Dissertations / Theses on the topic 'Runt related transcription factor'

The underlying inflammatory storm in renal or diabetic disease may induce expression of inducible nitric oxide synthase (iNOS). Similarly, expression of iNOS or nitric oxide (NO) production in vascular smooth muscle cells (VSMCs) in a calcifying environment, may promote vascular calcification (VC) (Zaragoza et al., 2006). However, emerging data suggests that NO generated by either endothelial nitric oxide synthase (eNOS) or iNOS may protect VSMCs from VC (Kanno et al., 2008). Thus, the role of NO and its associated enzymes in the development of VC is unclear. The aim of this study was to identify whether NO produced by iNOS regulates calcification in VSMCs, and to further understanding of potential mechanisms that may mediate the actions of NO/iNOS. A significant and sustained production of NO by iNOS, which peaked at day 3 and declined thereafter was found in rat aortic smooth muscle cells (RASMCs) that were preactivated with lipopolysaccharide (LPS; 100μg ml-1) and interferon gamma (IFN-γ;100U ml-1) in the presence of calcification buffer (CB) containing calcium chloride (CaCl2; 7mM) and β-glycerophosphate (β-GP; 7mM). This was associated with formation of hydroxyapatite crystals (HA) or calcification plaques, observed via alizarin red staining (ARS) and/or fourier transform infrared (FT-IR) analysis. However, when RASMCs were incubated with the iNOS inhibitor GW274150 at 10 μM, together with LPS + IFN-γ + CB, HA crystal formation was abolished. When RASMCs were pretreated with diethylenetriamine/nitric oxide adduct (NOC 18) at either 30 or 50 μM for an hour prior to addition of CB, to generate NO; calcium levels were elevated leading to form HA crystals. However, the elevation of calcium caused by the presence of NO generated via iNOS, did not result in phosphorylation of mitogen activated protein kinases (p38 MAPK), extracellular signal-regulated kinases (Erks), and protein kinase B. Furthermore, there was a reduction of Runx2 levels (pro-calcific factor) which could be another pro-calcific factor involved in this mechanism. These findings suggest that NO may indeed play a fundamental role in calcification, enhancing mineralisation of smooth muscle cells. Furthermore, the expression of iNOS/ NO appears to be enhanced under conditions that favour calcification and these together may contribute to enhanced calcification with potential detrimental consequences in vivo.

Bone mineral density (BMD) is a reflection of the action of osteoblasts compared to osteoclasts. An imbalance in the activity of osteoblasts or osteoclasts, results in bone disease such as osteoporosis caused by overactive osteoclasts. BMD is influenced by genetic and environmental factors as demonstrated through twin studies, association studies and linkage analysis (Ralston, 1999). Several polymorphisms involved in the determination of BMD have been identified, with Vitamin D receptor and Collagen Type 1 showing reproducible associations. To identify genes influencing BMD two distinct strategies have been employed: 1) To determine if DNA polymorphism within the runt related transcription factor (RUNX2) gene is a determinant of BMD and fracture in women. 2) The identification of RANKL target genes in osteoclastogenesis. RUNX2 is a runt domain transcription factor (Werner et al., 1999) essential for osteoblast differentiation (Lee et al., 1997). RUNX2 gene knock-out mice have no osteoblasts due to a failure in osteoblast differentiation and consequently unmineralised skeletons, (Komori et al., 1997; Otto et al., 1997). In humans, mutations in RUNX2 cause cleidocranial dysplasia (CCD), a disorder characterised by hypoplasia or aplasia of the clavicles, short stature, supernumerary teeth, patent fontanelles and other changes in skeletal patterning and growth (Mundlos et al., 1997). RUNX2 contains a poly-glutamine poly-alanine (polyQ/polyA) repeat where mutations causing cleidocranial dysplasia have been observed. BMD has not been routinely examined in CCD, two studies have identified CCD patients with lower BMD with one fracture case identified (Quack et al., 1999; Bergwitz et al., 2001). The central role of RUNX2 in determining osteoblast differentiation makes RUNX2 a prime candidate gene for regulating adult bone density. To determine if polymorphism was present in the polyQ/polyA tract the repeat was amplified within the upper and lower deciles of femoral neck (FN) BMD in the Geelong Osteoporosis study (GOS). The upper and lower deciles of FN BMD acted as a surrogate for genotyping the entire cohort. This study identified two common variants within the polyA repeat: an 18 base pair deletion (11Ala) and a synonymous alanine codon polymorphism with alleles, GCA and GCG (noted as A and G alleles, respectively). The 11Ala and SNP polymorphism are found on codon 64 and 66 respectively (RUNX2 MRIPV variant). A allele frequencies were significantly different in a comparison of the upper and lower deciles of FN BMD (p=0.019). In 495 randomly selected women of the Geelong Osteoporosis Study (GOS), the A allele was associated with higher BMD at all sites tested. The association was maximal at the ultra-distal radius (p=0.001). In a separate fracture study, the A allele was significantly protective against Colles' fracture in elderly women but not spine and hip fracture. The 11Ala polymorphism was not related to BMD in GOS. To further decipher the role of the RUNX2 A allele we genotyped 992 women from a Scottish cohort. The alleles of RUNX2 within the glutamine/alanine repeat were determined by MspA1I restriction digest. To examine the possible influence on estrogen related therapies or estrogen status on the potential genetic effect conferred by RUNX2, we divided the cohort by menopausal and hormone replacement therapy status. Within postmenopausal Scottish women the RUNX2 A allele was associated with significantly higher FN BMD (p=0.028, n=312) but not lumbar spine (LS) BMD. The A allele was associated with higher FN BMD (p=0.035) within a postmenopausal subgroup of the population (n=312). To investigate the effect of weight on the RUNX2 alleles the Scottish cohort was segregated into thin/normal (BMI ≥ 25 kg/m2) and overweight /obese (BMI > 25 kg/m2). RUNX2 A allele showed a stronger effect on FN BMD in postmenopausal women above the median BMI. The 11Ala RUNX2 deletion allele was significantly associated with decreased LS BMD (p=0.018) within overweight/obese women (n=546). The 11Ala allele was significantly associated with increased levels of pyridinoline (p=0.014) and deoxypyridinoline (p=0.038) in the HRT treated subgroup of the population (n=492). Glutamine variants and an alanine insertion were identified within the group. These data suggest that the RUNX2 11Ala and A alleles exert differing affects on BMD showing preference for different skeletal sites in a weight dependent manner. We genotyped 78 individuals from an osteoarthritic population to elucidate the role of the RUNX2 alleles on markers of bone turnover and inflammation. The RUNX2 11Ala allele was significantly associated with decreased osteocalcin (OC) serum levels (p = 0.01). The RUNX2 A allele was significantly related to reduced tumor necrosis factor alpha (TNF-alpha) serum levels (p = 0.004). RUNX2 is known to bind to the OC promoter. An OC promoter polymorphism is found 7bp upstream from a putative RUNX2 binding site. We hypothesized that OC polymorphism may effect the RUNX2 transactivation of the OC gene and thus affect OC serum levels. OC promoter polymorphism was not related to OC serum levels (n=78). These data present a novel link between RUNX2 alleles and OC and TNF serum levels, providing putative mechanisms of action for the RUNX2 alleles. Further studies in larger populations are required to confirm these findings. Ten individuals within the GOS and the Scottish cohort were found to carry rare mutations of the polyQ/polyA repeat. All polyQ variants had a normal polyA repeat (17 amino acids) and were heterozygous for a normal 23Q/17A allele. Variants observed were 15, 16, 24 and 30Q. One individual was observed with an extended polyA repeat (24A). Patient records indicated otherwise unremarkable clinical history except for fracture in 4/10 individuals from GOS (hip and spine). BMD data from the LS and the FN were expressed as T-scores, a measure that relates BMD in terms of standard deviations below the young normal value. In addition, BMD data were also expressed as Z-scores around the age-mean. Under the null hypothesis, where RUNX2 Q repeat variation has no effect on BMD, Z scores would be expected to be distributed around a mean of zero. However, when all variants were pooled the BMD was significantly lower than expected. This effect persisted when deletion variants were considered alone. The effect was stronger on FN BMD (p=0.001) rather than LS BMD (p=0.096), reflecting either difference in precision of BMD measurements at these sites or perhaps a differential genetic effect on different skeletal sites. These data suggest that polyQ and polyA variants are associated with significantly lower BMD, and may be an important determinant for fracture. Glutamine variants exist at high frequency (~0.7%): this rate of mutation could be important when considering large populations at risk of age related osteoporosis. Considering that these subjects are heterozygous for a normal allele, it suggests that a more severe phenotype might be expected in rare subjects homozygous for glutamine repeat variants. In summary, this study investigated the role of novel polymorphisms and rare variants of the RUNX2 gene in influencing BMD, fracture and markers of bone turnover. Two common polymorphisms were identified within the polyA repeat: an 18 base pair deletion and a synonymous alanine codon polymorphism with alleles, A and G. The A allele was associated with increased BMD and was protective against a common form of osteoporotic fracture within a Geelong population. To verify these findings the RUNX2 alleles were genotyped in 992 women from a Scottish cohort. The magnitude and the direction of the effect of the A allele was maintained in the Scottish cohort. Interestingly, the A allele was shown to exert a menopause specific effect, with postmenopausal women showing the strongest effect. On re-analysis of the GOS data the post-menopausal women were found to drive the significance identified in the cohort. The magnitude of the effect of the A allele on BMD was greater in overweight/obese postmenopausal women indicating a gene-weight interaction for RUNX2. The RUNX2 11Ala allele showed a significant relationship with decreased LS BMD in overweight/obese Scottish women. The 11Ala allele was also associated with higher levels of urinary PYD and DPD in women treated with HRT, indicating higher levels of bone turnover in carriers of the 11Ala allele. In contrast to the Scottish cohort, no significant association with heterozygous carriers of 11Ala was observed in GOS, although a significant association was detected for homozygous carriers and LS BMAD. The 11 Ala RUNX2 allele was significantly associated with decreased serum osteocalcin levels and the A allele was significantly associated with TNF in OA patients. Glutamine variants and an alanine insertion were identified within Geelong and Scottish cohorts, which showed low Z and T scores suggesting that RUNX2 variants may be related to genetic effects on BMD and osteoporosis. Polymorphism of the polyQ/polyA region of RUNX2 were identified within this study were shown to associate with significant differences in BMD. The A allele showed a significant association with increased BMD in postmenopausal women from a Geelong and Scottish cohort, with a decreased frequency of the A allele observed in Colles' fracture patients from Geelong. The 11Ala deletion allele was significantly associated with decreased LS BMD and increases in markers of bone turnover in the Scottish cohort. A significant decrease in OC serum levels was observed in OA patients suggesting a direct effect of the allele on the transactivation of the RUNX2 gene. Rare variants of RUNX2 were identified which showed low BMD. These studies have provided insight into the role of RUNX2 in influencing BMD, further studies are required to verify the role of the A allele on BMD and fracture, the role of the rare variants and to identify the precise mechanisms behind the observed changes in BMD. - 2) The identification of RANKL target genes in osteoclastogenesis. Osteoclastogenesis is regulated in vivo by the action of osteoblast/stromal cells that express membrane bound, receptor activator of NF-kB ligand (RANKL). Monocytes treated in vitro with a soluble form of RANKL and macrophage colony stimulating factor (M-CSF) differentiate to osteoclasts, whereas monocytes treated with M-CSF alone differentiate to macrophage-like cells. The gene expression profile of human osteoclasts has not been extensively explored. Genes highly expressed by rabbit osteoclasts were identified through random sequencing of an osteoclast cDNA library (Sakai et al., 1995). Differential gene expression of mouse osteoclastogenesis was elucidated by array analysis (Cappellen et al., 2002). To identify genes important for human osteoclastogenesis, total RNA was isolated from monocytes treated for three weeks with either M-CSF alone or with RANKL and M-CSF. RANKL treatment for 3 weeks and 12 hours was investigated in this study, to complement previous data. Differential display was performed on RNA (12 hour treatment with RANKL) and differential gene expression profiles examined. The differential display products were pooled to generate a probe for screening a gene array system derived from a human osteoclast cDNA library. cDNA (3 week treatment with RANKL) hybridisation experiments against the array revealed additional regulated genes. Gene clones that showed significant regulation in M-CSF and RANKL treated cells compared M-CSF treated cells represent genes that are targets for RANKL-specific regulation. Osteopontin, creatine kinase and various mitochondrial genes were up regulated by the treatment of RANKL. Changes in gene expression observed in the array data were confirmed with real-time PCR using mRNA derived from in vitro induced osteoclasts. Cathepsin K gene expression was more than 300 fold greater in osteoclasts compared to macrophage-like cells after one week treatment with RANKL and M-CSF. Cystatin C expression showed a six-fold induction at two weeks of RANKL and M-CSF treatment and cystatin B showed a steady increase in expression. Some of these regulated genes may provide useful targets for influencing BMD.

In summary, this study investigated the role of novel polymorphisms and rare variants of the RUNX2 gene in influencing BMD, fracture and markers of bone turnover. Two common polymorphisms were identified within the polyA repeat: an 18 base pair deletion and a synonymous alanine codon polymorphism with alleles, A and G. The A allele was associated with increased BMD and was protective against a common form of osteoporotic fracture within a Geelong population. To verify these findings the RUNX2 alleles were genotyped in 992 women from a Scottish cohort. The magnitude and the direction of the effect of the A allele was maintained in the Scottish cohort. Interestingly, the A allele was shown to exert a menopause specific effect, with postmenopausal women showing the strongest effect. On re-analysis of the GOS data the post-menopausal women were found to drive the significance identified in the cohort. The magnitude of the effect of the A allele on BMD was greater in overweight/obese postmenopausal women indicating a gene-weight interaction for RUNX2. The RUNX2 11Ala allele showed a significant relationship with decreased LS BMD in overweight/obese Scottish women. The 11Ala allele was also associated with higher levels of urinary PYD and DPD in women treated with HRT, indicating higher levels of bone turnover in carriers of the 11Ala allele. In contrast to the Scottish cohort, no significant association with heterozygous carriers of 11Ala was observed in GOS, although a significant association was detected for homozygous carriers and LS BMAD. The 11 Ala RUNX2 allele was significantly associated with decreased serum osteocalcin levels and the A allele was significantly associated with TNF in OA patients. Glutamine variants and an alanine insertion were identified within Geelong and Scottish cohorts, which showed low Z and T scores suggesting that RUNX2 variants may be related to genetic effects on BMD and osteoporosis. Polymorphism of the polyQ/polyA region of RUNX2 were identified within this study were shown to associate with significant differences in BMD. The A allele showed a significant association with increased BMD in postmenopausal women from a Geelong and Scottish cohort, with a decreased frequency of the A allele observed in Colles’ fracture patients from Geelong. The 11Ala deletion allele was significantly associated with decreased LS BMD and increases in markers of bone turnover in the Scottish cohort. A significant decrease in OC serum levels was observed in OA patients suggesting a direct effect of the allele on the transactivation of the RUNX2 gene. Rare variants of RUNX2 were identified which showed low BMD. These studies have provided insight into the role of RUNX2 in influencing BMD, further studies are required to verify the role of the A allele on BMD and fracture, the role of the rare variants and to identify the precise mechanisms behind the observed changes in BMD.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Health Sciences
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Thyroid Tumorigenesis is typically a well understood process, with well delineated oncogenic factors. Follicular and papillary thyroid cancers are typically survivable, with 5-year survival rates being >95% for Stage I-III of both cancer types. Anaplastic thyroid cancer, in contrast, lacks this prognosis, and is the most lethal of all endocrine-related cancers. The median survival time after a diagnosis is generally between 6-8 months, with a 5-year survival rate of <10%. Current treatment for anaplastic thyroid cancers routinely meet roadblocks, as resistance is quickly developed. Even non-discriminatory kinase inactivators, such as sorafenib, which are generally considered a drug of last resort, are unable to effect survival rates. As such, there is a clear need for further investigation of the causes of anaplastic thyroid cancer mechanisms. Previous work in the Carr lab revealed a novel regulatory pathway of an oncogene that is associated with several other endocrine-related cancers, as well as other non-endocrine-related cancers. Specifically, the Runt-related transcription factor 2 (Runx2) was found to be suppressed via direct binding of the thyroid hormone receptor beta 1 isoform (TRß1) to its proximal promotor. Runx2 was previously shown to be associated with increasing malignancy, with Runx2 occurring at low-levels in indolent cell lines, whilst occurring at high-levels in more malignant cell lines. TRß1, conversely, exhibited the opposite relationship. Endogenous levels of TRß1 were found to be high in indolent cell lines and were depleted in malignant cell lines. These findings were further confirmed via tissue microarrays. Restoration of TRß1 in malignant cell lines diminished Runx2 mRNA and protein levels, which was corroborated by evidence from electrophoretic mobility-shift assays, and chromatin immunoprecipitations that TRß1 was able to directly bind Runx2 promotor 1. Current studies have investigated the nuclear protein profile that associates with TRß1 to alter Runx2 transcription. Through EMSA-to-Mass Spectrometry methodologies, as well as novel DNA pulldown techniques, binding partners have been elucidated. Findings have also been confirmed via classical immunoprecipitations. Specifically, our findings show that TRß1 complexes with the brahma-related gene 1 (BRG1) protein, the nuclear co-repressor (NCOR), and BRG1-associated protein 60 (BAF60). BRG1 functions by preferentially recruiting histone deacetylases (HDAC), with BRG1 and the HDAC’s acting to alter chromatin, and thus transcription. Future studies aim at examining whether other proteins complex with TRß1 to alter Runx2 transcription, and whether these complexes are altered in aggressive cell lines.

A alveolite é uma complicação pós-operatória de carácter inflamatório que acomete alvéolos de dentes recém-extraídos. A incidência dessa complicação varia de 1 a 4% e pode chegar a 30%. O objetivo deste estudo foi analisar os mecanismos biológicos envolvidos no processo de reparo de alvéolos intencionalmente infectados, em ratos; comparar diferentes modalidades de tratamento e correlacionar os resultados encontrados através de duas análises (microscópica e molecular). Foram utilizados 84 ratos, divididos nos grupos: I: alvéolo não infectado; II: alvéolo infectado sem nenhum tratamento; III: alvéolo infectado tratado com irrigação de solução de iodeto de sódio a 2% e peróxido de hidrogênio a 3% na proporção de 1:1; e IV: alvéolo infectado submetido à curetagem, irrigação com soro fisiológico e preenchimento com uma pasta à base de metronidazol. Os animais foram eutanasiados aos 6, 15 e 28 dias pós-operatório. Foi realizada a análise quantitativa da expressão de genes envolvidos no processo de reparo [colágeno tipo I (COL-I), fator de crescimento do endotélio vascular (VEGF), osteocalcina (OCN), fosfatase alcalina (ALP), runt-related transcription factor 2 (RUNX2) e fator de necrose tumoral alfa (TNF-\'alfa\')], através da RealTimePCR, correlacionando sua expressão com as características microscópicas observadas qualitativa e quantitativamente. Com base nos resultados da análise microscópica e molecular, podemos concluir que os marcadores RUNX2, OCN e TNF-\'alfa\' podem ser usados como indicadores para avaliar a neoformação óssea e a quantidade de infiltrado inflamatório em alveolite. Os marcadores ALP e VEGF não representaram adequadamente o que se observou microscopicamente. Embora o tratamento da alveolite com a pasta à base de metronidazol promova maior densidade de neoformação óssea aos 28 dias, não há diferenças entre os tratamentos.
Dry socket is an inflammatory postoperative complication that undertakes sockets of recently extracted teeth. The incidence of such complication varies from 1 to 4% and might reach up to 30%. The objective of this study was to analyze the biological mechanisms involved in the repair process of intentionally infected sockets in mice; compare different treatment conditions and correlate the results of two different analysis (microscopic and molecular). 84 mice were used in this study, divided according the following groups: I: uninfected socket; II: infected socket without any treatment; III: infected socket treated with irrigation of 2% sodium iodide and 3% hydrogen peroxide solution at 1:1 proportion; and IV: infected socket submitted to curettage, physiological saline solution irrigation and fulfillment with metronidazole base paste. The animals were killed at a postoperative period of 6, 15 and 28 days. A quantitative analysis was performed using a RealTimePCR to evaluate the genes expression involved [Collagen Type I (COL-I), vascular endothelial growth factor (VEGF), osteocalcin (OCN), alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX2) and tumor necrosis factor-alpha (TNF-\'alpha\')], in the repair process, correlating its expression with the microscopic characteristics observed in both qualitative and quantitative manner. Based in the results of the microscopic and molecular analysis, it can be concluded that the RUNX2, OCN and TNF-\'alpha\' markers can be used as indicators to evaluate the dry socket bone neoformation and inflammatory infiltrate quantity. The ALP and VEGF markers did not represented appropriately what was observed microscopically. Although the dry socket treatment with metronidazole base paste promotes an increase in the bone neoformation density at 28 days, no difference was found among the treatments.

The malfunction of the transcriptional regulator RUNX1 is the major cause of several variants of acute human leukemias and its normal function is to regulate the development of the blood system in concert with other transcriptional co-regulators. RUNX1 belongs to a conserved family of heterodimeric transcription factors that share a conserved DNA binding domain, the Runt domain (RD), named after the first member of this group – Runt - found in Drosophila melanogaster. The binding partner CBFβ serves as a regulator of RUNX by enhancing its DNA binding affinity through an allosteric mechanism.

The main focus ofo my thesis work has been the crystallization and structural analysis of the RUNX1 RD and involved also more technical methodological aspects that can be applied to X-ray crystallography in general.

The high resolution crystal structure of the free RD shows that this immunoglobulin-like molecule undergoes significant structural changes upon binding to both CBFβ and DNA. This involves a large flip of the L11 loop from a closed conformation in the free protein to an open conformation when CBFβ and/or DNA are bound. We refer to this transition as the “S-switch”. Smaller but significant conformational changes in other parts of the RD accompany the “S-switch”. We suggest that CBFβ triggers and stabilizes the “S-switch” which leads to the conversion of the RD into a conformation enhanced for DNA binding.

During the structural analysis of the RD we identified two chloride ions that are coordinated by residues otherwise involved in DNA binding. In electrophoretic mobility-shift analyses (EMSA) we demonstrated a chloride ion concentration dependent stimulation of the DNA binding affinity of RUNX1. We further showed by NMR line width broadening experiments that the chloride binding occurred within the physiological range. A comparable DNA binding stimulation of RUNX1 was seen in the presence of negative amino acids. This suggests a regulation of the DNA binding activity of RUNX1 proteins through acidic amino acid residues possibly provided by activation domains of transcriptional co-regulators that interact with RUNX1.

The use of the anomalous signal from halide ions has become a powerful technique for obtaining phase information. By replacing the sodium chloride with potassium bromide in the crystallisation conditions of the RD, we could demonstrate in a single wavelength anomalous diffraction (SAD) experiment that the anomalous signal from 2 bromide ions were sufficient to phase a 16 kDa protein. Due to lack of completeness in the low-resolution shells caused by overloaded intensities, density modification schemes failed and the resulting electron density maps were not interpretable. By combining the highresolution

synchrotron data with low-resolution data from a native data set collected on a home X-ray source, the density modified bromide phases gave easily traceable maps.

Glaucoma is the leading cause of irreversible blindness and its prevalence is estimated to reach 79.6 million people by 2020. Wound contraction and scarring are the principal causes of blockage of aqueous flow at the drainage site in glaucoma filtration surgery. The cytotoxic antimetabolites, mitomycin-C and 5-fluorouracil, are widely used but have potentially blinding complications. There is thus a large unmet need for alternative agents with more targeted physiological effects and less cytotoxicity. The myocardin-related transcription factor/serum response factor (MRTF/SRF) pathway is a master regulator of cytoskeletal gene expression and thus represents a promising therapeutic target to prevent fibrosis. The MRTF/SRF pathway in human conjunctival fibroblasts responds to actin dynamics but as human conjunctival fibroblasts have constitutively high MRTF-A and MRTF-B nuclear concentrations, they show a relatively small response to serum stimulation and actin binding drugs, such as latrunculin B and cytochalasin D. The localisation of MRTF-A and MRTF-B is also conserved across species in mouse and rabbit conjunctival fibroblasts. As the MRTF/SRF pathway is a master regulator of many key cytoskeletal genes in fibrosis, we next studied the effects of downregulating the MRTF/SRF pathway in human conjunctival fibroblasts using three-dimensional in vitro collagen contraction assays. MRTF-A and MRTF-B siRNA silencing significantly decreased collagen matrix contraction, the cell protrusive activity, matrix degradation, and the expression of key matrix metalloproteinase genes. Liposome-peptide-siRNA nanoparticles also represent an efficient and safe siRNA delivery system for MRTF silencing in conjunctival fibrosis. We further compared the contractility of fibrotic and non-fibrotic human conjunctival fibroblasts from glaucoma patients with and without previous glaucoma surgery, respectively. Fibrotic human conjunctival fibroblasts were significantly more contractile than non-fibrotic human conjunctival fibroblasts, and the increased contractility was linked to the upregulation of alpha smooth muscle actin, a marker of myofibroblast differentiation. We finally validated in vivo the effects of inhibiting the MRTF/SRF pathway in conjunctival fibrosis using a rabbit model of experimental glaucoma filtration surgery. We have identified a new MRTF/SRF pathway inhibitor CCG-222740, which was more potent in the fibroblast-mediated collagen contraction assay, less cytotoxic, and a more potent inhibitor of alpha smooth muscle actin expression than inhibitor CCG-203971. Local delivery of CCG-222740 and CCG-203971 in a rabbit model of conjunctival fibrosis significantly increased the long-term success of the surgery and decreased scar tissue formation histologically. Unlike mitomycin-C, neither CCG-222740 nor CCG- 203971 caused any epithelial toxicity or systemic side effects with very low drug levels measured in the aqueous, vitreous and serum. In conclusion, our in vitro and in vivo results support that inhibiting the MRTF/SRF pathway represents a potential new therapeutic target to prevent conjunctival fibrosis in glaucoma and other contractile scarring conditions in the eye.

Activity based alterations in synaptic connectivity are thought to underlie the processes involved in learning and memory. Measurable changes in neuronal activation by long-term potentiation (LTP) are widely investigated as a possible cellular correlate of this phenomenon, as it can be induced quickly to elicit long-lasting modifications. These long-term changes in the activity of neuronal circuits are sustained by an altered pattern of gene expression and protein synthesis. The inducible transcription factor Zif268 has been implicated in almost all models of neuronal plasticity. Downstream targets of zif268 are widely believed to contribute to the duration and stabilisation of NMDA receptor dependent LTP which, in turn, can be linked to various models of learning and memory. However, these downstream targets are only just starting to receive attention. By utilising a wide range of contemporary neuroscience techniques covering molecular & cell biology approaches, this thesis proposes two known proteins, gephyrin and ubiquilin, as well as a novel gene (urma), as potential downstream targets of Zif268. Both gephyrin and ubiquilin are associated with GABAA receptors at inhibitory synapses. Gephyrin is thought to cluster and anchor GABAA receptors at postsynaptic sites whilst ubiquilin is reported to regulate receptor surface expression. We found that gephyrin mRNA and protein expression levels were downregulated in response to increased levels of zif268 by transient transfection in PC-12 cells and NMDA stimulation in primary cultured cortical neurones. In addition, ubiquilin mRNA and protein levels were also downregulated within the same experimental paradigms, implying that both gephyrin and ubiquilin are downstream transcriptional targets of this plasticity-related gene. A previously reported microarray experiment (James et al. 2005) contained 144 ESTs significantly affected by the transient transfection of Zif268 in PC-12 cells compared to control. Bioinformatic analyses of these tags revealed interesting genomic areas pertaining to little published information. After further investigation, EST AI169020 revealed a novel transcript that was downregulated in response to NMDA treatment of primary cortical neurones. Additional data mining suggests that urma may be a rarely expressed transcription factor. Basal levels of urma mRNA were also decreased in the Zif268 knockout mouse, as were ubiquilin mRNA levels. Zif268 is a regulatory immediate early gene, activating or suppressing downstream targets that play a role in the duration and stabilisation of LTP. These results indicate that gephyrin and ubiquilin are potential mediators of NMDA receptor-dependent plasticity, by modifying inhibitory-signalling. In addition, Zif268 may actively suppress a novel plasticity-related transcription factor, urma. These findings may underlie important processes in learning and memory.

La musculation est un exemple intuitif de mécanotransduction : lorsque nous exerçons nos muscles, ils se renforcent, au contraire s'ils sont immobilisés, ils s'atrophient. À un signal mécanique, les cellules apportent donc une réponse biologique. Au centre de cette réponse se trouve un facteur de transcription, Serum Response Factor (SRF), et son cofacteur Myocardin-Related Transcription Factor A (MRTF-A), qui contrôlent les gènes du cytosquelette et de la différenciation musculaire. La localisation dans la cellule de MRTF-A est régulée par l'état du cytosquelette : lorsque les monomères d'actine sont abondants, MRTF-A est dans le cytoplasme, s'ils manquent, MRTF-A s'accumule dans le noyau, où elle active SRF. Nous avons montré à l'aide d'expériences de pinces magnétiques et de substrats étirables que, soumis à des contraintes mécaniques, le cytosquelette d'actine se réorganise, provoquant des changements de localisation de MRTF-A dans les cellules. Lorsque la contrainte est modérée, l'actine polymérise et MRTF-A est accumulée dans le noyau. Lorsqu'elle est trop importante, le cytosquelette cède et MRTF-A est confinée dans le cytoplasme. Grâce à une version fluorescente de MRTF-A et à des techniques de visualisation de l'actine, nous avons pu observer ces évènements et déterminer les échelles de temps mises en jeu dans cette signalisation. De plus, nous avons constaté que les différentes techniques d'observation de l'actine perturbent toutes le cytosquelette de manière détectable sur la localisation de MRTF-A. Enfin, nous avons également étudié comment les propriétés rhéologiques des cellules changent sous une contrainte répétée
Our muscles show the most intuitive example of mechanotransduction : when trained they grow whereas immobilized they atrophy. To a mechanical signal, the cells give a biological response. The Sotiropoulos group showed that Serum Response Factor (SRF) and Myocardin-Related Transcription Factor (MRTF-A) play a central role in this phenomenon by regulating cytoskeletal genes and muscle development. The localization of MRTF-A in the cell is regulated by the state of the actin cytoskeleton : in the presence of monomeric actin, MRTF-A in confined to the cytoplasm and SRF inactive. Whereas ir case of monomeric actin scarcity, MRTF-A is accumulated in the nucleus, where it triggers SRF. Using magnetic tweezers and stretching experiments, we demonstrated that, when submitted tc mechanical cues, the actin cytoskeleton reorganizes and MRTF-A is relocated. In response to moderate cues, actin polymerizes and MRTF-A is transported to the nucleus. On the contrary, when the stress is too high, the cytoskeleton is damaged and MRTF-A is confined to the cytoplasm. Using a fluorescent MRTF-A and a panel of actin visualization techniques, we were able to observe the chronology of these events. Moreover, we showed that all the actin visualization techniques interfere with the actin cytoskeleton at a level detectable through MRTF-A localization. Finally we studied the changes in rheological properties of cells submitted to repeated steps of force

Understanding the connection between an organism’s genotype and its phenotype is a key question in evolutionary biology and genetics. It has been shown that many changes of morphological or other complex phenotypic traits result from changes in the expression pattern of key developmental genes rather than from changes in the genes itself. Such altered gene expression arises often from changes in the gene regulatory regions. That usually means the loss of important transcription factor (TF) binding sites within these regulatory regions, because the interaction between TFs and specific sites on the DNA is a key element of gene regulation. An established approach for the genome-wide mapping of genomic regions to phenotypes is the Forward Genomics framework. This approach compares the genomic sequences of species with and without the phenotype of interest based upon two ideas. First, the initial loss of a phenotype relaxes selection on all phenotypically related genomic regions and, second, this can happen independently in multiple species. Of interest are such regions that diverged specifically in phenotype-loss species. Although this principle is general, the current implementation is only well-suited for the identification of phenotype related gene-coding regions and has a limited applicability on regulatory regions. The reason is its reliance on sequence conservation as divergence measure, which does not accurately measure functional divergence of regulatory elements. In this thesis, I developed REforge, a novel implementation of the Forward Genomics principle that takes functional information of regulatory elements in the form of known phenotype-related TF into account. The consideration of the flexible organization of TF binding sites within a regulatory region, both in terms of strength and order, allows the abstraction from the region’s sequence level to its functional level. Thus, functional divergence of regulatory regions is directly compared to phenotypical divergence, which tremendously improves performance compared to Forward Genomics, as I demonstrated on synthetic and real data. Additionally, I developed TFforge which follows the same approach but aims at identifying the TFs relevant for the given phenotype. Given a multi-species alignment with a phenotype annotation and a set of regulatory regions, TFforge systematically searches for TFs whose changes in binding affinity between species fit the phenotype signature. The reported output is a ranking of the TFs according to their level of correspondence. I prove the concept of this approach on both biological data and artificially generated regions. TFforge can be used as a standalone analysis tool and also to generate the input set of TFs for a subsequent REforge analysis. I demonstrate that REforge in combination with TFforge is able to substantially outperform standard Forward Genomics, i.e. even without foreknowledge of relevant TFs. Overall, the in this thesis introduced methods are examples for the power of computational tools in comparative genomics to catalyze biological insights. I did not only show a detailed description of the methods but also conducted a real data analysis as validation. REforge and TFforge have a wide applicability on endless phenotypes, both on their own in the association of TF and regulatory region to a phenotype. Moreover, particularly their combination constitutes in respect to gene regulatory network analyses a valuable tool set for evo-devo studies.

The main hypotheses tested within this research focussed on the identification of polymorphisms within Runx2, and the classification of these with respect to bone mineral density (BMD). A separate set of hypotheses focussed upon the effects of calcium treatment in an elderly population, to identify any differences in BMD, bone mineral content (BMC) and bone area between the specific genotypes identified. The initial research strategy included taking subjects’ from the extremes of a population to identify alleles specifically related to the bone mineral density trait. The idea was tested using Runx2, the well known osteoblast transcription factor. From a population of 1300 subjects (from the Geelong Osteoporosis Study: the GOS) the age-weight adjusted femoral neck BMD was ranked and the upper and lower deciles taken to represent the adjusted extremes. After adjusting and ranking, the two groups (n=130 each) were not significantly different for age or weight. In these 260 subjects, we identified 16 allelic variations within the Runx2 gene and gene promoters (P1 and P2), and characterized these novel variations with respect to BMD strata by genotype using DHPLC.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Medical Science
Griffith Health
Full Text

Thesis (Ph.D.)--Southern Illinois University Carbondale, 2007.
"Department of Molecular Biology, Microbiology and Biochemistry." Includes bibliographical references (leaves 86-96). Also available online.

Thesis (M.A.)
Systemic sclerosis (SSc) is a clinically heterogenous chronic fibrotic disease which affects skin and internal organs. While the pathogenesis of SSc remains unknown, the hallmark of both localized and diffuse SSc in the skin is the replacement of normal dermal architecture with excessive deposition of collagen and other connective tissue macromolecules. Progressive replacement of tissue architecture by collagen-rich extracellular matrix (ECM) results in functional impairment of affected organs. Fibrotic damage to these affected organs accounts for much of the morbidity and mortality concomitant with SSc, particularly in the lungs. Myofibroblasts are the primary ECM-secreting cells during wound healing and fibrosis. Myocardin-related transcription factor A (MRTF-A), is an important regulator of myofibroblast differentiation, depending on serum response factor (SRF) for smooth muscle actin (SMA) and Sp1 in the regulation of collagen gene expression. MRTF-A continually shuttles between the nucleus and cytoplasm in unstimulated cells. Signals of stress, mechanical force, and migration control MRTF-A movement by a mechanism in which Rho-activated cytoskeletal actin polymerization induces its relocation from the cytoplasm to the nucleus. The major hypothesis in this thesis is that MRTF-A is dysregulated (impairment of a physiological regulatory mechanisms) and/or activated in SSc patients in part through transforming growth factor beta (TGF-β). To test this hypothesis, immunohistochemistry using MRTF-A antibodies was performed on SSc patient skin lesions and healthy control skin. Staining was observed in the epidermis, epidermal structures, vasculature and dermis of SSc and healthy control skin. In the epidermal layer of patients with SSc, there was significantly more nuclear localization of MRTF-A then in normal controls. Prominent staining is also present in endothelial, perivascular and some perivascular inflammatory cells of SSc patients. Perivascular staining was not seen in healthy controls. Interestingly, there was some accumulation of nuclear MRTF-A in areas typical of myofibroblasts in SSc skin, but this staining is not as striking as vascular staining. TGF-β activates MRTF-A in a cell-specific manner. As SSc typically begins within the skin, human dermal fibroblasts (HDF) were grown in culture. HDFs synthesize and secrete collagen to a greater extent when compared to human lung fibroblasts (IMR90 cells). Treatment with TGF-β enhances cytoplasmic localization of MRTF-A at 4-8 hours in HDFs and prolongs nuclear localization. Transgenic mouse lung cells were isolated from an MRTF-A loss-of-function mouse carrying the 3.6 kb proximal promoter of the rat COL1A1 gene driving topaz green fluorescent protein (GFP) (pOB3.6COLGFPtpz). Since angiotensin II (ANG II) may enhance TGF-β response or collagen transcription directly, wild type (WT) and MRTF-A knockout (KO) cells were treated with ANG II and TGF-β. Quantification of collagen transcription by GFP fluorescence and protein synthesis by Western and secretion by Sircol analysis revealed collagen gene expression is consistently lower in KO fibroblasts compared to WT. Total percentage of fluorescent KO cells were consistently lower in comparison to WT cells as well. KO cells do not respond to TGF-β or ANG II treatment, whereas TGF-β increased collagen gene expression by WT cells, but not KO cells. Furthermore, treatment with ANG II did not up-regulate transcription in WT mouse lung fibroblasts. However, TGF-β receptor kinase 1 (TβR-1) inhibitor SB431542 attenuated collagen transcription in both WT and KO fibroblasts regardless of treatment suggesting that the receptor is active with or without MRTF-A possibly with an endogenous ligand produced by these cells. The activation of MRTF-A is an important protein regulating collagen synthesis and may potentially serve as a therapeutic target in future treatments of fibrotic disease such as SSc.

The concept of cellular senescence is based on the notion that proliferation of normal diploid cells can only occur for a limited period of time after which cells slowly cease to divide and die. This limitation of lifespan for in vitro cell cultures was first described by Leonard Hayflick in 1961, going against the theory formulated by Alexis Carrel claiming that cells kept in culture have an unlimited potential for division (Carrel and Ebeling, 1921). Evidence from cell senescence studies have been used to explain the basis of healthy ageing as well as develop food supplements promoting the delay of ageing. Since these discoveries, there has been marked expansion of anti-stress gene response research with relation to the role of the transcription factor NF-E2-related factor-2 as well as healthy ageing with regards to dietary activators intake. This study examined the effects of sulforaphane (SFN) and hesperetin (HESP) on MRC-5 cell senescence in vitro as well as characterized gene expression and cell metabolism in cells escaping senescence by treatment with SFN and HESP and the mechanism of decrease glucose metabolism by SFN and HESP action linked to delay of fibroblast senescence. The effects of treatment of MRC-5 with the dietary bioactivators SFN and HESP was studied and shown to delay cellular senescence in these cells in vitro. Similar findings for SFN treatment of BJ cells were found previously by studies of the host team. Caloric restriction mimetic mechanisms by treatment with 1 μM SFN and 5 μM HESP was shown due to the decrease in culture glucose consumption increase with senescence in these treatment groups and this CR restriction mimetic effect and decrease in the flux of formation of D- and L-lactate in the SFN-treated group was consistent with previous studies done in CR and ageing in mice models (Hargopan, Ramsey and Weindruch, 2003). Moreover, in this study, SFN was shown to act as a CRM dietary bioactive through cellular contents of glycolytic intermediates with SFN-treatment (Bensaad et al., 2006). Finally, the mechanism by which SFN induces delay of senescence through CR was shown to be due to the extraction of Mondo A from the cell nucleus to the cytoplasm.

La Fibrose Pulmonaire Idiopathique (FPI), est une maladie pulmonaire chronique, sans aucune thérapeutique curative à l’heure actuelle. Les fibroblastes sont les cellules clés du développement de la fibrose. D’origine mésenchymateuse, la régulation de leur phénotype est toujours assez peu comprise et parmi les régulateurs possibles, peu de facteurs de transcription (FT) spécifiques de ces cellules ont été découverts. Nos travaux mettent en évidence pour la première fois, l’implication du FT mésenchymateux PRRX1 dans la régulation du comportement aberrant des fibroblastes et dans le développement de la fibrose pulmonaire. In vitro, PRRX1 favorise l’acquisition d’un phénotype pro-fibrosant des fibroblastes, avec l’induction de leur prolifération, de leur survie et de leur différenciation en myofibroblastes.In vivo, l’inhibition de PRRX1 par stratégie antisens diminue le développement des lésions fibrotiques et la déposition des protéines de collagène, de fibronectine et d’actine du muscle lisse dans le poumon des souris induites par la bléomycine. L’ensemble de ce travail a donc permis d’identifier PRRX1 comme un nouveau régulateur des fibroblastes au cours de la FPI et dont l’inhibition pourrait être une ouverture thérapeutique
Idiopathic Pulmonary Fibrosis (IPF) is a chronic pulmonary devastating disease, with no current therapeutic available. Fibroblasts are key cells driving fibrogenesis during IPF. From mesenchymal origin, their phenotype regulation is still poorly understood. And among the potential regulators, only few transcription factors (TF) are specific to these cells. Our work demonstrates for the first time, the implication of the mesenchymal TF PRRX1 in the regulation of fibroblast phenotype during IPF development. In vitro, PRRX1 induce proliferation, survival and myofibroblast differentiation. In vivo, the PRRX1 inhibition by antisens strategy lead to attenuate fibrotic lesions and extracellular matrix deposition of collagen, fibronectine, and smooth muscle actin of bleomycin lung mice. Here we identify PRRX1 as a new fibroblast transcription factor regulator during IPF, and inhibit it could be a promising therapeutic

As sequências dos genes dmrt1 e sox9 em pirarucu (Arapaima gigas), lambari (Astyanax altiparanae) e piapara (Leporinus elongatus) foram analisadas por técnicas de clonagem molecular. Fragmentos do gene dmrt1 foram clonados nas três espécies citadas, e do gene sox9 em lambari e piapara. A análise filogenética destes genes mostrou concordância com os dados morfológicos da filogenia destas espécies. Pelo acompanhamento do desenvolvimento gonadal de lambari por microscopia de luz, foi constatada a presença de células germinativas na região da futura gônada já no período de 5 dias após eclosão (dae), sendo também observado que a formação de ovário ocorre primeiro do que a formação de testículo. As expressões dos genes dmrt1 e sox9 por qRT-PCR em lambari mostraram maior especificidade para testículo do que para ovário, indicando que possivelmente estes genes ainda possuem função macho-específica nos adultos. Em lambari, os genes foram expressos em todos os períodos larvais, tendo uma diminuição no estágio de 12 dae, e aumentando sua expressão no estágio de 33 dae.
The sequences of dmrt1 and sox9 genes were analyzed in pirarucu (Arapaima gigas), lambari (Astyanax altiparanae) and piapara (Leporinus elongatus) using molecular cloning techniques. Fragments of dmrt1 gene were cloned in these three species, and sox9 gene fragments have been acquired only for lambari and piapara. Phylogenetic analysis of the genes was shown to be consistent with the morphological phylogeny of these species. The analysis of the gonadal development in lambari was followed by light microscopy, where it was found the presence of germ cells in the region of the future gonad in 5 days after hatching (dah), and that the formation of ovaries occurs first comparing to the formation of testis. The gene expression of dmrt1 and sox9 by qRT-PCR method show specificity for testis comparing to ovary, which indicates that these genes also have a male-specific role in the adult. In lambari, the genes were expressed in all larval periods, with a decrease of expression levels in 12 dah, and increasing its expression at stage of 33 dah.

Type 2 Diabetes (T2D) disproportionally affects ethnic minorities in the United States. The development of T2D involves complex interaction between environmental factors and genetic predisposition. The genetic associations of six single nucleotide polymorphisms (SNPs) in TCF7L2 gene with the risk of T2D were evaluated in three high risk minority populations: Cuban Americans, Haitian Americans, and African Americans. For Cuban Americans, four SNPs (rs7901695, rs4506565, rs7903146 and rs11225537) were significantly associated with the risk of T2D after multivariable adjustment (p=0.018, p=0.016, p=0.014, and p=0.0008, respectively). Among controls, risk allele carriers of SNPs rs7901695, rs4506565 and rs7903146 had significantly higher fasting glucose level, compared to non-risk allele carriers. Additionally, a significant interaction between dietary fiber intake and SNP rs7903146 for the risk of T2D (p= 0.044) was found in Cuban Americans. Similarly, for SNP rs7901695, significant interaction was also found for fiber intake (p=0.014) as well as glycemic load (p=0.040). Subgroup analysis revealed that significant associations were only found within higher intake groups of dietary factors for both SNPs. For Haitian Americans, SNPs rs11196205 (p=0.059) and rs7895340 (p=0.069) showed marginal significance for the risk of T2D. After stratification by gender, SNPs with marginal significance from the gender-combined analysis became statistically significant with the same trend for the risk of T2D when analysis were done in males: rs11196205 (p=0.034) and rs7895340 (p=0.024). For African Americans, SNP rs7903146 (p=0.065) showed a marginal significance with the risk of T2D in gender-combined analysis and a statistical significance (p=0.013) in males. Two additional SNPs rs7901695 and rs4506565 were found to be significantly associated with the risk of T2D in males. Risk allele carriers of these two SNPs had significantly higher mean level of the fasting glucose level, compared to non-risk allele carriers in controls. T2D related quantitative trait analysis also demonstrated that in controls, compared to non-minor allele carriers of SNP rs12255372, minor allele carriers had significantly higher means of BMI, diastolic blood pressure, numbers of components of Metabolic Syndrome, significantly lower mean values of HDL-cholesterol and adiponectin. Taken together, our studies demonstrated ethno-specific genetic associations between TCF7L2 gene and the risk of T2D and related phenotypes.

La mise en place de la lignée germinale au cours du développement constitue une des étapes fondamentales conditionnant la fertilité de l’individu adulte. Au cours des dernières décennies, le nombre croissant de couples consultant pour une aide médicale à la procréation a fait émerger l’hypothèse d’une altération des fonctions de reproduction chez l’Homme qui pourrait trouver son origine dans la perturbation du développement précoce. Dans l’ovaire fœtal, les cellules germinales s’orienteront vers la voie de l’ovogénèse, caractérisée entre autres par l’entrée en méiose de ces cellules. La majorité des données actuelles relatives à ces évènements sont issues du modèle murin alors que le développement de l’ovaire humain est significativement diffèrent de celui de la souris. Il est donc nécessaire d’approfondir nos connaissances du développement ovarien humain et d’identifier ses éventuelles perturbations. L’objectif de mon travail a été de mettre au point un outil d’étude du développement ovarien et d’identifier de nouvelles voies impliquées dans la régulation de l’entrée en méiose des cellules germinales fœtales humaines et leurs perturbations éventuelles.Nous avons mis au point un nouveau modèle de xénogreffe d’ovaires fœtaux humains du premier trimestre de gestation (au moment de l’apparition des premières cellules méiotiques). Ce modèle nous a permis d’observer un développement de l’organe et une différenciation des cellules germinales similaires à ceux observés in vivo. Ce modèle permettra des travaux à des âges auxquels le matériel d’étude est peu accessible. En couplant ce modèle de xénogreffe à une stratégie d’ARN-interférence, il nous a été possible d’inhiber l’expression d’un gène spécifiquement exprimé dans les cellules germinales ovariennes, DMRTA2, et de mettre en évidence un potentiel rôle de ce gène dans leur différenciation pré-méiotique. Nous avons observé une diminution du nombre de cellules ayant initié la méiose après inhibition de l’expression de ce gène. Par ailleurs, nous avons également identifié la présence dans l’ovaire fœtal de nombreux marqueurs décrits comme testiculaires chez la souris (PLZF, DNMT3L, FGF9, NANOS2 ou CYP26B1). L’expression de ces marqueurs pourrait expliquer la présence de cellules mitotiques tardives dans l’ovaire fœtal humain que nous avons pu observer jusqu’à 30 semaines de gestation. En parallèle de ces travaux, nous avons testé la sensibilité des cellules germinales à la dexaméthasone, glucocorticoïde pouvant être administré au cours de la grossesse. Il a été observé une augmentation de l’expression de PLZF, gène cible de l’activation des récepteurs aux glucocorticoïdes, pouvant expliquer la diminution du nombre de cellules germinales.En conclusion, ce travail de thèse a permis d’identifier un nouveau gène potentiellement régulateur de la transition mitose/méiose dans l’ovaire humain, et d’affiner nos connaissances sur le développement de l’ovaire humain et l’entrée en méiose des cellules germinales. Toutefois, de nombreuses questions restent posées ainsi de futures études devront clarifier si les cellules germinales mitotiques observées à des stades tardifs sont capables de se différencier en ovocytes compétents
Woman fertility is partially dictated by the set up of the human female germ line. During the last ten years, which saw an increased number of couples consulting for assisted reproductive cares, the hypothesis of an early alteration in reproduction functions has emerged.In the fetal ovary, germ cells enter the path of oogenesis differentiation characterized by meiotic initiation. On this subject, vast majority of the scientific data are obtained from the mouse model, even if differences with human ovarian physiology are widely acknowledged. Therefore it is necessary to extend our knowledge on human ovarian development and identify its perturbations. The objective of my work was to assess a new model to study ovarian growth, studying regulation of meiotic entry and perturbation of germ line differentiation.We sat up a new xenograft model of early human fetal ovaries, when very early meiotic germ cells appear. Organ growth and germ cells differentiation were comparable with in vivo observations. Using this model with an RNA-interference strategy, we inhibited the expression of an oogonia germ cell gene, DMRTA2. This inhibition conducted to a significantly reduced number of germ cells gene that initiated meiosis and DMRTA2 seemed to be required for mitotic-meiotic transition. In another hand, we identified, in the ovary, the expression of germ cells markers described as specifically male in rodent (PLZF, DNMT3L, FGF9, NANOS2 ou CYP26B1). The expression of these markers in the human ovary could explain the observation of mitotic germ cells in late fetal ovaries (30 wpf).In parallel, we tested germ cells sensibility to a synthetic glucocorticoid, dexamethasone, administrated during pregnancy in some justified pathologies. We observed an increased expression of PLZF that could explain the decreased number of germ cells observed in treated ovaries.In conclusion, we identified a new gene expressed in human fetal ovaries, potentially involved in the meiotic entry, and we extended our knowledge to characterized human germ line development. However, many points have to be clarified, as the possible competence of late mitotic germ cells to form oocytes

Skeletal development and homeostasis is comprised of complex events characterized by the presence of cell specific regulators and markers. Aberrant expression of any of the regulating factors usually results in metabolic bone disease. Therefore understanding the mechanisms of skeletal homeostasis may disclose new therapies for treating these disorders. While the complete pathway that defines normal osteoblast differentiation remains indeterminate, several regulatory factors have already been identified and characterized. Using gene targeting technology, the importance of parathyroid hormone-related peptide (PTHrP) in skeletal biology has been well established, as PTHrP^-/- and PTHrP^+/- mice demonstrate skeletal abnormalities. Several lines of evidence from in vivo and in vitro studies suggest that PTHrP has unique roles in osteoblast biology, independent from its well-established roles in chondrocytes. The presence of an intronic variable number tandem repeat sequence (VNTR) in the human PTHrP gene has been proposed to provide a novel mechanism of transcriptional gene regulation in osteoblasts. A preliminary survey indicated that osteoporotic patients with low bone mineral density have the shortest allele of the VNTR. In this work, we set out to investigate the role of the minisatellite in PTHrP expression and as a potential predictor of decreased bone mineral density. However, in a large sample of osteoporotic patients, a correlation between bone mineral density and VNTR length was not apparent. Also, the presence of the VNTR, or its length, seemed not to affect PTHrP promoter activity in osteoblast-like cells. EMSA experiments demonstrated the ability of the VNTR to bind proteins whose natures remain unclear. We also studied the effect of the newly identified zinc-finger transcription factor, Osterix, on the GC-rich PTHrP promoter. Osx did not bind the promoter or affect its transcriptional activity. However, unexpectedly, we observed that the transcription factor Cbfal, the master regulator of osteoblast differentiation, decreases PTHrP gene expression, likely through a molecular intermediate. Our investigations have shed some light on the role of PTHrP in osteoblast differentiation, and its regulation in this process by polymorphic elements and transcription factors specific to osteoblasts. The potential role of these parameters in the homeostatic regulation of the skeleton and the development of metabolic bone diseases such as osteoporosis warrants further investigation.

Introdução: O gene DMRT1 é um fator muito importante, o qual induz a determinação sexual masculina. Estudos mais recentes têm demonstrado que o Dmrt1 possui um papel significante no desenvolvimento ovariano. Deleções restritas ao gene DMRT1 têm sido raramente identificadas em pacientes com disgenesia gonadal (DG) sem outras características sindrômicas. Objetivo: Pesquisar a presença de haploinsuficiência do gene DMRT1 (deleções e/ou mutações inativadoras) em um grupo grande de pacientes não sindrômicos com distúrbios do desenvolvimento sexual (DDS) por anormalidades gonadais. Polimorfismos do DMRT1, como fatores potenciais pelas anormalidades gonadais, foram também identificados. Pacientes e Métodos: Foram avaliados cerca de 39 pacientes portadores de DDS por anormalidades do desenvolvimento gonadal 46,XY: 24 com disgenesia gonadal parcial e 15 pacientes com disgenesia gonadal completa. As regiões codificadoras do DMRT1 e o domínio DM (exon 1) foram amplificados e sequenciados. A análise de Multiplex ligation probe amplification (MLPA) do DMRT1 foi realizada usando um kit comercial. Resultados: Deleção parcial ou total do DMRT1 não foi identificada pela técnica de MLPA. Oito variantes alélicas do DMRT1 foram identificados. Uma nova variante c.968-15insTTCTCTCT foi identificada em 6,4% e em 14,3% dos alelos dos pacientes 46,XY e indivíduos controles, respectivamente. Conclusão: Este estudo sugere que deleções parciais ou completas no DMRT1 e mutações inativadoras não são frequentemente encontradas em pacientes com anormalidades do desenvolvimento gonadal. Além disso, nenhuma das variantes alélicas identificadas neste grupo de pacientes poderia ser considerada como um marcador potencial polimórfico para disgenesia gonadal
Introduction Dmrt1 gene is a very important factor in inducing male sex determination, and more recently it has been demonstrated that Dmrt1 plays a significant role in ovary development. DMRT1 deletions have rarely been identified in patients with 46,XY gonadal dysgenesis (GD) without syndromic features. Objective- To screen for the presence of DMRT1 haploinsufficiency (deletions and/or inactivating mutations) in a large cohort of non-syndromic patients with disorder of sex development (DSD) due to abnormalities of gonadal development. DMRT1 polymorphisms, as potential susceptibility factors for gonadal abnormalities, were also investigated. Subjects and Methods- We evaluated 39 patients with 46,XY GD: 24 patients with the partial, and 15 with the complete form. The entire coding region (éxons 2-5) of DMRT1 and the DM domain (exon 1) were PCR-amplified and direct sequenced. Multiplex ligation probe amplification (MLPA) analysis of DMRT1 was carried out using a commercial kit. Results- Partial or total deletion of DMRT1 was not identified by MLPA technique. Eight allelic variants of DMRT1 were identified. The novel variant c.968-15insTTCTCTCT was identified in 6.4% and in 14.3% of the alleles of 46,XY patients and control subjects, respectively Conclusion- This study suggest that complete or partial DMRT1 deletions and inactivating mutations are not frequently found in patients with abnormalities of gonadal development. Additionally, none of the allelic variants identified in this cohort of patients could be considered a potential polymorphic susceptibility marker for gonadal dysgenesis

Malignant cutaneous melanoma is the deadliest form of skin cancer, and a majority of melanoma diagnoses are a result of exposure to ultraviolet (UV) radiation. UV radiation causes DNA damage, which if not repaired correctly via nucleotide excision repair (NER) can result in mutations and melanomagenesis. The melanocortin 1 receptor (MC1R) is a Gs protein coupled receptor located on melanocyte plasma membranes and is involved in protecting the skin from UV induced damage. MC1R signaling results in the activation of two protective pathways: 1) induction of eumelanin synthesis downstream of micropthalmia-associated transcription factor (MITF) and 2) acceleration of NER downstream of ataxia telangiectaseia mutated and Rad3 related (ATR). MC1R signaling, however, also promotes melanocyte proliferation, therefore, the activation of the MC1R pathway must be regulated. The overall hypothesis of this dissertation is that the pathways downstream of MC1R can be manipulated to protect against UV induced damage. Chapter 2 investigates the regulation of the MC1R neutral antagonist human β-defensin 3 (βD3). UV damage did not induce βD3 mRNA expression in ex vivo human skin explants. The induction of βD3 expression instead correlated with inflammatory cytokines including TNF. Chapter 3 investigates the interdependence and cross talk between the two protective pathways downstream of MC1R. We directly tested the effect of MITF on the acceleration of NER and the effect of ATR on the induction of eumelanin synthesis following MC1R activation. MITF was not required for the acceleration of NER as mediated by ATR, however, the induction of transcription of enzymes involved in eumelanin synthesis was dependent upon ATR kinase activity. Finally, Chapter 4 investigates the mechanism by which MC1R promoted proliferation and whether the two UV protective pathways downstream of MC1R could be selectively activated without the risk of melanocyte proliferation. MC1R signaling resulted in activation of the mechanistic target of rapamycin complex 1 (mTORC1), a major regulator of cell growth and proliferation. Inhibition of mTORC1 signaling via rapamycin prevented MC1R induced proliferation in vitro. Rapamycin, however, did not prevent MC1R induced eumelanin synthesis or the acceleration of NER in vitro or in vivo suggesting it is possible to selectively activate the beneficial signaling pathways without the risk of melanocyte proliferation. The results of this dissertation suggest that MC1R signaling could be augmented in individuals to prevent UV induced damage.

No
Runt-related (RUNX) transcription factors are evolutionarily conserved either in vertebrate or invertebrate. Lozenge (Lz), a members of RUNX family as well as homologue of AML-1, functions as an important transcription factor regulating the hemocytes differentiation. In this paper, we identified and characterized RUNX family especially Lz in silkworm, which is a lepidopteran model insect. The gene expression analysis illustrated that BmLz was highly expressed in hemocytes throughout the whole development period, and reached a peak in glutonous stage. Over-expression of BmLz in silkworm accelerated the melanization process of hemolymph, and led to instantaneously up-regulation of prophenoloxidases (PPOs), which were key enzymes in the melanization process. Further down-regulation of BmLz expression by RNA interference resulted in the significant delay of melanization reaction of hemolymph. These findings suggested that BmLz regulated the melanization process of hemolymph by inducing PPOs expression, and played a critical role in innate immunity defense in silkworm.

The current study investigated the expression Runx2 and VEGF in the pulp, periodontal ligament and alveolar bone following hypothermal insult. Methods and Materials: Materials from a previous study performed by Tan (2011) were used for this research. The upper right first molars of fifteen eight-week-old male Sprague-Dawley rats were subjected to a single ten minute application of dry ice. The contralateral molar acted as an internal control. The animals were randomly divided into five groups of three and killed 0, 4, 7, 14 and 28 days post hypothermal insult. A further three Sprague-Dawley rats acted as an external control and were humanely killed on day 0 with no hypothermal insult. The maxilla was dissected out, fixed and embedded in paraffin. Coronal sections were cut to include the control and experimental teeth at 5-micron intervals through the furcation region. Sections were then stained with haematoxylin and eosin (H and E) and Runx2 and VEGF immunostains. Sections were scanned via a Nanozoomer Slide Scanner 2.0 series and viewed on a personal computer (MacBook Pro with 13 inch screen) using the Nanozoomer Digital Pathology (NDP) software. Semiquantitative counting was performed at a magnification of x20 via the ImageJ software. Data was analysed using SAS 9.3 (SAS Institute Inc., Cary, NC, USA). The level of significance was set at p<0.05.
Thesis (D.Clin.Dent.) -- University of Adelaide, School of Dentistry, 2013

碩士
臺中健康暨管理學院
資訊科學與應用學系碩士班
93
Abstract The purpose of transcription factors (TFs) is to regulate the expression of other genes. They are also the key-point to control if mutation will occur on promoter region or not? Current researchers on TFs mainly focus on predicting motifs using algorithms such as Multiple Em for Motif Elicitation (MEME), Genetic Algorithm (GA), and Gibbs Sampler. In this thesis, we propose a new approach to predict possible cancer-related genes based on transcription factor binding sites (TFBS). The experimented TFBS that are binding on promoter region and the known cancer-related genes have been collected from TFSEARCH and CHIP websites, respectively. The TFBS that result in mutation of genes are selected. We then analyze the occurrence frequencies of these TFBS to investigate the relations of TFBS and possible cancer-related genes. We also discuss the two-factor case of analyzing the relations of two TFBS and possible cancer-related genes. Our results show that the TFBS-based approach for predicting possible cancer-related genes is a reliable method to recognize possible cancer-related genes.

碩士
亞洲大學
生物資訊學系碩士班
96
The processes of oncogenesis are multiple steps. And the over-expression of oncogenes plays a very important role in the initial state of numerous cancer cases. Mining the human genome to identify genetic mutations that cause cancer is like looking for needles in a haystack, especially by using the traditional one-by-one analysis method. Hence, it is a very important issue in bioinformatics to find out any information about cancer-related factors from these huge relational databases. The purpose of this research is to analyze existed known cancer-related genes and focus on the investigation of their transcription factor binding sites (TFBS), and predict the possible influence of gene control of tuning normal genes into cancer ones. The method is that we collect the issued TFBS in human genes from TFBS databases over the Internet, and then analyze the occurrence frequencies of these TFBS to investigate the relations between TFBS and the cancer-related genes. Both cases of single TFBS and multiple TFBS are investigated in this thesis. The results of this research can be used to conclude that if higher occurrence frequencies of transcription factors have problem in the process of transcription, they will influence the performance of gene transcription, thus become important possible factors of tuning normal genes into cancer ones. This feature can be considered as a gene marker to identify if a gene is possible to be a cancerous gene, and it will be great helpful for promoting high-precision of searching cancer-related genes on medical.

CRTR-1 is a member of the CP2 family of transcription factors. Unlike other CP2 family members, CRTR-1 expression is regulated developmentally. Major sites of expression in the embryo include the pluripotent inner cell mass (ICM) of the pre-implantation blastocyst and the developing kidney. It is also expressed in embryonic stem (ES) cells, which are derived from the ICM of blastocysts, and is downregulated as these cells differentiate into early primitive ectoderm-like (EPL) cells. This expression pattern suggests that CRTR-1 plays a role in early pluripotent populations. This thesis aims to characterize the transcription factor CRTR-1 at the molecular level and analyses the role of sumoylation on CRTR-1 function to develop a better understanding of the molecular role of CRTR-1 in ES cells. Luciferase reporter assays show that CRTR-1 is able to regulate the activities of other CP2 family members: CP2, NF2d9 and altNF2d9. It enhances CP2- and NF2d9-mediated activation but suppresses altNF2d9-mediated activation. To map the functional domains in the CRTR-1 protein, transactivation studies using CRTR-1 deletion mutants fused to the GAL4 DNA binding domain and a GAL4-responsive reporter system were performed. These studies map repressor activity to amino acids 48-200, but fail to identify a transactivation domain within the CRTR-1 protein. In order to understand the mechanisms by which CRTR-1 regulates the transcriptional activities of CP2 family members, a number of approaches are taken, including co-immunoprecipitation to show that CRTR-1 interacts with other CP2-like proteins, EMSA which demonstrate that CRTR-1 forms DNA binding complexes with CP2 family members, and subcellular protein localisation studies which reveal the ability of CRTR-1 and other family members to shuttle between the nucleus and cytoplasm via a CRM1-dependent pathway. In addition, the subcellular localisation of CRTR-1 appears to be cell type specific, with an exclusively nuclear localisation pattern in ES cells, a predominantly cytoplasmic localisation pattern in HEK293T cells, and a cytoplasmic and nuclear speckle localisation pattern in COS-1 cells. Co-expression of CRTR-1 with CP2 or NF2d9 results in the re-localisation of CRTR-1 to the cytoplasm in ES cells. The sumoylation enzymes Ubc9 and PIAS1 have previously been identified as CP2-interacting proteins (Kang et al., 2005a). Given the identification of two potential sumoylation sites within CRTR-1, FK³⁰ QE and LK⁴⁶ ⁴AE, and the ability for sumoylation to regulate transcription factor function, the possibility that CRTR-1 is regulated by sumoylation is investigated in this thesis. Immunoprecipitation experiments show that CRTR-1 is modified by SUMO-1 and that lysine 30 is the critical residue for this modification. Mutation of lysine 30 to alanine, which abolishes CRTR-1 sumoylation, results in enhancement of transactivation by CRTR-1, suggesting that sumoylation of CRTR-1 blocks maximal activation. Unexpectedly, however, overexpression of Ubc9, PIAS1, or SUMO-1 results in enhancement of CRTR-1 transcriptional activity, indicating that a more complex mechanism of regulation of CRTR-1 activity is likely. This thesis presents several novel properties of CRTR-1 and other CP2 family members, including the ability of CRTR-1, previously characterized as a repressor, to activate transcription. It is also the first demonstration that CP2 proteins are regulated by sumoylation and that they shuttle between the nucleus and cytoplasm via a CRM1-dependent mechanism.
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Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2009

CRTR-1 is a member of the CP2 family of transcription factors. Unlike other CP2 family members, CRTR-1 expression is regulated developmentally. Major sites of expression in the embryo include the pluripotent inner cell mass (ICM) of the pre-implantation blastocyst and the developing kidney. It is also expressed in embryonic stem (ES) cells, which are derived from the ICM of blastocysts, and is downregulated as these cells differentiate into early primitive ectoderm-like (EPL) cells. This expression pattern suggests that CRTR-1 plays a role in early pluripotent populations. This thesis aims to characterize the transcription factor CRTR-1 at the molecular level and analyses the role of sumoylation on CRTR-1 function to develop a better understanding of the molecular role of CRTR-1 in ES cells. Luciferase reporter assays show that CRTR-1 is able to regulate the activities of other CP2 family members: CP2, NF2d9 and altNF2d9. It enhances CP2- and NF2d9-mediated activation but suppresses altNF2d9-mediated activation. To map the functional domains in the CRTR-1 protein, transactivation studies using CRTR-1 deletion mutants fused to the GAL4 DNA binding domain and a GAL4-responsive reporter system were performed. These studies map repressor activity to amino acids 48-200, but fail to identify a transactivation domain within the CRTR-1 protein. In order to understand the mechanisms by which CRTR-1 regulates the transcriptional activities of CP2 family members, a number of approaches are taken, including co-immunoprecipitation to show that CRTR-1 interacts with other CP2-like proteins, EMSA which demonstrate that CRTR-1 forms DNA binding complexes with CP2 family members, and subcellular protein localisation studies which reveal the ability of CRTR-1 and other family members to shuttle between the nucleus and cytoplasm via a CRM1-dependent pathway. In addition, the subcellular localisation of CRTR-1 appears to be cell type specific, with an exclusively nuclear localisation pattern in ES cells, a predominantly cytoplasmic localisation pattern in HEK293T cells, and a cytoplasmic and nuclear speckle localisation pattern in COS-1 cells. Co-expression of CRTR-1 with CP2 or NF2d9 results in the re-localisation of CRTR-1 to the cytoplasm in ES cells. The sumoylation enzymes Ubc9 and PIAS1 have previously been identified as CP2-interacting proteins (Kang et al., 2005a). Given the identification of two potential sumoylation sites within CRTR-1, FK³⁰ QE and LK⁴⁶ ⁴AE, and the ability for sumoylation to regulate transcription factor function, the possibility that CRTR-1 is regulated by sumoylation is investigated in this thesis. Immunoprecipitation experiments show that CRTR-1 is modified by SUMO-1 and that lysine 30 is the critical residue for this modification. Mutation of lysine 30 to alanine, which abolishes CRTR-1 sumoylation, results in enhancement of transactivation by CRTR-1, suggesting that sumoylation of CRTR-1 blocks maximal activation. Unexpectedly, however, overexpression of Ubc9, PIAS1, or SUMO-1 results in enhancement of CRTR-1 transcriptional activity, indicating that a more complex mechanism of regulation of CRTR-1 activity is likely. This thesis presents several novel properties of CRTR-1 and other CP2 family members, including the ability of CRTR-1, previously characterized as a repressor, to activate transcription. It is also the first demonstration that CP2 proteins are regulated by sumoylation and that they shuttle between the nucleus and cytoplasm via a CRM1-dependent mechanism.
Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2009

INTRODUCTION: A recent area of interest has become adipose tissue of the bone marrow. Unlike fat found elsewhere in the body, it is able to respond to its microenvironment by expanding, contracting, and even releasing hormones of its own. The marrow adipose tissue (MAT) increases with age and even within anorexic patients. Changes in MAT may be directly linked to bone tissue, since both adipocytes and osteoblasts share a common progenitor cell population. This is supported by recent results showing that the constitutive deletion of the MRTF-A (Myocardin related transcription factor A) gene not only led to leaner mice leaner but also had a bone phenotype. The bones were shorter and had decreased bone mass; the female mice were more susceptible to osteopenia. It is unclear as to how this bone phenotype will respond to fracture repair, a coordinated process that is dependent upon differentiation toward the osteoblast lineage. Therefore, both male and female MRTF-A knockout (KO) and wild type (WT) mice were examined in a fracture study. METHODS: Male and female mice (KO and WT) aged 8-16 weeks were fractured using a closed, stabilized fracture model. Tissues were harvested as post-operative day 14, 21, and 37. Radiographic films and histological assessment were completed at each time point to visualize the progression of fracture repair. Gene expression studies using RT-qPCR were all done at time points 14 and 37. Results were compared between genotype and sex. RESULTS: Fracture calluses between WT and KO male and female mice appeared nearly identical via X-ray, suggesting that MRTF-A did not affect fracture repair. Only at the later time points did the histology show females had increased MAT regardless of genotype. Immunofluorescence with perilipin further confirmed this. Adipogenic, chondrogenic, and osteogenic markers also yielded differences in relative mRNA expression between the sexes. CONCLUSIONS: The MRTF-A KO mice did not show delayed or altered fracture repair compared to the WT mice. However, the data suggest difference in MAT deposition between male and female mice. Further work is necessary to fully understand this sex difference.

Thermogenic brown adipose tissue generates heat via mitochondrial uncoupling protein-1 (UCP-1), increases whole-body energy expenditure and may protects against obesity and metabolic disorders. White adipocytes store excess energy in the form of triglycerides. UCP-1 positive adipocytes develop within white adipose tissue (beige or brite adipocytes) in response to cold exposure or β3 adrenergic agonists. It was known that beige adipocytes arise from a distinct lineage compared with brown adipocytes, but the developmental origin of the beige adipocytes is still unclear. Signaling pathways that control beige adipocyte determination and formation are essentially unknown. Here, we identified a novel signaling pathway that regulates the lineage specification of beige adipocytes. Bone morphogenetic protein 7 (BMP7), a known brown adipogenesis inducer, suppresses Rho-GTPase kinase (ROCK) and depolymerizes F-actin (filamentous actin) into G-actin (globular actin) in mesenchymal stem cells. G-actin regulates myocardin-related transcription factor A (MRTFA) that co-transactivates serum response factor (SRF) and promotes smooth muscle cell differentiation in various organs. Subcutaneous white adipose tissue from MRTFA-/- mice had enhanced accumulation of UCP-1+ adipocytes and elevated levels of brown-selective proteins. Compared with wild type (WT) controls, MRTFA-/- mice exhibited improved metabolic profiles and were protected from diet-induced obesity and insulin resistance, suggesting that the beige adipocytes are physiologically functional. Compared to WT mice, stromal vascular cells from MRTFA-/- mice expressed higher levels of distinct beige progenitor markers and reduced levels of smooth muscle markers. Our studies demonstrate a novel ROCK-actin-MRTFA/SRF pathway that contributes to the development of beige adipocytes.

Nitrogen is an essential nutrient for plant growth and is normally obtained from the soil medium. Legumes are a unique group of plants that acquired the ability to form a symbiosis with nitrogen-fixing bacteria, called rhizobia, enabling growth in nitrogen-poor soils. GmSAT1, a predicted bHLH transcription factor from soybean, is essential for nitrogen fixation; however the role of this protein remains elusive (Kaiser et al., 1998; Loughlin, 2007). In this work, a further functional characterization of GmSAT1 was undertaken. Using the promoters of known upregulated genes in yeast upon expression of GmSAT1, it was found that purified GmSAT1 directly interacts with DNA. Further, GFP-fusion analysis in onion epidermal cells, found that GmSAT1 localizes to the nucleus, as well as peripheral vesicles, demonstrating that GmSAT1 is a likely a transcription factor. Residues from both the N- and C-termini required for GmSAT1 activity were also identified by exchanging domains with GmSAT2, a protein that arose during the relatively recent whole-genome duplication in soybean. Recently, GmSAT1 was shown to be essential for proper nodule development in soybean (Loughlin, 2007). Therefore, a DNA microarray analysis was conducted to identify transcripts that are differentially expressed after silencing of GmSAT1 by RNA interference (RNAi) in soybean nodules. Of the ninety-five genes downregulated, twelve were associated with the circadian clock, potentially explaining the GmSAT1 RNAi phenotype. Investigations were also initiated in the model legume Medicago truncatula to identify and characterize GmSAT1 orthologues. Two genes, MtSAT1 and MtSAT2 were cloned and analyzed. MtSAT1 and MtSAT2 are expressed in roots and the inner cortex of nodules, similar to GmSAT1. By in planta GFP-fusion analysis, both MtSAT1 and MtSAT2 were found to associate with vesicles and the nucleus. Insertional mutations in either gene alone did not render a phenotype, however downregulating both genes by RNAi disrupted nodule formation. Using the sequence of a newly discovered ammonium channel protein from yeast (ScAMF1), which is activated by GmSAT1, a novel subfamily of major facilitator transporter proteins (MFSs) from plants was identified. Interestingly, members of the MFS gene family are found linked with GmSAT1 loci in soybean, as well as M. truncatula and many sequenced dicots. GmMFS1.3, a representative from soybean, was cloned and characterized. GmMFS1.3 expression was localized to the root stele and the inner cortex of nodules. Further, expression of GmMFS1.3 in yeast induced the uptake of methylammonium. Interestingly, GmMFS1.5 was found to be downregulated in GmSAT1 RNAi nodules. The link between GmSAT1 and the MFS transporters in planta will be the focus of future experiments. A novel receptor-like kinase protein was also characterized from soybean nodules. GmCaMK1 was identified in a protein interaction screen using conserved calmodulin as bait. The calmodulin-binding domain overlaps the GmCaMK1 kinase subdomain XI, however it was found that GmCaMK1 is able to auto-phosphorylate independent of calmodulin. Therefore, calmodulin binding may influence the interaction of GmCaMK1 with its phosphorylation targets. Taken together, these studies have enriched our knowledge of nitrogen fixation, a critically important component of agricultural practice.
Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2012

碩士
慈濟大學
分子生物及細胞生物研究所
92
Tumorgenesis is a multi-step process involving genetic alterations that lead to tumor formation and tumor invasion. Tumor metastasis is the most severe stage in the tumorgenesis process. However, the molecular mechanism of metastasis remains unclear. Recent evidence has indicated that the expression of the transcription factor Snail plays an important role in tumor metastasis. Snail is a zinc-finger transcription factor, which represses the expression of E-cadherin and induces epithelial-mesenchymal transition (EMT) associated with tumor progression and an invasive phenotype. Thus, it is important to understand the mechanism how Snail is upregulated in metastatic cancer cells. In the present study, we isolated the regulatory elements of the human Snail gene by polymerase chian reaction (PCR) and inserted these serially deleted regulatory fragments of Snail into a luciferase reporter vector, respectively. In order to select the model cell line, the reverse transcription polymerase chian reaction (RT-PCR) was carried out to examine the cDNA expression level of Snail in various cancer cell lines. In a functional assay, analysis of these constructs in melanoma cell line (A2058) revealed the strongest promoter activity among the regions is located between —262 and —91. This region was further investigated by electrophoresis mobility shift assay (EMSA) and was found to contain some protein complexes in region between —111 and —91. Preliminary database analysis showed it contained putative SP1 binding sites. The co-transfection experiment showed that the SP1 could slightly up regulate the activity of Snail expression. In addition, the EMSA experiments using recombinant SP1 protein revealed that SP1 interacts with the fragment, which is between —111 and —91. Our results suggest that SP1 is functional in regulating the Snail gene

Single-minded 1 (SIM1) is a basic Helix-Loop-Helix/PER-ARNT-SIM (bHLH/PAS) transcription factor essential for survival in mice. The early post-natal lethality exhibited by Sim1⁻ʹ⁻ mice is believed to be the consequence of severely compromised hypothalamus development, although the contribution of reduced SIM1 signalling in the numerous other tissues in which it is expressed has never been formally investigated. The presence of a single Sim1 allele is sufficient to avoid this perinatal lethality, and instead confers an early onset, hyperphagic obesity phenotype, potentially via disruption of critical intracellular signalling pathways that are activated in Sim1-expressing hypothalamic neurons in response to food intake. Similar correlations between reduced SIM1 gene dosage and severe obesity have also been documented in humans. Alterations in SIM1 expression and/or function therefore have important implications in health and disease, and warrant a detailed investigation into the downstream target genes and regulatory behaviours of this critical transcription factor, which are thus far almost entirely lacking in the literature. The studies presented in this thesis describe a twofold approach to dissecting the gene regulatory properties of the SIM1 protein. Firstly, we optimised and performed a range of functional assays, including a cell-based luciferase reporter gene assay, a subcellular localisation assay, a co-immunoprecipitation assay, and an electrophoretic mobility shift assay, which were designed to assess the contribution of nineteen unique point mutations within the SIM1 protein sequence to altered SIM1 expression and behaviour. These nineteen mutations were identified in multiple cohorts of severely obese humans, and therefore represent potentially pathogenic alterations in the SIM1 sequence. Indeed, we observed a significant loss of function for many of these variants in luciferase reporter gene assays relative to wild type SIM1. The severe loss of function observed for one of these variants, SIM1 T292A, could be further attributed to altered subcellular localisation, thus impacting on its ability to form a stable heterodimer with ARNT2 in co-immunoprecipitation experiments. Secondly, we performed microarray studies on cultured kidney-derived cells inducibly overexpressing Myc-tagged SIM1 and its obligate partner factor ARNT2, and subsequently identified several genes that selectively responded to SIM/ARNT2 overexpression in this context. Further validation in hypothalamus-derived cultured cells highlighted Myomesin 2 (Myom2) as a potentially genuine downstream SIM1 target gene in both kidney and hypothalamus. We also present data that are the first to indicate Somatostatin (Ss) as a hypothalamic target gene regulated by SIM1 in a cell-autonomous manner. These data are among the first to dissect the downstream target genes and regulatory properties of the SIM1 protein, and therefore make an important contribution to our understanding of the molecular basis to the hyperphagic obesity exhibited by Sim1⁺ʹ⁻ mice. They are also the first to link reduced activities of mis-sense mutations in the SIM1 coding sequence to increased weight gain in humans, and give further credence to the possibility that SIM1 represents a novel genetic contributor to obesity disorders in the wider population. This knowledge may inform future attempts to develop therapies for obese phenotypes in humans, and broaden our understanding of the molecular events that underpin Sim1-mediated survival and maintenance of homeostasis.
Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2011

Abiotic stress is one of the major factors that affect food production worldwide and, therefore understanding stress responsive proteins and engineering plants for abiotic stress tolerance is very important. In the present study, the biological role of pea pathogenesis-related 10.4 (PR-10.4; also known as abscisic acid responsive 17; ABR17) in abiotic stress tolerance has been investigated. Our investigation on ribonuclease (RNase) activity of ABR17 suggested that highly conserved histidine-69 and glutamic acid-148 are important for RNase activity. In order to further investigate the biological role(s) of ABR17, transcriptional profiling of pea ABR17-mediated gene expression changes in ABR17-transgenic Arabidopsis thaliana plants was carried out using microarrays. Our results indicated that pea ABR17 modulates many plant growth/development genes most of which are cytokinin (CK) responsive. These results agree very well with previously reported enhanced endogenous CKs in these transgenic plants. However, no significant changes in transcript abundance of CK biosynthetic genes were observed between transgenic and wild-type plants, suggesting an alternate source of CK in ABR17-transgenic plants. It is speculated that ABR17 may act as either a CK reservoir (through its reported CK binding property) or may be responsible for isopentenylated-tRNA degradation (through its demonstrated RNase activity) thereby increasing endogenous CK pools. Furthermore, microarray analysis of salinity stressed ABR17-Arabidopsis indicated that ABR17 modulates many stress responsive genes that included four putative AP2 family genes (RAP2.6-At1g43160, RAP2.6L-At5g13330, DREB26-At1g21910 and DREB19-At2g38340). Functional characterization of these genes suggested that they are transcription factors and they play very important roles in abiotic stress response in addition to growth and development. Moreover, overexpression of RAP2.6L and DREB19 genes enhanced salinity and drought tolerance in Arabidopsis. Taken together, our results suggest that pea ABR17 proteins are important in abiotic stress responses as they may act as source of enhanced CKs and they may also modulate expression of stress responsive genes to enhance stress tolerance in plants. However, additional research aimed at deciphering the links between ABR17 and CK biosynthesis as well as the mechanism of ABR17-mediated gene expression changes should be conducted in order to get more insights into the biological roles of PR10 proteins in planta.
Plant Science

The differentiation of osteoblasts and bone marrow adipocytes are closely associated yet mutually exclusive processes that are essential for maintaining bone homeostasis. Various diseases have been shown to develop once the delicate balance between adipogenesis and osteoblastogenesis is disrupted. Investigating the underlying molecular mechanisms of the osteoblasto-adipogenic switch under osteoporotic conditions will facilitate our understanding of the pathogenesis of osteoporosis and may eventually lead to the development of clinical therapeutic approaches for this life-threatening disease. While changes in cell morphology and cytoskeletal integrity can alter pre-committed mesenchymal stem cell (MSC) differentiation of certain lineages, previous studies have shown that cellular morphological changes can affect the early commitment of pluripotent MSCs via modulation of Ras homolog gene family, member A (RhoA) activity. The RhoA pathway regulates actin polymerization to promote the incorporation of globular-actin (G-actin) into filamentous-actin (F-actin). Actin polymerization releases G-actin bound myocardin-related transcription factors (MRTFs), which translocate to the nucleus and co-activate serum response factor (SRF) target gene expression. Exactly how the RhoA-actin-MRTF-SRF circuit is involved in the regulation of early commitment of MSCs remains poorly understood. Here we show that global MRTFA knockout mice (MRTFA KO) exhibited lower body weight, shorter femur and tibia lengths, and decreased trabecular bone volume. Furthermore, bone marrow MSCs isolated from MRTFA KO mice showed increased adipogenesis and brown fat gene expression as well as compromised osteoblastogenic differentiation as compared to WT controls. Treatment of WT bone marrow MSCs with the SRF inhibitor, CCG1423, mimicked these effects in that the compound inhibited osteoblastogenesis and promoted adipogenesis. Over-expression of MRTFA or SRF inhibited adipogenesis and enhanced osteoblastogenesis in C3H/10T1/2 cell lines, whereas over-expression of dominant-negative MRTFA or SRF variants had the opposite effects. In conclusion, our study identified MRTFA as a crucial regulator of skeletal homeostasis via regulating the balance between adipogenic and osteoblastogenic differentiation of the MSCs. Furthering our understanding of how the RhoA-actin-MRTFA-SRF circuit is involved in regulating the fate commitment of MSCs may ultimately lead to novel therapeutic strategies for treating osteoporosis and obesity.