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1
Rosales-Alday, Javier. "Mexican simmental-brahman genetic characterization, genetic parameters and genetic trends". [Gainesville, Fla.] : University of Florida, 2004. http://purl.fcla.edu/fcla/etd/UFE0004581.
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Shareck, Julie. "Isolation and characterization of a cryptic plasmid from Lactobacillus plantarum". Thesis, McGill University, 2005. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=84072.
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Lactic acid bacteria (LAB), a group of generally recognized as safe (GRAS) organisms that metabolize sugars into primarily lactic acid, have traditionally been used for the fermentation and preservation of various foods and beverages. There is increasing interest in the genetic manipulation of LAB to improve existing characteristics or introduce novel, industrially pertinent phenotypes. However, because these bacteria have food-related applications, their genetic modification requires the use of food-grade genetic engineering tools. LAB plasmids, self-replicating extrachromosomal DNA molecules, can be used to derive food-grade cloning vectors. The rationale of this research was to develop a food-grade cloning vector using a lactobacilli cryptic plasmid and to investigate its cloning and expression properties. The main objectives were to (i) screen Lactobacillus spp. for plasmids, (ii) isolate and characterize a plasmid, and (iii) use the plasmid replicon to construct a cloning vector and express heterologous genes in various hosts. This is the first step in the development of a new family of food-grade cloning vectors for the genetic modification of lactobacilli.
3
Ezpeleta, Jessica. "The characterization of the ABF-1 promoter". Scholarly Commons, 2001. https://scholarlycommons.pacific.edu/uop_etds/559.
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The basic helix-loop-helix (bHLH) family oftranscription factors consists of proteins involved in cellular proliferation and differentiation. The HLH structure plays a key role in protein-protein dimerization and with the DNA target sites, referred to as E boxes containing the consensus DNA sequence CANNTG. One class of mammalian class I bHLH proteins includes products of the E2A gene, which result from alternative splicing (E12, E47, and ITF), E2-2 and HEB. E2A proteins have also been detected in most cell lines with high levels of expression in lymphoid- and pancreatic cells. It has also been demonstrated that E2A is required for B cell maturation, T cell development and has been shown to function as tumor suppressors. To date, an E2A-interacting bHLH transcription factor largely restricted to activated B lymphocytes, called ABF -1, was isolated using the two-hybrid system. ABF -1 is the only B cell restricted bHLH protein isolated. ABF-1/E2A heterodimers have been detected in B lymphocytes. In these studies, the mapping of the ABF-1 promoter and the critical 5' regulatory elements that control ABF-1 gene expression were analyzed through 5' deletional analysis. 5' -DNA flanking pieces of the promoter region were created through PCR and inserted into a promoterless cloning vector containing the firefly luciferase reporter gene. RT -PCR analysis and anchored PCR was utilized to demonstrate the transcriptional activity of the - promoter region of the ABF-1 gene. Transient transfections were completed to determine critical regulatory sequences. The promoter location was confirmed through computer analysis of the nucleotide sequence and deletional analysis.
4
Kjellman, Magnus. "Genetic characterization of adrenocortical tumors /". Stockholm, 1999. http://diss.kib.ki.se/1999/91-628-3358-8/.
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Camp, Darius. "Genetic characterization of clk genes". Thesis, McGill University, 2006. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=100781.
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clk-1 encodes an enzyme involved in the biosynthesis of ubiquinone (UQ), a redox active lipid that is found in all cellular membranes, including in the mitochondrial respiratory chain. In the nematode Caenorhabditis elegans clk-1 mutants display slow and deregulated physiological rates, including slow embryonic and post-embryonic development, retarded germline development, slow behaviours, and an increased lifespan. clk-1 slow germline development phenotype was previously linked to clk-1 altered ROS metabolism and its effect on lipid oxidization and the let-60/ras pathway. I have taken a genetic suppressor approach to further investigate the causes of the slow germline development phenotype of the clk-1 mutants.Through this approach one mutant that suppresses the germline phenotype of clk-1 was identified. This suppressor, gsc-1(qm216), restored clk-1 germline development to slightly faster rates than wild type worms. This effect was specific to clk-1 and gsc-1 did not speed germline development rates in wild type worms. Furthermore, this effect appeared to be additive to lowering cholesterol levels but not to increasing cytoplasmic ROS levels. gsc-1 by itself appeared to have a deleterious effect on brood size and to increase lifespan. Neither of these effects were additive to the clk-1 phenotype and were therefore believed to affect similar mechanisms. The genetic mapping of gsc-1 precisely located the mutation to the center of chromosome II and linked it tightly to the lin-5 mutation. However, none of the transgenic lines managed to complement the gsc-1 mutation and its identity was not discovered.
In addition, to determine the role of reactive oxygen species (ROS) in the Clk phenotype, I have been analyzing all clk mutants (clk-1 to -10), by increasing ROS levels through the disruption of sod-1 and sod-2 genes, and scoring Clk phenotypes. I found that, although several clk mutants appear to have altered ROS levels, the phenomenon does not apply to all clk worms and does not correlate with lifespan. The disruption of either sod-1 or -2 affects growth and embryonic viability: sod-2 tends to exacerbate the mutant phenotypes, while sod-1 shows both weakly enhancing or weakly suppressing effects. Interestingly, only one mutant, clk-4, and only one phenotype of this mutant, slow post-embryonic development, is suppressed by sod-2 (but not sod-1). Furthermore, disrupting the expression of the sod-1 gene has only moderate or no effect on the lifespan of wild type worms, while sod-2 was shown to extend lifespan. On the whole, our results suggest that low superoxide levels do not participate in extending lifespan and are not the common process inducing the Clk phenotype in these mutants. Yet, several of the mutants analyzed have a dramatically increased lifespan and specifically behave like mutants which affect mitochondrial electron transport such as isp-1. Thus, our findings suggest that electron transport has a crucial role in longevity and developmental rates that is independent of superoxide generation.
6
Fourie, Mariesa. "Molecular characterization and further shortening of recombinant forms of the Lr19 translocation". Thesis, Link to the online version, 2005. http://hdl.handle.net/10019/189.
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Johnstone, Oona. "Characterization of the Vasa-eIF5B interaction during Drosophila development". Thesis, McGill University, 2004. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=84265.
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Translational control is an important means of regulating gene expression. Development of the Drosophila germ line relies on translational regulation to differentially express maternal mRNAs, allowing it to develop distinctly from the soma. One of the critical factors required for germ cell development and function is the conserved DEAD-box RNA helicase Vasa (Vas). The research presented in this thesis examines the role of Vas in translational regulation during Drosophila germ line development. A two-hybrid screen conducted with Vas identified a translation initiation factor eIF5B (dIF2), as a direct interactor. Mutations were created in eIF5B and were found to enhance the vas mutant phenotypes of reduced germ cell numbers, and posterior segmentation defects, suggesting a functional interaction between these factors in vivo. In order to further understand the biological significance of the Vas-eIF5B interaction, the region of Vas required for eIF5B-binding was mapped and then specifically disrupted. Reduction of Vas-eIF5B binding using a transgenic approach, virtually eliminated germ cell formation, while having only a moderate effect on the somatic requirement of Vas in posterior segmentation. In addition, Vas-eIF5B interaction was found to be required for the establishment of polarity within the egg during oogenesis, likely through direct regulation of gurken (grk) mRNA. We concluded that through interaction with eIF5B, Vas plays a critical role in translational regulation in the germ line. In addition, another Drosophila DEAD-box protein, highly similar to Vas, called Belle (Bel) was characterized. Mutations in bel were found to also affect the germ line, leading to both female and male sterility. Like Vas, Bel is implicated in translation initiation, however bel is an essential gene, with a requirement for growth, whose function is not restricted to the germ line. Our data suggest that Bel may be a nucleocytoplasmic shuttling protein,
8
Stephens, Alexandre, i N/A. "Genetic and Functional Characterization of RUNX2". Griffith University. School of Medical Science, 2007. http://www4.gu.edu.au:8080/adt-root/public/adt-QGU20070823.100953.
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RUNX2 belongs to the RUNT domain family of transcription factors of which three have been identified in humans (RUNX1, RUNX2 and RUNX3). RUNX proteins are vital for metazoan development and participate in the regulation of cellular differentiation and cell cycle progression (Coffman, 2003). RUNX2 is required for proper bone formation by driving the differentiation of osteoblasts from mesenchymal progenitors during development (Ducy et al, 1997; Komori et al, 1997; Otto et al, 1997). RUNX2 is also vital for chondrocyte maturation by promoting the differentiation of chondrocytes to the hypertrophic phenotype (Enomoto et al, 2000). The consequences of completely disrupting the RUNX2 locus in mice provided compelling and conclusive evidence for the biological importance of RUNX2 where knockout mice died shortly after birth with a complete lack of bone formation (Komori et al, 1997; Otto et al, 1997). A further indication of the requisite role of RUNX2 in skeletal development was the discovery that RUNX2 haploinsufficiency in humans and mice caused the skeletal syndrome Cleidocranial Dysplasia (CCD) (Mundlos et al, 1997; Lee et al, 1997). A unique feature of RUNX2 is the consecutive polyglutamine and polyalanine tracts (Q/A domain). Mutations causing CCD have been observed in the Q/A domain of RUNX2 (Mundlos et al, 1997). The Q/A domain is an essential part of RUNX2 and participates in transactivation function (Thirunavukkarasu et al, 1998). Previous genotyping studies conducted in our laboratory identified several rare RUNX2 Q/A variants in addition to a frequently occurring 18 base pair deletion of the polyalanine tract termed the 11Ala allele. Analysis of serum parameters in 78 Osteoarthritis patients revealed the 11Ala allele was associated with significantly decreased osteocalcin. Furthermore, analysis of 11Ala allele frequencies within a Geelong Osteoporosis Study (GOS) fracture cohort and an appropriate age matched control group revealed the 11Ala allele was significantly overrepresented in fracture cases indicating an association with increased fracture risk. To further investigate the 11Ala allele and rare Q/A variants, 747 DNA samples from the Southeast Queensland bone study were genotyped using PCR and PAGE. The experiment served two purposes: 1) to detect additional rare Q/A variants to enrich the population of already identified mutants and 2) have an independent assessment of the effect of the 11Ala allele on fracture to either support or refute our previous observation which indicated the 11Ala allele was associated with an increased risk of fracture in the GOS. From the 747 samples genotyped, 665 were WT, 76 were heterozygous for the 11Ala allele, 5 were homozygous for the 11Ala allele and 1 was heterozygous for a rare 21 bp deletion of the polyglutamine tract. Chi-square analysis of RUNX2 genotype distributions within fracture and non-fracture groups in the Southeast Queensland bone study revealed that individuals that carried at least one copy of the 11Ala allele were enriched in the fracture group (p = 0.16, OR = 1.712). The OR of 1.712 was of similar magnitude to the OR observed in the GOS case-control investigation (OR = 1.9) providing support for the original study. Monte-Carlo simulations were used to combine the results from the GOS and the Southeast Queensland bone study. The simulations were conducted with 10000 iterations and demonstrated that the maximum probability of obtaining both study results by chance was less than 5 times in two hundred (p < 0.025) suggesting that the 11Ala allele of RUNX2 was associated with an increased fracture risk. The second element of the research involved the analysis of rare RUNX2 Q/A variants identified from multiple epidemiological studies of bone. Q/A repeat variants were derived from four populations: the GOS, an Aberdeen cohort, CAIFOS and a Sydney twin study. Collectively, a total of 20 rare glutamine and one alanine variants were identified from 4361 subjects. All RUNX2 Q/A variants were heterozygous for a mutant allele and a wild type allele. Analysis of incident fracture during a five year follow up period in the CAIFOS revealed that Q-variants (n = 8) were significantly more likely to have fractured compared to non-carriers (p = 0.026, OR 4.932 95% CI 1.2 to 20.1). Bone density data as measured by quantitative ultrasound was available for CAIFOS. Analysis of BUA and SOS Z-scores revealed that Q-repeat variants had significantly lower BUA (p = 0.031, mean Z-score of -0.79) and a trend for lower SOS (p = 0.190, mean Z-score of -0.69). BMD data was available for all four populations. To normalize the data across the four studies, FN BMD data was converted into Z-scores and the effect of the Q/A variants on BMD was analysed using a one sample approach. The analysis revealed Q/A variants had significantly lower FN BMD (p = 0.0003) presenting with a 0.65 SD decrease. Quantitative transactivation analysis was conducted on RUNX2 proteins harbouring rare glutamine mutations and the 11Ala allele. RUNX2 proteins containing a glutamine deletion (16Q), a glutamine insertion (30Q) and the 11Ala allele were overexpressed in NIH3T3 and HEK293 cells and their ability to transactivate a known target promoter was assessed. The 16Q and 30Q had significantly decreased reporter activity compared to WT in NIH3T3 cells (p = 0.002 and 0.016, for 16Q and 30Q, respectively). In contrast 11Ala RUNX2 did not show significantly different promoter activation potential (p = 0.54). Similar results were obtained in HEK293 cells where both the 16Q and 30Q RUNX2 displayed decreased reporter activity (p=0.007 and 0.066 for 16Q and 30Q respectively) whereas the 11Ala allele had no material effect on RUNX2 function (p = 0.20). The RUNX2 gene target reporter assay provided evidence to suggest that variation within the glutamine tract of RUNX2 was capable of altering the ability of RUNX2 to activate a known target promoter. In contrast, the 11Ala allele showed no variation in RUNX2 activity. The third feature of the research served the purpose of identifying potential RUNX2 gene targets with particular emphasis on discovering genes cooperatively regulated by RUNX2 and the powerful bone promoting agent BMP2. The experiment was conducted by creating stably transfected NIH3T3 cells lines overexpressing RUNX2 or BMP2 or both RUNX2 and BMP2. Microarray analysis revealed very few genes were differentially regulated between standard NIH3T3 cells and cells overexpressing RUNX2. The results were confirmed via RT-PCR analysis which demonstrated that the known RUNX2 gene targets Osteocalcin and Matrix Metalloproteinase-13 were modestly induced 2.5 fold (p = 0.00017) and 2.1 fold (p = 0.002) respectively in addition to identifying only two genes (IGF-II and SCYA11) that were differentially regulated greater than 10 fold. IGF-II and SYCA11 were significantly down-regulated 27.6 fold (p = 1.95 x 10-6) and 10.1 fold (p = 0.0002) respectively. The results provided support for the notion that RUNX2 on its own was not sufficient for optimal gene expression and required the presence of additional factors. To discover genes cooperatively regulated by RUNX2 and BMP2, microarray gene expression analysis was performed on standard NIH3T3 cells and NIH3T3 cells stably transfected with both RUNX2 and BMP2. Comparison of the gene expression profiles revealed the presence of a large number of differentially regulated genes. Four genes EHOX, CCL9, CSF2 and OSF-1 were chosen to be further characterized via RT-PCR. Sequential RT-PCR analysis on cDNA derived from control cells and cells stably transfected with either RUNX2, BMP2 or both RUNX2/BMP2 revealed that EHOX and CSF2 were cooperatively induced by RUNX2 and BMP2 whereas CCL9 and OSF-1 were suppressed by BMP2. The overexpression of both RUNX2 and BMP2 in NIH3T3 fibroblasts provided a powerful model upon which to discover potential RUNX2 gene targets and also identify genes synergistically regulated by BMP2 and RUNX2. The fourth element of the research investigated the role of RUNX2 in the ascorbic acid mediated induction of MMP-13 mRNA. The study was carried out using NIH3T3 cell lines stably transfected with BMP2, RUNX2 and both BMP2 and RUNX2. The cell lines were grown to confluence and subsequently cultured for a further 12 days in standard media or in media supplemented with AA. RT-PCR analysis was used to assess MMP-13 mRNA expression. The RT-PCR results demonstrated that AA was not sufficient for inducing MMP-13 mRNA in NIH3T3 cells. In contrast RUNX2 significantly induced MMP-13 levels 85 fold in the absence of AA (p = 0.0055) and upregulated MMP-13 mRNA levels 254 fold in the presence of AA (p = 0.0017). The results demonstrated that RUNX2 was essential for the AA mediated induction of MMP-13 mRNA in NIH3T3 cells. The effect of BMP2 on MMP-13 expression was also investigated. BMP2 induced MMP-13 mRNA transcripts a modest 3.8 fold in the presence of AA (p = 0.0027). When both RUNX2 and BMP2 were overexpressed in the presence of AA, MMP-13 mRNA levels were induced a massive 4026 fold (p = 8.7 x 10-4) compared to control cells. The investigation revealed that RUNX2 was an essential factor for the AA mediated induction of MMP-13 and that RUNX2 and BMP2 functionally cooperated to regulate MMP-13 mRNA levels.
9
Stephens, Alexandre. "Genetic and Functional Characterization of RUNX2". Thesis, Griffith University, 2007. http://hdl.handle.net/10072/365677.
Pełny tekst źródłaStreszczenie:
RUNX2 belongs to the RUNT domain family of transcription factors of which three have been identified in humans (RUNX1, RUNX2 and RUNX3). RUNX proteins are vital for metazoan development and participate in the regulation of cellular differentiation and cell cycle progression (Coffman, 2003). RUNX2 is required for proper bone formation by driving the differentiation of osteoblasts from mesenchymal progenitors during development (Ducy et al, 1997; Komori et al, 1997; Otto et al, 1997). RUNX2 is also vital for chondrocyte maturation by promoting the differentiation of chondrocytes to the hypertrophic phenotype (Enomoto et al, 2000). The consequences of completely disrupting the RUNX2 locus in mice provided compelling and conclusive evidence for the biological importance of RUNX2 where knockout mice died shortly after birth with a complete lack of bone formation (Komori et al, 1997; Otto et al, 1997). A further indication of the requisite role of RUNX2 in skeletal development was the discovery that RUNX2 haploinsufficiency in humans and mice caused the skeletal syndrome Cleidocranial Dysplasia (CCD) (Mundlos et al, 1997; Lee et al, 1997). A unique feature of RUNX2 is the consecutive polyglutamine and polyalanine tracts (Q/A domain). Mutations causing CCD have been observed in the Q/A domain of RUNX2 (Mundlos et al, 1997). The Q/A domain is an essential part of RUNX2 and participates in transactivation function (Thirunavukkarasu et al, 1998). Previous genotyping studies conducted in our laboratory identified several rare RUNX2 Q/A variants in addition to a frequently occurring 18 base pair deletion of the polyalanine tract termed the 11Ala allele. Analysis of serum parameters in 78 Osteoarthritis patients revealed the 11Ala allele was associated with significantly decreased osteocalcin. Furthermore, analysis of 11Ala allele frequencies within a Geelong Osteoporosis Study (GOS) fracture cohort and an appropriate age matched control group revealed the 11Ala allele was significantly overrepresented in fracture cases indicating an association with increased fracture risk. To further investigate the 11Ala allele and rare Q/A variants, 747 DNA samples from the Southeast Queensland bone study were genotyped using PCR and PAGE. The experiment served two purposes: 1) to detect additional rare Q/A variants to enrich the population of already identified mutants and 2) have an independent assessment of the effect of the 11Ala allele on fracture to either support or refute our previous observation which indicated the 11Ala allele was associated with an increased risk of fracture in the GOS. From the 747 samples genotyped, 665 were WT, 76 were heterozygous for the 11Ala allele, 5 were homozygous for the 11Ala allele and 1 was heterozygous for a rare 21 bp deletion of the polyglutamine tract. Chi-square analysis of RUNX2 genotype distributions within fracture and non-fracture groups in the Southeast Queensland bone study revealed that individuals that carried at least one copy of the 11Ala allele were enriched in the fracture group (p = 0.16, OR = 1.712). The OR of 1.712 was of similar magnitude to the OR observed in the GOS case-control investigation (OR = 1.9) providing support for the original study. Monte-Carlo simulations were used to combine the results from the GOS and the Southeast Queensland bone study. The simulations were conducted with 10000 iterations and demonstrated that the maximum probability of obtaining both study results by chance was less than 5 times in two hundred (p < 0.025) suggesting that the 11Ala allele of RUNX2 was associated with an increased fracture risk. The second element of the research involved the analysis of rare RUNX2 Q/A variants identified from multiple epidemiological studies of bone. Q/A repeat variants were derived from four populations: the GOS, an Aberdeen cohort, CAIFOS and a Sydney twin study. Collectively, a total of 20 rare glutamine and one alanine variants were identified from 4361 subjects. All RUNX2 Q/A variants were heterozygous for a mutant allele and a wild type allele. Analysis of incident fracture during a five year follow up period in the CAIFOS revealed that Q-variants (n = 8) were significantly more likely to have fractured compared to non-carriers (p = 0.026, OR 4.932 95% CI 1.2 to 20.1). Bone density data as measured by quantitative ultrasound was available for CAIFOS. Analysis of BUA and SOS Z-scores revealed that Q-repeat variants had significantly lower BUA (p = 0.031, mean Z-score of -0.79) and a trend for lower SOS (p = 0.190, mean Z-score of -0.69). BMD data was available for all four populations. To normalize the data across the four studies, FN BMD data was converted into Z-scores and the effect of the Q/A variants on BMD was analysed using a one sample approach. The analysis revealed Q/A variants had significantly lower FN BMD (p = 0.0003) presenting with a 0.65 SD decrease. Quantitative transactivation analysis was conducted on RUNX2 proteins harbouring rare glutamine mutations and the 11Ala allele. RUNX2 proteins containing a glutamine deletion (16Q), a glutamine insertion (30Q) and the 11Ala allele were overexpressed in NIH3T3 and HEK293 cells and their ability to transactivate a known target promoter was assessed. The 16Q and 30Q had significantly decreased reporter activity compared to WT in NIH3T3 cells (p = 0.002 and 0.016, for 16Q and 30Q, respectively). In contrast 11Ala RUNX2 did not show significantly different promoter activation potential (p = 0.54). Similar results were obtained in HEK293 cells where both the 16Q and 30Q RUNX2 displayed decreased reporter activity (p=0.007 and 0.066 for 16Q and 30Q respectively) whereas the 11Ala allele had no material effect on RUNX2 function (p = 0.20). The RUNX2 gene target reporter assay provided evidence to suggest that variation within the glutamine tract of RUNX2 was capable of altering the ability of RUNX2 to activate a known target promoter. In contrast, the 11Ala allele showed no variation in RUNX2 activity. The third feature of the research served the purpose of identifying potential RUNX2 gene targets with particular emphasis on discovering genes cooperatively regulated by RUNX2 and the powerful bone promoting agent BMP2. The experiment was conducted by creating stably transfected NIH3T3 cells lines overexpressing RUNX2 or BMP2 or both RUNX2 and BMP2. Microarray analysis revealed very few genes were differentially regulated between standard NIH3T3 cells and cells overexpressing RUNX2. The results were confirmed via RT-PCR analysis which demonstrated that the known RUNX2 gene targets Osteocalcin and Matrix Metalloproteinase-13 were modestly induced 2.5 fold (p = 0.00017) and 2.1 fold (p = 0.002) respectively in addition to identifying only two genes (IGF-II and SCYA11) that were differentially regulated greater than 10 fold. IGF-II and SYCA11 were significantly down-regulated 27.6 fold (p = 1.95 x 10-6) and 10.1 fold (p = 0.0002) respectively. The results provided support for the notion that RUNX2 on its own was not sufficient for optimal gene expression and required the presence of additional factors. To discover genes cooperatively regulated by RUNX2 and BMP2, microarray gene expression analysis was performed on standard NIH3T3 cells and NIH3T3 cells stably transfected with both RUNX2 and BMP2. Comparison of the gene expression profiles revealed the presence of a large number of differentially regulated genes. Four genes EHOX, CCL9, CSF2 and OSF-1 were chosen to be further characterized via RT-PCR. Sequential RT-PCR analysis on cDNA derived from control cells and cells stably transfected with either RUNX2, BMP2 or both RUNX2/BMP2 revealed that EHOX and CSF2 were cooperatively induced by RUNX2 and BMP2 whereas CCL9 and OSF-1 were suppressed by BMP2. The overexpression of both RUNX2 and BMP2 in NIH3T3 fibroblasts provided a powerful model upon which to discover potential RUNX2 gene targets and also identify genes synergistically regulated by BMP2 and RUNX2. The fourth element of the research investigated the role of RUNX2 in the ascorbic acid mediated induction of MMP-13 mRNA. The study was carried out using NIH3T3 cell lines stably transfected with BMP2, RUNX2 and both BMP2 and RUNX2. The cell lines were grown to confluence and subsequently cultured for a further 12 days in standard media or in media supplemented with AA. RT-PCR analysis was used to assess MMP-13 mRNA expression. The RT-PCR results demonstrated that AA was not sufficient for inducing MMP-13 mRNA in NIH3T3 cells. In contrast RUNX2 significantly induced MMP-13 levels 85 fold in the absence of AA (p = 0.0055) and upregulated MMP-13 mRNA levels 254 fold in the presence of AA (p = 0.0017). The results demonstrated that RUNX2 was essential for the AA mediated induction of MMP-13 mRNA in NIH3T3 cells. The effect of BMP2 on MMP-13 expression was also investigated. BMP2 induced MMP-13 mRNA transcripts a modest 3.8 fold in the presence of AA (p = 0.0027). When both RUNX2 and BMP2 were overexpressed in the presence of AA, MMP-13 mRNA levels were induced a massive 4026 fold (p = 8.7 x 10-4) compared to control cells. The investigation revealed that RUNX2 was an essential factor for the AA mediated induction of MMP-13 and that RUNX2 and BMP2 functionally cooperated to regulate MMP-13 mRNA levels.Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Medical Science
Faculty of Health
Full Text
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Zhou, Gaoying, i 周高英. "Molecular characterization of chicken SOCS2 gene". Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2005. http://hub.hku.hk/bib/B31480263.
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Bech, Linda. "Genetic and phenotypic characterization of trypanosomas". Thesis, Mälardalen University, School of Sustainable Development of Society and Technology, 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:mdh:diva-6435.
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Trypanosoma theileri, of the subgenus Megatrypanum, a non-pathogenic cosmopolitan blood dwelling parasite of bovine. T. theileri can be cultured at room temperature in several culture media.
Blood samples were collected from deer's. To see if the blood was infected with trypanosomes it was cultivated in 2 ml sheep blood or cell cultivation medium DMEM with antibiotics.
Growth was detected by microscopy to see if there were any trypanosomes.
To determine the species of trypanosomes that was in the deer blood a DNA-preparation was done before a Polymerase Chain Reaction (PCR) could be done. With sequencing the trypanosomes where determined to be Trypanosoma theileri.
Different tests were made to see in what way the trypanosomes best were caught to the objective slides.
Forty samples of borrelia positive serum from forty different patients were tested with the fluorescent microscopy. Forty different samples from blood donors were tested the same way.
Blood samples from 16 different fissiped were taken and to see if they were infected with trypanosomes. Three different PCR's were done on the 16 blood samples.
A small test on human blood was also performed.
Protein identification by immunoblot with western blot and silver staining was done.
With the electron microscopy tests were done in the ordinary way and Critical Dry Point to see if both of the techniques worked.
Enzyme-Linked Immuno Sorbent Assay (ELISA) test were accomplished on two 96 well plates. The wells on the plates were diluted in different ways before they were processed.
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Prior, Heather Marie. "Genetic characterization of human SOX genes". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0029/NQ59655.pdf.
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Houston, Peter Louis. "Biochemical characterization of genetic recombination proteins /". Digital version accessible at:, 1998. http://wwwlib.umi.com/cr/utexas/main.
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Santos, Tânia Raquel Martins dos. "Genetic characterization of Portuguese Fasciola hepatica isolates". Master's thesis, Faculdade de Ciências e Tecnologia, 2012. http://hdl.handle.net/10362/8689.
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Dissertation presented to obtain the Master Degree in Molecular, Genetics and BiomedicinePart of the results discussed in this dissertation was presented in the following communications: R. Santos, M. Calado, J. Sampaio, C. Ferreira, A. Afonso and S. Belo. Contribution to the genetic characterization of Fasciola hepatica populations in Portugal. XXXVII Portuguese Genetic Conference, Lisbon, Portugal, May 28th-30th 2012 [poster communication] R. Santos, M. Calado, J. Sampaio, C. Ferreira, A. Afonso and S. Belo. Contribution to the genetic characterization of Fasciola hepatica populations in Portugal. Arquivos Portugueses das Ciências Biológicas. Tomo XXXVI (in press)
Fasciola hepatica is a parasitic trematode with debilitating and socio-economically devastating effects. At present near to 600 million animals and 2.4 million people in the entire world suffer from fascioliasis. Genetic characterization is of the utmost importance to an efficient epidemiologic control of helminth infections. In the present study we aimed to provide the first insights into the genetic variability of F. hepatica in Portugal. 47 isolates from different hosts (cattle and sheep) and geographical locations (Beja, Castelo Branco, Coimbra, Évora, Faro, Leiria, Lisboa, Portalegre, Santarém and Setúbal) were analyzed through Random Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR), Restriction Fragment Length Polymorphism (RFLP) and sequencing of NADH dehydrogenase subunit 1 (nad1) gene, cytochrome c oxidase subunit 1 (cox1) gene and Internal Transcribed Spacers (ITS) region. RAPD-PCR and RFLP patterns were similar for all the analyzed samples, despite their host and geographical origin. Nucleotide sequencing revealed low levels of genetic diversity within Portuguese isolates and no direct correlation was observed between haplotype and geographical location or host. Phylogenetic analysis revealed a high similarity within samples from Mediterranean countries, such as Portugal, Spain, Tunisia, Algeria and Egypt, possibly due to livestock import/export trade between these countries. Moreover, Portugal presents a low risk of fascioliasis drug-resistance.
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Hibbets, Eric Matthew. "Molecular Characterization of Hybridization Between Magellanic (Spheniscus magellanicus) and Humboldt (Spheniscus humboldti) Penguins in the Wild". Bowling Green State University / OhioLINK, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1562071641076803.
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Lam, Ho-yin, i 林灝賢. "The prevalence and genomic characterization of HIV-1 unique recombinant forms in Hong Kong". Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2010. http://hub.hku.hk/bib/B45719007.
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Delaney, Deborah A. "Genetic characterization of U.S. honey bee populations". Online access for everyone, 2008. http://www.dissertations.wsu.edu/Dissertations/Summer2008/d_delaney_070108.pdf.
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GUILLEN, JEFF MAYNARD. "STUDIES ON RESERVOIR CHARACTERIZATION VIA GENETIC PROGRAMMING". PONTIFÍCIA UNIVERSIDADE CATÓLICA DO RIO DE JANEIRO, 2015. http://www.maxwell.vrac.puc-rio.br/Busca_etds.php?strSecao=resultado&nrSeq=25771@1.
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PONTIFÍCIA UNIVERSIDADE CATÓLICA DO RIO DE JANEIROCOORDENAÇÃO DE APERFEIÇOAMENTO DO PESSOAL DE ENSINO SUPERIOR
CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO
PROGRAMA DE EXCELENCIA ACADEMICA
Na área de exploração e produção de petróleo são alocados grandes investimentos para conseguir diminuir os riscos associados à baixos níveis de produção, que podem ser minimizados mediante a acertada caracterização do reservatório de petróleo. Uma valiosa fonte de informação pode ser extraída de dados sísmicos 3D, obtidos do campo em estudo. O custo econômico de aquisição de esta base de dados para o reservatório completo é relativamente baixo, se comparado com uma amostragem direta por meio de perfurações de poços. Embora, a relação entre os dados sísmicos e as propriedades de reservatório seja considerada ambígua, esta deve ser integrada com informação confiável, como aquela obtida mediante perfilagem de poços. Fazendo uso dos abundantes dados sísmicos e das escassas, mas, precisas medições em perfurações existentes, foi desenvolvido neste trabalho um sistema baseado no algoritmo de Programação Genética (PG) para caracterizar geologicamente um reservatório de petróleo. PG é uma técnica de computação evolucionária capaz de estimar relações não lineares entre um conjunto de entrada e de saída, mediante uma expressão simbólica explícita. Para extrair informação adicional nos registros sísmicos são calculados atributos sísmicos, que facilitam a identificação de características estratigráficas ou estruturais do subsolo representadas indiretamente pela sísmica. Adicionalmente, é utilizado o método de inversão sísmica para o cálculo da impedância acústica, que é uma variável auxiliar derivada de sísmica calibrada com perfis de poço. Os atributos sísmicos junto com a impedância acústica servirão para a estimação de propriedades geológicas. Esta metodologia de trabalho foi testada em um reservatório real de grande complexidade geológica. Por meio de PG, foi representada satisfatoriamente a relação entre dados derivados da sísmica e a porosidade do campo, demostrando assim que PG é uma alternativa viável para a caracterização geológica de reservatórios. Posteriormente, foi realizada uma clusterização do campo baseada em características geofísicas que permitiram a construção de estimadores por PG especializados para cada zona.
In the field of oil exploration and production a great deal of investment is allocated in reducing the risks associated to low production levels that can be minimized through an accurate oil reservoir characterization. A valuable source of information can be extracted from 3D seismic data, obtained from the studied reservoir. The economic cost of the acquisition of this data base for the whole reservoir is relatively low, if compared to the direct sampling method of well drilling. Being that the relationship between seismic data and reservoir properties is considered ambiguous, it must be integrated with reliable information, such as that obtained by well logging. Making use of abundant seismic data and scarce, yet accurate, measurements from the existing drillings, it was developed in this study a system based in the algorithm of Genetic Programming (GP), to geologically characterize an oil reservoir. GP is an evolutionary computational technique capable of estimating the non-linear relationships between input and output parameter, through an explicit symbolic expression. In order to extract additional information from seismic records, seismic attributes are calculated, which facilitate tasks of identifying stratigraphic and structural characteristics of the subsurface, represented indirectly by seismic data. Moreover, a seismic inversion method is used to estimate the acoustic impedance, an auxiliary variable derived from seismic data calibrated by well logs. The seismic attributes along with the acoustic impedance will be used to estimate geological properties. This workflow was tested on a real reservoir, thus presenting geological complexity. Through GP, the relationship between seismic derived data and the field porosity was represented satisfactorily, demonstrating that GP is a viable alternative for geologic reservoir characterization. Afterwards, the reservoir was divided in clusters according to geophysical properties, this allowed the construction of GP based estimators for each zone.
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Dilawari, Mridull. "Genetic Characterization of Dormancy in Durum Wheat". Diss., North Dakota State University, 2012. https://hdl.handle.net/10365/26476.
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Two populations derived by crossing LDN x LDN Dic-3A (Population I) and LDN x LDN Dic-3B (Population II) were genetically characterized for the seed dormancy present on chromosome 3A and 3B of durum wheat. The genes for seed dormancy in these two populations were contributed by the wild parent T. dicoccoides. Although the populations showed transgressive segregants for both dormant as well as nondormant parent, the populations were similar to the dormant parent at Langdon and Prosper 2006 field locations for Population I and at Langdon 2007 and Autumn greenhouse season for Population II. Genotypic and phenotypic analysis over the combined populations showed an environmental effect on expression of the trait. Different QTL were identified for both field and greenhouse season for the population derived from the cross between LDN x LDN Dic-3A. Five QTL for seed dormancy were identified on chromosome 3A for the QTL analysis performed over combined field locations. One QTL ranging between marker interval Xcfa2193 and Xcfd2a was consistently present for the 30 day period of seed germination and was also found to be linked to red grain color trait. The QTL analysis performed on the population derived from the cross between LDN x LDN Dic-3B identified only one major QTL on the long arm of chromosome 3B between the marker interval Xbarc84 and Xwmc291. This QTL was consistently present for all the field and spring greenhouse season for the seed germination period of 30 days. The QTL x E effect was also observed for this QTL, however it was very small.
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Zulu, Dackson Nkonje. "Genetic Characterization of Zambian Native Cattle Breeds". Thesis, Virginia Tech, 2008. http://hdl.handle.net/10919/35210.
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Breed characterization is a primary step in designing appropriate management and conservation programs of livestock in developing countries. Since cattle represent a major food animal species in Zambia, its conservation is a major goal for both the government and non-governmental organizations. To support the conservation effort, the objective of this thesis research was to assess the phenotypic and molecular characteristics of indigenous Zambian cattle breeds including Angoni, Barotse, Tonga, and Baila based on body measurements and randomly amplified polymorphic DNA (RAPD) markers, respectively. A total of 100 animals, 25 from each of the four breeds associated with different tribes and region of Zambia, were used in the molecular analysis research. Additionally, 10 Holstein x Jersey crossbred animals were used as a reference and to test the extent of cross-breeding, if any, of the indigenous stock with exotic breeds. To further compare the Zambian indigenous breeds, morphometric measurements including body length, heart girth, and height at withers on 50 animals of each breed were measured. Blood was collected from animals at randomly selected farms and DNA isolated by standard protocols in Zambia. A total of 10 primers, of the 20 evaluated for informativeness, were used in the RAPD-PCR analyses. Differences among the four breeds for all the three morphometric measurements were significant with the Barotse significantly higher than the other three (P<0.05). The average number of bands per primer was 7.1 and the percentage of polymorphic bands per primer ranged from 40 to 71.4 with an average of 64.8%. Breed divergence was highest between the Tonga and the Barotse and lowest between the Tonga and Baila breeds. Both the morphometric measurements and RAPD-based distance estimates suggest that the Barotse may be different from the other indigenous breeds while the Tonga and Baila were more closely related. In addition, the genetic distance estimates imply that the Holstein x Jersey crosses are different from the four Zambian indigenous cattle breeds evaluated. This thesis research provides, for the first time, the basic genetic information necessary for conservation of Zambian cattle breeds and the use of these populations for effective crossbreeding. The data suggest that though there is isolated by geographic distance and cultural differences among the tribes, two of the breeds are significantly related.Master of Science
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Marques, Vanessa Alexandra Freire. "Genetic and epigenetic characterization of laryngeal carcinoma". Master's thesis, Universidade de Aveiro, 2015. http://hdl.handle.net/10773/15016.
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Mestrado em Biomedicina MolecularLaryngeal carcinomas belong to a bigger family of tumours known as Head and Neck Cancer (HNC). HNC is the sixth most malignant type of cancer in the world and it can arise from several anatomical sites. Among them, the larynx is the second most common affect organ. The incidence of laryngeal carcinoma is 1,9% worldwide and it presents a high mortality rate (1,6%). Despite technological advances in diagnosis and treatment fields, the 5 year-survival rate did not improved significantly. The low survival rates are mainly explained by a late diagnosis, tumour aggressiveness and the fact that laryngeal carcinoma metastasize easily. Taking this into consideration, it is essential to identify biomarkers with significant diagnostic and prognostic value in order to anticipate the detection of laryngeal carcinoma in an early stage. This study arises mainly for characterize the genetic and epigenetic profile of laryngeal squamous cell carcinoma (LSCC). Eight LSCC samples and seven non-tumour samples contralateral to the primary tumour were collected upon resection surgery and characterized by MLPA, MS-MLPA and array CGH. The results showed that gain of genetic material was mainly present in chromosomes 3q, 8q, 11q, 14q13.1, Xp22.31 and Xq21.1 while genetic loss occurred mainly in chromosomes 3p, 9p23.1 and Y. Gain of MYC and TNFRSF1A was the most common event among the tumour samples included in this study. Regarding the methylation profile, the genes CDKN2A, CHFR, RARβ e RASSF1 were the only ones which were methylated in this samples. In conclusion, this study allowed to identify genetic alterations associated with LSCC that have already been reported in scientific papers as well as alterations that have been associated with tumour development and progression. In addition, a few genetic alterations which have never been reported as being associated with human cancer before were identified. Nevertheless, new studies must be carried out, with a higher number of samples. Ultimately, the main goal would be to identify genetic alterations significantly associated with LSCC progression and establish a correlation with clinicopathological data.
O carcinoma da laringe pertence a uma grande família de tumores conhecida como Cancro da Cabeça e do Pescoço que é considerado o sexto tipo de tumor mais maligno em todo o mundo. Dentro desta família, os tumores podem ter origem em diversos locais anatómicos, sendo a laringe o segundo órgão mais comummente afetado. O cancro da laringe apresenta uma incidência mundial de 1,9% e uma taxa de mortalidade elevada de 1,6%. Apesar dos avanços tecnológicos na área do diagnóstico e da terapêutica, a taxa de sobrevivência ao fim de 5 anos não apresentou melhorias significativas. As baixas taxas de sobrevivências são explicadas essencialmente pelo diagnóstico tardio, pela agressividade do tumor e pela sua propensão a desenvolver metástases. Desta forma, torna-se essencial a identificação de biomarcadores com valor de diagnóstico e prognóstico a fim de detetar a presença do tumor numa fase mais precoce. Este estudo surge com o objetivo principal de caracterizar o perfil genético e epigenéticos do carcinoma das células escamosas da laringe com recurso às técnicas de MLPA, MS-MLPA e array CGH, usando oito amostras tumorais e sete amostras não-tumorais contra laterais ao tumor, ambas coletadas após cirurgia A análise genética revelou uma maior taxa de ganho de material genético nos cromossomas 3q, 8q, 11q, 14q13.1, Xp22.31, Xq21.1 e perda de material genético nos cromossomas 3p, 9p23.1 e Y. O ganho dos genes MYC e TNFRSF1A revelou ser o evento mais comum entre as amostras analisadas. Relativamente ao perfil epigenético, observou-se que os genes CDKN2A, CHFR, RARβ e RASSF1 se encontravam metilados nas amostras em estudo. Em suma, este trabalho permitiu identificar algumas alterações genéticas e epigenéticas descritas na literatura como estando associadas ao CCEL, assim como alterações associadas ao desenvolvimento tumoral. Foram ainda identificadas alterações que ainda não foram reportadas como estando associadas ao cancro. Desta forma, este estudo piloto permitiu dar início ao estudo de potenciais biomarcadores associados ao CCEL. Porém, novos estudos devem ser realizados, com um número de amostras superior, de forma a identificar alterações genéticas significativas no desenvolvimento e progressão do CCEL e associa-las às características clinico patológicas dos doentes.
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Kim, Eejung. "Functional characterization of genetic alterations in cancer". Thesis, Harvard University, 2016. http://nrs.harvard.edu/urn-3:HUL.InstRepos:33493591.
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The comprehensive identification of genetic alterations is critical to understanding the pathophysiology of cancer. Recent advances in sequencing technology have enabled the detailed description of cancer genomes. However, to translate these findings into a deeper understanding of cancer biology, analyzing the functional impact of cancer-associated genetic aberration is essential. Here I investigate how to accelerate the functional characterization of two classes of genetic alterations, point mutations and amplifications.
The wide spectrum of point mutations that arise in cancer makes them challenging to study comprehensively. I have developed a scalable systematic method to experimentally infer the functional impact of cancer-associated gene variants. I performed pooled in vivo tumor formation assays and gene expression profiling using 474 mutant alleles curated from 5,338 human tumors. I identified 12 transforming alleles including two in genes (PIK3CB, POT1) that have not been previously shown to be tumorigenic. One rare KRAS allele, D33E, displayed tumorigenicity and constitutive activation of RAS effector pathways. By correlating gene expression changes induced upon expression of wild type and mutant alleles, I could infer the activity of specific alleles. These approaches enable the interrogation of cancer-associated alleles at scale and demonstrate that rare alleles may be functionally important.
Frequently amplified regions in cancer often harbor oncogenic drivers. However, identifying the driver gene among many other amplified genes is challenging. In high-grade serous ovarian cancer (HGSOC), 1,825 genes are amplified across 63 amplicons. We employed systematic loss-of-function RNAi data to identify amplified genes that were essential in the ovarian lineage. We identified 50 amplified and essential genes and validated FRS2, an adaptor protein in FGFR pathway. FRS2-amplified cancer cell lines were dependent on FRS2 expression and FRS2 overexpression in immortalized cell lines was sufficient to promote anchorage independent growth and tumorigenesis in nude mice. This approach demonstrates that intersecting structural genomics with functional genomics can facilitate the discovery of driver genes in recurrently amplified regions. Collectively, the methods I present here provide a framework to study point mutations and amplifications to accelerate the interpretation of the cancer genome.Medical Sciences
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Haußig, Joana. "Genetic characterization of Plasmodium berghei apicoplast proteins". Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2013. http://dx.doi.org/10.18452/16808.
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Malaria wird durch den einzelligen Parasiten Plasmodium verursacht. Hierbei handelt es sich um einen obligat intrazellulären, eukaryotischen Erreger, der zum Phylum der Apicomplexa gehört. Apicomplexa zeichnen sich durch das einzigartige Vorhandensein eines ungewöhnlichen Plastids, genannt Apicoplast, aus. Die Exklusivität dieser Organelle und ihre metabolische Notwendigkeit für das Parasitenwachstum haben sie als attraktives pharmakologisches Ziel bestätigt. In dieser Arbeit wurden, unter Anwendung des Nagetier-Malariaerregers Plasmodium berghei, zwei verschiedene Aspekte von Apicoplast Proteinfunktionen untersucht. Zum Ersten wurde ein bislang unbeschriebenes Plasmodium Apicoplast Protein, Plasmodium-specific Apicoplast protein important for Liver Merozoite formation (PALM), charakterisiert. Drei voneinander unabhängige palm— Parasitenlinien, wurden durch zielgerichtete Gendeletion generiert. Die PALM Knockout-Mutanten entwickelten sich während eines Großteils des Lebenszyklus normal, jedoch war die Abgabe von Merozoiten in den Blutstrom und die Fähigkeit eine Blutstadien-Infektion zu etablieren signifikant beeinträchtigt. Experimentelle Immunisierung von Mäusen mit palm— Sporozoiten bewirkte einen starken und langanhaltenden Schutz gegen Reinfektion mit Malaria. Diese Ergebnisse lassen darauf schließen, dass Parasiten mit einem Arrest in den finalen Schritten der Bildung von Leberstadien-Merozoiten einen Vorteil gegenüber genetisch attenuierten Parasiten der ersten Generation haben, die in der frühen Leberstadienentwicklung arretiert sind. Zum Zweiten wurden die sechs Nucleus-kodierten Komponenten der [Fe-S] Cluster Biosynthese im Apicoplast systematisch durch experimentelle Genetik analysiert. Insgesamt zeigen meine Studien, dass bisher unbekannte Ziele im Plasmodium Apicoplast für Interventionsstrategien gegen Malaria geeignet sind.Malaria is caused by Plasmodium, an obligate intracellular eukaryotic pathogen that belongs to the phylum Apicomplexa. Apicomplexan parasites harbor an unusual plastid organelle, termed apicoplast. Because this unique organelle is indispensable for parasite growth it is a validated and attractive drug target. Using the rodent malaria parasite Plasmodium berghei, two different aspects of apicoplast protein functions were analyzed in this study. Firstly, a previously uncharacterized Plasmodium apicoplast protein, Plasmodium-specific Apicoplast protein important for Liver Merozoite formation (PALM), was investigated. Three independent palm— knockout parasite lines were generated by targeted gene deletion. While the resulting knockout mutants developed normally for most of the life cycle, merozoite release into the blood stream and the ability to establish an infection was severely impaired. Experimental immunization of mice with palm— sporozoites elicited unprecedented potent and long-lasting protection against malaria re-infection. The results indicate that a tailor-made arrest in the final steps of hepatic merozoite formation could be an improvement over first-generation early liver-stage genetically arrested parasites (GAPs). Secondly, the six nuclear-encoded components of the apicoplast [Fe-S] cluster biosynthesis pathway were systematically targeted by experimental genetics. Together, my studies show that the Plasmodium apicoplast harbors previously unrecognized targets for anti-malaria intervention strategies.
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Labreche, Karim. "Genetic Susceptibility and Molecular Characterization of Glioma". Thesis, Université Paris-Saclay (ComUE), 2018. http://www.theses.fr/2018SACLS161/document.
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Les gliomes constituent les plus fréquentes des tumeurs malignes primaires du système nerveux central. Les liens qui existent entre ces tumeurs et un certain nombre de cancers rares héréditaires, comme les Neurofibromatoses I et II ou les syndromes de Turcot et de Li-Fraumeni, attestent d’une prédisposition génétique aux gliomes. L’observation d’un risque deux fois plus élevé de développer un gliome chez les parents de premier degré de patients atteints suggère aussi une possible prédisposition génétique dans les gliomes sporadiques. Par ailleurs, l’analyse à haut débit permet de préciser le profil somatique des gliomes et d’identifier des biomarqueurs pronostiques voire prédictifs et s’inscrire dans une démarche de traitement personnalisé du patient. Durant ma thèse, je me suis focalisé sur deux axes de recherches complémentaires; l’identification de gènes de susceptibilité et la découverte de nouveaux gènes fréquemment mutés dans les gliomes, afin de déterminer les voies de signalisation contribuant à la gliomagenèse. Dans leur ensemble, les résultats obtenus dans cette thèse apportent non seulement des informations importantes sur la nature de la prédisposition génétique aux gliomes mais également de son association spécifique pour les différents sous-types de tumeurs. La découverte d’un nouveau gène muté, offre la perspective à plus long terme d’un traitement personnalisé pour chaque patient sur la base du profil génétique de sa tumeurGliomas are the most common adult malignant primary tumour of the central nervous system. Thus far, no environmental exposures has been linked to risk except for ionizing radiation, which only accounts for a very small number of cases. Direct evidence for inherited predisposition to glioma is provided by a number of rare inherited cancer syndromes, such as Turcot's and Li–Fraumeni syndromes, and neurofibromatosis. Even collectively, these diseases however account for little of the twofold increased risk of glioma seen in first-degree relatives of glioma patients. My research was centred on two complementary research activities: Identifying susceptibility genes for glioma to delineate key biological pathways contributing to disease pathogenesis and to identify new recurrent mutated genes for glioma to provide for further insights into glial oncogenesis and suggesting targets for novel therapeutic strategies. Collectively the findings in this thesis provide increased insight into the nature of genetic predisposition to glioma and substantiate the often distinct associations between susceptibility variants and glioma molecular groups. In addition the discovery of a new mutated gene in glioma offers the potential to support drug development and advance precision medicine for this tumours
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Tarlarini, C. "MOLECULAR AND GENETIC CHARACTERIZATION OF ALS PATIENTS". Doctoral thesis, Università degli Studi di Milano, 2014. http://hdl.handle.net/2434/232574.
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Amyotrophic lateral sclerosis (ALS) is an adult‑onset, rapidly progressive and ultimately fatal
neurodegenerative disorder characterised by degeneration of upper and lower motor neurons.
This leads to weakness, muscular atrophy and spasticity, which relentlessly progress to complete
paralysis with a low survival rate beyond 5 years from symptom onset. In 10% of cases the
disease is considered to be genetically transmitted (FALS) while in the remaining cases it occurs
sporadically in the population (SALS). To date cases of hereditary ALS have been attributed to
mutations in more than 16 different genes, the most common being SOD1, FUS, TARDBP and
C9ORF72; mutations in other genes are rare. The above genes explain 60% of the cases of
familial ALS and 15% of sporadic cases.
The disease can be subdivided into bulbar (25%) and spinal‑onset (75‑80%) forms. Nevertheless,
it is currently recognized that pathological changes are not limited to the motor system: patients
with ALS may exhibit cognitive abnormalities ranging from impaired frontal executive dysfunction to
overt frontotemporal dementia (FTD).
In spite of the above evidence, ALS is regarded as a complex disease in which multiple
environmental and genetic risk factors contribute to disease susceptibility. Furthermore it is
possible that the phenotypic variability, which is frequently detected within families, could be due to
multiple genetic factors as devised in the oligogenic disease model.
In the present study we analysed 285 SALS and 17 FALS cases. Globally, our molecular analysis
explained 10.3% of all ALS cases (31/302). The genes screened were SOD1, TARDBP, FUS and
C9ORF72. Genomic DNA was extracted from peripheral blood through a salting out procedure;
coding regions and exon‑intron boundaries of SOD1 (5 exons), TARDBP (1 exon), and FUS
(5 exons) genes were amplified from genomic DNA and sequenced. Otherwise the repeat‑primed
PCR assay, used for C9ORF72 gene, was performed in order to screen for the presence of the
GGGGCC hexanucleotide repeats expansion in ALS patients. The repeat unit of 6 nucleotides
expands up to more than several hundred times in the affected individuals. Fewer than 10 repeats
are not associated with a pathological phenotype, while more than 30 repeats are associated.
However we still do not know the meaning of intermediate repeat sizes (11‑29). This fact
complicates the attribution of the size expansion to the pathological phenotype observed.
According to the oligogenic disease model, all patients were screened for the most common
ALS‑associated genes and all mutated subjects were tested also for ANG. The affected/unaffected
family members, when available for the study, were screened for SOD1, TARDBP, FUS and
C9ORF72 in order to identify their genetic difference from the proband and to evaluate if this
difference could explain an heterogeneous phenotypic expression of the disease.
Following these analyses we selected and described in detail 5 FALS and 2 SALS cases and
one SETX case, with different intrafamilial phenotypic expression. In one of our SOD1 mutation
carriers, the proband manifested the disease after the delivery; together with a specific angiogenin
genetic variant this condition seems to have anticipated the age of disease onset and contributed
to the aggressive clinical course observed in the proband compared to other family members.
We also found one case in which we observed the phenomenon of anticipation, which could be
due to hormonal treatments together with the simultaneous presence of 2 mutations (C9ORF72/
TARDBP), as suggested in the oligogenic disease model. Indeed, the neuroprotective effects of
estrogens could account for the later age at onset in women as we have tested in another family
harbouring the R521C mutation. In this case a male subject developed the disease, whereas his
older sister didn’t show any neurological signs. In another family we observed a wide spectrum
of clinical symptoms associated with A382T TARDBP mutation. Some family members showed
cognitive impairment without signs of ALS, conversely, other relatives showed a typical ALS without
any signs of dementia. In addition we have evidence of TARDBP mutation carriers without a
neurological phenotype. Surprisingly, two subjects harbouring the mutation in homozygous state
display different spectra of clinical symptoms. The screening of SOD1, FUS, C9ORF72 and ANG
in the family members, to identify other mutations that could explain this intrafamilial phenotypic
heterogeneity, resulted negative; indicating that other genes/conditions could play a role in
ALS disease.
These data support the hypothesis that phenotypic variability seen in patients carrying the same
mutation, could be attributed to additional genetic and/or environmental factors, which could
modify the penetrance of the disease. Furthermore we identified a clinical subgroup of patients
that develop the disease during pregnancy (child-bearing age), thus indicating an additional
modifier conditions linked to hormonal changes. For all these reasons predictive or presymptomatic
testing should be undertaken with caution, especially in unaffected family members. Indeed, it is
impossible for the genetic test to predict clinical outcomes based on the genotypic results only.
A number of uncertainties remain, due to variability in penetrance, expressivity, and modifying
factors, such as gene‑gene interactions and environmental influences. This adds to the debate for
the ethical and psychological dilemmas about genetic testing, since still more has to be discovered
related to this devastating disease.
26
Zhang, Ling 1962. "Molecular cloning and characterization of the chicken ornithine decarboxylase gene". Thesis, McGill University, 1994. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=22831.
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Ornithine decarboxylase (ODC) is the rate determining enzyme in the biosynthesis of polyamines which are essential for cell growth. In chickens, significantly higher bioactivity is reported in broiler than in egg layer strains of chickens (Bulfield et al., 1988). To characterize the genetic differences in growth rates and ODC levels in chickens, an ODC cDNA and genomic gene were cloned and sequenced. Sequencing of ODC cDNA revealed that this clone (pODZ3: 2,052 bp) was not a full length of ODC cDNA and contained 2 putative introns. The open reading frame (introns deleted) coded for a protein of 404 amino acids which had about 85% amino acid identity with human ODC. Sequencing of genomic ODC clone (pODG2-8: 5098 bp) represented the 3$ prime$ end of ODC gene from downstream of intron 7. Northern blotting of chicken RNA probed with the insert of pODZ3 revealed 2 hybridizable messages of 1.6 and 2.1 kb, respectively. In addition, analysis of MspI restriction fragment length polymorphism (RFLP) using the 3$ prime$ end of ODC gene as a probe suggested that two MspI RFLPs present in the lean line of broiler chickens was related to selection of high lean body mass.
27
葉志遠 i Chi-yuen Ip. "Characterization of the 5'flanking transcriptional regulation region of the chicken growth hormone gene". Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2001. http://hub.hku.hk/bib/B31226115.
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Lau, Suk-ling Joanna, i 劉淑玲. "Molecular characterization of the chicken growth hormone receptorgene". Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2005. http://hub.hku.hk/bib/B45015430.
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García, Eusebi Paulina. "Genetic characterization of the Mexican Bovine Lidia Breed". Doctoral thesis, Universitat Autònoma de Barcelona, 2018. http://hdl.handle.net/10803/666836.
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El de la raza de Lidia ha sido seleccionado durante siglos por caracteres relacionados al comportamiento, una peculiaridad que la distingue del resto de las razas vacunas, principalmente seleccionadas por características de interés productivo, como carne y leche. En España, la población de Lidia originaria ha sido estudiada por medio de información genómica, permitiendo conocer que la riqueza genética de ésta raza se debe al aporte proporcionado por cada uno de los múltiples encastes o linajes en los que se subdivide. En México la raza de Lidia representa un legado histórico y cultural importante y actualmente, su población no ha sido caracterizada genéticamente.
En esta tesis analizamos la diversidad y estructura genética de la población Mexicana y la comparamos con información proveniente de la población originaria Española utilizando información genómica mediante diferentes tipos de marcadores moleculares.
Primero analizamos los parámetros de diversidad genética en ambas poblaciones con marcadores autosómicos de tipo Microsatélite y Polimorfismos de nucleótido único, encontrando valores similares de heterocigosis esperada con ambos tipos de marcadores moleculares. Encontramos también valores elevados en términos de FIS en ambas poblaciones. Tanto los valores elevados de FIS en los encastes así como el comportamiento que presentan las Carreras de Homocigosis son consecuencia del bajo censo de los encastes, contribuyendo por ende a incrementar la tasa de endogamia. También encontramos una alta diferenciación genética entre poblaciones con ambos tipos marcadores moleculares; microsatélites y SNPs. La partición de la variabilidad genética total analizada con SNPs mostró que el 19% de la variación se explica por las diferencias genéticas entre linajes. Curiosamente, la estructura genética de la población mexicana reveló que comparte escasos orígenes genéticos en común con la población originaria española, ubicando a ambas poblaciones en grupos diferentes.
El análisis de cromosoma Y mostró que la Casta Navarra ha dejado huella paterna en la población mexicana mediante una frecuencia elevada en el haplotipo H6, exclusivo de ésta casta así como del encaste de Miura. Los análisis de ADN mitocondrial, por otro lado, revelaron patrones de haplotipos similares en ambas poblaciones.
Por último, considerando la peculiaridad en la selección de esta raza, realizamos un análisis para detectar huellas de selección que pudieran afectar caracteres asociados a comportamiento de tipo agonista, utilizando dos razas mansas españolas como referencia. Utilizando dos métodos que se basan en inferencias bayesianas, identificamos en común dos regiones genómicas seleccionadas. A demás, la dirección e intensidad en la frecuencia del alelo seleccionado en la raza de Lidia es opuesto a los de las razas mansas. En éstas regiones detectamos genes asociados a rutas metabólicas como las de la serotonina y la dopamina, así como genes expresados en corteza cerebral, los cuáles han sido relacionados con patrones de comportamiento agresivo en humanos y animales de laboratorio.The cattle of the Lidia breed have been selected during centuries for behavioral related traits, a peculiarity that distinguishes it from the rest of the bovine breeds, selected mostly for characteristics of productive interest, such as meat and milk. In Spain, the original Lidia population has been studied through genomic data, allowing to know that the genetic richness of the breed is owed to the contribution of each of the multiple lineages or encastes in which it is subdivided. In Mexico, the Lidia breed represents an important historical and cultural legacy and currently, its population has not been genetically characterized. In this thesis we analyze the genetic diversity and structure of the Mexican population and compared it with data from the original Spanish population by using genomic information derived from different types of molecular markers. First, we analyzed parameters of genetic diversity in both populations using Microsatellite and Single Nucleotide Polymorphisms autosomal markers, finding similar values of expected heterozygosities with both types of molecular markers. We found also high values in terms of FIS in both populations. Both, the high values of FIS in the lineages and the behavior of the Runs of Homocigosity are a consequence of the lineages´ low census, contributing hence to increase the inbreeding rate. Furthermore, we detected high genetic differentiation between populations with both types of molecular markers: microsatellite and SNP, and the partition of the total genetic variability analyzed with SNPs showed that 19% of the variation is explained by the genetic differences among lineages within populations. Curiously, the genetic structure of the Mexican population revealed that it shares few common genetic origins with the original Spanish population, placing both populations in different groups. The Y chromosome analysis evidenced the paternal footprint that Casta Navarra has left in the Mexican population through a high frequency of the H6 Haplotype, exclusive of this lineage. Mitochondrial DNA analyzes, on the other hand, revealed similar haplotype patterns in both populations. Finally, considering the peculiarity of the selection performed in this breed, we carried out an analysis to detect signatures of selection that could affect agonistic behavioral related traits, using as a reference two tamed Spanish breeds. Using two methods based on Bayesian inferences, we jointly identified two selected genomic regions. Also, the direction and intensity in the frequency of the allele selected of the Lidia breed is opposite to that of the tame breeds. In these regions were detected genes associated to metabolic pathways such as serotonin and dopamine, as well as genes expressed in the brain cortex, which have been related to patterns of aggressive behavior in humans and laboratory animals.
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Sahlqvist, Anna-Stina. "Genetic Characterization of Chicken Models for Autoimmune Disease". Doctoral thesis, Uppsala universitet, Autoimmunitet, 2012. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-182843.
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Autoimmune diseases are endemic, but the disease mechanisms are poorly understood. A way to better understand these are to find disease-regulating genes. However, this is difficult as the diseases are complex, with several genes as well as environmental factors influencing the development of disease. A way to facilitate the search for genes responsible for the diseases is to use comparative genomic studies. Animal models are relatively easy to analyze since control of environment and breeding are obtained. The University of California at Davies – line 200 (UCD-200) chickens have a hereditary disease that is similar to systemic sclerosis. Using a backcross between UCD-200 chickens and red junglefowl (RJF) chickens we identified three loci linked to the disease. The loci contained immune-regulatory genes suggested to be involved in systemic sclerosis in humans, as well as a previously unidentified linkage between systemic sclerosis in UCD-200 chickens and IGFBP3. The Dark brown (Db) gene enhances red pheomelanin and restricts expression of eumelanin in chickens. The Db phenotype is regulated by an 8 kb deletion upstream of SOX10. Pigmentation studies are potentially useful when trying to identify pathogenic mechanisms and candidate genes in vitiligo The Obese strain (OS) of chickens spontaneously develops an autoimmune thyroiditis which closely resembles human Hashimoto’s thyroiditis. By using an intercross between OS chickens and RJF chickens, we found several disease phenotypes that can be used in an ongoing linkage analysis with the goal to find candidate genes for autoimmune disease. An important phenotype to record and add to the linkage analysis is autoantibodies against thyroid peroxidase, since this phenotype is a key feature in Hashimoto’s thyroiditis. Previous attempts to measure these titres in OS chickens have failed, hence an assay was developed for this purpose.
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Garrine, Carmen Maria Lucas Pedro. "Genetic characterization of indigenous goat populations of Mozambique". Diss., University of Pretoria, 2007. http://upetd.up.ac.za/thesis/available/etd-05082008-145341.
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Wallis), Adolfsen William W. (William. "Molecular genetic characterization of the Drosophila synaptotagmin family". Thesis, Massachusetts Institute of Technology, 2005. http://hdl.handle.net/1721.1/31189.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2005.Includes bibliographical references.
Proper functioning of the nervous system requires fast, spatially-restricted neuron- neuron communication at synapses. Classic physiology studies have demonstrated the importance of calcium in regulating synaptic communication; however the molecular events underlying basic synaptic transmission and plasticity have only recently become the subject of intense investigation in neuroscience. The synaptotagmin family of vesicle proteins has been implicated in calcium- dependent neurotransmitter release, although Synaptotagmin 1 (Syt 1) is the only isoform demonstrated to control synaptic vesicle fusion. We have characterized the six remaining synaptotagmin isoforms encoded in the Drosophila genome, including homologs of mammalian Synaptotagmins 4, 7, 12 and 14. Using immunolocalization and in situ hybridization experiments (Chapter 2), we demonstrate that each isoform has a unique subcellular localization and expression pattern, suggesting only Synaptotagmin 1 functions in synaptic vesicle exocytosis. Consistent with their distinct localizations, overexpression of Synaptotagmin 4 (Syt 4) or Synaptotagmin 7 (Syt 7) cannot functionally substitute for the loss of Syt 1 in synaptic transmission and loss-of-function mutations in Syts 4 and 7 do not have defects in neurotransmitter release (Chapter 4). Rather, Syt 4 and Syt 7 likely function in novel regulated-exocytosis pathways within neurons, distinct from synaptic vesicle cycling. The unique ability of Syt 1, but not other Syt isoforms, to localize to synaptic vesicles prompted us to determine the domains within Syt 1 responsible for its trafficking to synaptic vesicles (Chapter 3). We find the trafficking of Syt 1 is complex, likely requiring several sorting signals present at the N-terminus and in the C2 domains.
(cont.) The N-terminus was required for proper targeting to presynaptic terminals, while the C2 domains were essential for internalization at synapses. Furthermore, we demonstrate the C2 domains of Syts 4,7, [alpha] and [beta] can not promote localization to synaptic vesicles, even if mislocalized to presynaptic terminals, further arguing only Syt 1 is present on synaptic vesicles in vivo (Chapter 3). Like Synaptotagmin 1, Syt 4 is ubiquitously present at most, if not all synapses, but localizes to the postsynaptic compartment (Chapter 2). Syt 4 homologs have been identified in all metazoan genomes sequenced to date, suggesting this isoform may mediate an evolutionarily conserved role in postsynaptic vesicle trafficking. To elucidate the function of Syt 4-dependent postsynaptic vesicle trafficking we have generated and analyzed null mutations in syt 4. Although Syt 4 is not required for viability, embryonic neuromuscular junctions in mutant animals show a developmental delay in the formation of varicosities, a reduction in neurotransmitter release, and loss of a particular form of synaptic plasticity following high frequency stimulation, we have termed HFMR (High Frequency-induced Miniature Release). Postsynaptic expression of a syt4 transgene can rescue the presynaptic defects (Chapter 4), indicating Syt 4 mediates a retrograde signaling pathway at synapses. In addition, we demonstrate Syt 4 cycles through the postsynaptic plasma membrane (Chapter 4), suggesting it may regulate secretion of retrograde signals in a manner analogous to Syt 1 regulation of neurotransmitter release, presynaptically. There is mounting evidence in several experimental systems for a regulated form of postsynaptic vesicular trafficking.
(cont.) Dendritic release of a number of neuromodulators such as dopamine, ATP, GABA, and neuropeptides has been documented, while postsynaptic vesicles within dendritic spines and shafts have been directly visualized using electron microscopy. Studies in hippocampal culture neurons indicate that long-term labeling with FMI-43 loads dendritic organelles that undergo rapid calcium-triggered exocytosis. The localization and evolutionary conservation of Syt 4 makes it an attractive candidate for mediating a postsynaptic trafficking pathway common to all metazoan synapses. Indeed, localization studies of Syt 4 in mammals have noted the presence of the protein within dendrites and soma, similar to our studies in Drosophila. Utilizing the exceptional genetic tools available to Drosophila, we expect the characterization of Syt 4 and this novel retrograde signaling pathway will lead to new and exciting insights into the role of this protein family in fundamental synapse biology.
by William W. Adolfsen.
Ph.D.
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Whichard, Jean Marie. "Bacteriophage Felix O1: Genetic Characterization and Bioremedial Application". Diss., Virginia Tech, 2000. http://hdl.handle.net/10919/29591.
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Bacteriophage Felix O1 was studied for applicability as a Salmonella intervention. Felix O1's potential as a Salmonella therapeutic was explored, as was its utility as a food application. Felix O1 is specific for and infects most serovars within the genus Salmonella. The entire 86.155-kb sequence of the phage's linear, double-stranded chromosome was determined. 213 open reading frames (ORFs) were found, including 23 homologues of phage genes (e<0.008). Homology searches do not indicate genes that would be expected to increase virulence of Salmonella. Thirteen T4 homologues were found, including rIIA and rIIB, rapid lysis genes of T-even phages. Site-directed mutagenesis of the rIIB region was attempted by homologous recombination with plasmids containing luxAB of Vibrio harveyi. No DrIIB luxAB+ recombinants resulted from the methods tried.
Serial in vivo passage was used to select for a longer-circulating Felix O1 mutant using the modified methods of Merril et al., (1996). No difference was found in the clearance of wild-type (WT) and Felix O1 following nine serial passages. Injection of 109pfus yielded 24-hour concentrations of 6.5 and 4.9 log10 pfus/ml plasma for WT and 9th passage, respectively. Both isolates were undetectable in plasma by 72 hours, but remained in spleens at 96 hours.
A large-plaque Felix O1 variant (LP) isolated during in vivo serial passage was compared with WT for Salmonella growth suppression. Spectrophotometric measurement of BHI cultures indicated greater suppression of S. typhi by LP than by WT, a difference not seen with S. typhimurium DT104. Both isolates suppressed 24-hour S. typhimurium DT104 growth on experimentally-contaminated chicken frankfurters at 22°C. Untreated frankfurters yielded 6.81 log10 Salmonella cfus/g, whereas WT and LP-treated samples yielded 5.01 and 4.70 log10 cfus/g, respectively. Both phages suppressed the Salmonella typhimurium DT104 growth (p<0.0001), but the isolates did not perform differently (p=0.5088). Presence of Salmonella caused a higher yield of WT phage than from the uninoculated group (p=0.0011), but did not affect LP yield (p=0.4416). With Salmonella present, the 24-hour LP concentration was lower than WT concentration. This supports the surmised LP rapid-lysis phenotype since T4 rapid-lysis mutants typically exhibit lower burst sizes than wild-type phage.Ph. D.
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Schop, Roelandt François Johannes. "Studies on the genetic characterization of Waldenström macroglobulinemia". [S.l.] : Rotterdam : [The Author] ; Erasmus University [Host], 2008. http://hdl.handle.net/1765/13519.
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ANNUNZIATA, SILVIA. "Genetic and phenotypic characterization of Autism Spectrum Disorders". Doctoral thesis, Università degli studi di Pavia, 2022. http://hdl.handle.net/11571/1452943.
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SALSANO, ETTORE. "Clinical and Genetic Characterization of Leukoencephalopathies in Adults". Doctoral thesis, Università degli Studi di Milano-Bicocca, 2022. http://hdl.handle.net/10281/374739.
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Retroterra scientifico Negli adulti circa il 30-40% delle leucoencefalopatie (LKEN) (= malattie della sostanza bianca) non sono diagnosticati. I pazienti che restano non diagnosticati dopo indagini approfondite possono avare forme atipiche di malattie note, sia genetiche che acquisite, oppure nuove malattie che sono più probabilmente di origine genetica. Nel nostro lavoro abbiamo voluto esplorare l’efficienza di un approccio sistematico che include il sequenziamento del DNA con tecniche di nuova generazione (NGS) nella diagnosi di pazienti adulti con LKEN a causa ignota, e descrivere le loro caratteristiche cliniche.
Pazienti e Metodi In questo studio osservazionale analitico, abbiamo inizialmente revisionato le caratteristiche cliniche e paracliniche dei pazienti adulti (>=18 anni) con LKEN a causa ignota valutati nell’Unità di Malattie Neurodegenerative e Neurometaboliche Rare dell’Istituto Neurologico “C. Besta”, Milano, Italia, dal 2012 al 2018. Successivamente abbiamo usato un pannello genico per il sequenziamento di 142 geni associati a leucoencefalopatie ereditarie e, in un caso familiare rimasto non diagnosticato abbiamo studiato l’intero esoma.
Risultati Abbiamo identificato 57 adulti con LKEN a causa ignota (età media 43 anni, intervallo 18-72; 23 maschi, 53 con esordio tardo-adolescenziale o adulto). Trenta di loro, che abbiamo chiamato «ipomielinizzanti», presentavano un pattern di MRI suggestivo di ipomielinizzazione (lieve iperintensità in T2 e segnale T1 normale), mentre i restanti 27, che abbiamo chiamato «demielinizzanti», avevano un pattern di MRI suggestivo di demielinizzazione (marcata iperintensità in T2 e ipointensità in T1). In 13 soggetti ipomielinizzati il pannello genico ha identificato i seguenti geni: POLR3A (n = 2), POLR1C, TUBB4A, RARS1, GJA1, PLP1, GJC2, TBCD, CYP7B1, SPG11, PEX3, e PEX13, mentre in due altri pazienti lo studio dell’esoma ha portato all’identificazione di un nuovo gene malattia. Al contrario, il pannello genico ha permesso la diagnosi di un solo paziente demielinizzante (aciduria metilglutaconica associata al gene AUH). In due altri pazienti la diagnosi è stata posta o con l’analisi diretta di un singolo gene-malattia dopo revisione dei dati clinico-neuroradiologici (leucodistrofia metacromatica associata al gene PSAP) o con una biopsia di cute dopo la negatività del pannello genico (malattia da inclusi intranucleari neuronali, NIID). Tre pazienti (uno ipomielinizzante, due demielinizzanti) avevano malattie acquisite che mimavano una leucodistrofia: una vasculite primaria diagnostica con la biopsia cerebrale senza analisi genetiche e rare varianti di sclerosi multipla, diagnosticate dopo la negatività del pannello. Infine, in otto pazienti con una LKEN scoperta incidentalmente che sono rimasti senza manifestazioni cliniche per anni, il pannello genico ha dato esito negativo.
Conclusioni Negli adulti un pattern ipomielinizzante caratterizza un gran numero (circa il 50%) di LKEN a causa ignota. Le LKEN ipomielinizzanti ad esordio tardivo sono molto più comunemente dovute a geni associati alle forme precoci, come POLR3A e TUBB4A, o possono essere dovute a geni associati a paraplegie spastiche ereditarie, come CYP7B1 e SPG11, a geni associati a disordini perossisomiali, come PEX3 e PEX13, o anche a nuovi geni malattia. Fra le forme demielinizzanti solo molto poche sono diagnosticate con un pannello genico se i dati clinico-paraclinici non orientano verso una diagnosi specifica. Occasionalmente, varianti atipiche di malattie acquisite della sostanza bianca possono mimare una LKEN genetica con caratteristiche demielinizzanti o ipomielinizzanti alla risonanza. Infine, un sottogruppo di LKEN demielinizzanti caratterizzato dalla mancanza di manifestazioni cliniche e dall’assenza di mutazioni in geni associati a forme note di LKEN può costituire una nuova entità che abbiamo chiamato leucoencefalopatia diffusa subclinica.Background In adults, many cases (about 30-40%) of leukoencephalopathies (LKENs), i.e. white matter (WM) diseases, are without definitive diagnosis. Patients who remain undiagnosed despite extensive investigations may have atypical forms of known acquired or genetic diseases, or novel diseases more likely genetic in nature. Aims of our work were to explore the efficiency of a systematic approach, including next generation sequencing (NGS), in the diagnosis of a cohort of adult patients with LKEN of unknown cause, and to describe their clinical features. Patients and Methods In this analytical observation study, we first reviewed the clinical and laboratory features of the adult patients (age >= 18 years) with undiagnosed LKEN assessed at the Unit of Rare Neurodegenerative and Neurometabolic Diseases of the Istituto Neurologico “C. Besta”, Milan, Italy, from 2012 to 2018. A targeted-gene panel sequencing (TGPS) was subsequently used to investigate 142 genes responsible for genetic LKENs, and a whole-exome sequencing (WES) was performed in one familial case remained undiagnosed. Results We identified 57 adult patients with LKEN of unknown cause (mean age 43 years, range 18-72; 23 males; 53 with late-adolescence or adult-onset). Thirty of them, henceforward called hypomyelinating leukoencephalopathies (HypoLKENs), presented an MRI pattern suggestive of hypomyelination (mild T2-hyperintensity and normal T1 signal), whereas the remaining 27 (henceforward called demyelinating leukoencephalopathies, DemLKENs) had an MRI pattern suggestive of demyelination (prominent T2-hyperintensity and prominent T1-hypointensity). In 13 HypoLKENs, TGPS identified the disease-causing genes, i.e., POLR3A (n = 2), POLR1C, TUBB4A, RARS1, GJA1, PLP1, GJC2, TBCD, CYP7B1, SPG11, PEX3, and PEX13, while in two further patients, WES led to the identification of a novel disease-causing gene (preliminarily called GENE_A). In contrast, TGPS identified the disease-causing gene (i.e., AUH) in only one (out of 27) DemLKEN patient affected by methylglutaconic aciduria type 1. In two other DemLKEN patients, the diagnosis was made on the basis of their clinical and MRI features directly by single gene analysis (PSAP-related metachromatic leukodystrophy), or by skin biopsy after negative results of TGPS (neuronal intranuclear inclusion disease, NIID). Three patients (one with HypoLKEN and two with DemLKEN) had acquired diseases mimicking a leukodystrophy, i.e., a primary cerebral vasculitis (diagnosed by brain biopsy without genetic analyses) and rare variants of multiple sclerosis, diagnosed after negative results of TGPS. Finally, in eight subjects with an incidentally found DemLKEN who remained without clinical manifestations over a long period of time, no mutation was found by TGPS. Conclusions In adults, a hypomyelinating pattern characterizes a large number (about 50%) of LKENs of unknown cause. HypoLKENs are most commonly due to genes causing severe early-onset hypomyelinating leukodystrophies (HLDs), such as POLR3A and TUBB4A, or can be due to genes associated with hereditary spastic paraplegias, such as CYP7B1 and SPG11, peroxisomal biogenesis disorders, such as PEX3 and PEX13, or even novel disease-causing genes. Among the DemLKENs of unknown cause, only very few are diagnosed by TGPS if clinical and paraclinical data pointing toward specific diagnoses are lacking. Occasionally, atypical variants of acquired WM diseases can mimic a genetic leukoencephalopathy with demyelinating or hypomyelinating features on MRI. Finally, a subset of DemLKENs characterized by lack of neurological manifestations and no mutation after comprehensive NGS testing may constitute a novel entity we termed subclinical diffuse leukoencephalopathy (SDL).
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Woo, Andrew Jonghan. "Characterization and identification of transcription factors that bind to the tumor necrosis factor -308 polymorphism". University of Western Australia. School of Biomedical and Chemical Sciences, 2003. http://theses.library.uwa.edu.au/adt-WU2004.0044.
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[Formulae and special characters can only be approximated. Please see the pdf version of this abstract for an accurate reproduction.] Tumor necrosis factor (TNF) is a pleiotropic cytokine that mediates a long list of immunological and pathophysiological processes. TNF is produced by a wide variety of cells including immune and non-immune cells, however in most cell types TNF is not expressed prior to stimulation. The function of TNF is mediated via its trimeric domain by binding to TNF receptors that are found on most types of cells, especially of the haematopoietic systems, hence transpiring its effects on a wide variety of cells and organ systems. The cytotoxic (apoptosis) and pro-inflammatory (differentiation, proliferation and activation) functions of TNF are protective but can also result in pathological or deleterious consequences. A biallelic G to A transition polymorphism in the promoter region of TNF at nucleotide position 308 from the transcription start site is suggested to be involved in differential transcriptional regulation of TNF expression. The high TNF producing 308A allele is associated with susceptibility to or worse outcome of many infectious diseases in addition to autoimmune and other pathophysiological conditions. A previous study in our laboratory observed a selective affinity towards the polymorphic 308A allele by an EMSA protein(s) complex, named E. Several other protein complexes were found along with complex E and one of them was identified as Sp1. The identification of complex E was unsuccessful but it was hypothesized to play a major role as transcriptional activator in 308A allele individuals hence transpiring its effect in various pathophysiological states. In this study, the EMSA complexes observed in the TNF promoter region between nucleotides 322 to 283, encompassing the 308 polymorphism, is characterized. EMSA using mutated oligonucleotides mapped the binding sites of complexes B, C, D and E. TRANSFAC database search in addition to previous work revealed the identity of complex C as Sp1 but the rest of complexes remained unknown. Moreover, in contrast to our previous study, the protein(s) in the complex E was found to preferentially bind 308G nucleotide hence posing as a transcriptional repressor, resulting in decreased production state of TNF in 308G allele individuals than 308A allele individuals. In order to characterize putative transcription factors binding to the promoter region, first the biochemical characteristics such as the effects of temperature, salts and cations on DNA binding ability of EMSA complexes were studied. EMSA complexes B, C, DI and E required cations, probably Zn+2, to bind DNA. By optimizing a technique that couples EMSA with SDS-PAGE, the molecular weight of C, DI and E was determined. A novel technique that couples EMSA with IEF determined the pI of complexes B, C, D, DI and E. Although a commonly used technique of identifying unknown DNA-binding protein of interest, Yeast One-Hybrid assay, did not identify complex E, the novel identification method involving chromatography, two-dimensional electrophoresis, EMSA, mass spectrometry and database interrogation successfully identified TNF EMSA complex E as transcription factor Ying Yang 1 (YY1). Supershift EMSA confirmed complex E as YY1. In addition, the supershift assay showed presence of Sp1 and Sp3 in complex C. Similarly, complex DI is identified as Sp3. The novel method in identifying DNA-binding proteins is particularly useful as this technique allows identification of protein seen in EMSA without the need of extensive identification process. YY1 binds to a 6 base pair sequence, 5? TTGAGG 3?, from nt 295 to 290 of TNF promoter. The loss of affinity in 308A allele is caused by transition of underlined G nucleotide to A. The determined and described molecular weight of YY1 in literature is 60 kDa while the theoretical weight is 45 kDa. Both the determined and theoretical pI of YY1 is 5.8. YY1 is a multifunctional transcription factor implicated in both positive and negative regulation of gene expression as well as in initiation of transcription. It is ubiquitously expressed in growing, differentiated, and growth-arrested cells. Although future experiment is yet to establish in vivo presence of YY1 in TNF promoter, our study so far provides convincing evidence that the putative transcription factor that has selective affinity towards 308G allele is indeed YY1.
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Zhang, Xue-Cheng. "Functional characterization of the Arabidopsis disease resistance gene RPS4". Diss., Columbia, Mo. : University of Missouri-Columbia, 2005. http://hdl.handle.net/10355/5826.
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Thesis (Ph.D.)--University of Missouri-Columbia, 2005.The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file viewed on (November 27, 2006) Vita. Includes bibliographical references.
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Chu, Ying-ying Jamie. "Characterization of the promoter of dehalogenase IVa gene of Burkholderia sp. MBA4". Click to view the E-thesis via HKUTO, 2006. http://sunzi.lib.hku.hk/hkuto/record/B37840150.
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Chu, Ying-ying Jamie, i 朱盈盈. "Characterization of the promoter of dehalogenase IVa gene of Burkholderia sp. MBA4". Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2006. http://hub.hku.hk/bib/B37840150.
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Hui, Koon-chun Eleanor, i 許冠珍. "Characterization of PML/RARA fusion in acute promyelocytic leukemia: molecular cytogenetics study". Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2005. http://hub.hku.hk/bib/B45010080.
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Williams, Bruce. "Isolation and characterization of abscisic acid-responsive, embryo specific genes from Zea mays". Thesis, McGill University, 1993. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=41786.
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Embryogenesis in plants, as in animals, requires the regulated expression of sets of genes involved in developmental processes. To gain insight into the processes regulating gene expression during embryogenesis differential screening was used to identify embryo-specific sequences in a cDNA library constructed from Zea mays embryo RNA. Four embryo-specific sequences and one constitutive sequence were characterized further by RNA blot hybridization and DNA sequence determination. The constitutive sequence and two of the embryo-specific sequences were found to encode parts of the previously-reported chloroplast 23S rRNA, Oleosin KD-18, and RAB-17 genes. Two sequences, named Emb5 and Emb564, were found to encode novel maize homologs of a gene expressed during late embryogenesis in a wide range of seed plants. These 5 genes exhibited differential temporal and spatial accumulation during development. Moreover, analysis of RNA from cultured embryos suggested that 4 of these genes were regulated by abscisic acid. The ABA-responsive genes could be divided into 3 classes, based on their developmental expression, tissue-specificity, and sensitivity to ABA. Antibodies raised against a $ beta$-galactosidase:EMB564 fusion protein were used to analyze the accumulation of the EMB564 and/or EMB5 proteins. These polyclonal antibodies detected one or several polypeptides with a molecular weight less than 14 kD which exhibited patterns of developmental accumulation and regulation similar to Emb5 and Emb564 transcripts.
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Greberg, Maria Hellqvist. "Cloning and characterization of FREACs, human forkhead transcription factors". Göteborg : Dept. of Cell and Molecular Biology, Göteborg University, 1997. http://catalog.hathitrust.org/api/volumes/oclc/39751934.html.
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Thaha, Fathuma Zuleikha. "Characterization of acetate metabolism genes in Sinorhizobium, Rhizobium, meliloti". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape7/PQDD_0016/MQ55093.pdf.
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Simmons, Mary Kecia Rigsby. "Genetic characterization of ribosomal protein L10 in Saccharomyces cerevisiae". College Park, Md. : University of Maryland, 2005. http://hdl.handle.net/1903/2659.
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Thesis (M.S.) -- University of Maryland, College Park, 2005.Thesis research directed by: Cell Biology & Molecular Genetics. Title from t.p. of PDF. Includes bibliographical references. Published by UMI Dissertation Services, Ann Arbor, Mich. Also available in paper.
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Wu, Tsung-Sheng. "Functional characterization of sex hormone-binding globulin genetic polymorphism". Thesis, University of British Columbia, 2015. http://hdl.handle.net/2429/53980.
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Human plasma SHBG is produced by the liver, and it transports biologically active sex steroids and determines their availability to target tissues. The 4.3 kb human SHBG transcriptional unit encoding a signal polypeptide for secretion followed by two laminin G (LG)-like domains is utilized by hepatocytes. The N-terminal LG domain of SHBG contains a region responsible for homodimer formation, and a steroid ligand-binding site that accommodates androgens and estrogens in opposite orientations. Among over 250 genetic polymorphisms identified in human SHBG, few are functionally characterized. In this research, I have performed a comprehensive analysis of functionally relevant SHBG single nucleotide polymorphisms (SNPs). Nine out of nineteen non-synonymous SNPs within the coding region of SHBG N-terminal LG domain were shown to encode SHBG mutants with abnormal properties in steroid ligand binding, calcium coordination, fibulin-2 interaction, glycosylation or secretion. In particular, SHBG R123H (encoded by rs143269613) has a general reduction in affinity for steroid ligands, whereas SHBG E176K (encoded by rs372114420) has a higher affinity specifically for estradiol. Crystallography revealed that instead of losing the structural integrity of the steroid-binding site, reduced flexibility of the loop region that covers the steroid-binding site, and conformational changes at the opening rim of a putative estradiol entrance, likely account for the abnormal steroid-binding affinities of SHBG R123H and SHBG E176K, respectively. Among eight SNPs within SHBG regulatory sequences selected for analysis, only rs138097069 increases SHBG promoter activity. In silico prediction revealed that rs138097069 is located within a putative FXR binding site, while rs6257, which is linked to low plasma SHBG concentrations, is located within a putative FOXA2 binding element. In HepG2 cells, GW4064-activated FXR and overexpressed FOXA2 both suppress SHBG expression by direct binding to their corresponding binding elements in an HNF4⍺-independent manner. By contrast, knock-down of FXR reduces, while knock-down of FOXA2 induces, HNF4⍺ expression and SHBG production. Characterization of functional SHBG SNPs has provided molecular explanations of how genetic differences contribute to SHBG production and function, and has identified possible roles for two novel regulators, FXR and FOXA2, in a more complex regulatory network that determines SHBG expression.Medicine, Faculty of
Obstetrics and Gynaecology, Department of
Graduate
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Laramée, Louise. "Genetic characterization of a diclofop-methyl-degrading bacterial consortium". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp04/mq29733.pdf.
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Kennett, Jennifer Yvonne. "Molecular genetic characterization of retinoblastoma tumors lacking RB1 mutations". Thesis, University of British Columbia, 2012. http://hdl.handle.net/2429/43791.
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Retinoblastoma is a rare childhood cancer of the retina and is the most common intraocular tumor in children. Classically, retinoblastoma results from biallelic loss of the RB1 tumor suppressor gene. As with other cancer types, dysregulation of a single gene alone is not considered sufficient for complete transformation to malignancy. Frequent regions of genetic alteration harbouring additional genes, implicated in retinoblastoma oncogenesis and progression, include chromosomes 1q, 2p, 6p, 13q and 16q.
Sensitive molecular genetic screening techniques are capable of identifying RB1 mutations in 98% of unilateral retinoblastoma tumors. The remaining 2% harbour no identifiable RB1 inactivating alterations, and therefore molecular interrogation of these cases would likely reveal alternative genetic events driving retinoblastoma tumorigenesis in the absence of RB1 inactivation. Towards this objective, in this thesis work, I describe genetic alterations identified by tiling path array comparative genomic hybridization in a rare sample set composed of 23 RB1⁺/⁺ tumors. In addition to gene disruption by copy number alteration, mechanisms of gene disruption resulting in no overall change in copy number or change in copy number with allelic imbalance were also investigated utilizing genome-wide SNP array analysis on five of the RB1⁺/⁺ tumors.
The most striking recurrent genetic alteration identified in retinoblastoma tumors lacking RB1 inactivating mutations, was focal high-level MYCN amplification, which occurred at a frequency of approximately 48%. The MYCN amplified RB1⁺/⁺ tumors also exhibited a statistically significant lower proportion of their genome affected by genomic instability when compared with the RB1 ⁻/⁻ tumors. In a subset of five matched tumor and blood normal samples the occurrence of copy neutral LOH was explored, although none was observed. Allele specific copy number analysis identified instances of allelic imbalance, including all five MYCN amplifications.
Amplification of MYCN may represent a rare and novel alternate mechanism of retinoblastoma tumorigenesis. This work provides insight into the role of mutational events driving tumorigenesis in retinoblastoma in the absence of RB1 inactivation.
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Trottier, Oliver. "Genetic characterization of gamma-aminobutyrate metabolism in Sinorhizobium meliloti". Thesis, McGill University, 2008. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=111551.
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Transcriptional fusion mutants and Tn5-B20 transposon mutants were isolated where the only genes affected are believed to either be involved in the hypothetical GABA shunt or code for subunits of the alpha-ketoglutarate dehydrogenase enzyme complex of Sinorhizobium meliloti. The growth phenotypes of Rm30222 (gabT) and eight mutants in gabD1, 2, 3, and 4 on minimal media were comparable to that of the wild-type. Compared to wild-type, Rm30222 (gabT) lacked alpha-ketoglutarate-dependent gamma-aminobutyrate transaminase activity showed high induction of gabT on GABA but produced green plants indicative of being Fix+. Mutants in gabD alleles maintained wild-type levels of succinic semialdehyde dehydrogenase activity and could fix nitrogen as well as the wild-type in symbiosis.Mutation of sucB encoding a subunit of a-ketoglutarate dehydrogenase produced a mutant, Rm30230, that initially had difficulty growing on minimal media supplemented with either arabinose or glutamate. In symbiosis with alfalfa, Rm30230 had a fix- phenotype and was also devoid of alpha-ketoglutarate dehydrogenase activity. The ability of Rm30230 to grow on arabinose or glutamate, without alpha-ketoglutarate dehydrogenase activity, strengthens the hypothesis that S. meliloti has a functional GABA shunt allowing it to circumvent the forward-direction TCA cycle from alpha-ketoglutarate to succinate. Mutation of the second potential dihydrolipoamide succinyltransferase component (E2) of alpha-ketoglutarate dehydrogenase yielded Rm30267 (SMb20019) with wild-type growth on minimal media and a Fix+ phenotype in plants. The introduction or a sucB mutation into the SMb20019 mutant background (Rm30275) was comparable to the sole sucB mutation. This finding shows that the locus SMb20019 cannot be substituted for sucB in the alpha-ketoglutarate dehydrogenase enzyme complex.
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Doyle, Caitlin. "Isolation and characterization of genetic modifiers of Arabidopsis RHD3". Thesis, McGill University, 2012. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=110637.
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AbstractRoot hair cells of Arabidopsis are a model system for investigations of polarized cell expansion. Root hairs are long, cellular extensions, produced by tip growth on trichoblast cells of the root epidermis. Many genes necessary for proper tip growth have been identified in Arabidopsis including RHD3. The Arabidopsis root hair defective3 (rhd3) mutant produces short, misshapen root hairs. RHD3 was cloned as a large GTP binding protein, and recently it was shown to be involved in the formation of the tubular ER network. To date, the role of RHD3 in ER formation and the cause of the defective root hairs in rhd3 mutants are still not clear. To uncover genetic interactions of RHD3, a genetic modifier screen was conducted in an rhd3 mutant background. Through this experiment 10 genetic modifiers of rhd3 were isolated. Characterization of these modifiers, revealed that 9 of them carry single, recessive mutations which, in combination with the rhd3 mutation, disrupt the growth of root hairs. Three of the mutations cause mutant root hair phenotypes without the rhd3 mutation. The remaining mutations fall into two groups; those that appear to be epistatic to mutations in RHD3 and those that are only able to affect root hair development in an rhd3 mutant background. A test for genetic interactions between RHD3 and two known genes, SHV2 and BOT1, was also conducted. The SHV2 gene encodes a GPI-anchored, COBRA-like protein, and mutations in SHV2 impair root hair elongation. Analysis of shv2 rhd3 double mutants, revealed that mutations in SHV2 can synthetically enhance the rhd3 mutant root hair phenotype.BOT1 encodes a Katanin-like, microtubule severing protein. Mutations in BOT1 lead to the growth of ectopic root hairs. Double mutant analysis revealed that mutations in BOT1 can partially suppress the effects of mutations in RHD3 on the growth of root hairs.Résumé Les poils absorbants sont de longues extensions cellulaires produits dans la racine au niveau de la zone pilifère. Le mutant root hair defective3 (rhd3) d'Arabidopsis produit des poils absorbants courts et malformé. RHD3 est une grande protéine de liaison de GTP. Récemment il a été démontré que RHD3 est impliquée dans la formation du réseau tubulaire du réticulum endoplasmique (RE), mais l'implication de RHD3 dans la malformation des poils absorbants reste à élucider. Pour mieux comprendre le rôle de RHD3 lors de l'élongation des poils absorbants, nous avons crée d'autres mutants à partir d'Arabidopsis thaliana rhd3. Ce criblage nous a permis d'identifier 10 nouveaux gènes impliqués dans la croissance de l'apex racinaire, nommés de ren1 à ren10. L'analyse de ces mutants a révélé qu'à l'exception de ren5 les 9 autres mutants sont des mutations ponctuelles récessives qui associées la mutation rhd3 affectent la formation des poils absorbants. Trois de ces mutations (ren6, ren7 et ren9) causent des phénotypes anormaux des poils absorbant en l'absence de la mutation rhd3. Les mutations restantes se répartissent en deux groupes : ren1, ren2 et ren8, qui sont difficile à interpréter et ren3, ren4 et ren10, qui ont la capacité d'affecter le développement des poils des racines seulement dans le cadre du mutant rhd3.Nous avons également étudié l'interaction entre rhd3 et deux autres gènes connus, shv2 et bot1, pour leur implication dans la formation des poils absorbants. Le gène shv2 code pour une protéine ancrée à un GPI (Glycosylphosphatidylinositol), semblable à la protéine COBRA. Le mutant shv2 ralentit l'allongement des poils. L'analyse du double mutant shv2/rhd3 a révélé que la mutation shv2 accentue le phénotype de rhd3. Le gène bot1 code pour une protéine semblable à la katanine qui sert à sectionner les microtubules. Le mutant bot1 est connu pour induire la formation ectopique de poils absorbants. Le double mutant bot1/rhd3 présente quant à lui un phénotype atténué de rhd3.