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1
Nguyen, Tam Hong. "Pex13 Mutant Mice as Models for the Peroxisome Biogenesis Disorders." Thesis, Griffith University, 2008. http://hdl.handle.net/10072/366797.
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Zellweger syndrome (ZS) is the most severe form of a spectrum of disorders resulting from mutations in PEX genes, genes that encode proteins necessary for peroxisome biogenesis. Loss of functional peroxiosmes leads to disruption of multiple metabolic pathways involving the peroxisome, including very long chain fatty acid oxidation and plasmalogen and bile acid synthesis. ZS patients exhibit a range of clinical abnormalities, including facial dysmorphism, cataracts, hypotonia, seizures, psychomotor retardation, and hearing impairment. In terms of tissue pathology, there are also wide ranging effects, including neuronal migration defects, hepatomegaly, retinopathy, and renal cysts. Pex13 encodes a peroxisomal membrane protein that is essential for peroxisome biogenesis. Previous work in this laboratory resulted in the generation of a Pex13-null mouse model for the purpose of investigating the pathogenesis of Zellweger syndrome. The work in the first part of this thesis extends these studies and describes the generation and initial characterisation of tissue-specific Pex13 mouse models. These tissue-specific models are expected be useful tools for analysis of the impact of localised, brain- and liver-specific elimination of peroxisomes on the pathogenesis of ZS. In addition, in the second part of the thesis, a separate and novel hypothesis is addressed as an explanation for the molecular pathogenesis of ZS, through investigating the relationship between reduced peroxisome abundance and microtubule-mediated peroxisome trafficking.
Pex13 brain mutant mice were generated by mating the previously generated Pex13-floxed mice with mice expressing Cre recombinase under the control of the neuron-specific rat nestin promoter. Pex13 brain mutant mice displayed growth retardation beginning at day 5 postnatal, with gradual deterioration until death at approximately day 22 postnatal. Other clinical features included contracted postures, under-developed lower body mass, abnormal and unsteady gait, and abnormal motor coordination. In terms of brain metabolic function, these mice exhibited significant defects in plasmalogen synthesis, but, surprisingly, VLCFA levels were similar to those of littermate control mice. Significantly, peroxisome elimination in brain resulted in increased levels of plasmalogen levels in liver of Pex13 brain mutant mice. Consistent with the expected pathology resulting from deficiency of brain peroxisomes, brain mutants exhibited defective neuronal migration characterised by increased cellular density in the intermediate zone of the neocortex. Microarray analysis of total brain RNA from Pex13 brain mutants revealed several functionally-linked pathways associated with the differentially expressed genes, including cell-cell signalling, cell compromise/death, lipid metabolism, cell movement, and serotonin synthesis.Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Biomolecular and Physical Sciences
Science, Environment, Engineering and Technology
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Maxwell, Megan Amanda, and n/a. "PEX1 Mutations in Australasian Patients with Disorders of Peroxisome Biogenesis." Griffith University. School of Biomolecular and Biomedical Science, 2004. http://www4.gu.edu.au:8080/adt-root/public/adt-QGU20040219.100649.
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The peroxisome is a subcellular organelle that carries out a diverse range of metabolic functions, including the b-oxidation of very long chain fatty acids, the breakdown of peroxide and the a-oxidation of fatty acids. Disruption of peroxisome metabolic functions leads to severe disease in humans. These diseases can be broadly grouped into two categories: those in which a single enzyme is defective, and those known as the peroxisome biogenesis disorders (PBDs), which result from a generalised failure to import peroxisomal matrix proteins (and consequently result in disruption of multiple metabolic pathways). The PBDs result from mutations in PEX genes, which encode protein products called peroxins, required for the normal biogenesis of the peroxisome. PEX1 encodes an AAA ATPase that is essential for peroxisome biogenesis, and mutations in PEX1 are the most common cause of PBDs worldwide. This study focused on the identification of mutations in PEX1 in an Australasian cohort of PBD patients, and the impact of these mutations on PEX1 function. As a result of the studies presented in this thesis, twelve mutations in PEX1 were identified in the Australasian cohort of patients. The identified mutations can be broadly grouped into three categories: missense mutations, mutations directly introducing a premature termination codon (PTC) and mutations that interrupt the reading frame of PEX1. The missense mutations that were identified were R798G, G843D, I989T and R998Q; all of these mutations affect amino acid residues located in the AAA domains of the PEX1 protein. Two mutations that directly introduce PTCs into the PEX1 transcript (R790X and R998X), and four frameshift mutations (A302fs, I370fs, I700fs and S797fs) were identified. There was also one mutation found in an intronic region (IVS22-19A>G) that is presumed to affect splicing of the PEX1 mRNA. Three of these mutations, G843D, I700fs and G973fs, were found at high frequency in this patient cohort. At the commencement of these studies, it was hypothesised that missense mutations would result in attenuation of PEX1 function, but mutations that introduced PTCs, either directly or indirectly, would have a deleterious effect on PEX1 function. Mutations introducing PTCs are thought to cause mRNA to be degraded by the nonsense-mediated decay of mRNA (NMD) pathway, and thus result in a decrease in PEX1 protein levels. The studies on the cellular impact of the identified PEX1 mutations were consistent with these hypotheses. Missense mutations were found to reduce peroxisomal protein import and PEX1 protein levels, but a residual level of function remained. PTC-generating mutations were found to have a major impact on PEX1 function, with PEX1 mRNA and protein levels being drastically reduced, and peroxisomal protein import capability abolished. Patients with two missense mutations showed the least impact on PEX1 function, patients with two PTC-generating mutations had a severe defect in PEX1 function, and patients carrying a combination of a missense mutation and a PTC-generating mutation showed levels of PEX1 function that were intermediate between these extremes. Thus, a correlation between PEX1 genotype and phenotype was defined for the Australasian cohort of patients investigated in these studies. For a number of patients, mutations in the coding sequence of one PEX1 allele could not be identified. Analysis of the 5' UTR of this gene was therefore pursued for potential novel mutations. The initial analyses demonstrated that the 5' end of PEX1 extended further than previously reported. Two co-segregating polymorphisms were also identified, termed 137 T>C and 53C>G. The -137T>C polymorphism resided in an upstream, in-frame ATG (termed ATG1), and the possibility that the additional sequence represented PEX1 coding sequence was examined. While both ATGs were found to be functional by virtue of in vitro and in vivo expression investigations, Western blot analysis of the PEX1 protein in patient and control cell extracts indicated that physiological translation of PEX1 was from the second ATG only. Using a luciferase reporter approach, the additional sequence was found to exhibit promoter activity. When examined alone the -137T>C polymorphism exerted a detrimental effect on PEX1 promoter activity, reducing activity to half that of wild-type levels, and the -53C>G polymorphism increased PEX1 promoter activity by 25%. When co-expressed (mimicking the physiological condition) these polymorphisms compensated for each other to bring PEX1 promoter activity to near wild-type levels. The PEX1 mutations identified in this study have been utilised by collaborators at the National Referral Laboratory for Lysosomal, Peroxisomal and Related Genetic Disorders (based at the Women's and Children's Hospital, Adelaide), in prenatal diagnosis of the PBDs. In addition, the identification of three common mutations in Australasian PBD patients has led to the implementation of screening for these mutations in newly referred patients, often enabling a precise diagnosis of a PBD to be made. Finally, the strong correlation between genotype and phenotype for the patient cohort investigated as part of these studies has generated a basis for the assessment of newly identified mutations in PEX1.
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Maxwell, Megan Amanda. "PEX1 Mutations in Australasian Patients with Disorders of Peroxisome Biogenesis." Thesis, Griffith University, 2004. http://hdl.handle.net/10072/366184.
Full textAbstract:
The peroxisome is a subcellular organelle that carries out a diverse range of metabolic functions, including the b-oxidation of very long chain fatty acids, the breakdown of peroxide and the a-oxidation of fatty acids. Disruption of peroxisome metabolic functions leads to severe disease in humans. These diseases can be broadly grouped into two categories: those in which a single enzyme is defective, and those known as the peroxisome biogenesis disorders (PBDs), which result from a generalised failure to import peroxisomal matrix proteins (and consequently result in disruption of multiple metabolic pathways). The PBDs result from mutations in PEX genes, which encode protein products called peroxins, required for the normal biogenesis of the peroxisome. PEX1 encodes an AAA ATPase that is essential for peroxisome biogenesis, and mutations in PEX1 are the most common cause of PBDs worldwide. This study focused on the identification of mutations in PEX1 in an Australasian cohort of PBD patients, and the impact of these mutations on PEX1 function. As a result of the studies presented in this thesis, twelve mutations in PEX1 were identified in the Australasian cohort of patients. The identified mutations can be broadly grouped into three categories: missense mutations, mutations directly introducing a premature termination codon (PTC) and mutations that interrupt the reading frame of PEX1. The missense mutations that were identified were R798G, G843D, I989T and R998Q; all of these mutations affect amino acid residues located in the AAA domains of the PEX1 protein. Two mutations that directly introduce PTCs into the PEX1 transcript (R790X and R998X), and four frameshift mutations (A302fs, I370fs, I700fs and S797fs) were identified. There was also one mutation found in an intronic region (IVS22-19A>G) that is presumed to affect splicing of the PEX1 mRNA. Three of these mutations, G843D, I700fs and G973fs, were found at high frequency in this patient cohort. At the commencement of these studies, it was hypothesised that missense mutations would result in attenuation of PEX1 function, but mutations that introduced PTCs, either directly or indirectly, would have a deleterious effect on PEX1 function. Mutations introducing PTCs are thought to cause mRNA to be degraded by the nonsense-mediated decay of mRNA (NMD) pathway, and thus result in a decrease in PEX1 protein levels. The studies on the cellular impact of the identified PEX1 mutations were consistent with these hypotheses. Missense mutations were found to reduce peroxisomal protein import and PEX1 protein levels, but a residual level of function remained. PTC-generating mutations were found to have a major impact on PEX1 function, with PEX1 mRNA and protein levels being drastically reduced, and peroxisomal protein import capability abolished. Patients with two missense mutations showed the least impact on PEX1 function, patients with two PTC-generating mutations had a severe defect in PEX1 function, and patients carrying a combination of a missense mutation and a PTC-generating mutation showed levels of PEX1 function that were intermediate between these extremes. Thus, a correlation between PEX1 genotype and phenotype was defined for the Australasian cohort of patients investigated in these studies. For a number of patients, mutations in the coding sequence of one PEX1 allele could not be identified. Analysis of the 5' UTR of this gene was therefore pursued for potential novel mutations. The initial analyses demonstrated that the 5' end of PEX1 extended further than previously reported. Two co-segregating polymorphisms were also identified, termed –137 T>C and –53C>G. The -137T>C polymorphism resided in an upstream, in-frame ATG (termed ATG1), and the possibility that the additional sequence represented PEX1 coding sequence was examined. While both ATGs were found to be functional by virtue of in vitro and in vivo expression investigations, Western blot analysis of the PEX1 protein in patient and control cell extracts indicated that physiological translation of PEX1 was from the second ATG only. Using a luciferase reporter approach, the additional sequence was found to exhibit promoter activity. When examined alone the -137T>C polymorphism exerted a detrimental effect on PEX1 promoter activity, reducing activity to half that of wild-type levels, and the -53C>G polymorphism increased PEX1 promoter activity by 25%. When co-expressed (mimicking the physiological condition) these polymorphisms compensated for each other to bring PEX1 promoter activity to near wild-type levels. The PEX1 mutations identified in this study have been utilised by collaborators at the National Referral Laboratory for Lysosomal, Peroxisomal and Related Genetic Disorders (based at the Women's and Children's Hospital, Adelaide), in prenatal diagnosis of the PBDs. In addition, the identification of three common mutations in Australasian PBD patients has led to the implementation of screening for these mutations in newly referred patients, often enabling a precise diagnosis of a PBD to be made. Finally, the strong correlation between genotype and phenotype for the patient cohort investigated as part of these studies has generated a basis for the assessment of newly identified mutations in PEX1.Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Biomolecular and Biomedical Sciences
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Sawyer, Rosalind Mary. "Isolation of a vicilin gene from pea (Pisum sativum L.), and nuclease sensitivity of seed storage protein genes in pea chromatin." Thesis, Durham University, 1986. http://etheses.dur.ac.uk/6873/.
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A library of pea genomic DNA in the bacteriophage vector EMBL3 was screened by hybridisation to cDNAs encoding vicilin, a major storage protein of pea (Pisum sativum L.) seeds. A vicilin gene, vic A, was isolated and characterised by restriction mapping and DNA sequencing. The nucleotide and predicted amino acid sequences of vic A were compared to those of vicilin cDNAs, and the gene was found to encode a 50,000M(_r) non-glycosylated vicilin subunit that does not undergo post-translational proteolytic cleavage. The introns in vic A were typical of those in plant genes, being small and high in A+T content, and the nucleotide sequences at the splice sites showed good homology to the plant consensus. The positions of the introns in vic A were similar to those in a gene encoding a subunit of phaseolin, a related protein from French bean (Phaseolus vulgaris). Methods were developed for the analysis of nuclease sensitivity of specific genes in pea chromatin. The DNAase I sensitivity of the seed storage protein genes was found to be greater in developing cotyledons, where the genes were transcriptionally active, than in leaves, where they were inactive. The pea ribosomal genes showed relative resistance to DNAase I in both tissues. The nucleosome repeat length, determined by digestion of chromatin with micrococcal nuclease, was similar in both tissues. No evidence was obtained for DNAase I hypersensitive sites in pea chromatin. This result supports the findings of two other studies, and suggests that such sites are absent from plant chromatin.
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Liu, Xiaoguang. "Characterization of the pea pathogenicity (PEP) gene cluster in the fungal pathogen Nectria haematococca." Diss., The University of Arizona, 2002. http://hdl.handle.net/10150/279972.
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The fungus Nectria haematococca is a broad host range pathogen. Isolates pathogenic on pea are able to detoxify the phytoalexin pisatin using the enzyme pisatin demethylase. When the gene (PDA1 ) encoding this enzyme was mutated via gene disruption, the mutants were less virulent but still pathogenic on pea. Additional studies demonstrated that PDA1 was on a 1.6-Mb conditionally dispensable (CD) chromosome and that loss of this CD chromosome resulted in the complete loss of pea pathogenicity. This leads to the hypothesis that there are other pea p̱athogenicity (PEP) genes in addition to the PDA1 gene on the CD chromosome. One of the major goals of this work was to test this hypothesis by isolating and characterizing these PEP genes. The results identified three novel PEP genes: PEP1, PEP2, and PEP5, each of which can confer disease-causing ability independently when introduced into a nonpathogenic isolate lacking the CD chromosome. The predicted product of PEP5 is related to members of the major facilitator superfamily, including proton-dependent multidrug export systems, and the predicted product of PEP2 contains conserved RNA-binding motifs. PEP1 shows no significant similarity to any known gene in the public databases. The three PEP genes and PDA1 are organized into a functional cluster, termed the PEP cluster, within 25 kb that conditions full pathogenicity on pea. The PEP cluster contains two additional genes, cDNA3 and cDNA4, and neither gene by itself is able to confer disease-causing abilities. The open reading frame (ORF) for cDNA3 gene is small. The predicted cDNA4 product exhibits significant similarities to the transposon impala of Fusarium oxysporum. The sequences of other portions of the PEP cluster predict four ORFs showing strong similarities to other fungal transposases. Several features of the PEP cluster, such as possession of multiple virulence genes, presence of DNA mobile elements, and differences in both codon usage and G+C content compared with other portions of the genome, resemble those of the pathogenicity islands identified in plant and animal bacterial pathogens. These properties raise the possibility that the PEP gene cluster may represent a fungal pathogenicity island. The second major goal of my work was to quantify the expression of PDA1, PEP1, PEP2, and PEP5 in vitro and in planta, and to characterize the regions flanking the PEP cluster. A real-time quantitative RT-PCR approach was used to measure the mRNA levels of these pathogenicity genes. In a glucose-based growth medium, mRNA levels of PDA1, PEP1, PEP5 were very low, while expression of PEP2 was undetectable. Starvation in vitro strongly stimulated PDA1 gene expression, whereas expression of PEP1 and PEP5 increased only moderately. In contrast, starvation had no effect on expression of PEP2 as indicated by an undetectable mRNA level during the 12 hr time course tested. In vitro pisatin strongly induced the expression of all four pathogenicity genes and the PDA1 experienced the highest level of induction (∼300-fold increase). Finally, marked induction of PDA1, PEP1 and PEP2 was observed during infection of pea roots, whereas the expression of PEP5 was only moderately induced. The flanking regions of the PEP cluster were sequenced and the sequences predict six ORFs that display significant similarities to various fungal genes. The G+C content, gene density, and codon preference of the PEP cluster and its flanking regions are similar. All six of the predicted genes in the flanking regions of the PEP cluster are expressed during infection of pea.
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Ferreira, Briana Cardoso. "Prospecção de genes pif (per os infectivity factor) em variantes genotípicos de Anticarsia gemmatalis MNPV e construção de recombinante com interrupção do gene pif-1." reponame:Repositório Institucional da UnB, 2008. http://repositorio.unb.br/handle/10482/2610.
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Os baculovirus são vírus patogênicos a insetos, principalmente aos da ordem Lepidoptera. É comum o aparecimento de mutantes defectivos em populações de campo, com ausência de genes essenciais, que são mantidos pela co-infecção de células por diferentes genótipos virais. Esses genótipos quando purificados podem perder a capacidade de infectar a larva hospedeira, devido a deleções em genes pif (per os infectivity factor), essenciais para a infecção por via oral. Sabe-se que as proteínas PIF estão associadas ao envelope da partícula ODV, necessária para o estabelecimento da infecção primária no inseto. O genoma do Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) foi recentemente seqüenciado sendo então relatada a presença dos genes pif-1 e pif-2 no vírus. Neste trabalho, genótipos de AgMNPV derivados do isolado de campo AgMNPV-79 foram selecionados e, através de análise de perfil de restrição do DNA viral, foi confirmada a existência de variantes genotípicos na população. Para a investigação da possível presença de mutantes defectivos, amplificações das regiões de pif-1 e pif-2 por PCR foram realizadas em cada variante sendo que todos os clones da população apresentaram amplificações dos dois genes. Todos os clones mostraram-se altamente infecciosos por ingestão oral, porém o genótipo Ag79-01 apresentou maior virulência entre eles. A presença de um terceiro gene pif (pif-3) foi identificada recentemente no genoma do Autographa californica multiple nucleopolyhedrovirus (AcMNPV). Da mesma forma, esse gene é também essencial para o estabelecimento da infecção primária por via oral. Por análise de BLAST o gene foi então identificado como a ORF 114 do genoma do vírus AgMNPV, a qual apresentou uma identidade de aminoácidos de 67% com a ORF do vírus AcMNPV. Com base nessa informação, primers para o gene pif-3 foram desenhados e amostras de DNA dos diferentes genótipos virais foram submetidas a amplificações por PCR. Novamente todos os clones virais apresentaram amplificação de um fragmento correspondente à região de pif-3. Uma vez que os genes pif estavam presentes em todos os variantes genotípicos analisados, foi elaborada uma estratégia para a construção de um vírus recombinante com deleção do gene pif-1, visando o estudo de seu papel na infecção oral do inseto. O vírus recombinante vAgGFP?pif-1, obtido por recombinação homóloga, teve o gene pif-1 substituído por um cassete gênico contendo um gene repórter (gfp) sob comando do promotor constitutivo hsp70. Esse vírus foi selecionado a partir da visualização de células infectadas emitindo fluorescência, devido à expressão da proteína GFP. O vírus vAgGFP?pif-1 encontra-se sob processo de purificação.
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Harris, Robert. "The regulation of the embryonic transcription factor Pax-3." Thesis, University of Southampton, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.342629.
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Zasiura, Colette. "Characterisation and expression of pea lipoxygenase genes." Thesis, University of East Anglia, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.365059.
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Wright, David. "The role of pax genes in vertebrate segmentation." Thesis, University of Dundee, 2010. https://discovery.dundee.ac.uk/en/studentTheses/15d5c38e-b000-4dcb-be35-49a46feb4664.
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Pax genes are involved in a range of processes in the developing embryo. Pax3 and Pax7 in particular are associated with the paraxial mesoderm, especially the development of the myogenic lineage. However, recent studies have suggested that Pax3 and Pax7 may have earlier, morphogenetic roles in the segementation of the paraxial mesoderm. Here we examine the expression of Pax3 and Pax7 in the chicken presomitic mesoderm, and investigate their function using an in ovo electroporation transfection technique. We find that Pax3/7 drives precocious differentiation of PSM towards the myogenic lineage, as well as inducing a range of morphological changes in PSM tissue. These changes include alterations in the cytoskeleton, cell adhesion, and extracellular matrix formation.
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Garrett, Christine. "Functional mapping of pea legumin upstream regulatory elements using TI plasmid vectors." Thesis, Durham University, 1991. http://etheses.dur.ac.uk/6041/.
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The leg A gene from Pisum sativum L. has been extensively characterised and a distinct pattern of developmental and organ-specific gene expression demonstrated. Homology between legumin genes from other species has given some indication of those sequences which may be responsible for the regulation at the level of transcription. This study was designed to provide a functional analysis of the upstream sequences. A number of plasmid vectors containing a maximum of 1.2 kb of upstream sequence from the leg A gene of Pisum sativum I., ligated to the coding region of the nopaline synthase (nos) gene, were constructed. The use of smaller promoter fragments and the insertion of spacer DNA within the promoter region was employed in an effort to localise the regions of 5' flanking sequence which may play a role in tissue specific expression. In a minority of tumours derived from tissue transformed with the vector containing the ' full-length ' leg A promoter, low levels of nopaline were detected, but not with those containing a shorter promoter fragment. Results from the analysis of Seed tissue indicates that 800 bp of the leg A promoter was insufficient to direct tissue-specific expression of the fused nopaline synthase gene in transgenic Nicotiana tabacum, although one individual plant showed a constitutive pattern of nopaline synthesis. However, published results obtained with legumin and other storage protein gene promoters would suggest that this promoter fragment should have been sufficient to confer seed-specific expression. This suggests that there may have been undesirable secondary structures, or small undetected rearrangements, introduced during the construction of the transcriptional fusions between leg A and nos. Alternatively the marker gene may be inadequately sensitive to permit detection of low levels of expression.
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Huttly, A. K. "Genes for ATP synthase subunits in pea chloroplast DNA." Thesis, University of Cambridge, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.356654.
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Dursun, Umut. "Pax genes and neurogenic placode development in the chicken embryo." Thesis, University of Cambridge, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.610089.
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Drew, Janice Elizabeth. "Cloning and characterisation of genes determining pod morphology in pea." Thesis, Durham University, 1994. http://etheses.dur.ac.uk/5882/.
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Genes expressed in developing pea pods were isolated as cDNAs by differential screening techniques. The cDNAs were characterised by DNA sequencing and expression studies were used to investigate the role of isolated cDNAs in pod development. A clone isolated from a pea {Piswn sativum L.) pod cDNA library was shown to contain the complete coding sequence of a polypeptide with considerable homology to various members of the Rab subfamily of small ras-related GTP- binding proteins. Conserved sequences in the isolated clone include the GTP-buiding site, GDP/GTP hydrolysis domain and C-terminal Cys residues involved in membrane attachment. The high percentage amino acid identity suggests that this cDNA may be the product of a gene, designated Psa-rai?, which is the plant counterpart of Rab7. Rab/Ypt proteins are thought to be involved in intracellular transport from the endoplasmic reticulum to the Golgi apparatus and in vesicular transport. If Psa-ra6 is a functional counterpart of yeast YPT7 (RabT) it should be able to complement a yeast YPT7 mutant. An attempt was made to demonstrate that this was the case. Northern analysis showed invariant expression of Psa-rab in developing pods 'with different phenotypes, indicating an essential function for Psa-rab in developing pods. Hybridisation of the Psa-rab cDNA to pea genomic DNA showed that this protein is probably encoded by a single gene. Nearly isogenic pea lines were selected to investigate the genetic basis for lignification of the pea {Pisum sativum L.) pod endocarp. The development of the pod endocarp in the normal and mutant pea pod phenotypes was examined by histochemical staining and light microscopy. The effect of plant growth regulators on endocarp development was also investigated. A pea pod cDNA library representing poly (A)+ RNA purified from L59 pea pods (genotype, PV; phenotype, lignified endocarp) was differentially screened with total cDNA probes prepared from total pod RNA from L59 and LI390 (genotype, PV; phenotype, no lignification of endocarp) pods 4-6 days after flowering (DAP). Two clones, designated pLP18 and pLP19, were selected for further characterisation on the basis of hybridisation to the L59 cDNA probe, but not the LI390 cDNA probe. Northern blotting was used to show that pLP18 represented a mRNA of 0.95 kb. The predicted polypeptide from the LP18 cDNA encoded a putative blue type I copper protein. The expression pattern of LP 18 mRNA in pods and tissues of the experimental pea lines was determined using RT-PCR quantitation. Hybridisation of the cDNA to pea genomic DNA showed that this protein is probably encoded by a single gene. Clone pLP19 yielded a 1.02 kb cDNA fragment encoding the C-terminal portion of an Hsp70 homologue belonging to a highly conserved family of proteins found in a number of eukaryotic species. Northern analysis of RNA from lignified and unlignified pods showed the presence of differentially expressed LP19 transcripts of varying lengths, which may represent differently processed transcripts. Southern analysis confirmed the presence of a single hybridised band in genomic digests of L59, L58 arid LI390. Several mRNA transcripts of the LP19 gene were isolated which differ in the length of their- 3' untranslated regions.
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Hellens, Roger Paul. "Structure and regulation of the CHS-1 genes of pea." Thesis, University of East Anglia, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.240369.
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Gourlay, Campbell William. "An analysis of genes involved in pea compound leaf development." Thesis, University of East Anglia, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.302214.
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Levasseur, M. D. "Comparative studies of the nucleotide sequences of pea seed storage protein genes." Thesis, Durham University, 1988. http://etheses.dur.ac.uk/9529/.
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Nucleotide sequence data from several pea (Piswn sativum L.) seed storage protein genes was obtained. Of two legumin genes sequenced, one was shown to be a pseudogene, apparently once coding for a polypeptide belonging to the 'major' legumin class, whilst the other was shown to be a functional gene coding for a polypeptide of the 'minor' legumin class. Sequence data was also obtained for two vicilin genes. Complete sequencing of one revealed it to be truncated by sequence of unknown origin at its 3' end, whilst partial sequence for the other suggested the presence of a stop codon in the coding region. These findings implied that both vicilin genes are no longer functional. Additionally, various comparisons of nucleic acid and amino acid sequence data were made between these genes and also with other legume seed storage protein genes. Results showed these genes conform with the major structural features of eukaryotic genes, and also revealed the presence of potential tissue -specific regulatory elements in the 5' flanking regions of the genes. Dendrograms for legume 11S and 7S classes of globulin seed storage protein genes clearly supported the model theory of each class having arisen by successive duplications from a common ancestral gene.
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Purton, Saul. "Genes for components of the transcription-translation apparatus of pea chloroplasts." Thesis, University of Cambridge, 1988. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.257299.
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Lloyd, James. "Effect and interactions of rugosus genes on pea (Pisum sativum) seeds." Thesis, University of East Anglia, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.633047.
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Mutant alleles at five rugosus loci (r, rb, rug-3, rug-4 and rug-5) were examined for their effects on the growth and starch content of the pea seed. In order to study gene interaction double mutants between pairs of rugosus loci were produced. In addition, starch extracted from the single mutant lines was examined for structural differences. The primary effect of the r mutation was to reduce the production of amylopectin in the developing embryo. This led to decreased starch in the embryo, but an increased proportion of amylose in the starch. Starch grains in the mutant embryo differentiated from 'simple' to 'compound' during development. The primary effect of both the rb and rug-4 mutations was to reduce carbon flux through the starch biosynthetic pathway. This led to a decreased starch content in the embryo during development and in the mature seed. The proportion of amylose in the starch also was reduced, in relation to the reduction in starch. In addition the rb mutation caused a reduction in the amount of starch accumulated in the testa during development. The rug-3 mutation acted as a complete block on starch synthesis in both the embryo and testa throughout development. Reciprocal F, crosses indicated that there was a maternal effect of both the rb and rug-3 mutations on final seed size. The rug-5 mutation also blocked starch synthesis, but only late in development when the embryo began to dry. In addition, the mutation altered the appearance of the starch grains, which were irregular in appearance throughout embryo development. The fine structure of amylopectin was studied by enzymically debranching amylopectin and separating the constituent chains by HPLC. Only the rand rug-5 genes were demonstrated to affect amylopectin structure. The effect of the r gene was minor, however, the rug-5 mutation caused a fundamental alteration in amylopectin structure.
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Van, den Boogaart Tom. "The mechanism of replicase-derived resistance to pea early browning virus and pea seed-borne mosaic virus." Thesis, University of East Anglia, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.327603.
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Chua, Y. L. "Chromatin structure of the pea plastocyanin gene." Thesis, University of Cambridge, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.597674.
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The pea plastocyanin gene (PetE) is a single-copy, nuclear photosynthesis gene. Pea PetE is flanked by an enhancer/5' matrix attachment region (MAR) and a 3' MAR. When linked upstream to uidA directed by the CaMV 35S promoter, the enhancer/5' MAR increased reporter gene expression in transgenic tobacco plants. In contrast, the 3' MAR increased expression only when linked downstream of the reporter gene. The 3' MAR, but not the 5' MAR, decreased variation in reporter gene expression. These results indicate that the two MARs surrounding PetE have different effects on transgene expression. The chromatin structure of PetE was examined at three different transcriptional states by investigating the nuclease accessibility of the gene in pea roots, etiolated shoots and green shoots. Time-course digestions of nuclei with micrococcal nuclease and DNaseI indicated that the enhancer/5' MAR and promoter regions were more resistant to digestion in the inactive gene in pea roots than the same regions in the active gene in shoots, whereas the transcribed region of PetE was digested similarly amongst the tissues. PetE transcription is hence accompanied by changes in the nuclease accessibility of the enhancer/5' MAR and promoter regions only. The acetylation states of histone H3 and H4 proteins associated with PetE were analysed by chromatin immunoprecipitation with antibodies specific for acetylated or non-acetylated histone tails followed by polymerase chain reaction quantification. Comparison of pea tissue indicated that histone acetylation was associated with increased PetE transcription in green shoots. Moreover, acetylation of both histone H3 and H4 proteins was targeted to the enhancer/5' MAR and promoter regions in green shoots, suggesting that only specific nucleosomes along the gene were modified.
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Castro, i. Català Marta de. "Candidate genes for psychosis and their interaction with environmental factors: impact on psychosis-proneness = Gens candidats per psicosi i la seva interacció amb factors ambientals: impacte sobre la vulnerabilitat per a psicosi." Doctoral thesis, Universitat de Barcelona, 2017. http://hdl.handle.net/10803/456371.
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Schizophrenia is a severe and complex mental disorder that results in an inability to cope with life’s ordinary demands and routines. Normally, it appears in late adolescence, more frequently and developing earlier in males. It seems that disruption of the factors and pathways involved in brain development may cause modifications in neural structure, function or connectivity, leading to a final brain more susceptible to develop psychotic symptoms. The high diversity of symptoms intra- and inter-patient, makes the exploration of the aetiology and biological pathways underlying this illness, as well as its treatment, substantially difficult and suggests that alternative strategies may be more helpful for the study of such disease.
Despite the psychotic phenotype has traditionally been thought as a dichotomous entity (i.e. “healthy” and “ill” subjects), evidence shows that psychosis exists as a distribution of symptoms expressed across a continuum ranging from nonclinical, subclinical to clinical manifestations with shared underlying etiological and developmental processes. Schizotypy and psychotic-like experiences are intermediate phenotypes of psychosis that measure the liability for psychosis-proneness. They can be found in healthy people and, although they are non-destabilising in the daily life, represent an increased risk for psychosis and are believed to share risk factors with the illness.
The predisposition to develop psychopathology is determined by a complex combination and/or interplay between genetic, environmental, personal and epigenetic factors. The genes responsible for schizophrenia susceptibility have been tried to identify using classic candidate-gene research strategies and, more recently genome-wide association studies (GWAS), but despite interesting genes have been reported, the variance in liability explained by these genes is low. It seems thus, that these approaches are be missing epistatic and gene-environment interactions (GxE), which could explain part of the phenotype. Additionally, the study of intermediate phenotypes related with the disorder, instead of the illness itself, seems to be a good strategy that allows a better definition of the phenotype under study.
Environmental factors also play a significant role in the aetiology of the disorder. Exposure to childhood trauma is a robust risk factor for psychosis that has been associated to increased schizotypy and psychotic-like experiences. It seems to affect mental health though alterations in the stress-related systems (i.e. HPA axis), however, not all subjects who experience these adverse events will develop psychosis, pointing out that other factors are modulating this response.
Gene-environment interaction may be an important variable explaining the observed differential risk to develop psychosis. In this sense, genetic vulnerability predisposes an individual to the development of psychopathology and, when a level of stressors exceeds this vulnerability threshold, symptoms emerge. In this sense, polymorphic variants in genes involved in stress-response related genes (e.g. FKPB5), as well as those involved in neurodevelopment or neurotransmitter systems (e.g. COMT, BDNF, RGS4, ZNF804A), are interesting candidate genes.
According to this, the present thesis aimed to explore the involvement of several schizophrenia candidate gens (i.e. COMT, RGS4 and ZNF804A) on the development of psychosis-related phenotypes in nonclinical samples, as well as the modulating role of some of these genes (i.e. BDNF, FKBP5, COMT) in the association between childhood trauma and psychosis-proneness phenotypes.
In line with previous published research, the results of the present thesis showed interesting associations between COMT, RGS4 and ZNF804A genes and the psychosis-proneness phenotype (i.e. schizotypy and/or psychotic-like experiences) in nonclinical subjects, which seem to be sex-specific in some cases. Moreover, BDNF and FKBP5 show a modulating role on the association between childhood trauma and psychopathology in nonclinical subjects. In the case of FKBP5 we found that it modulates this association in relation to depressive-anxious symptoms.
This thesis, shows the interest of exploring schizophrenia candidate genes in relation to psychosis-related phenotypes in nonclinical samples to study the underlying mechanisms involved in the development of psychopathology. The findings support the existence of a continuum psychotic phenotype with a multifactorial aetiology involving genes, environment and possibly other factors that are active agents in the formation of an individuals’ level of vulnerability. Moreover, they encourage future studies in samples across the psychosis continuum considering the multifactorial nature of the disorder and related traits by implementing the new developed methodologies and considering adequate sample sizes, appropriate statistical models, accurate phenotypic and environmental measures and also sexual differences.L’objectiu de la present tesi doctoral era estudiar el paper de diversos gens candidats per esquizofrènia (COMT, RGS4 i ZNF804A) en la vulnerabilitat a patir psicosis en individus no clínics, així com en estudiar al paper modulador d’alguns d’aquests ganes (BDNF, FKBP5, COMT) en l’associació entre el trauma durant la infància i els fenotips de vulnerabilitat a psicosi. D’acord amb estudis previs, els resultats obtinguts mostraren una associació entre els gens COMT, RGS4 i ZNF804A i els fenotips de vulnerabilitat estudiats. A més, vam detectar que els gens BDNF i FKBP5 tenien un paper modulador en l’associació entre el trauma i la psicopatologia en els individus no clínics estudiats. En el cas de l’FKBP5 vam observar que estava modulant aquesta associació en relació amb símptomes depressius-ansiosos. Aquesta tesi mostra l’interès d’explorar gens candidats per esquizofrènia en relació a fenotips de vulnerabilitat pe psicosi en mostres no clíniques per estudiar els mecanismes subjacents al desenvolupament de psicopatologia. Els nostres resultats recolzen l’existència d’un fenotip contínuum de les psicosis que presenta una etiologia multifactorial, incloent gens, ambient i probablement altres factors que són agents actius en la formació del nivell de vulnerabilitat de cada individu. Addicionalment, mostren la necessitat de realitzar nous estudis futurs en mostres al llarg del contínuum (no clíniques, subclíniques i clíniques) considerant la naturalesa multifactorial de les psicosis i fenotips relacionats mitjançant l’ús de les noves metodologies desenvolupades i considerant mides mostrals adequades, mesures fenotípiques i ambientals acurades, així com diferències sexuals.
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Hammarström, Alva. "Hur genus förmedlas av barn och till barn : En studie om genus i bilderboken som en genre." Thesis, Högskolan i Halmstad, Akademin för lärande, humaniora och samhälle, 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:hh:diva-27735.
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Genus är ett begrepp som beskriver hur det socialt och kulturellt är att vara kvinna eller man. Inom litteraturvetenskapen växer intresset för genusinriktade studier där man tittar på hur genus konstrueras i litteraturen. Den här studien undersöker hur genus förmedlas till barn genom en bilderbok. En bilderbok idag är något som är självklart för många men samtidigt oklart. Bilderboken har sin plats på biblioteket och i bokhandeln men det finns inte alla gånger en klar definition av vad bilderböcker är. Syftet med studien är att studera bilderboken som en egen genre och hur en nutida bilderbok kan skildra genus. Analysen kommer att gå in på de valda författarna och illustratörernas, Pija Lindenbaum och Per Gustavsson, uppbyggnad av en bilderbok. Diskussionen kring analysen kommer att vara genusfokuserad och där kommer flicka mot flicka och pojke mot pojke i Lindenbaum och Gustavssons bilderböcker att sättas emot varandra. Studien visar på att Lindenbaum och Gustavsson är normbrytande bilderboksförfattare som med olika typer av huvudkaraktärer bryter normen.
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Endres, Thomas [Verfasser], and Thomas [Akademischer Betreuer] Kissel. "Biodegradable amphiphilic PEG-PCL-PEI triblock copolymers designed for the self-assembly of multifunctional gene carriers / Thomas Endres. Betreuer: Thomas Kissel." Marburg : Philipps-Universität Marburg, 2013. http://d-nb.info/103231396X/34.
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Bown, David Philip. "Characterisation of genes expressed in various tissues of PEA (Pisum sativum L.) : correlation of genotype and phenotype." Thesis, Durham University, 1992. http://etheses.dur.ac.uk/2216/.
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Genes encoding representatives of two subfamilies from the vicilin storage protein family in pea (Pisum sativum L.) have been sequenced and characterised. One, encoding convicilin, shows that this protein differs from vicilin by the insertion of a hydrophilic region near the N-terminus. The transcription start point has been determinedand the pattern of expression in developing seeds elucidated. By theexpression of this gene in tobacco, the specific polypeptide product ofthe gene was identified as a minor component of convicilin , with a lowerMr than the major species . The other gene subfamily investigated wasThat encoding the vicilin 47,000 Mr polypeptide. A gene and a cDNA weresequenced, and the gene found to diverge from the cDNA in the 3" regionof the coding sequence. No product from this divergent gene could beidentified.A member of the legumin gene family (legK) was sequenced and found to be inactive due to a mutation of the start codon. The region of DNAencoding the start codon of this gene was amplified by polymerase chainreaction from a pea line in which the gene was known to be active . Thesequence of this revealed the presence of a normal start codon. Two dimensional protein gels were run with seed extracts from these twolines , and the product of (legK) demonstrated by its occurrence in theline with the functional gene. A method for the extraction and purification of the major pea root protein was established. The protein was shown to have a Mr of 16,000 and not to be susceptible to cleavage by cyanogen bromide. Partial amino acid sequence data was obtained from the purified protein .A differential screen of cDNA from purple and green poddedvarieties of pea was conducted, and differentially expressed cDNAsisolated . The nature of the expression of these cDNAs was studied in thetwo lines and the cause of instability in the purple podded phenotypeinvestigated . A genomic library was constructed from the purple poddedline . Two genes were selected by the differentially expressed cDNAs andtheir DNA sequences determined. A gene encoding a pectinestera- likesequence, and the pod expressed cDNA used to select it , were found to betwo members of a small multigene family in pea. The second gene selectedproved to encode a protein containing two distinct domains; the N-terminal region being of a repetitive proline-rich nature and the C-terminal region being hydrophobic and cysteine rich . This gene waspresent as a single copy in the pea genome and its expression appearedto be linked to pigmentation.
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Khoubehi, Bijan. "The expression pattern of class III PAX genes in human prostate cancer." Thesis, Imperial College London, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.251704.
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Gilbert, Erin V. "Characterization of the Circadian Clock in Pet-1 Knockout Mice." Kent State University / OhioLINK, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=kent1290535254.
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Thompson, Andrew John. "Regulation of gene expression in developing pea seeds." Thesis, Durham University, 1989. http://etheses.dur.ac.uk/6486/.
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Three classes of legumin, encoded by the gene sub-families legA, legJ and legS, and a lectin encoded by a single gene, lecA, accumulate in the developing cotyledons of Pisum sativum L. Transcription rates for the genes encoding these proteins were measured in nuclei isolated from cotyledons at 12 and 16 days after flowering (DAF). The steady-state levels of the corresponding mRNA species were also measured, in absolute terms, throughout cotyledon development. It was found that the different legumin gene sub-families are not coordinately expressed and, in addition, members within the legJ sub-family show differential temporal expression. Also, it was demonstrated that the length of the poly (A) tail of the lectin mRNA is reduced during the period when the steady-state level of this mRNA is in decline. When transcription rates and steady-state mRNA levels of the different gene families are compared, there is little correlation. This suggests a posttranscriptional regulation of the quantitative level of expression of these genes. Expression of the legumin genes is known to be seed-specific, whereas expression of the lectin gene occurs in the root as well as the seed. When transcription rates were measured in leaf nuclei the levels of legumin and lectin transcripts detected approached background levels, indicating that these genes are either inactive or transcribed at very low levels in leaf; however, the rate of transcription of the chlorophyll a/b binding-protein gene was high. This suggests transcriptional control as the major factor in the organ-specificity of legumin and lectin expression. The apparent posttranscriptional regulation of the quantitative level of expression of different seed-protein genes was investigated further by pulse-chase labelling the RNA of pea cotyledons grown in culture. Also, the possibility of using cell-free extracts to assay the cytoplasmic stability of specific polysomal mRNAs was investigated.
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Helliwell, Christopher Andrew. "Regulation of expression of the pea plastocyanin gene." Thesis, University of Cambridge, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.338311.
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Last, David Ian. "Structure and expression of the pea plastocyanin gene." Thesis, University of Cambridge, 1988. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.256631.
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Nordstrom, Karin. "Evolution of eyes: Pax, gene duplications & morphology." Thesis, Karin Nordstrom, Department of Cell and Organism Biology, Lund University, 2003. http://hdl.handle.net/2440/34750.
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During the course of evolution animal complexity and diversity is generated. The last couple of years it has become increasingly clear that morphologically diverse animals share a great deal of their genomic contents, and it must be the expression of regulatory genes in new setting, rather than the generation of new genes, that are fundamental for the generation of diversity. Basal phyla hold a key position for explaining the generation of diversity. About 10 years ago it was realized that eyes that had previously been believed to constitute classical examples of convergent evolution, share the genes patterning their development. We have here studied one of these patterning genes (pax), and the morphology of evolutionary basal animals. Cnidarians have nervous systems and simple ocelli despite being overall morphologically simple. We isolated three pax genes from Aurelia, Polyorchis and Chiropsalmus. With a re-evaluation of the early evolution of the Pax family, we concluded that the last ancestor to cnidarians and Bilateria had a repertoire of at least 4 pax genes. Pax genes are recognized by a highly conserved paired domain, which binds to conserved DNA sequences. We showed that the coral PaxD paired domain does not bind to these conserved sequences in vitro. This is surprising considering the fact that PaxD is closely related to the Bilateria Pax3/7 subfamily - members of which all bind to conserved DNA sites. With mutagenesis we identified two adjacent residues to be primarily responsible for the observed lack of binding. Cnidarian larvae are of the planula type. The larva’s main purpose is to find the optimal settling location for metamorphosis into a sessile polyp. We have showed that cubozoan larvae, although morphologically simple, have distinct ocelli that might aid in settling. Finally we have studied vertebrate evolution. Vertebrate rods and cones provide night and day vision respectively. Not only opsins, but also several of the phototransduction proteins differ between the two cell types. It is believed that the vertebrates underwent two whole-genome duplications after the divergence from early chordates. We have showed that the generation of rod and cone specific phototransduction paralogs took place at the time of the early vertebrate tertaploidizations.http://www.lu.se/o.o.i.s?id=12588&postid=465922
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Kuznetsova, Elena. "Characterization of Pea (Pisum Sativum L.) genes implicated in arbuscular mycorrhiza formation and function." Phd thesis, Université de Bourgogne, 2010. http://tel.archives-ouvertes.fr/tel-00583434.
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The arbuscular mycorrhizal (AM) association results from a successful interaction between the genomes of the two symbiotic partners. In this context, the aim of my research was to better characterize the role of the late stage symbiosis-related pea genes PsSym36, PsSym33 and PsSym40 in the functional AM (i) by investigating the effect of mutations in the three genes on fungal and plant gene responses and (ii) by creating conditions for the localization of two of the genes, PsSym36 and PsSym40, on the pea genetic map for future map-based cloning. The expression of a subset of ten fungal and eight plant genes,previously reported to be activated during mycorrhiza development, was compared in Glomus intraradices-inoculated roots of wild type and Pssym36, Pssym33 and Pssym40 mutant pea plants. Most of the fungal genes were down-regulated in roots of the Pssym36 mutant where arbuscule formation is defective, and several were upregulated with more rapid fungal development in roots of the Pssym40 mutant. Microdissection of mycorrhizal PsSym40 roots corroborated preferential expression of the three G. intraradices genes SOD, DESAT and PEPISOM in arbuscule-containing cells. Inactivation of PsSym36 also resulted in down regulation of plant genes whilst mutation of the PsSym33 and PsSym40 genes affected plant gene responses in a more time-dependent way. Results thus indicate an implication of the investigated pea SYM genes in the modulation of plant and fungal molecular interactions linked to signaling, nutrient exchange or stress response regulation during AM symbiosis formation and functioning. Conditions for localization of the PsSym36 and PsSym40 genes on the pea genetic map were developed for their future map-based cloning. Based on the molecular markers obtained, it was possible to conclude that localization of the PsSym40 gene most likely resides outside the linkage groups I, II, III or V of the genetic map of pea.
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Wang, Fangjing. "Biomedical Imaging of Stem Cells Using Reporter Genes." Case Western Reserve University School of Graduate Studies / OhioLINK, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=case1261441999.
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Li, Ye. "MULTIFACTOR DIMENSIONALITY REDUCTION WITH P RISK SCORES PER PERSON." UKnowledge, 2018. https://uknowledge.uky.edu/statistics_etds/34.
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After reviewing Multifactor Dimensionality Reduction(MDR) and its extensions, an approach to obtain P(larger than 1) risk scores is proposed to predict the continuous outcome for each subject. We study the mean square error(MSE) of dimensionality reduced models fitted with sets of 2 risk scores and investigate the MSE for several special cases of the covariance matrix. A methodology is proposed to select a best set of P risk scores when P is specified a priori. Simulation studies based on true models of different dimensions(larger than 3) demonstrate that the selected set of P(larger than 1) risk scores outperforms the single aggregated risk score generated in AQMDR and illustrate that our methodology can determine a best set of P risk scores effectively. With different assumptions on the dimension of the true model, we considered the preferable set of risk scores from the best set of two risk scores and the best set of three risk scores. Further, we present a methodology to access a set of P risk scores when P is not given a priori. The expressions of asymptotic estimated mean square error of prediction(MSPE) are derived for a 1-dimensional model and 2-dimensional model. In the last main chapter, we apply the methodology of selecting a best set of risk scores where P has been specified a priori to Alzheimer’s Disease data and achieve a set of 2 risk scores and a set of three risk scores for each subject to predict measurements on biomarkers that are crucially involved in Alzheimer’s Disease.
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Peres, Regina Celia Rocha. "Analise dos polimorfismos na região promotora dos TGF 'Beta' -1, MMP-9, TIMP-2 e PAX-9 : correlação com a hipodontia." [s.n.], 2004. http://repositorio.unicamp.br/jspui/handle/REPOSIP/288493.
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Orientador: Pedro Duarte NovaesTese (doutorado) - Universidade Estadual de Campinas. Faculdade de Odontologia de Piracicaba
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Resumo: Hipodontia, a ausência congênita de um ou mais dentes, é uma das alterações mais comuns da dentição humana. Os dentes mais atingidos são os terceiros molares, segundos pré-molares superiores, e incisivos laterais superiores. Embora hipodontia não represente um sério problema de saúde pública, pode causar disfunção na mastigação e fala, além de problemas estéticos. Os genes TGFBETA1, MMP9, TIMP2 e PAX9 estão expressos em várias regiões do germe dentário durante as diversas fases da odontogênese. Estudos recentes mostram que polimorfismos em regiões reguladoras da transcrição parecem ser freqüentes, e que estas variações são responsáveis por características fenotípicas individuais. No entanto, pouco se sabe sobre o papel de polimorfismos genéticos no surgimento da agenesia dental de forma isolada. O objetivo do presente estudo foi analisar a associação existente entre os polimorfismos na região promotora dos genes TGF BETA1, MMP9, TIMP2 e PAX9, e a agenesia dental de terceiros molares, segundos pré-molares e incisivos laterais. A amostra foi composta pelo DNA genômico de 50 indivíduos afetados e 50 indivíduos controles, com idade acima de 16 anos, para os três primeiros marcadores. Com o objetivo de aumentar a amostra, a análise de polimorfismos no gene PAX9 foi realizada utilizando-se o DNA genômico de 100 indivíduos afetados e 100 indivíduos controles. Após a obtenção e extração do DNA, as regiões de interesse foram amplificadas por reação em cadeia da polimerase (PCR) e os polimorfismos foram analisados por digestão com enzima de restrição. Os géis foram corados pelo nitrato de prata. A análise estatística foi realizada através das Simulações de Monte Carlo (programa Clump) e teste Qui-quadrado ao nível de significância de 5%. O programa ARLEQUIN foi utilizado para verificar combinações de haplótipos nos genes do TGF- BETA1 e PAX9. As análises mostraram que o polimorfismo da região promotora do TIMP2 não está presente na população estudada, que os polimorfismos na região promotora dos genes TGF BETA1 e MMP9 não estão associados com agenesia dental, e que os polimorfismos no promotor do PAX9 estão associados com a agenesia dental, podendo ser considerados marcadores genéticos para a hipodontia
Abstract: Hypodontia, the congenital absence of one or a few teeth, is one of the most common alterations of the human dentition. The most common permanent missing teeth are the third molars, maxillary second premolars and maxillary lateral incisors. Although hypodontia does not represent a serious public health problem, it may cause masticatory and speech dysfunctions and esthetic problems. TGFBETA1, MMP9, TIMP2 and PAX9 genes are expressed in many regions of the tooth germ during the diverse phases of odontogenesis. Recent studies show that polymorphisms in transcription regulator regions seem to be frequent and that these variations are responsible for individual phenotypic features. However, the role of genetic polymorphisms in the development of sporadic tooth agenesis is not established. The aim of this study was to analyse the association between TGF BETA1, MMP9, TIMP2 and PAX9 genes promoter polymorphisms and hypodontia in humans. Samples consisted of genomic DNA of 50 affected individuals and 50 control subjects, with age above 16. For PAX9 promoter polymorphisms analysis, test and control groups were composed of 100 individuals. After DNA extraction, the regions of interest were amplyfied by polymerase chain reaction (PCR). The polymorphic sites were analysed by restriction length fragment polymorphism (RLFP). The gel bands were stained by silver nitrate. Monte Carlo simulations (Clump software) and Chi-square test (x2) were used for statistical analysis. Differences were considered significant when p<0.05. ARLEQUIN computer program was used to analyse haplotypic combinations in TGF BETA1 and PAX9 genes. The analysis showed that TIMP2 promoter polymorphism was not present in the studied population and that TGF BETA1 and MMP9 promoter polymorphisms are not associated with hypodontia. There was a positive correlation between the two PAX9 promoter polymorphisms and hypodontia
Doutorado
Histologia e Embriologia
Doutor em Biologia Buco-Dental
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Brown, Naomi Jane. "Post-transcriptional regulation of the pea plastocyanin gene (PetE)." Thesis, University of Cambridge, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.597002.
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Expression of the pea plastocyanin gene (PetE) is regulated both by light and by signals from the chloroplast. Previous work has indicated that the light and chloroplast-controlled regulation operates post-transcriptionally in transgenic tobacco, requiring the correct 5’ terminus of the transcript and elements within the plastocyanin-coding region. The post-transcriptional light and chloroplast-controlled regulation of pea PetE has now been demonstrated to operate in transgenic Arabidopsis plants, indicating that the regulation is conserved in an additional plant species. The overall aim of the research described in this dissertation was to investigate the mechanisms by which light and plastid signals influence the stability of PetE transcripts. PetE constructs containing premature stop codons in the coding region were generated to investigate whether translation has a role in the light or chloroplast-controlled regulation. RNA-gel-blot analysis of transgenic plants containing these constructs was used to examine the effects of light and plastid inhibitors on pea PetE transcript accumulation in 7-day-old tobacco seedlings. The results obtained suggested that translation of the start of the PetE coding region is required for both light and plastid-regulated transcript stability. Constructs containing progressive 3’ deletions of the PetE coding region, fused to the Luc reporter gene, were generated to examine how much of the coding sequence is necessary for the regulation. Luciferase assays and RNA-gel-blot analysis were carried out on transgenic tobacco seedlings containing the constructs, to examine the effects of light and plastid inhibitors on the regulation. The results indicated that an element important in the light and chloroplast-controlled regulation is located in the first 12% of the coding region, corresponding to the first 60 nucleotides. The start of the plastocyanin-coding region therefore appears to contain sequences important in the regulation by light and plastid signals, and these sequences may need to be translated in order for the regulation to operate.
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Bergström, Niclas, and Sara Arvini. "PAX9-genens betydelse för tandstorleken -En systematisk litteraturöversikt." Thesis, Malmö högskola, Odontologiska fakulteten (OD), 2013. http://urn.kb.se/resolve?urn=urn:nbn:se:mau:diva-19800.
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SammanfattningSyfte/frågeställning: Tandutvecklingen involverar en rad olika genetiska interaktioner som innefattar uttryck av bland annat transkriptionsfaktorer, tillväxtfaktorer och signalmolekyler. Syftet med den här uppsatsen var att systematiskt undersöka dagens vetenskapliga litteratur utifrån frågeställningen: Har uttryck av PAX9-genen en direkt påverkan på den slutliga tandstorleken hos människor?Material och metod: En systematisk litteratursökning av studier gjordes via databasen PubMed med hjälp av MeSH-termer, fritextsökning och handsökning via referenslistor. Två granskare selekterade, oberoende av varandra, kontrollerade studier på engelska samt extraherade data och bedömde kvaliteten.Resultat: Sökningen resulterade i 274 artiklar och efter genomgång av inklusions- och exklusionskriterier kvarstod 4 artiklar.Slutsats: Denna systematiska litteraturöversikt visar att PAX9-genen har en roll i tandutvecklingen, men att det vetenskapliga underlaget är bristande för att avgöra ifall genen har en direkt påverkan på den slutgiltiga tandstorleken. Studierna visar dock att en mutation av genen kan leda till avvikelser, så som avsaknad av tänder och mindre tandstorlek hos befintliga tänder. Det vetenskapliga underlaget för kopplingen mellan uttryck av PAX9-genen och tandstorlek saknar emellertid tillförlitligt bevisvärde. Ytterligare forskning är nödvändigt för att fastställa PAX9-genens påverkan av tandstorleken.
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Kuznetsova, Elena Vladislavovna. "Characterization of Pea (Pisum Sativum L.) genes implicated in arbuscular mycorrhiza formation and function." Thesis, Dijon, 2010. http://www.theses.fr/2010DIJOS023/document.
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L’association mycorhizienne à arbuscules (AM) est le résultat d’une interaction compatible entre les génomes des deux partenaires symbiotiques. Dans ce contexte, le but de mes recherches a été de mieux caractériser le rôle des gènes de pois liés aux stades tardifs de la symbiose, PsSym36, PsSym33 and PsSym40, dans le fonctionnement de la symbiose MA (i) en étudiant l’effet des mutations de ces trois gènes sur l’expression des gènes de la plante et du champignon, et (ii) en créant les conditions pour positionner deux de ces gènes, PsSym36 and PsSym40, sur la carte génétique afin d’envisager leur clonage futur. L’expression d’un groupe de dix gènes fongiques et de huit gènes de plante, déjà décrits pour être activés durant le développement de la mycorhize, a été comparée dans les racines de pois inoculées avec G. intraradices chez les plantes de génotypes sauvages, ou les mutants Pssym36, Pssym33 et Pssym40. L’expression de la plupart des gènes fongiques a été inhibée dans les racines du mutant Pssym36 où la formation des arbuscules est avortée, tandis que l’expression de plusieurs d’entre eux a été activée lorsqu’il existe un développement plus rapide du champignon dans les racines du mutant Pssym40. Des microdisséquats obtenus à partir de racines mycorhizées du mutant PsSym40 confirment l’expression préférentielle de trois gènes de G. intraradices (SOD, DESAT et PEPISOM) dans les cellules contenant les arbuscules. L’inactivation du gène PsSym36 provoque également une inhibition des gènes de plante alors que la mutation des gènes PsSym33 and PsSym40 affecte l’expression des gènes de plante plutôt de façon temporelle. Les résultats indiquent ainsi une implication des gènes SYM de pois dans la modulation des interactions moléculaires entre la plante et le champignon impliquées au niveau de la signalisation, des échanges nutritifs ou de la régulation des réponses au stress durant la formation et/ou le fonctionnement de la symbiose AM. Les conditions pour la localisation des gènes PsSym36 and PsSym40 sur la carte génétique du pois ont été développées pour leur clonage basé sur la cartographie. En utilisant les marqueurs moléculaires obtenus, il a été possible de conclure que la localisation du gène PsSym40 réside vraisemblablement à l’extérieur des groupes de liaison I, II, III ou V de la carte génétique du poisThe arbuscular mycorrhizal (AM) association results from a successful interaction between the genomes of the two symbiotic partners. In this context, the aim of my research was to better characterize the role of the late stage symbiosis-related pea genes PsSym36, PsSym33 and PsSym40 in the functional AM (i) by investigating the effect of mutations in the three genes on fungal and plant gene responses and (ii) by creating conditions for the localization of two of the genes, PsSym36 and PsSym40, on the pea genetic map for future map-based cloning. The expression of a subset of ten fungal and eight plant genes,previously reported to be activated during mycorrhiza development, was compared in Glomus intraradices-inoculated roots of wild type and Pssym36, Pssym33 and Pssym40 mutant pea plants. Most of the fungal genes were down-regulated in roots of the Pssym36 mutant where arbuscule formation is defective, and several were upregulated with more rapid fungal development in roots of the Pssym40 mutant. Microdissection of mycorrhizal PsSym40 roots corroborated preferential expression of the three G. intraradices genes SOD, DESAT and PEPISOM in arbuscule-containing cells. Inactivation of PsSym36 also resulted in down regulation of plant genes whilst mutation of the PsSym33 and PsSym40 genes affected plant gene responses in a more time-dependent way. Results thus indicate an implication of the investigated pea SYM genes in the modulation of plant and fungal molecular interactions linked to signaling, nutrient exchange or stress response regulation during AM symbiosis formation and functioning. Conditions for localization of the PsSym36 and PsSym40 genes on the pea genetic map were developed for their future map-based cloning. Based on the molecular markers obtained, it was possible to conclude that localization of the PsSym40 gene most likely resides outside the linkage groups I, II, III or V of the genetic map of pea
Формирование арбускулярной микоризы (АМ) является результатом успешного взаимодействия между геномами двух симбиотических партнёров. Целью моего исследования являлось изучение роли поздних симбиотических генов гороха PsSym36, PsSym33 и PsSym40 в формировании функционального АМ симбиоза. Для этого было проведено исследование эффекта мутаций в генах PsSym36, PsSym33 и PsSym40 на экспрессию грибных и растительных генов, предположительно (по литературным данным) вовлечённых в процессы формирования АМ, а так же проведена работа по локализации генов PsSym36 и PsSym40 на генетической карте гороха для последующего более точного картирования и позиционного клонирования данных генов. Экспрессия десяти грибных и восьми растительных генов была определена в корнях растений дикого типа и PsSym36, PsSym33 и PsSym40 мутантов, инокулированных G. intraradices. В корнях PsSym36 мутанта, имеющего дефект развития арбускул, большая часть грибных генов была супрессирована, в то время как в корнях PsSym40 мутанта, для которого характерна более быстрая по сравнению с диким типом микоризация, был отмечен более высокий уровень экспрессии грибных генов. Использование метода микродиссекций позволило выделить клетки, содержащие арбускулы, из микоризованных корней мутанта PsSym40 и подтвердить, что гены G. intraradices SOD, DESAT и PEPISOM преимущественно экспрессируются в клетках, содержащих арбускулы. Мутация в гене PsSym36 также привела к подавлению экспрессии большинства вовлечённых в анализ растительных генов, тогда как мутации в генах PsSym33 и PsSym40 оказали влияние на ксперессию растительных генов в меньшей степени. Полученные результаты свидетельствуют о роли исследуемых SYM генов гороха в контролировании растительно-грибных молекулярных взаимодействий, связанных с сигналингом, обменом питательными веществами и стрессовыми реакциями в процессе формирования и функционирования АМ симбиоза. Проведённое генетическое картирование не привело к локализации генов PsSym36 и PsSym40 на генетической карте гороха. Однако разработка и использование молекулярных маркеров для картирования позволили исключить локализацию гена PsSym40 в I, II, III и V группах сцепления с высокой долей вероятности
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Laverty, Edward. "The molecular basis of gene expression variability in transgenic tobacco plants." Thesis, Durham University, 1996. http://etheses.dur.ac.uk/5238/.
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An extensive investigation into and charactaisation of factors influencing transgene expression following introduction of the transgoie into tobacco via Agrobacterium- mediated transformation was carried out. Characterisation of material supplied at the outset of this project revealed that this material was unacceptable for further analysis. It was thus deemed necessary to obtain large populations of transgenic tobacco heterogenous for levels of transgene expression. Characterisation of these populations (CaMV-lecA and ssRubisco-lecA plants) showed that all plants fell into one of four segregation classes based on segregation of the kanamycin-resistance selectable marker. Results showed that the majority of regenerants contained multiple nptII-containing inserts, while the presence of one or two such inserts was also found, albeit at a much lower frequency. Segregation analysis based on detection of the lecA transgene agreed, in the majority of cases, with these results. However, in a few cases it was found that data obtained from both segregation analyses did not agree, with the presence of a single lecA-containing transgene being detected in plants shown to contain two copies of the nptII-contaning transgene. This result indicates the occurrence of T-DNA rearrangement either within the tobacco genome or during T-DNA transfer and integration. Southern blot analyses allowed a detailed characterisation of T-DNA structure, copy number and number of integration sites to be undertaken. Results from these analyses revealed a higher frequency of T-DNA rearrangement within plants containing multiple inserts. However, such rearrangements did not correlate with a significant reduction in levels of transgene expression since all detected rearrangements were found to occur at or towards the left hand border of the T-DNA, that border distant to the lecA transgene. Plants containing more than one T-DNA were also frequently found to contain these T-DNAs arranged as an inverted repeat at a single locus although no significant relationship between copy number and the presence of such structures was found. Correlating transgene expression levels, as determined by radioimmunoassay-based quantitation of lectin protein in tissues of transgenic plants, with T-DNA copy number, organisation and structure revealed no significant relationship. It is thus feasible to conclude that the major contributory factor influencing levels of transgene expression is the location of T-DNA integration within the plant genome. Subsequent work concerned with investigating the nature of those integration site-specific factors i.e. 'position effect' indicated a possible role for methylation-induced modulation of gene expression. Results presented in this thesis provide an insight into the fate of transgenes following introduction into the plant genome and clearly demonstrate the importance of further exploring the molecular mechanisms underlying transgene expression variability.
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Oliveira, Kezia Gomes de. "Amplificação isotérmica de DNA mediada por loop (LAMP) em microchip de poliéster-toner." Universidade Federal de Goiás, 2016. http://repositorio.bc.ufg.br/tede/handle/tede/6663.
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Outro
The several advantages of miniaturization of DNA amplification and coupling with sample preparation and detection steps on the same chip are well known. Currently, most miniaturized systems for nucleic acids analysis are based on polymerase chain reaction (PCR). PCR amplification requires precise temperature control, switching between heating and cooling solution in three specific temperatures. Therefore, the adaptation of PCR for microchip is relatively complex and presents some limitations particularly for use in remote locations. Without the need for heating cycles, isothermal microsystems for DNA amplification can be designed to be simple and low energy consumption and hence can overlap the portable PCR detection systems. The loop-mediated isothermal amplification (LAMP) is a novel technique which has emerged as a simple and fast tool for DNA amplification which can be used for the detection and identification of several pathogens. The LAMP using Bst DNA polymerase enzyme which is an enzyme having strand displacement activity and uses a set of four primers designed from six individual segments of the sequence to be amplified. In this study, we developed a simple and rapid LAMP reaction for the E. coli malB gene amplification in the reaction was thermally controlled with a thermoblock for 60 minutes at 66 ° C. The PeT microdevices demonstrated compatibility with all reagents used in the LAMP and the success of the isothermal amplification was observed by agarose gel electrophoresis, yielding detectable amount amplicons as few as starting with 1 copy of DNA. Moreover, the success of the nucleic acid amplification reaction was evaluated by visual detection of the amplicons in the microchip by the use of fluorescent DNA intercalators, which yielded fluorescence in positive reactions. The LAMP in PeT microdevice is a simple and inexpensive method, that allowed a rapid detection (62 minutes) of E. coli. Because of simple operation and without the need for sophisticated instrumentation, LAMP held in microchip PeT has proven to be a valuable tool for molecular diagnostics, with great potential for applications in point-of-care.
As diversas vantagens da miniaturização das reações de amplificação de DNA e o acoplamento com as etapas de preparo da amostra e de detecção no mesmo chip já são bem conhecidas. Até o presente momento, a maioria dos sistemas miniaturizados para a análise de ácidos nucleicos são baseados na reação em cadeia da polimerase (PCR). A PCR necessita de controle preciso de temperatura, alternando entre aquecimento e resfriamento da solução em três temperaturas específicas. Desta forma, a adaptação da PCR em microchips é relativamente complexa apresentando algumas limitações relacionadas principalmente a utilização em lugares remotos. Sem a necessidade de ciclos de aquecimento, os microssistemas isotérmicos podem ser projetados para serem simples e de baixo consumo de energia e, portanto, pode sobrepor a PCR em sistemas de detecção portáteis. A amplificação isotérmica mediada por loop (LAMP) é uma técnica recente e inovadora que surgiu como uma ferramenta simples e rápida de amplificação de DNA que pode ser utilizada para detecção e identificação de diversos patógenos. A LAMP utiliza a enzima Bst DNA polimerase que é uma enzima com atividade de deslocamento de fita e utiliza um conjunto de quatro iniciadores desenhados a partir de seis segmentos específicos da sequência a ser amplificada. Neste trabalho foi desenvolvida uma metodologia simples e rápida para detecção de E.coli através da amplificação isotérmica do gene malB em dispositivos descartáveis de poliéster-toner (PeT) contendo um microcâmara com capacidade para 5 μL, e a reação foi incubada a 66 ºC em um termobloco por 60 minutos. Os microchips de PeT demonstraram compatibilidade com todos os reagentes utilizados na LAMP e o sucesso da amplificação isotérmica foi observado por eletroforese em gel de agarose, obtendo quantidade de amplicons detectáveis no gel em reações que partiram de 1 cópia de DNA. Além disso, o sucesso da reação de amplificação do ácido nucleico também foi avaliado através da detecção visual dos produtos amplificados no microchip através do uso de intercaladores fluorescentes de DNA, que produziram fluorescência nas reações positivas. A LAMP realizada em microdispositivos de PeT representa um método simples e de baixo custo, que permitiu a detecção rápida (62 minutos) da E.coli. Devido a simples operação, e sem a necessidade de instrumentação sofisticada, a LAMP realizada no microchip de PeT demonstrou ser uma ferramenta valiosa para diagnósticos moleculares, apresentando grande potencial para aplicações no point-of-care.
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Gilmartin, P. M. "The isolation and characterisation of nuclear-encoded light-regulated genes from Pisum sativum and their expression in transgenic plants of Nicotiana tabacum." Thesis, University of Warwick, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.378261.
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Zhuma, Talgat M. "CIS and TRANS elements that influence hCD2 gene expression in transgenic mice." Thesis, University College London (University of London), 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.313396.
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Bustamante, Pineda Mariona. "Estudis d'associació i funcionals en gens candidats per a l'osteoporosi." Doctoral thesis, Universitat de Barcelona, 2007. http://hdl.handle.net/10803/1887.
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L'osteoporosi, una malaltia complexa determinada tant per factors genètics com ambientals, està caracteritzada per una reduïda densitat mineral òssia (DMO), un deteriorament de la microarquitectura òssia i un augment del risc de patir fractures osteoporòtiques. En el present treball ens vam plantejar estudiar les causes genètiques que determinen l'aparició de l'osteoporosi mitjançant la realització d'estudis d'associació i estudis funcionals. Els gens candidats clàssics per a l'osteoporosi que hem analitzat han estat el gen COL1A1, el gen ESR1, el gen VDR i el gen TGFB1. Els estudis d'associació s'han dut a terme en la cohort BARCOS formada per dones postmenopàusiques de l'àrea de Barcelona. Els resultats obtinguts en la cohort BARCOS han estat incorporats en el projecte GENOMOS (metaanàlisi prospectiva amb 20.000 mostres). En la cohort BARCOS vam observar que polimorfismes de canvi puntual de nucleòtid (SNP) del gen COL1A1 així com també determinats haplotips i la interacció entre ells o bé amb SNPs del gen VDR es trobaven associats a la DMO. Pel que fa al gen ESR1, vam trobar que l'haplotip LPX es trobava associat a la DMO femoral. Els resultats del projecte GENOMOS, van mostrar que només el polimorfisme Sp1 del gen COL1A1 es trobava associat a la DMO. En el projecte GENOMOS, els polimorfismes XbaI (ERSR1), Cdx2 (VDR) i Sp1 (COL1A1) es van trobar associats al risc de patir fractures.
En la cohort BARCOS també vam analitzar l'efecte de SNPs situats en gens candidats no clàssics (RUNX2 i IL6R). Pel que fa a RUNX2 vam trobar que un SNP del promotor 2, però no un SNP del promotor 1 es trobava associat a la DMO femoral. Aquesta ha estat la primera vegada que SNPs del gen IL6R s'han analitzat en relació a fenotips osteoporòtics. Vam observar que SNPs i haplotips en aquest gen es trobaven associats a la DMO femoral i a l'índex de massa corporal (IMC).
Com a segon objectiu ens vam plantejar completar l'estudi funcional dels SNPs del promotor del gen COL1A1 (-1997 G/T i -1663 indelT) en relació a les proteïnes CIZ/NMP4 i BMP2. Prèviament s'havia descrit que la proteïna CIZ/NMP4, un factor de transcripció arquitectònic que inhibeix la via osteoblastogènica de la BMP2, s'unia a la regió nucleotídica on es troba el SNP -1663 indelT. En les cèl·lules Saos-2 vam observar que el patró de transcripció de diferents construccions del promotor del gen COL1A1 fusionat al gen de la luciferasa era similar tant si aquestes eren tranfectades de manera estable o bé de forma transitòria. En estudiar l'efecte de la BMP2 sobre l'activitat transcripcional del gen COL1A1, vam observar que mentre el promotor basal disminuïa la seva activitat en presència de BMP2, les altres construccions analitzades l'augmentaven. Les construccions que eren estimulades en major grau presentaven la regió del promotor on es troben els polimorfismes. Tot i això, analitzant els diferents haplotips, vam observar que aquests no participaven en l'estimulació induïda per BMP2. In silico la regió estimulada per BMP2, presenta dues possibles caixes d'unió de factors de transcripció de la via de la BMP2. En mutagenitzar-les per separat, però, aquestes no van semblar participar en l'estimulació induïda per la BMP2. Seguidament vam analizar el patró d'expressió de CIZ/NMP4 en diversos teixits humans i vam observar que diverses isoformes s'expressaven simultàniament en tots ells i també en les cèl·lules d'osteosarcoma humanes Saos-2 i MG-63. Es van identificar les isoformes 11H, CIZ6.1, 21H i 21H-I1. Aquestes dues últimes van ser clonades i sobrexpressades de manera estable en les cèl·lules Saos-2, les quals es van transfectar transitòriament amb el promotor del gen COL1A1 i es van tractar amb BMP2. Els resultats van indicar que CIZ/NMP4 augmentava lleugerament la transcripció del gen COL1A1 en absència de BMP2. En presència de BMP2, i contràriament al què s'esperava, vam observar que la sobrexpressió de CIZ/NMP4 no tenia cap efecte.
"ASSOCIATION AND FUNCTIONAL STUDIES WITH CANDIDATE GENES FOR OSTEOPOROSIS"
TEXT:
Osteoporosis, a complex disease determined by genetic and environmental factors, is characterized by a reduced bone mineral density (BMD) and an increased risk of fracture. In this thesis we studied the genetic component of osteoporosis through association and functional studies.
The classical candidate genes for osteoporosis we analyzed were COL1A1, ESR1, VDR and TGFB1. The association studies were performed with the BARCOS cohort, a group of postmenopausal women from Barcelona. The results obtained in this cohort were included in a prospective metaanalysis called GENOMOS (20,000 samples). In the BARCOS cohort we observed that SNPs in the COL1A1 gene, their haplotypes and interactions, and interactions with SNPs in the VDR gene were associated with BMD. The haplotype LPX (ESR1) was also associated with BMD. In GENOMOS, although several SNPs were found to be associated with fracture risk, the Sp1 polymorphism (COL1A1) was the only one associated with BMD. In the BARCOS cohort we also found that one SNP situated in promoter 2 of RUNX2 gene, but not one situated in promoter 1, was associated with BMD. Finally, this was the first time that SNPs in IL6R gene were analyzed in relation to osteoporosis and we found that they were associated with BMD and also with body mass index (BMI).
The second aim of this thesis was the functional study of two SNPs situated in the promoter of COL1A1 gene in relation to CIZ/NMP4 and BMP2 proteins. It was known that CIZ/NMP4, an architectural transcription factor that inhibits the BMP2 osteoblastogenic pathway, binded to the COL1A1 promoter. We observed that the transcription pattern of differnt constructs of COL1A1 promoter was similar between transient and stable transfections. BMP2 stimulated all these constructs, except for the basal promoter. The constructs that were stimulated more, contained the region where the SNPs lie, but when the haplotypes were analyzed no differences were observed between them. In this region there are two putative transcription factor sites for the BMP2 pathway, but they seemed not to participate in the BMP2 stimulation. Several isoforms of CIZ/NMP4 were simultaneously expressed in different tissues. We identified 11H, CIZ6.1, 21H and 21H-I1 isoforms. These two last were stably overexpressed in Saos-2 cells, which were then transiently tranfected with COL1A1 promoter and treated with BMP2. In absence of BMP2, overexpression of CIZ/NMP4 isoforms slightly increased COL1A1 transcription.
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Mayer, Melinda Jane. "Gene expression during late embryogenesis in pea (Pisum sativum L)." Thesis, Durham University, 1993. http://etheses.dur.ac.uk/5722/.
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A thesis submitted by Melinda Jane Mayer, B.Sc.(Bristol) in accordance with the requirements of the University of Durham for the degree of Doctor of Philosophy. Department of Biological Sciences, August 1993.Two cDNA libraries were constructed from desiccating pea cotyledons. Differential screening of the libraries with cDNA from an earlier developmental stage (physiological maturity) demonstrated that the abundant message population during dehydration shows some noticeable differences to the message populations present before desiccation. Clones hybridising to a polyubiquitin probe were isolated from a cDNA library. These clones were identified as messages for the two types of ubiquitin extension proteins (with 52 and 79 residue tails), already characterised in other species as being involved in ribosome biogenesis. The pea ubiquitin extension tail amino acid sequences showed considerable homology to tails from other plants, animals, yeast and protozoa, including a nuclear localisation site and a putative zinc-binding nucleic acid binding domain, the positions of which are conserved within the tail sequences. Sequencing of a second polyubiquitin cDNA from pea leaf demonstrated that pea contains a ubiquitin multigene family of at least four members. The expression of several genes associated with plant response to stress and two abundant seed messages (Leg A and J) was examined in developing and dehydrating cotyledons and axes. This confirmed conspicuous variations in the message levels of the genes examined as the cotyledons aged, with different members of the ubiquitin and legumin multigene families showing differential expression with age. It was also demonstrated that the expression pattern of certain messages in the cotyledons was different to that in the axes and other seed tissues. This was confirmed by an analysis of total and albumin protein fractions in cotyledons and axes. The effect on specific message and protein levels of premature desiccation treatments indicated that the temporal expression of several seed genes is related to the state of hydration of the seed, artificial desiccation leading to premature maturation. Seed storage protein message and protein levels were especially increased by premature desiccation. Legumin seed storage protein messages were also shown to be responsive to exogenous ABA applied to immature cotyledons during the seed filling stage. However, the other stress-related messages examined in pea (ubiquitin and a pea putative metallothionein) were not responsive to exogenous ABA at this developmental stage.
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Hufnagel, Hansjörg. "Overcoming barriers in non-viral gene transfection by PEI-25." Thesis, University of Cambridge, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.608478.
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Fella, Carolin. "Dynamic and effective gene vectors via pH-sensitive PEG-shielding." Diss., kostenfrei, 2008. http://edoc.ub.uni-muenchen.de/8592/.
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Gibbon, Marjorie Jane. "Molecular characterisation of an avirulence gene from race 2 of Pseudomonas syringae pathovar pisi." Thesis, University of the West of England, Bristol, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.385396.
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Radosevic, Marija. "Spatial control of inner ear neurogenesis by retinoic acid, Tbx1 and her genes." Doctoral thesis, Universitat Pompeu Fabra, 2011. http://hdl.handle.net/10803/38436.
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Sensory neurons are key mediators of the transduction of external stimuli from the ear to the brain, essential for the sense of balance and hearing. Understanding when, where and how the sensory nervous system is assembled during development can provide insights on deafness and balance disorders. Here, I show in zebrafish that Her9 transcription factor is a key element in the regulation of the otic neurogenesis. Loss of Her9 function leads to the ectopic expression of neurogenic genes neurod and neurod4. Moreover, I show that Her9 acts downstream of Tbx1, and both genes are activated by retinoic acid signaling emanating from the paraxial mesoderm and negatively regulated by Hedgehog signaling. Altogether, the data demonstrates a role of retinoic acid in axial patterning and the establishment of a neurogenic domain through Tbx1 and Her9. At later stages, retinoic acid has an additional role by regulating neuronal differentiation in the statoacoustic ganglion.Les neurones sensorials de l’oïda interna són mediadores claus en la transducció dels estímuls externs des de l’oïda interna al cervell. Entendre a on, quan i com el sistema nerviós sensorial s’organitza durant el desenvolupament embrionari pot ajudar en l’estudi de les malalties neurosensorials. En el present treball, mostro en peix zebra que el factor de transcripció Her9 és un element clau en el control de la neurogènesi òtica i que Her9 es troba sota el control directe del factor Tbx1. A més, ambdos factors estan regulats de manera positva per la via de senyalització de l’àcid retinoic i negativament per la vía de hedgehog. En resum, la tesis demostra un paper de l’àcid retinoic en la regionalització axial del primordi òtic en l’eix anteroposterior i l’establiment d’un domini neurogènic a través de Tbx1 i Her9. En estadis tardans, l’àcid retinoic regula la diferenciació neuronal en el gangli estato-acústic.
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Mulugeta, Wubet Edwards Kevin A. "Functional analysis of the Pez-Cpr genomic region in Drosophila." Normal, Ill. : Illinois State University, 2005. http://wwwlib.umi.com/cr/ilstu/fullcit?p3196653.
Full textAbstract:
Thesis (Ph. D.)--Illinois State University, 2005.Title from title page screen, viewed September 26, 2006. Dissertation Committee: Kevin Edwards (chair), David Borst, Laura Vogel, David Rubin, Jon Friesen. Includes bibliographical references (leaves 133-143) and abstract. Also available in print.
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Adshead, James Michael. "The expression of class III PAX genes in transitional cell carcinoma of the human bladder." Thesis, Imperial College London, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.248409.
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Dhankher, Om Parkash. "Cloning and characterisation of heat shock and wound-induced genes in pea (Pisum sativum L.)." Thesis, Durham University, 1997. http://etheses.dur.ac.uk/4704/.
Full textAbstract:
Plant productivity in many regions of the world is limited primarily as a result of environmental stresses. High temperature and wounding caused by pest and pathogen infection are among the main factors accounting for unpredictable and often severe yield losses worldwide. These stresses, force the plants to alter then gene expression in order to adapt to the changed environment. Attempts were made in the study to isolate and characterise the differentially expressed heat shocked and wound-induced genes to understand the underlying molecular mechanism of heat shock and wounding response. The isolation of the promoters and their use to derive the tissue-specific and high expression of the linked coding sequences will be proved practically more significant. A cDNA clone designated LP 19 was isolated from a differential screening of a cDNA library prepared from lignifying pods of pea line L59. Sequence homology analysis showed that LP19 belongs to the hsp70 gene family. Northern analysis of RNA from pods from pea lines of different genotypes, showing the presence or absence of pod lignification, showed that LP 19 expression was specifically associated with lignification. Several cDNA species derived from transcripts of the LP 19 gene were subsequently isolated, which showed varying positions of poly (A) addition to the 3' untranslated region. Southern blotting of genomic DNA indicated the presence of single gene corresponding to LP 19.The pea hsp70 gene corresponding to LP 19 was isolated from a pea genomic library using LP 19 as a probe. The pea hsp70(LP19) gene predicts an open reading frame encoding a polypeptide of 648 amino acid residues. This sequence is similar to other plant hsp70 proteins. However, unlike most other plant hsp70 genes, the pea hsp70(LP19) gene lacks an intron. 1.8 kb of 5' flanking sequence of hsp70(LP19) gene was also sequenced. The promoter region contains 6 putative consensus heat shock elements (HSEs) as well as 4 A-T rich sites upstream from TATA box. Induction of gene expression of the pea hsp70(LP19) was observed in all organs of the plant after heat shock; the highest level of expression was observed in root, followed by stem and least in leaves. A similar expression pattern for a corresponding gene was observed in chickpea (Cicer arietinum L.). Other stress conditions such as salt stress and wounding failed to induce the expression of hsp70LP(19) gene both in pea and chickpea. The pea hsp70(LP19) promoter region, including 1.8 kb 5'-flanking sequence, and the first 18 amino acids of the coding region, was fused with coding sequence for P- glucuronidase (GUS). Tobacco plants were transformed with this chimaeric gene in order to study tissue specific and developmental expression of the hsp70(LP19) promoter. Histological staining of GUS activity in transgenic tobacco plants showed that protein was present predominantly in the phloem tissue in stem, root and petioles In addition, developmental expression of the hsp70(LP19) gene promoter, without heat shock, was observed in petals, pollen grains, developing seeds as well as in germinating seeds and seedlings at different stages of growth. Quantitative assay of GUS activity by fluorometric assay was used to follow the time course of protein accumulation. Activity was detected within few minutes of the start of heat shock and increased to a maximum after 6 hrs. A high level of GUS activity was observed only in the heat shocked parts of the plant; no endogenous signal that spread systemically from the heat shocked areas to the rest of the plant was observed.Pea and chickpea plants showed a transient increase of polyphenol oxidase (PPO) with maximum level at 48 hrs after wounding. No systemic induction of PPO was observed in unwounded parts in response to both wounding and MeJA treatment. In order to isolate transcripts expressed differentially in response to wounding, a pea subtractive cDNA library was made. 21 subtracted cDNA clones were partially sequenced. Most of the subtracted cDNA clones showed homology with wound or pathogen induced sequences. Northern analysis of the genes corresponding to the subtracted cDNA clones (SC3, SC7, SC12, SC33, SC57 and SC58), indicated differential expression in response to wounding. Full length or nearly full length cDNAs corresponding to 4 subtracted cDNA clones, designated SC10, SC15, SC57 and SC58, were isolated and sequenced. These cDNA clones will be further studied and efforts will be made to isolate their promoters. The tissue-specific expression will be carried out by using promoter-reporter system. These isolated cDNA clones were partially characterised and will be available for further studies to isolate their respective promoters. The tissue specific expression will be carried out by using promoter-reporter system.