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1
RIGAMONTI, AURORA. « Functional characterization of SMARCA2 gene ». Doctoral thesis, Università degli Studi di Milano-Bicocca, 2016. http://hdl.handle.net/10281/130275.
Texte intégralRésumé :
My PhD project is based on the characterization of the SMARCA2 gene. On one hand, I focused my attention on the role of SMARCA2 product, the protein brahma (BRM), in the regulation of alternative pre-mRNA splicing. On the other hand, I investigated the transcriptional regulation of SMARCA2 expression.
My first project derived from a comparative evaluation and validation of microarray data from two mitochondrial stress models. The first model represented by an acute mitochondrial stress is constituted by human SH-SY5Y neuroblastoma cells treated with Paraquat (PQ), while the second model is a chronic model constituted from the same cell line stably overexpressing the Superoxide Dismutase 1 carrying the most common mutation found in familiar ALS (SOD1 G93A). The merge of these microarrays data showed that oxidative stress affects the choice of specific alternative last exons (ALEs) increasing the production of transcripts variants terminating at a more proximal ALE. Moreover, oxidative stress induces the transcriptional downregulation of the SMARCA2 gene product BRM, one of the two alternative ATPase subunits of the SWI/SNF complex. I found that in normal condition BRM is enriched on the proximal ALE. In addition, I observed the accumulation of BARD1, a protein that forms a functional heterodimer with BRCA1, which has E3 ubiquitin-ligase activity and interacts with the 50 kDa subunit of CstF inhibiting 3’ end processing. Consistent with these observations, I detected an ubiquitinated pool of CstF50 and showed that ubiquitination is mediated by BARD1/BRCA1. Taken together, these results suggested that the presence of BRM on the proximal exon leads to the BARD1/BRCA1-mediated ubiquitination of CstF50 and the inhibition of 3’ end processing at the proximal poly(A). This in turn allows transcription to proceed to the distal terminal exon.
In the same microarray data used as a starting point for my first project we detected a shift in SMARCA2 expression towards shorter mRNA isoforms upon oxidative stress. Thus, my second project dealt with the characterization of these transcripts. Bioinformatic analysis revealed that the shorter mRNA variants are evolutionarily conserved and are most likely generated from an internal promoter. Interestingly, in zebrafish the short isoform is produced as an independent gene on the same chromosome of the long isoform but in its reverse strand. This peculiar genomic organization hints to a potentially relevant function for this alternative isoform of the BRM protein. Bioinformatic analyses revealed that the short isoform encode a protein that lacks the N-terminal, catalytic ATP-ase domain but shares the C-terminal region that contains a Bromodomain, a protein motif that is known to bind to acetylated histones.
First, I identified the potential alternative promoter region using bioinformatic tools and cloned this region. Using a luciferase reporter system I demonstrated the existence of the alternative promoter. Next, I cloned the short, most conserved isoform and I tested it for interaction with known partners of the full-length BRM protein by Co-Immunoprecipitation (Co-IP). I discovered that BRM-s interacts with histone H3 but not with core component of the SWI/SNF complex. Considering that short isoform does not display an ATPase domain, the Co-IP suggests a possible “dominant negative” role for this protein. If short isoforms works as negative dominant of Brm, the ratio between long and short expressed proteins could become very important for BRM target genes in development and differentiation. In particular, this alteration of ratio could have a negative effect on the cells since several tumor cell lines show a very low level of the long protein. (i.e. human lung tumor cell lines).
2
Lee, Ka Young. « Functional characterization of gene regulation by nhr-49 ». Thesis, University of British Columbia, 2016. http://hdl.handle.net/2429/58365.
Texte intégralRésumé :
Nuclear hormone receptors (NHRs) are transcription factors that regulate a wide variety of developmental and physiological processes. NHRs are targets of numerous drugs. However, due to limited knowledge on NHR specificity, many such drugs activate multiple biological pathways downstream of NHRs, leading to undesired side effects. To study NHR specificity in vivo, I used the model organism Caenorhabditis elegans. One C. elegans NHR is NHR-49, which regulates various aspects of lipid metabolism. Specifically, it activates genes involved in fatty acid desaturation and fatty acid β-oxidation by binding to a subunit of the Mediator multiprotein complex, MDT-15. Vice versa, NHR-49 represses genes involved in sphingolipid breakdown by heterodimerizing with another C. elegans NHR, NHR-66. Recently, three point mutations in nhr-49 were identified that promote fatty acid desaturation, but whether these alleles act specifically in this pathway or also affect other nhr-49 regulated processes is not clear. To test whether the mutated residues are linked to specific biological functions, I studied how they affect gene expression and protein-protein interactions by real time quantitative PCR and Yeast 2 Hybrid assays. I found that the three point mutations have different effects on nhr-49 dependent metabolic processes. While all three alleles broadly promoted nhr-49 dependent activation, only one allele affected nhr-49 dependent repression. This shows that the mutations and the corresponding amino acid residues have some association with specific nhr-49 dependent biological processes.Medicine, Faculty of
Medical Genetics, Department of
Graduate
3
Pomerleau, Véronique. « Functional characterization of the BHD tumor suppressor gene ». Thesis, McGill University, 2006. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=101734.
Texte intégralRésumé :
Birt-Hogg-Dube syndrome is a hereditary cancer syndrome that increases the risk of developing kidney cancer. The gene responsible for this disease was recently identified but the protein encoded by this gene has yet to be characterized. In this thesis, we present an initial characterization of the BHD protein. To do so, a polyclonal antibody against the human BHD protein was produced that detects both the exogenous and endogenous protein. Moreover, several BHD putative interacting proteins were identified by multidimensional protein identification technology (MudPIT) analysis following large scale purification of epitope-tagged BHD. The interaction of BHD with one candidate was investigated by co-immunoprecipitation and by in vitro binding assays but could not be validated. In parallel, a Far Western was attempted to discover BHD putative interacting proteins but with no success. Furthermore, the intracellular localization of BHD revealed to be mostly cytoplasmic as shown by differential centrifugation and by indirect immunofluorescence. A BHDwt-GFP construct was also engineered to perform further studies on BHD subcellular localization. Finally, the effect of BHD disease-associated mutations on protein expression was investigated by Western blotting.
4
Dennis, Megan Young. « Functional characterization of the dyslexia candidate gene KIAA0319 ». Thesis, University of Oxford, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.504385.
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5
Le, Hang Thi Thu. « Functional characterization of IGF2BP2, a diabetes-susceptibility gene ». Thesis, University of Cambridge, 2011. https://www.repository.cam.ac.uk/handle/1810/283871.
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6
Wahab, S. M. Riajul. « Molecular and functional characterization of GAEC1 gene in human colorectal cancer ». Thesis, Griffith University, 2018. http://hdl.handle.net/10072/377621.
Texte intégralRésumé :
Background:
Colorectal cancer is now one of the most common causes of death in Australia, with an estimated 1486 new cases in the country in 2010, accounting 12.7% of all cancer deaths (ACIM, 2014). In addition to its significance in Australia, it is one of the most common global health concerns. At present colorectal cancer is the third most common cancer worldwide, which cost more than 600,000 lives every year. Most of the colorectal cancer is diagnosed at a late stage but if it is diagnosed at an early stage, the five-year survival rate exceeds in 90% cases. This is the reason there is a need to find out biomarker for early detection and the exact underlying cause for designing a better treatment for colorectal cancer.
GAEC1 (Gene amplified in esophageal cancer 1) showed a series of amplifications and deletions in oesophageal cancer. The gene is located at 7q22.1. GAEC1 has tumorigenic potential approximately equal to the Ras gene family and overexpression of this gene played a pivotal role in the cancer transformation of oesophageal squamous cell carcinoma. GAEC1 has higher amplification in colorectal adenocarcinoma tissues when compared to non-cancer colorectal tissues. In this study, we focused on finding out the oncogenic properties of GAEC1, correlation with clinical and pathological features and its underlying mechanism in colorectal cancer initiation and progression.
Materials and method:
Human colon cancer cell lines (SW480, SW48, HCT116 cells) and non-neoplastic colonic epithelium cell (FHC cells) were purchased from American Type Culture Collection (ATCC). SW480, SW48 and HCT116 cell lines were maintained in Dulbecco's Modified Eagle Medium (DMEM) (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum at 37 ℃ in 5% CO2. FHC cells were maintained in DMEM: F-12 (1:1) with 10% fetal bovine serum with containing an extra 10 mM N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES) (Thermo Fisher Scientific) (for a final concentration of 25 mM), 10 ng/ml cholera toxin, 0.005 mg/ml insulin, 0.005 mg/ml transferrin, 100 ng/ml hydrocortisone. Fresh frozen human colorectal cancer tissues and adjacent non-cancer tissues were collected with no selection bias. Expression levels of mRNA and protein were measured by real-time PCR and western blot analysis respectively. Immunocytochemistry, immunohistochemistry and immunofluorescence assay were used to identify the localization of GAEC1 protein in colon cancer cells and colon cancer tissues. Flow cytometry was used for the detection of apoptotic cells and cell cycle alteration. Co-immunoprecipitation followed by mass spectrometry analysis was used to identify the protein-protein interaction. Severe combined immunodeficiency (SCID) mice were used for tumour xenograft experiment.
Results:
We found differential expression of GAEC1 protein and mRNA in different pathological stages of colon cancer cells (SW480-Stage II, SW48-Stage III and HCT116-Stage IV) when compared to non-neoplastic colon cells (FHC cells). GAEC1 protein was predominantly expressed in the cytoplasm of colon cancer cells (SW480, SW48, and HCT116) and the nucleus of non-neoplastic colon epithelial cells (FHC). The transient knockdown of GAEC1 using siRNA induced apoptosis in SW480 and SW48 cells, which was associated with G2/M phase arrest and decreased expression of Bcl-2 and K-ras proteins and increased expression of p53. In addition, down-regulation of GAEC1 significantly inhibited cell proliferation, reduced migration capacity and decreased clonogenic potentiality of colon cancer cells (SW480 and SW48 cells). Furthermore, a xenotransplantation model showed that stable knockdown of GAEC1 using shRNA constructs in colon cancer cells entirely suppressed xenograft tumour growth in mice.
Approximately 52.5% of patients with colorectal cancers showed high expression of GAEC1 mRNA whereas 47.5% exhibited low expression compared to their matched non-neoplastic tissues. Similarly, ~ 66% (53/80) of colorectal cancer tissues showed high GAEC1 protein expression (positive staining), while the remaining colorectal cancer cases were noted with no GAEC1 protein (negative) expression. GAEC1 protein was predominantly located in the cytoplasm and showed low to no expression in normal colon tissues.
High expression of GAEC1 mRNA was predominantly seen among patients below 60 years compared to those patients over 60 years of age (78%, versus 44%, p=0.008). Patients with synchronous colorectal adenocarcinomas mostly exhibited with low expression of GAEC1 mRNA. On the other hand, compared to poorly differentiated colorectal carcinomas (grade III), patients with well and moderately differentiated colorectal carcinomas (grade I+II) colorectal cancers showed a high expression of GAEC1 mRNA. Similarly, high GAEC1 mRNA expression was frequently noted among patients presented without any pre-neoplastic adenomas in their colorectal cancer tissues compared to patients with an adenoma in their colorectal cancer tissues.
By co-immunoprecipitation followed by mass spectrometry analysis 31 interacting protein was identified. The interaction between GAEC1 and four proteins (HIGD1A, Rhotekin, Granulin and eIF3J) was further confirmed. Western blot analysis detected reduced expression of these proteins following stable knockdown of GAEC1 in colon cancer cells.
GEAC1 endogenously interacts with p53 in SW480 and SW48 colon cancer cells. In this study, we have noted that overexpression of GAEC1 increased cell proliferation, migration, and reduced apoptosis in colon cancer cells. Also, these cells showed cell cycle arrest at the synthetic phase, activation of Bcl-2, K-ras, pAKT proteins as well as inhibition of p53, PUMA, p21 and BAX proteins. Furthermore, silencing of GAEC1 reduces the nuclear import of MDM2 and increase the expression of p53 in the nucleus suggesting that GAEC1 expression is essential for interaction of p53-MDM2 and nuclear translocation of MDM2 in colon cancer cells.
Conclusion:
In summary, the expression analysis, in vitro and in vivo data indicated that GAEC1 is differentially expressed in cancer cells and act as an oncogene in colon cancer progression. The high expression of GAEC1 mRNA/protein, as well as its correlation with multiple clinical and pathological characteristics in patients with colorectal carcinoma, strongly, suggests that GAEC1 is a key regulator in the initiation of colorectal carcinogenesis. In addition, the protein-protein interaction with a number of proteins and the effect of GAEC1 modulation on the expression of interacting proteins indicates the potential role of GAEC1 in the signalling pathway of colon cancer pathogenesis.Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Medicine
Griffith Health
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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.
Texte intégralRésumé :
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.
8
Salehin, Mohammad. « Molecular and Functional Characterization of Medicago Truncatula Npf17 Gene ». Thesis, University of North Texas, 2013. https://digital.library.unt.edu/ark:/67531/metadc407747/.
Texte intégralRésumé :
Legumes are unique among plants for their ability to fix atmospheric nitrogen with the help of soil bacteria rhizobia. Medicago truncatula is used as a model legume to study different aspects of symbiotic nitrogen fixation. M. truncatula, in association with its symbiotic partner Sinorhizobium meliloti, fix atmospheric nitrogen into ammonia, which the plant uses for amino acid biosynthesis and the bacteria get reduced photosynthate in return. M. truncatula NPF1.7 previously called MtNIP/LATD is required for symbiotic nitrogen fixing root nodule development and for normal root architecture. Mutations in MtNPF1.7 have defects in these processes. MtNPF1.7 encodes a member of the NPF family of transporters. Experimental results showing that MtNPF1.7 functioning as a high-affinity nitrate transporter are its expression restoring chlorate susceptibility to the Arabidopsis chl1-5 mutant and high nitrate transport in Xenopus laevis oocyte system. However, the weakest Mtnip-3 mutant allele also displays high-affinity nitrate transport in X. laevis oocytes and chlorate susceptibility to the Atchl1-5 mutant, suggesting that MtNPF1.7 might have another biochemical function. Experimental evidence shows that MtNPF1.7 also functions in hormone signaling. Constitutive expression of MtNPF1.7 in several species including M. truncatula results in plants with a robust growth phenotype. Using a synthetic auxin reporter, the presence of higher auxin in both the Mtnip-1 mutant and in M. truncatula plants constitutively expressing MtNPF1.7 was observed. Previous experiments showed MtNPF1.7 expression is hormone regulated and the MtNPF1.7 promoter is active in root and nodule meristems and in the vasculature. Two potential binding sites for an auxin response factors (ARFs) were found in the MtNPF1.7 promoter. Chromatin immunoprecipitation-qRT-PCR confirmed MtARF1 binding these sites. Mutating the MtARF1 binding sites increases MtNPF1.7 expression, suggesting a mechanism for auxin repression of MtNPF1.7. Consistent with these results, constitutive expression of an ARF in wild-type plants partially phenocopies Mtnip-1 mutants’ phenotypes.
9
Crimmins, Stephen Lewis. « Characterization and functional analysis of Usp14 ». Thesis, Birmingham, Ala. : University of Alabama at Birmingham, 2007. https://www.mhsl.uab.edu/dt/2007p/crimmins.pdf.
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10
Yan, Kaiping. « Characterization and functional analyses of the transcriptional cofactor TIF1γ gene ». Université Louis Pasteur (Strasbourg) (1971-2008), 2003. http://www.theses.fr/2003STR13144.
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11
Jain, Nishant Rajkumar. « Characterization and functional analysis of the P2Y₂R gene promoter ». Diss., Columbia, Mo. : University of Missouri-Columbia, 2006. http://hdl.handle.net/10355/4629.
Texte intégralRésumé :
Thesis (M.S.)--University of Missouri-Columbia, 2006.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 April 21, 2009) Includes bibliographical references.
12
Islam, Md Farhadul. « Molecular and Functional Characterization of JK1 (FAM134B) Gene in Human Colorectal Cancer ». Thesis, Griffith University, 2017. http://hdl.handle.net/10072/371217.
Texte intégralRésumé :
Family with sequence similarity 134, member B (FAM134B) (also called
JK1/RETREG1) was first identified in 2001 using comparative genomic hybridization analysis
in cancer. Subsequent studies characterized this gene in biological processes and human
diseases. FAM134B regulates endoplasmic reticulum-turnover by selective autophagy and loss
or dysregulation of FAM134B impairs endoplasmic reticulum-turnover. In addition, aberrant
FAM134B expression encompasses in the pathogenesis of a number of human diseases,
including, neuronal disorder, vascular disease, allergic rhinitis, viral infections and cancer. In
cancer, FAM134B acts as a tumour suppressor or promoter in different types of cancer. For
example, in oesophageal squamous cell carcinoma, FAM134B acts as an oncogene and
stimulates cancer cell growth and proliferation, whereas in breast and colorectal cancer (CRC)
it acts as a tumour inhibitor. In CRC, FAM134B copy number deletion, loss or reduced mRNA
and protein expressions associated with the biological aggressiveness of cancer. However, indepth
functional insights, genetic and epigenetic regulation, interacting signalling pathways
and expression pattern of FAM134B in different stages of CRC have never been studied. Thus,
the present study aims to explore in vitro and in vivo functional role, and molecular
characterization of FAM134B through mutational, micro-RNAs, promoter methylation
screening and identifying partners to which FAM134B interacts in CRC. The clinical
significance and validation of FAM134B in a large cohort of patients with CRC also
investigated.
Detection of FAM134B expression and quantified at the protein and mRNA levels in
cell lines derived from different stages of colon cancer and non-neoplastic colon epithelial cells
was performed using immunocytochemistry, immunofluorescence, western blot and real-time
PCR. Functional assays and mouse xenotransplantation model were used to investigate the role
of FAM134B in cancer cell biology followed by FAM134B silencing with shRNA lentiviral
particles. In cells derived from stage IV colon cancer, FAM134B expression was remarkably
reduced when compared to non-neoplastic colon cells and cells derived from stage II colon
cancer. Suppression of FAM134B increased the proliferation of colon cancer cells following
lentiviral transfection (p < 0.05). Additionally, FAM134B silencing induced increased
clonogenic capacity (34-52 %; p < 0.05), wound healing potential and enhanced the proportion
of cells in S-phase of the cell cycle (p < 0.01). In vivo xenotransplantation mouse model showed
that larger and higher-grade tumours were formed in mice receiving FAM134B knockdown cells.
Following in vitro, in vivo functional studies, we have screened FAM134B mutations
in CRC tissue samples (n = 88) and cell lines (n = 4) using high-resolution melt-curve (HRM)
analysis and subsequently confirmed by Sanger sequencing. The FAM134B mutation was
noted in 46.5 % (41/88) of patients with CRC. Thirty-one novel pathogenic mutations were
identified in coding and intronic sites of FAM134B in CRC. Of the 31 mutations, eight novel
frameshift mutations showed the potential to cause nonsense-mediated mRNA decay (NMD)
in computational analysis. The identified mutations were associated with different
clinicopathological factors, including, sex of the patients, the presence of metachronous
cancer, the larger size of a tumour, advanced T stages, the presence of distant metastases and
positivity of microsatellite instability (MSI) in cancer (p < 0.05). Also, the expressions of
FAM134B mRNA and protein were downregulated in FAM134B mutated cancers.
Afterwards, this study investigated the epigenetic (methylation and micro-RNA
mediated) regulation of FAM134B expression in CRC. Methylation-specific high-resolution
melt-curve (MS-HRM) analysis followed by sequencing was used to identify FAM134B
promoter methylation in colorectal adenomas (n = 32), CRC (n = 164), adjacent non-neoplastic
(n = 83) tissue samples and cell lines (n = 4). FAM134B promoter methylation was more
frequent in CRC (52 %; 85/164) compared to that of adenomas (28 %; 9/32) and non-neoplastic
(35 %; 29/83) tissue samples. FAM134B promoter methylation was inversely correlated with
low FAM134B copy numbers and mRNA/protein expressions, whereas in vitro demethylation
has restored FAM134B expression in colon cancer cells. FAM134B promoter methylation was
associated with adverse clinicopathological factors, including, high histological grade (p =
0.025), presence of peri-neural infiltration (p = 0.012), lymphovascular invasion (p = 0.021),
lymph node metastasis (p = 0.0001), distant metastasis (p = 0.0001) and advanced pathological
stages (p = 0.0001). Also, FAM134B promoter methylation correlated with cancer recurrence
(p = 0.001) and poor survival (p = 0.009) of patients with CRC. Furthermore, the present study
investigated micro-RNA mediated repression of FAM134B in CRC. It was noted that
microRNA-186-5p (miR-186-5p) represses the endogenous FAM134B expression in colon
cancer cells and tissues, which in turn, modulated various cellular events such as cell
proliferation, colony formation, cell migration and altered cell cycle kinetics. Therefore, this
project explored the epigenetic silencing of FAM134B in CRC, which might lead the initiation
and progression of CRC.
Simultaneously, the current study identified potential binding proteins partner of
FAM134B in colon cancer cells using liquid chromatography coupled with tandem mass
spectrometry (LC-MS/MS) and western blot analysis followed by co-immunoprecipitation (co IP). Interacting partners, including, CAP1, RPS28, FTH1, KDELR2, PSMD6, MAP4,
MAPRE1/EB1, PPIB/CYPB of FAM134B were detected by LC-MS/MS analysis. Subsequent
western blot analysis followed by co-IP confirmed the interactions of FAM134B with CAP1,
EB1, CYPB (onco-proteins), and KDELR2 (ER-resided protein). In addition,
immunofluorescence microscopic analysis exhibited co-localization of FAM134B with CAP1,
KDELR2, EB1, CYPB proteins in colon cancer cells. Furthermore, upregulation of EB1 as
well as downregulation of KDELR2 proteins followed by knockdown of FAM134B in colon
cancer cells further indicated the potential interactions between themselves. Activation of EB1
via FAM134B downregulation could inactivate tumour inhibitor APC or activate aurora B
kinase, thereby could modulate the WNT/β-catenin pathway in adenoma-carcinoma sequence
of colon cancer pathogenesis.
Finally, this study developed an electrochemical approach for rapid, sensitive, and
specific detection of FAM134B protein in biological and clinical samples. The method usages
a differential pulse voltammetry (DPV) in the presence of the [Fe(CN)6]3-/4- redox system to
quantify the FAM134B protein in a two-step approach that involves (i) initial attachment of
anti-FAM134B antibody on the surface of extravidin-modified screen-printed carbon
electrode, and (ii) subsequent detection of FAM134B protein present in the samples. This
method has detected FAM134B protein at a concentration down to 10 pg μl-1 in phosphate
buffered saline (pH 7.4) with a good inter-assay reproducibility (% RSD = <8.64, n = 3). Also,
this method showed excellent sensitivity and specificity for the detection of FAM134B protein
in a panel of colon cancer cell lines and serum samples of patients with CRC. The assay
performance was validated with ELISA and it is believed that this method could potentially
lead a low-cost alternative to conventional immuno-based assays for target antigens analysis
in point-of-care applications.
Altogether, from genetic to epigenetic, functional to molecular studies herein, has
provided critical insights of tumour suppressive properties of FAM134B in CRC pathogenesis.
FAM134B executes cancer inhibitory function by interacting with oncoproteins and proteins
involved in vesicle-mediated transport in cells. Genetic and epigenetic alterations of FAM134B
regulates its expression in colon cancer cells and clinical samples of patients with CRC, which
in turn contributes to the biological aggressiveness of carcinomas. The association of the
FAM134B promoter methylation and mutations, as obvious loss or reduced FAM134B
expression, with adverse clinicopathological factors are in concur with the tumour suppressive
property of the gene, which in turn, might act as a potential marker in predicting clinical prognosis in colorectal carcinomas.Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Medicine
Griffith Health
Full Text
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Aigner, Johanna 1981. « Genetical, structural and functional characterization of the human BTNL gene cluster ». Doctoral thesis, Universitat Pompeu Fabra, 2013. http://hdl.handle.net/10803/127222.
Texte intégralRésumé :
La família B7 de proteïnes és àmpliament reconeguda per jugar un paper important en els processos inflamatoris mitjançant l'alteració de la capacitat de resposta de les cèl•lules T. La unió d’aquestes proteïnes als seus receptors situats a la superfície de cèl•lules T pot promoure (per exemple, B7-1, B7-2, ICOS- L) o inhibir (per exemple, PD-L1, PD-L2, B7-H3, B7x) l'activació, la proliferació, la maduració i la producció de citoquines en cèl•lules T . A més, s’ha identificat l’expressió de diversos membres d’aquesta família de proteïnes tant en diferents tipus de tumors com en el microambient tumoral. Degut a les capacitats immunosupressores de diversos membres de la família B7, es creu que l'expressió aberrant d'aquestes molècules a interferit negativament amb la resposta immune de l' hoste, el que porta a la progressió de la malaltia. En efecte, l'expressió de proteïnes de la família B7 en molts tumors hematològics malignes s'associa sovint amb un mal pronòstic i un comportament agressiu dels tumors. Actualment, diversos membres de la família B7, com CTLA-1 i PD-1 són usats per el tractament del càncer i, més recentment , el primer agent que focalitza la via de B7, el anti-CTLA-4 MAB (ipilimumab) ha estat aprovat per l'Administració d'Aliments i Medicaments (FDA) per al tractament del melanoma metastàsic. A més, els estudis en curs dirigits a membres recentment descrits de la família B7: B7-H3, B7x, B7-H6 són prometedors , però una major clarificació del seu paper patogènic en malalties hematològiques ajudarà a identificar el seu paper més actiu com a adjuvants immunes al tractament convencional.
Tot i els grans grans progressos en aquest camp , es coneix poc de les proteïnes homòlogues butyrophilin-like (BTNL). La família butyrophilin (BTN) comparteix homologia estructural amb membres de la família B7 i similars a les proteïnes B7. Gairebé tots els BTNS / BTNLs estudiats fins al moment han demostrat ser capaços d'esmorteir la resposta immune mitjançant la co-estimulació negativa en l’activació del les cèl•lules T, fent-les candidates importants en la immunitat antitumoral. Per tant, en aquest estudi, caracteritzem un clúster que conté tres gens BTNL, situats al cromosoma humà 5q35.3, a nivell genòmic, transcripcional i funcional per obtenir una visió més clara en la funció de les proteïnes BTNL.
En el primer capítol, presentem la identificació d’una deleció d’una variant en nombre de còpia (CNV en anglès) de 56 kb donant com a producte un nou gen quimèric (BTNL8*3). Aquesta deleció és responsable de la sobre-expressió del tercer gen del clúster, BTNL9. Posteriorment, es desenvolupà un assaig de genotipació i es va dur a terme un anàlisi poblacional d’aquesta variant en mostres de diferents poblacions pertanyents al HapMap i el panell de diversitat humana (HGDP-CEPH). Aquest assaig de genotipació ens va permetre identificar clares diferències en l’estratificació de l’aŀlel BTNL8_BTNL3-del entre grups continentals majors. A més, presentem tagging SNPs en diverses poblacions, facilitant una genotipació futura de la variant de deleció BTNL8-BTNL3. Finalment mostrem la influència de la deleció CNV en els nivells d’expressió de diferents gens involucrats en càncer i en la resposta immune, suggerint la involucració d’aquesta CNV en rutes biològiques específiques.
En el segon capítol d’aquesta tesi s’investiguen les conseqüències funcionals de la CNV trobant una sobre-expressió de BTNL9 en leucèmia limfoblàstica aguda (ALL en anglès) després del subministrament de glucocorticoides (GC). S’havia mostrat ja prèviament que uns nivells elevats de BTNL9 correlacionen amb un elevat risc en pacients de ALL amb reorganització de MLL-AF4. Per comprovar si aquesta observació és deguda a la implicació de BTNL9 en apoptosi induïda per GC, es varen analitzar diferents línies ceŀlulars pre-B ALL trobant-se una clara correlació entre els nivells d’expressió de BTNL9 i resistència a GC en ALL amb reorganització de MLL i nivells més baixos en MLL en ALL germinal. Aquests resultats suggereixen un paper completament nou i inesperat de la proteïna BTNL que podrien resultar en el desenvolupament de inhibidors específics de BTNL9 per millorar la prognosi de ALL amb reorganització de MLL.
En resum, en aquesta tesi proporcionem un anàlisi del clúster de gens humà BTNL. Identifiquem un nou gen de fusió BTNL8*3 amb implicacions potencials en rutes genètiques involucrades en la regulació i proliferació immune i mostrem una clara funció de BTNL9 en la resistència a GC el la leucèmia amb reorganització de MLL. Aquestes observacions proporcionen un nou coneixement sobre la família de gens BTNL i podria proporcionar la base per noves teràpies basades en BTNL9 en ALL amb reorganització de MLL.In this thesis, we undertook a broad genomic, evolutionary, transcriptomic and functional analysis of a cluster containing three BTNL genes, namely BTNL8, BTNL3 and BTNL9, located on human chromosome 5q35.3. In the first chapter we report the identification of a 56 kb deletion copy number variant (CNV), which results in the formation of a novel chimeric gene, BTNL8*3, and leads to an upregulation in the expression-level of the third gene in the cluster, BTNL9. Next, we developed a genotyping assay and undertook a population analysis of this variant in several Hap Map and human diversity panel (HGDP-CEPH) populations. With this genotyping assay we could identify clear differences in the stratification of the BTNL8_BTNL3-del allele amongst major continental ethnic groups. In addition we report tagging SNPs in several population, facilitating the genotyping process of the BTNL8-BTNL3 deletion variant in the future. Moreover, we show an influence of the deletion CNV in the expression-level of several genes involved in cancer and immune response, suggesting an involvement of this CNV in specific biological pathways. In the second chapter we look for functional consequences of this CNV and found an upregulation of BTNL9 in acute lymphoblastic leukemia (ALL) after glucocorticoid (GC) treatment. Previously, it was shown that high-level BTNL9 correlates with high-risk in MLL-AF4 rearranged acute lymphoblastic leukemia (ALL) patients. To check whether this might be due to in involvement of BTNL9 in GC-induced apoptosis, we analyzed several pre-B ALL cell-lines and found a clear correlation between BTNL9 expression-level and resistance to GC in MLL rearranged ALL and at a lower level in MLL germ-line ALL. These results suggest a completely new and unexpected role for a BTNL protein and may led to the development of specific BTNL9 inhibitors to improve outcome of MLL rearranged ALL. Overall, we provide a comprehensive analysis of a BTNL gene cluster. We identified a new BTNL8*3 fusion-gene with potential implication in genetic pathways involved in immune regulation and proliferation, and show a clear function for BTNL9 in GC-resistance in MLL rearranged leukemia. This knowledge sheds more light on the BTNL family and may provide the basis for novel approaches using BTNL9 in MLL rearranged ALL therapy.
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Woitecki, Anne [Verfasser]. « Functional characterization of the synaptic-activity regulated gene Synaptotagmin10 / Anne Woitecki ». Bonn : Universitäts- und Landesbibliothek Bonn, 2013. http://d-nb.info/1077288840/34.
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Papaconstantinou, Maria Campos Ana Regina. « Functional characterization of the Mnn1 tumour suppressor gene in Drosophila melanogaster ». *McMaster only, 2007.
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Fugelstad, Johanna. « Functional characterization of cellulose and chitin synthase genes in Oomycetes ». Doctoral thesis, KTH, Glykovetenskap, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-34012.
Texte intégralRésumé :
Some species of Oomycetes are well studied pathogens that cause considerable economical losses in the agriculture and aquaculture industries. Currently, there are no chemicals available that are environmentally friendly and at the same time efficient Oomycete inhibitors. The cell wall of Oomycetes consists of b-(1à3) and b-(1à6)-glucans, cellulose and in some species minute amounts of chitin. The biosynthesis of cellulose and chitin in Oomycetes is poorly understood. However, cell wall synthesis represents a potential target for new Oomycete inhibitors. In this work, cellulose and chitin synthase genes and gene products were analyzed in the plant pathogen Phytophthora infestans and in the fish pathogen Saprolegnia monoica. A new Oomycete CesA gene family was identified, containing four subclasses of genes designated as CesA1 to 4. The gene products of CesA1, 2 and 4 contain pleckstrin homology (PH) domains located at the N-terminus, which is unique to the Oomycete CesAs. Our results show that the SmCesA2 PH domain binds to phosphoinositides, F-actin and microtubules in vitro and can co-localize with F-actin in vivo. Functional characterization of the CesA genes by gene silencing in P. infestans led to decreased cellulose content in the cell wall. The cellulose synthase inhibitors DCB and Congo Red inhibited the growth of the mycelium of S. monoica and had an up-regulating effect on SmCesA gene expression. Zoospores from P. infestans treated with DCB were unable to infect potato leaves. In addition, two full-length chitin synthase genes (Chs) were analyzed from S. monoica. Expression of SmChs2 in yeast yielded an active recombinant protein. The biochemical characterization of the in vitro product of SmChs2 confirmed that the protein is responsible for chitin formation. The chitin synthase inhibitor nikkomycin Z inhibited the SmChs2 both in vivo and in vitro. Altogether these results show that at least some of the CesA1-4 genes are involved in cellulose biosynthesis and that synthesis of cellulose is crucial for infection of potato by P. infestans. The PH domain is involved in the interaction of CesA with the cytoskeleton. In addition, we firmly demonstrate that the SmChs2 gene encodes a catalytically active chitin synthase.QC 20110531
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Ng, Tak-pan. « Expression significance and functional characterization of homeoprotein Six 1 in hepatocellular carcinoma ». Click to view the E-thesis via HKUTO, 2008. http://sunzi.lib.hku.hk/hkuto/record/B41508798.
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Yu, Ka-yin. « Functional and metabolic characterization of a stilbene synthase gene (SbSTS1) from sorghum ». Click to view the E-thesis via HKUTO, 2007. http://sunzi.lib.hku.hk/hkuto/record/B38689406.
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Tang, Yue-bun Alan, et 鄧裕斌. « Molecular and functional characterization of a testis-specific TRS4 gene in spermatogenesis ». Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2010. http://hub.hku.hk/bib/B43572169.
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Zhou, Ke. « Functional characterization of GPI-anchored proteins of the SKU5/SKS gene family ». Phd thesis, Université Paris Sud - Paris XI, 2013. http://tel.archives-ouvertes.fr/tel-01066888.
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ABP1 (Auxin Binding Protein1), who can bind auxin, is essential for the development of plants. It was proved to have the ability to bind auxin and transduce auxin signal into the cells. It is supposed to be localized and functions at the outer surface of plasma membrane through unknown component. In my thesis, we tried to invesitgate the interaction between ABP1 and the candidate of the unknown component, CBP1 (From maize), which is GPI-acnhored and already identified as the binding ability to synthesized C-terminus peptide of ABP1 in 2006. The orthologous of CBP1 in arabidopsis belongs to a gene family with 19 members, in which only three of them were prediceted to be GPI anchored. We did the functional characterisation of these three GPI-anchored members. Data suggested that GPI-anchored SKS were involved in cell orientation, gametophyte and embryo development.
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Tang, Yue-bun Alan. « Molecular and functional characterization of a testis-specific TRS4 gene in spermatogenesis ». Click to view the E-thesis via HKUTO, 2010. http://sunzi.lib.hku.hk/hkuto/record/B43572169.
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Jin, Shao-Bo. « Molecular Cloning and Functional Characterization of Factors Involved in Post-transcriptional Gene Expression ». Doctoral thesis, Stockholm : Institutionen för molekylärbiologi och funktionsgenomik, Univ, 2004. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-24.
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Wunderlich, Kris Rakowitz. « Structural and functional characterization of the polled interval on bovine chromosome 1 ». Texas A&M University, 2008. http://hdl.handle.net/1969.1/85933.
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The horned condition in cattle is believed to be the wild type with morphogenesis
primarily occurring after birth. The polled condition has existed since domestication and
has been selected for its economic importance. The polled locus has previously been
mapped by genetic linkage analysis to the proximal region of bovine chromosome 1. In
order to help us eventually identify the causative mutation, the objective of the study was
to structurally and functionally characterize the polled interval from IFNAR1 to SOD1
on BTA1. Our hypothesis was that the polled locus is a tissue specific transcription
factor that is expressed in the developing horn buds and acts directly or indirectly upon
SOX9.
A 2.5 Mb BAC contig and STS content map of the polled interval was
constructed. Three candidate genes encoding transcription factors were identified within
this region but only C21orf66 was expressed in the horn buds from 1 d old Bos indicus
influenced calves. The C21orf66 gene has 18 exons, spans 30,976 bp of genomic DNA,
and 144 SNP were identified. No single SNP discovered in C21orf66 can be attributed
as the causative mutation. None of the genes from the polled interval were differentially expressed in skin
and horn from 1 d old Bos indicus influenced calves. However, there were significant
differences in the levels of expression of RUNX2, SOX9, BMP4, PRKCA, and FOXL2 in
these samples. Expression of RUNX2 was localized to the osteoblasts, and both RUNX2
and SOX9 were expressed in sebaceous glands of the horn at 1 d of age. Histological
examination of horns and scurs from newborn, 5 to 6 mo, and ~1.5 yr old Bos indicus
influenced cattle suggest that horns form through intramembranous ossification.
Based on the data presented herein, we propose that the polled locus is upstream
of RUNX2 and SOX9 in the osteogenic pathway, and could have its primary effect on
the differentiation of mesenchymal condensations. The genes IL10RB, SFRS15,
C21orf66, OLIG1, OLIG2 and HUNK remain candidates for the polled locus and
warrant further investigation.
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Ng, Tak-pan, et 吳德斌. « Expression significance and functional characterization of homeoprotein Six 1 in hepatocellular carcinoma ». Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2008. http://hdl.handle.net/10722/210303.
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Treacy, Brian K. « Molecular characterization and functional analysis of a pollen-specific gene in Brassica napus ». Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp02/NQ28378.pdf.
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Ho, Meng-Hsuan. « CHARACTERIZATION AND FUNCTIONAL ANALYSIS OF A COTTON RING-TYPE UBIQUITIN LIGASE (E3) GENE ». MSSTATE, 2009. http://sun.library.msstate.edu/ETD-db/theses/available/etd-11032009-165510/.
Texte intégralRésumé :
A cotton fiber cDNA, GhRING1, and its corresponding gene have been cloned and characterized. The GhRING1 gene encodes a RING-type ubiquitin ligase (E3) containing 337 amino acids (aa). The GhRING1 protein contains a RING finger motif with conserved cysteine and histine residues at the C-terminus and is classified as a C3H2C3-type RING protein. Blast searches show that GhRING1 has the highest homology to At3g19950 from Arabidopsis. Real time RT-PCR analysis indicates that the GhRING1 gene is highly expressed in cotton fiber in a developmental manner. The transcript level of the GhRING1 gene reaches a maximum in elongating fibers at 15 DPA. In vitro auto-ubiquitination assays using wheat germ extract and a reconstitution system demonstrate that GhRING1 has the ubiquitin E3 ligase activity. A fiber specific lipid transfer protein 4 (FSltp4) is identified as the target substrate of GhRING1 by using a bacterial two-hybrid system. The binding of GhRING1 and FSltp4 is confirmed by using an in vitro pull down assay and a yeast two-hybrid system. The histochemical GUS assay was performed to analyze tissue specificity of the GhRING1 and At3g19950 promoters in transgenic Arabidopsis plants. The GUS assay shows that the promoter of At3g19950 is highly activated in leaves, roots, trichomes and also in anthers and stigma of flowers. In contrast, the GUS expression directed by the promoter of GhRING1 is only located at stipules and anthers and stigma of flowers. The GhRING1 is the first ubiquitin E3 gene isolated and studied from cotton. Based on the expression pattern of GhRING1, FSltp4, and GhUBC E2s and the identification of a fiber-specific target protein, FSltp4, we propose that protein ubiquitination occurs in fiber and the ubiquitin-proteasome pathway regulates fiber development.
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Frumerie, Clara. « Functional characterization of a phage integrase and its possible use in gene therapy / ». Stockholm : Department of genetics, microbiology and toxicology, Stockholm university, 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-397.
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Ferdous, Zannatul. « Functional and phenotypic characterization of the stearoyl CoA desaturase gene of Anopheles coluzzii ». Thesis, Imperial College London, 2016. http://hdl.handle.net/10044/1/50707.
Texte intégralRésumé :
Malaria is an infectious disease caused by Plasmodium parasites that are transmitted by the bite of female Anopheles mosquitoes. Successful acquisition and transmission of malaria parasites requires a female mosquito obtaining a blood meal from human hosts. The blood meal, which is rich in protein, is required for egg development. Most of the ingested protein is converted to lipid and stored in the fat body where vitellogenesis takes place. In this process, saturated fatty acids are converted to unsaturated fatty acids by the stearoyl-CoA desaturase (SCD1). Unsaturated fatty acids are also essential for maintaining cell membrane fluidity and other housekeeping functions. The main aim of this thesis was to functionally and phenotypically characterize the function of SCD1 during blood meal metabolism in the African mosquito vector Anopheles coluzzii. RNA interference (RNAi) silencing of the SCD1 gene and administration of a small molecule inhibitor of SCD1 had a significant impact on the survival of female mosquitoes following a blood meal. SCD1 knockdown (KD) caused a 100% mortality within 48 h after a human blood meal, while addition of the SCD1 small molecule inhibitor sterculic acid (SA) in the blood meal caused a 50% mortality within 72 h of blood meal. Microscopic analysis showed that SCD1 KD mosquitoes failed to develop eggs in response to the blood meal, while their thorax was filled with blood at 24 h post blood meal. These findings were highly consistent with electron microscopy data that showed increased plasma membrane rigidity and depletion of lipid droplets in the midgut epithelial cells. Transcriptional profiling using A. coluzzii oligonucleotide DNA microarrays showed that genes involved in protein, lipid and carbohydrate metabolism, as well as a large number of immunity genes were the most affected in blood-fed SCD1 KD versus control mosquitoes. Metabolomics profiling highlighted the biochemical framework by which the SCD1 KD phenotype is manifested after a blood meal, revealing increased amounts of saturated fatty acids and TCA cycle (and other interlinked pathway) intermediates in SCD1 KD and SA-treated mosquitoes. The data reported in this thesis reveal that silencing of SCD1 in female A. coluzzii mosquitoes leads to a metabolic syndrome primarily associated with the increase of saturated fatty acids and TCA cycle intermediates, which affects important biological functions leading to premature mosquito death. The accumulation of saturated fatty acids is also the likely cause of a potent immune response observed in the absence of infection, which resembles an auto-inflammatory reaction. These data provide important leads for the development of novel interventions aiming to block transmission of mosquito-borne diseases.
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Wright, Kenneth Lynn. « Functional and Structural Characterization of a Human H4 Histone Gene Promoter : a Thesis ». eScholarship@UMMS, 1990. https://escholarship.umassmed.edu/gsbs_diss/52.
Texte intégralRésumé :
Expression of the cell cycle dependent FO10S human H4 histone gene is regulated at both the transcriptional and post-transcriptional levels. We have investigated the 5' promoter elements mediating the transcriptional aspects of its regulation. A detailed in vivo and in vitro transcriptional analysis of promoter deletion mutants from this gene has identified three positive regulatory elements and two potentially negative regulatory elements within the first 1000 base pairs upstream of the transcription initiation site. In addition, the minimal promoter located within the first 70 base pairs is required for accurate transcription initiation and contains one of two in vivo identified protein-DNA interactions, site II. Binding of the nuclear factor HiNF-D to this region was correlated with the turn-on of histone gene transcription following stimulation of quiescent normal diploid fibroblasts to re-enter the proliferative phase. The most proximal positive regulatory element contains the other in vivo identified protein-DNA interaction, site I. Results from a series of in vitroprotein-DNA interaction studies revealed the binding of two nuclear factors to this element. One protein, HiNF-C, is indistinguishable from the transcription factor Sp1 while the other, HiNF-E, is a novel, potentially histone-specific member of the ATF transcription factor family. Binding of HiNF-C was required to stabilize the interaction of HiNF-E and together this region stimulated transcription 5 fold.
The near-distal transcription activator region lies between -418 and -213 base pairs and forms a single protein- DNA complex, H4UA-1. The interaction domain for H4UA-1 contains recognition sequences for both the thyroid hormone receptor and the nuclear factor CTF/NF-1. The far-distal activator region (-730 and -589 base pairs) was the strongest positive regulatory element identified in the H4 promoter. This region increased transcription 10 fold and contains three protein-DNA interactions. One of the factors, H4UA-2, is an ATF transcription factor closely related to the HiNF-E interaction in the proximal positive element.
These studies have defined the functional human H4 histone promoter to be a complex, modular structure extending at least 1000 base pairs.
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Neuillé, Marion. « Identification and functional characterization of gene defects underlying congenital stationary night blindness (csnb) ». Thesis, Paris 6, 2016. http://www.theses.fr/2016PA066139/document.
Texte intégralRésumé :
Le processus visuel débute lorsque les photorécepteurs transforment la lumière en un signal biochimique qui est ensuite traité et transmis via la rétine. Notre groupe s'intéresse à élucider les défauts génétiques et les mécanismes à l'origine de pathologies rétiniennes comme la cécité nocturne congénitale stationnaire (CNCS), conséquence d'un défaut de transmission du signal entre les photorécepteurs et les cellules bipolaires. Cette thèse apporte de nouvelles connaissances sur la physiologie de cette première synapse visuelle. Nous avons identifié quatre nouvelles mutations dans SLC24A1, un échangeur ionique intervenant dans l'homéostasie du calcium dans les bâtonnets, à l'origine de la CNSC de type Riggs. Nous avons également identifié LRIT3 comme étant un nouveau gène impliqué dans la forme complète de CNCS. Nous avons décrit un modèle de souris invalidé pour Lrit3 avec un phénotype visuel similaire à celui des patients. Nous avons confirmé la localisation de LRIT3 aux extrémités dendritiques des cellules bipolaires ON, suggérant un rôle dans la cascade de signalisation mGluR6. Nous avons montré que LRIT3 était nécessaire à la localisation fonctionnelle de TRPM1. Nous avons de plus démontré un rôle additionnel de LRIT3 dans la formation de la synapse du cône n'impactant probablement que faiblement les voies OFF. Nous avons également réussi à détecter LRIT3 par spectrométrie de masse, ouvrant la voie à l'identification de ses partenaires. La meilleur connaissance de la physiologie et de la physiopathologie rétinienne doit mener non seulement à un meilleur diagnostic et conseil génétique des patients mais également au développement de nouvelles approches thérapeutiquesThe first steps in vision occur when rod and cone photoreceptors transform light into a biochemical signal, which gets processed through the retina. Our group investigates genetic causes and mechanisms involved in inherited retinal diseases as congenital stationary night blindness (CSNB), which reflects a signal transmission defect between photoreceptors and bipolar cells. This thesis gives several insights on the retinal physiology at this first visual synapse. We identified four novels mutations in SLC24A1 underlying the Riggs-type of CSNB, which has a role in calcium balance in rods. We subsequently identified a novel gene, LRIT3, which is mutated in the complete form of CSNB. We delivered a knock-out mouse model lacking Lrit3 which displays a phenotype similar to patients. We confirmed the localization of LRIT3 at the dendritic tips of ON-bipolar cells, suggesting a role of LRIT3 in the mGluR6 signaling cascade. We showed that LRIT3 is necessary for the functional localization of TRPM1. We also revealed that LRIT3 has an additional role in formation of the cone synapse but with probably only a minor effect on OFF-pathway functionality. We finally succeeded in immunoprecipitating and detecting LRIT3 by mass spectrometry, opening the way for the identification of LRIT3 partners. Improving knowledge about retinal physiology and physiopathology will lead to a better diagnosis and genetic counseling of the patients and to the development of novel therapeutic approaches
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Zukunft, Jörg. « Functional characterization of promoter polymorphisms in the human Cytochrome P450 2B6 gene (CYP2B6) ». [S.l. : s.n.], 2005. http://www.bsz-bw.de/cgi-bin/xvms.cgi?SWB11947836.
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Fan, Cui. « Evolutionary and functional characterization of Os-POLLUX, a rice gene orthologous to a common symbiosis gene in legume ». Lexington, Ky. : [University of Kentucky Libraries], 2008. http://hdl.handle.net/10225/942.
Texte intégralRésumé :
Thesis (M.S.)--University of Kentucky, 2008.Title from document title page (viewed on December 10, 2008). Document formatted into pages; contains: vii, 56 p. : ill. (some col.). Includes abstract and vita. Includes bibliographical references (p. 47-55).
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Kiesler, Eva. « Isolation and functional characterization of Hrp65-binding proteins in Chironomus tentans ». Doctoral thesis, Stockholm : Institutionen för molekylärbiologi och funktionsgenomik, Univ, 2004. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-218.
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Bullard, Daniel Charles. « Functional and molecular characterization of a candidate gene family for thet-complex responder locus ». Case Western Reserve University School of Graduate Studies / OhioLINK, 1992. http://rave.ohiolink.edu/etdc/view?acc_num=case1055961951.
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Ohlhoff, Colin Walter. « Biopolymer gene discovery and characterization using metagenomic libraries ». Thesis, Link to the online version, 2008. http://hdl.handle.net/10019/1801.
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Orhan, Le Gac De Lansalut Elise. « From gene identification and functional characterization to genome editing approaches for inherited retinal disorders ». Thesis, Paris 6, 2015. http://www.theses.fr/2015PA066218/document.
Texte intégralRésumé :
La rétine est un tissu spécialisé dans le traitement de l'information visuelle par l'intermédiaire des photorécepteurs, cônes et bâtonnets, et des neurones de deuxième ordre, les cellules bipolaires et les cellules ganglionnaires dont les axones forment le nerf optique. Notre groupe s'intéresse à élucider les mécanismes génétiques impliqués dans les maladies rares stationnaires, comme dans la cécité nocturne congénitale stationnaire (CNCS), ou progressives comme dans la dystrophie de type bâtonnet-cône (DBC). Cette thèse apporte de nombreuses connaissances sur la physiologie rétinienne. D'une part, nous avons identifié GPR179, un nouveau gène impliqué dans la CNCS complète, étudié la localisation de la protéine et la physiopathologie des protéines mutantes. Nous avons également créé et caractérisé fonctionnellement un nouveau modèle souris invalidé pour GPR179 qui nous a permis de mieux approcher la première synapse rétinienne entre les photorécepteurs et les cellules bipolaires adjacentes. D'autre part, nous avons caractérisé le génotype et le phénotype de l'un des modèles les plus utilisés de la DBC, le rat P23H. Nous avons ensuite développé une approche d'édition génomique pour invalider les mutants RHO ayant un effet dominant négatif en testant in vitro, ex vivo et in vivo les meganucleases, TALEN (Transcription Activator-Like Effector Nuclease) puis le système CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9)The first steps in vision occur in the retina when rod and cone photoreceptors transform light into a biochemical signal, which gets processed by bipolar cells, ganglion cells and finally by the brain. Our group investigates genetic causes and mechanisms involved in inherited stationary and progressive retinal diseases as congenital stationary night blindness (CSNB), and rod-cone dystrophy (RCD), also called retinitis pigmentosa. This thesis gives several insights on the retinal physiology. On one hand, we identified GPR179, a new gene mutated in complete CSNB, studied the localization and the physiopathology of missense and splice-site mutations. We also delivered a new knock-out mouse model which we functionally characterized, and studied GPR179 partners to provide a better understanding of the first visual synapse between photoreceptors and ON-bipolar cells. On the other hand, we genotypically and phenotypically characterized one of the most popular RCD model, the P23H rat model. There is currently no treatment for RCD and different therapeutic strategies are under investigation. We wanted to deliver the basis for a genome editing approach for RHO mutations, acting as a dominant negative effect, which cannot be addressed by current gene replacement strategies. We opened the field by performing in vitro, ex vivo and in vivo genome editing experiments using meganucleases, TALEN (Transcription Activator-Like Effector Nuclease) and finally CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9) and revealed how challenging the setting of genome editing strategies was
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Porter, Charmaine L. Locy Robert D. « Functional identification and characterization of an Arabidopsis thaliana gene involved in vitamin B6 biosynthesis ». Auburn, Ala., 2005. http://hdl.handle.net/10415/1261.
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Lin, Chun-Hung, et 林俊宏. « Functional characterization of human NIT gene ». Thesis, 2007. http://ndltd.ncl.edu.tw/handle/24619524845166914059.
Texte intégralRésumé :
博士國立陽明大學
生化暨分子生物研究所
95
The mammalian Nit is a member of the nitrilase superfamily whose function remains to be characterized. Two human nitrilase homologs have been identified and named as Nit1 and NIT. Functions of Nit1 have been revealed to be similar to tumor supppressor Fhit. However, the biological function of human NIT is still unknown. In the present study, we conducted experiments to elucidate the biological role of NIT expressed in human cancer cell line. We confirmed the expression of NIT mRNA by Northern analysis, and NIT was found to be a ubiquitously expressed gene by screening of cDNA in multiple tissues. Confocal image and Western blot analysis revealed that NIT protein is distributed mainly in cytoplasm of cells. Based on Rosetta stone hypothesis with NitFhit in C.elegans, we proposed that NIT may exhibit growth-inhibitory effect. Overexpression of NIT in HeLa cells inhibited cell growth and colony formation. Flow cytometry analysis revealed that NIT transiently transfected cells were arrested in G2 phase. However, neither synergistic effect on growth-inhibitory effect nor physical interaction could be found between NIT and Fhit protein in HeLa cells. The result suggested that interaction with Fhit may not be essential for NIT-mediaed cell cycle arrest. Proteomic analysis illustrated that 14-3-3 family proteins were expressed in response to NIT-induced growth arrest. Overexpressed NIT up- and down-regulated both mRNA and protein levels of 14-3-3σ and 14-3-3β, respectively. It has been documented that 14-3-3σ is involved in cell cycle arrest while 14-3-3β has oncogenic potential, thereby, NIT-induced G2-arrest of cells could be the effect resulting from upregulation of 14-3-3σ and downregualtion of 14-3-3β in HeLa cells. Genotype analysis in four types of primary tumor tissues showed 12.5% to 38.5% allelic imbalance surrounding the NIT locus. In conclusion, these findings suggested that NIT may play an important role in the regulation of human cell growth.
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Wang, Hai-Fan, et 王海帆. « Functional characterization of human NAP gene ». Thesis, 2011. http://ndltd.ncl.edu.tw/handle/38488436393450826604.
Texte intégralRésumé :
碩士國立陽明大學
生化暨分子生物研究所
100
Abstract The Nap protein is ubiquitously and exists in plant, animal and bacteria. Based on sequence homology, the protein was assumed to have enzymatic activity. In eukaryotes, little is known about the function of mammalian Nap and its substrates. Recent studies demonstrated that loss of Nap expression was observed in 48% of esophageal adenocarcinomas. Previous studies indicated that: 1)Nap-deficient mouse kidney cells may increase cyclin D1 expression and accelerate cell proliferation; 2) Nap overexpression induced caspase-dependent apoptosis in mouse cells; 3) Nap-knockout mice are more susceptible to carcinogen-induced murine forestomach tumors; 4) Nap is a cell cycle progression regulator in the T cell homeostasis. However, the previous studies only showed the Nap function based on the mouse model, but for the human cancer cell line. To this end, I assessed the biological functions of Nap in human cancer cell lines. In this study, I analyzed the effect of Nap overexpression on cell growth in three different human cancer cell lines. And knockdown of endogenous Nap expression by siRNA in HeLa cells. Furthermore, overexpression of Nap in HeLa cells and detect mitochondrial membrane potential by JC-1 dye. In the end, I analyzed the ATP levels after overexpression Nap in HeLa cell.
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王家芳. « Functional characterization of a novel gene- FLJ10468 ». Thesis, 2003. http://ndltd.ncl.edu.tw/handle/46543262604189290509.
Texte intégralRésumé :
碩士國立陽明大學
遺傳學研究所
91
Hepatocellular carcinoma (HCC) is one of the most common forms of human malignancy worldwide, and it poses a serious threat to the public health, particularly, among Asian population. To search for the mechanism of carcinogenesis of HCC, we have identified a novel gene FLJ10468. FLJ10468 maps to human chromosome 1p34.2. The completed coding sequence comprises 843 nucleotides, and encodes a 280-amino-acid protein. Two NLS, two coiled-coil domain, and one D box were predicted through genome database search, and only one homolog in human and murine genome were found. By using RT-PCR technique, we found that FLJ10468 was overexpressed in the liver tissue of HCC patients, and it was cell cycle-related and upregulated during G2/M phase in synchronous HEK293T and regenerating mouse liver. Using indirected immunofluorescence, FLJ10468 was mainly localized on nucleolus of HEK293T, HEK293, HeLa, and Huh7, respectively. Some signals were also detected as associated with nuclear envelope. The localization of C-terminal truncated forms (the first 1-26, 1-92, or 1-254 aa of the N-terminus) was similar to the FLJ10468 full length, but the localization of N-terminal deletion mutants (ΔN88 or ΔN180) was mainly localized in the cytoplasm of HEK293T, suggests that there was probably a nucleolus localization signal (NoLS) at the N-terminus. Overexpression of FLJ10468 caused G2/M phase accumulation and a defect on mitosis-G1 phase transition in HEK293T. However, this G2/M arrested cells were not gone to apoptosis. This phenomenon is consistent with the observation that cyclin B1 was upregulated when FLJ10468 was overexpressed. Finally, the G2/M arrest caused by overexpression of FLJ10468 could be override by the overexpression of HURP, another HCC overexpressed gene, suggest that FLJ10468 may play a novel regulatory role on G2/M transition in the cell cycle progression.
41
Wang, Yi-Fang, et 王怡方. « Functional Characterization of Human Homeobox Gene OTEX ». Thesis, 2004. http://ndltd.ncl.edu.tw/handle/08638470721052811290.
Texte intégralRésumé :
碩士臺北醫學大學
醫學研究所
92
A pair-like homeobox gene OTEX (Ovary-Testis-Epididymis-homeoboX) was identified through genomic DNA sequence alignment with a previously isolated homeobox gene TSX1. OTEX gene is localized on the human X chromosome and expressed in testis, epididymis, prostate, ovary and brain. No ortholog of OTEX gene has been identified in mouse genome through intensive GenBank search and cDNA library screening. Zoo blot analysis further confirmed that OTEX is only present in human genome. The results suggested that OTEX is appeared in a later period of the evolution, and may be involved in an important genetic regulation of the human physiology. OTEX expression is also identified in both LNCaP and PC3 prostate cancer cell lines, and the expression was stimulated dramatically following androgen treatment. Proscran was used to search binding motif of upstream and intron 1 of OTEX gene, and an ARE site was identified in the upstream region. Since androgen is crucial to the development of prostate cancer, we change the expression level of OTEX in the LNCaP cell lines, to elucidate the potential role of OTEX in prostate cancer. Both transgenic overexpression and RNA interference were induded in our strategy. OTEX was transfected into PC-3 and COS 7 cell lines to understand the effect of OTEX in other cell lines. From the preliminary result, we found that OTEX overexpression seem enhanced the cell proliferation, which imply a correlation between OTEX and prostate cancer.
42
Chang, Ya-Chin, et 張雅津. « Functional Characterization of Human TSX1 Homeobox Gene ». Thesis, 2002. http://ndltd.ncl.edu.tw/handle/95282001626509945172.
Texte intégralRésumé :
碩士台北醫學院
醫學研究所
90
Several lines of evidence suggest that homeobox genes encode transcription factors to regulate developmental events in philologically diverse organisms. To identify novel homeobox genes involved in testis development, we employed a PCR strategy using homeodomain degenerate primers to screen human testis cDNA libraries. We have isolated a human testis specific homeobox gene, TSX1 (testis-specific homeobox gene). In situ hybridization analysis revealed that TSX1 gene is expressed in spermatocytes and spermatids during spermatogenesis. Analysis of the phenotypes of TSX1 knockout mice is a straight-forward approach to understand the in vivo function of a gene. We have not been able to isolate mouse Tsx1 ortholog after several times screening of mouse testis cDNA libraries with human TSX1 cDNA as probes. We employed Southern blot analysis by using a 12-species-DNA Zoo blot membrane, and found TSX1 is only present in human genome. Since there is no mouse TSX1 gene for making knockout construct, we therefore generated transgenic mice to further study the effect of TSX1 overexpression in the animal. TSX1 expressions were identified in testis, oviduct and uterus in some of the transgenic lines by Western analysis. We have also found some interesting phenotypes in some of the transgenic mice. Whether these phenotypes are correlated to the TSX1 expression or phenotypes related to reproduction are induced by TSX1 overexpression will need further analysis. In the study of protein level, we constructed expression plasmids to generate TSX1-fusion protein from the bacteria. TSX1-fusion protein will be used to raise antibody, identify the target-binding site and immunoprecipitate the TSX1-interacting proteins. Results from both in vitro and in vivo studies will further imply the function of TSX1 during spermatogenesis.
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Godinho, Lia Raquel Marques. « Characterization of genes of unknown function in Bacillus subtilis : gene regulation and functional analysis ». Doctoral thesis, 2016. http://hdl.handle.net/10362/19080.
Texte intégral
44
Kao, Gour-Shenq, et 高國勝. « Functional characterization of the KIAA1866 gene in human ». Thesis, 2004. http://ndltd.ncl.edu.tw/handle/75592674186493642001.
Texte intégralRésumé :
碩士國立成功大學
分子醫學研究所
92
Deletions of the chromosome 6 q were observed in several human tumor types, especially in many different types of lymphoma. Our previous study in nasal NK/T cell lymphomas has mapped the smallest region of overlapping (SRO) deletion to a 2.6Mb region on 6q25.3 interval with 86% deletion rate. The neighbor regions also have high frequency of DNA copy number loss; for example, the KIAA1866 gene located in 6q25.3 exist 43% loss in examined nasal NK/T cell lymphoma cases. The KIAA1866 gene has been cloned from the human fetal brain cDNA library. The role of this hypothetical protein remains unknown. We had examined the expression profiles in human normal and tumor tissues and results indicate a low abundance in all tested tissues with relatively higher expression in normal heart, bladder, testes, brain, and thymus tumor. Quantitative measurement of expression in cDNAs of bladder cancer cell lines (RT4, TSGH8301, J82, and T24) demonstrated that the KIAA1866 expresses trace amount in J82 cells and this only counts 1/10 of the expression of normal bladder tissue. The constructed KIAA1866 fusion proteins were successfully expressed in E. coli system as well as in mammalian cell lines (HeLa and HEK293). The KIAA1866ORF-EGFP protein showed cytoplasm localization but not in the nuclear as predicted by pSORT II program. Bioinformatics predications have revealed many functional domains in KIAA1866 protein including phosphorylation sites, N-myristoylation sites, cleavage sites, interaction motifs and binding motifs. However, the exact physiological role of KIAA1866 protein in human cells remains to be determined empirically.
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Wei-YuHuang et 黃偉瑜. « Functional Characterization of yeast Saccharomycopsis fibuligera MIG1 gene ». Thesis, 2015. http://ndltd.ncl.edu.tw/handle/35214070566349899389.
Texte intégralRésumé :
碩士國立成功大學
生命科學系
103
Glucose is the most preferred carbohydrate source in yeast. When glucose is present, genes are responsible for metabolizing other sugars will be suppressed and this phenomenon has been termed glucose repression. Mig1p is the major transcription factor that is a Cys2-His2 type of zinc-finger protein and responsible for the glucose repression in yeast. When S. cerevisiae is exposed to high glucose concentrations, Mig1p is not phosphorylation state and is located in the nucleus to represses the target genes by binding to the promoters of the genes. When S. cerevisiae is exposed to low glucose concentrations, Mig1p is phosphorylated through the Snf1p mediated signal transduction cascades and is translocated from nucleus to cytoplasm to cause the genes expression which are repressed by Mig1p. Otherwise, Mig1p is not only suppressing alternative sugars but also suppressing other genes. Mig1p is global regulation transcriptor in S. cerevisiae. Previous studies indicated that glucose repression phenomenon was observed in S. fibuligera. However, the role played by Mig1p homologous of S. fibuligera is still not clear. In this study, we compared the sequence similarity between Saccharomyces cerevisiae MIG1 and S. fibuligera MIG1 genes, and we replaced the S. cerevisiae MIG1 gene with S. fibuligera MIG1 gene to gain more information on the functionality of S. fibuligera MIG1 gene. Doubling time is no difference between wild type, KO mutants (knock out S. cerevisiae mig1), and KI mutants (knock in S. fibuligera MIG1). Spot assay in YPS or YPG contains 2-deoxyglucose; KO grew well than wild type and KI mutants. In real-time PCR, KO mutants’ gene expressions are higher than KI mutants or wild type for SUC2, HXT4 and EMI2. But it’s no difference between KO mutants and KI mutants for REG2. Based on the above, there are some different functions between S. cerevisiae Mig1p (scMig1p) and S. fibuligera Mig1p (sfMig1p).
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Lin, Wan-Shiuan, et 林宛萱. « Functional Characterization of Metallophosphoesterase Domain Containing Gene 1 ». Thesis, 2008. http://ndltd.ncl.edu.tw/handle/05264319071999760404.
Texte intégralRésumé :
碩士國立陽明大學
生命科學暨基因體科學研究所
96
Metallophosphoesterase domain containing gene 1 (MPPED1) is a novel gene expressed mainly in the brain. To address the biochemical function of Mpped1, study of MPPED1 interacting proteins may provide a basis. Using yeast two-hybrid system, MPPED1 interaction proteins were identified, including transcription factors such as COUP-TFI, COUP-TFIII, TSG101, CFP1, a member of MAP kinase, ERK5 and several ECM or cystoskeleton related proteins. Based on the protein conserved domain prediction, MPPED1 belongs to calcineurin-like phosphoesterase family and enzymes of this family require histidine or aspartate residues for metal chelating in maintaining their structure and phosphatase function. In order to investigate on the potential phosphatase function of MPPED1, mutant variants at histidine residues were generated. We found that some MPPED1 protein-protein interactions were histidine-dependent and some were histidine-independent, suggesting a differential interaction mechanism of MPPED1. Since COUP-TFI plays an important role in developing brain where MPPED1 is specifically expressed, further analysis of the MPPED1-COUPTFI interaction was performed. Luciferase reporter system revealed that MPPED1 acted as an antagonist of COUP-TFI mediated transcriptional activation of NGFI-A promoter. Notably, MPPED1 conserved histidine residues did not seem to play important role in the function of MPPED1. Subcellular localization suggested that MPPED1 might alter COUP-TFI cellular distribution by retarding COUP-TFI mainly in the cytoplasm and thus provided a mechanism of MPPED1 inhibition on COUP-TFI function.
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Hsieh, Yi-Chen, et 謝宜真. « Cloning and functional characterization of the HRASLS2 geneCloning and functional characterization of the HRASLS2 geneCloning and functional characterization of the HRASLS2 gene ». Thesis, 2005. http://ndltd.ncl.edu.tw/handle/59721003245325865786.
Texte intégralRésumé :
碩士國防醫學院
微生物及免疫學研究所
93
英文摘要 H-REV107 gene family belongs to the type Ⅱ tumor suppressor genes, which includes H-REV107, RIG1, HRASLS, HRASLS2 and HRLP5. Proteins of this gene family contain the N-terminal NC domain and the C terminal hydrophobic transmembrane domain. Presence of the C terminal transmembrane domain is essential for functions of the proteins. The H-REV107 family proteins exhibit activities to suppress cell growth and to induce cellular apoptosis, and/or differentiation. The anti-tumor activities of the protein family may be mediated through suppressing of Ras-mediated signal pathways. Although displays high sequence homology with H-REV107 family proteins, functions of the HRAS-like suppressor 2 (HRASLS2) have not be investigated. This study analyzed expression profiles and biological functions of the human HRASLS2. The HRASLS2 was expressed at high levels in normal tissues of the colon, small intestine, stomach, kidney and trachea. However, it was expressed at low levels in breast (MCF-7) and colorectal (HCT116) cancer cell lines. Expression vectors that synthesize HRASLS2 fusion proteins were constructed by subcloning of the HRASLS2 cDNA amplified from the SW480 colorectal cancer cells. Successful expression of HRASLS2 fusion proteins was detected in transfected HtTA cells by Western bloting and immunocytochemical staining. The full length HRASLS2-DsRed fusion protein predominantly expressed on the Golgi apparatus but not the plasma membrane nor the nucleus. Expression of the HRASLS2-myc fusion protein in MCF-7 breast cancer cells resulted in suppression of colony formation. Also, transient HRASLS2 transfection induced the release of lactate dehydrogenase and chromatin condensation in concentration and time dependent mannars in HtTA cervical cancer cells. The carboxyl-terminal truncated HRASLS2 fusion proteins expressed as the diffuse pattern in cytoplasm and had no effect on colony formation and the release of lactate dehydrogenase. In conclusion, HRASLS2 protein exhibited growth suppressive and proapoptotic activities on cancer cells. The carboxyl-domain was indispensable for endomembrane localization and biological functions of the HRASLS2 protein.
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Chuan-Nien et 姚俊年. « Functional characterization of an oocyte specific homeobox Gene, Cphx ». Thesis, 2009. http://ndltd.ncl.edu.tw/handle/40165753368829043666.
Texte intégralRésumé :
碩士中山醫學大學
生物醫學科學學系碩士班
97
The homeobox gene products act as transcription factors during animal developmental process. From previous results, we identified a novel homeobox-containing gene, Cphx (Cytoplasmic Polyadenylation Homeobox gene), which is preferentially expressed in embryonic stem cells and oocytes. De novo expression of Cphx started at 13.5 days postcoitum in the ovaries, which coincides with the initiation of oogenesis. Since oocyte maturation is a complex process and the mechanism of gene regulation during oogenesis is unclear, studies of Cphx functions in vivo may have a great help to decipher them. Here, we used anti-Cphx polyclonal antibodies to analyze Cphx protein expression pattern. The results of Western blotting and immunohistochemistry indicated that Cphx proteins were increasingly expressed from 3-week ovaries and only in secondary follicles. We further used RNA interference technique to explore the in vivo functions of the Cphx gene. We generated transgenic mice expressing a double-stranded interfering Cphx RNA, drived by an oocyte-specific Zp3 promoter, to interfere in vivo Cphx gene expression. Among five transgenic lines, only the line M50D with the highest copy of transgenes (eight copies) showed significant decrease in female fertility than control littermates. Results of real-time PCR also showed decrease in Cphx gene expression in adult ovaries of these four transgenic lines M31, M47, M48, M50 and M50D. Compared the oocyte numbers of M50D transgenic mice with control littermate, there is only 16% decrease in M50D line. We also found Figlα might directly regulate Cphx expression. The significant decrease of Cphx expression in Figlα knockout mice was recently identified. Figlα is a gene which only expresses in germline cell and can regulate the development of oocytes and follicles. Thus, we constructed a Figlα expression vector and cotransfect with reporter genes drived with different lengths of Cphx promoter. Reporter genes with Cphx promoter were upregulated by Figlα expression. Therefore, we presumed that Figlα directly regulates Cphx expression. Since the expression of Cphx and Gdf9 was decreased at the same time in Cphx RNAi transgenic mice, we suggest that Gdf9 expression was regulated by Cphx.
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Lin, Li, et 林立. « Functional characterization of a NCED gene from Phaius tankervilliae ». Thesis, 2015. http://ndltd.ncl.edu.tw/handle/50651174789606459308.
Texte intégralRésumé :
碩士國立中興大學
生物科技學研究所
103
Orchid seeds are characterized by their tiny size, with a globular stage embryo and without endosperm. Seed germination in vitro of several terrestrial orchids is difficult while the seed matured. Previous studies have shown that the endogenous ABA level remained high in the mature seeds, suggest a cause for their low germination rate. Isolated from Phaius tankervilliae by Dr. Lee, a gene putatively encode 9-cis-epoxycarotenoid dioxygenase (NCED) that catalyze conversion of 9-cis xanthophylls to xanthoxin, an ABA precursor, was named as PtNCED1. Southern hybridization confirmed its single gene status in Phaius tankervilliae. As PtNCED1 was expressed in early seeds and persistent till seeds maturation, it is speculated that the un-descending PtNCED1 transcripts may cause an accumulation of ABA, so to arrest the embryo development and block the seed germination in Phaius. To demonstrate that the PtNCED1 encode enzyme with NCED activity, and to identify its orthologous gene in Arabidopsis, 11 T-DNA knock-out lines of AtNCED1, 2, 5, 6, or 9 were obtained from Arabidopsis Biological Resource Center (ABRC). As AtNCED6 and -9 were highly expressed in the developing seeds, their homozygotic T-DNA insertion lines were chosen for functional complementation test and the PtNCED1 were expressed under controlled by 35S promoter. However, the complemented transgenic plants grown weakly and barely produced seeds, therefore could not be further analyzed. Coincided with other species, PtNCED1/mGFP5 was localized mainly in chloroplast. Finally, a significantly increased ABA content was detected in tobacco leaves that transiently overexpressed PtNCED1, demonstrating that PtNCED1 is involved in ABA biosynthesis.
50
Li, Guo-Bin, et 李國斌. « Characterization of zebrafish KLF4a gene and its functional analysis ». Thesis, 2005. http://ndltd.ncl.edu.tw/handle/04301660238043234655.
Texte intégralRésumé :
碩士國立臺灣海洋大學
生物科技研究所
93
Abstract Krüppel-like factors (KLFs) encoding transcription factors have been found in many organisms. They contain three highly conserved tandem zinc finger DNA binding motifs in the C-terminal region and unique functional domain in the N-terminal coding region. Studies indicated that KLFs are multi-functional protein and play key roles in cell proliferation, differentiation, as well as regulation of cell cycle and repression of tumor. Recently, zebrafish becomes a model organism for studying vertebrate embryogenesis. However, derailed molecular mechanisms regarding morphogenesis of digestive tract development are still lacking. Therefore, in this thesis we investigated possible roles of zebrafish KLF in digestive tract development during embryogenesis. We have previously cloned a zebrafish KLF4a cDNA, which contains 2119bp and encodes 396 amino acids. Amino acid sequence comparison showed that zebrafish KLF4a shares 65.8％ sequence similarity with mouse KLF4. Zebrafish KLF4a also contains acidic activation domain in the N-terminus and nuclear localization signal and three highly conserved zinc finger DNA binding domains in the C-terminal region. Phylogenetic tree analysis grouped zebrafish KLF4a with human and mouse KLF4 in the same branch. Whole mount in situ hybridization revealed KLF4a mRNA first detected in 1 cell stage and was expressed in blastula period. KLF4a was expressed in the dorsal shield and axial mesendoderm during gastrula. During segmentation period, KLF4a was expressed in the otic primordial, brain and somite. After 24 hpf KLF4a was expressed in the eyes, ventral region of brain, midbrain-hindbrain boundary (MHB), ventricular zone, otic vesicle, somites, pharynx, liver, exocrine pancreas and gut. Frozen sectioning of 96 hpf embryos hybridized with KLF4a RNA probe showed KLF4a was mainly localized in epithelial cell of gut. Using KLF4a antisense morpholino oligomer to inhibit the function of KLF4a, development of MHB and dorsal-ventral axis of brain in morphant were affected in embryos form 24 hpf to 72 hpf. In the meantime, the development of eyes and semi-circular canal was delayed. After 72 hpf, cavity of foregut, folding of gut and development of swim-bladder were affected. Conparison of expression pattern of different digestive tract marker genes in KLF4a morphant and wild-type embryos showed that the expression level of dlx7 in esophagus and insulin in endocrine pancreas were not alterd. However, the expression level and domain of IFABP in the liver and foregut, LFABP in the liver and trypsin in the exocrine pancreas were decreased. On the contrary, expression of shh in the month and pharynx were inhibited by KLF4a. Overall, our results showed that KLF4a is homologue of mammalian KLF4 and it plays an important role in the development of liver, exocrine pancreas and gut.