC model.
These data suggest that RANKL-induced chemokines are involved in osteoclast differentiation at the stage of multinucleation of osteoclast precursors and provides a rationale for increased osteoclast activity in inflammatory conditions where chemokines are abundant. Furthermore, MCP-I induced TRAP positive, CTR positive multinuclear cells appear to represent an arrested stage in osteoclast differentiation, afler NFATcI induction and cellular ftision, but prior to the development of bone resorption activity and therefore, could be termed 'preosteoclasts'.Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Medical Science
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