Academic literature on the topic 'RUNT domain family'

ABSTRACT Osf2/Cbfa1, hereafter called Osf2, is a member of the Runt-related family of transcription factors that plays a critical role during osteoblast differentiation. Like all Runt-related proteins, it contains a runt domain, which is the DNA-binding domain, and a C-terminal proline-serine-threonine-rich (PST) domain thought to be the transcription activation domain. Additionally, Osf2 has two amino-terminal domains distinct from any other Runt-related protein. To understand the mechanisms of osteoblast gene regulation by Osf2, we performed an extensive structure-function analysis. After defining a short Myc-related nuclear localization signal, a deletion analysis revealed the existence of three transcription activation domains and one repression domain. AD1 (for activation domain 1) comprises the first 19 amino acids of the molecule, which form the first domain unique to Osf2, AD2 is formed by the glutamine-alanine (QA) domain, the second domain unique to Osf2, and AD3 is located in the N-terminal half of the PST domain and also contains sequences unique to Osf2. The transcription repression domain comprises the C-terminal 154 amino acids of Osf2. DNA-binding, domain-swapping, and protein interaction experiments demonstrated that full-length Osf2 does not interact with Cbfβ, a known partner of Runt-related proteins, whereas a deletion mutant of Osf2 containing only the runt and PST domains does. The QA domain appears to be responsible for preventing this heterodimerization. Thus, our results uncover the unique functional organization of Osf2 by identifying functional domains not shared with other Runt-related proteins that largely control its transactivation and heterodimerization abilities.

Runt domain proteins are transcriptional regulators that specify cell fates for processes extending from pattern formation in insects to leukemogenesis in humans. Runt domain family members are defined based on the presence of the 128-amino-acid Runt domain, which is necessary and sufficient for sequence-specific DNA binding. We demonstrate an evolutionarily conserved protein-protein interaction between Runt domain proteins and the corepressor Groucho. The interaction, however, is independent of the Runt domain and can be mapped to a 5-amino-acid sequence, VWRPY, present at the C terminus of all Runt domain proteins. Drosophila melanogaster Runt and Groucho interact genetically; the in vivo repression of a subset of Runt-regulated genes is dependent on the interaction with Groucho and is sensitive to Groucho dosage. Runt's repression of one gene, engrailed, is independent of VWRPY and Groucho, thus demonstrating alternative mechanisms for repression by Runt domain proteins. Unlike other transcriptional regulatory proteins that interact with Groucho, Runt domain proteins are known to activate transcription. This suggests that the Runt domain protein-Groucho interaction may be regulated.

The Drosophila runt gene is the founding member of the Runt domain family of transcriptional regulators. Mammalian Runt domain genes encode the alpha subunit of the heterometric DNA-binding factor PEBP2/CBF. The unrelated PEBP2/CBF beta protein interacts with the Runt domain to increase its affinity for DNA. The conserved ability of the Drosophila Runt protein to respond to the stimulating effect of mammalian PEBP2/CBF beta indicated that flies were likely to have a homologous beta protein. Using the yeast two-hybrid system to isolate cDNAs for Runt-interacting proteins, we identified two Drosophila genes, referred to as Brother and Big-brother, that have substantial sequence homology with PEBP2/CBF beta. Yeast two-hybrid experiments as well as in vitro DNA-binding studies confirmed the functional homology of the Brother, Big-brother, and PEBP2/CBF beta proteins and demonstrated that the conserved regions of the Runt and Brother proteins are required for their heterodimeric interaction. The DNA-bending properties of Runt domain proteins in the presence and absence of their partners were also examined. Our results show that Runt domain proteins bend DNA and that this bending is influenced by Brother protein family members, supporting the idea that heterodimerization is associated with a conformational change in the Runt domain. Analysis of expression patterns in Drosophila embryos revealed that Brother and Big-brother are likely to interact with runt in vivo and further suggested that the activity of these proteins is not restricted to their interaction with Runt.

Brother and Big brother were isolated as Runt-interacting proteins and are homologous to CBF(beta), which interacts with the mammalian CBF(alpha) Runt-domain proteins. In vitro experiments indicate that Brother family proteins regulate the DNA binding activity of Runt-domain proteins without contacting DNA. In both mouse and human there is genetic evidence that the CBF(alpha) and CBF(beta) proteins function together in hematopoiesis and leukemogenesis. Here we demonstrate functional interactions between Brother proteins and Runt domain proteins in Drosophila. First, we show that a specific point mutation in Runt that disrupts interaction with Brother proteins but does not affect DNA binding activity is dysfunctional in several in vivo assays. Interestingly, this mutant protein acts dominantly to interfere with the Runt-dependent activation of Sxl-lethal transcription. To investigate further the requirements for Brother proteins in Drosophila development, we examine the effects of expression of a Brother fusion protein homologous to the dominant negative CBF(beta)::SMMHC fusion protein that is associated with leukemia in humans. This Bro::SMMHC fusion protein interferes with the activity of Runt and a second Runt domain protein, Lozenge. Moreover, we find that the effects of lozenge mutations on eye development are suppressed by expression of wild-type Brother proteins, suggesting that Brother/Big brother dosage is limiting in this developmental context. Results obtained when Runt is expressed in developing eye discs further support this hypothesis. Our results firmly establish the importance of the Brother and Big brother proteins for the biological activities of Runt and Lozenge, and further suggest that Brother protein function is not restricted to enhancing DNA-binding.

Abstract Background: AML with RUNX1 mutation is a provisional entity in the WHO classification. RUNX1 variants generally consist of truncating mutations throughout the gene, as well as SNVs, insertions, and deletions in the RUNT domain. In the germline setting, these mutations may lead to familial platelet disorders and have profound implications in donor selection for allogeneic stem cell transplant. Approximately half of mutations in RUNX1 are classified as variants of unknown clinical significance (VOUS), underscoring the unmet needs of this patient population. Methods: Bone marrow, peripheral blood, or FFPE tissue samples from 10,118 patients with suspected myeloid disease were sequenced using a 303 gene myeloid NGS panel. A total of 1209 RUNX1-mutated patients formed the cohort for this study. While pair-matched samples were not available and germline status not established, these patients were likely to be in the 40-60% variant allele frequency (VAF) range. Statistics were performed using Fishers exact test. Results: Among RUNX1-mutated patients, 277 (22.9%) had a RUNX1 variant at a subclonal VAF (<20%), 505 (41.7%), 352 (29.1%), & 75 (6.2%) had variants at VAFs of 20-39%, 40-60%, & >60%, respectively. While all truncating mutations were classified as pathogenic, 84.3% of non-truncating RUNT domain mutations were classified as VOUS. RUNT domain mutations included 272 non-truncating and 220 truncating mutations. There were 599 (49.5%) truncating mutations throughout the entire gene. Non-truncating RUNT domain-mutated patients most frequently harbored mutations in ASXL1 (31.6%), SRSF2 (31.3%), TET2 (29.4%), STAG2 (19.1%), RAS (18.3%), and BCOR/BCORL1 (17.3%). The frequency of RUNT domain co-mutations in the ASXL1 (36.8% [35] vs. 10.4% [5]; p=0.0007) and RAS families (28.4% [26] vs. 10.4% [5]; p=0.03) were significantly higher in the 40-60% VAF group compared to subclonal populations, while BCOR/BCORL1 mutations (10.5% [10] vs. 29.1% [14]; p=0.008) were significantly lower. These results were recapitulated in a cohort of VOUS-only RUNT domain patients with a VAF of 40-60% compared to subclonal populations: ASXL1 (36.8% [21] vs. 22.2% [10]), RAS family (24.5% [14] vs. 10.9% [5]), and BCOR/BCORL1 (8.8% [5] vs. 31.1% [14]; p=0.005). Among patients with truncating mutations in RUNX1, mutations in ASXL1 (34.4% [44] vs 20.3% [42]; p=0.0047) and RAS family members (25.8% [33] vs. 10.1% [21]; p=0.0002) were more frequent in patients with VAFs of 40-60% when compared to subclonal populations, and mutations in BCOR/BCORL1 were similar (28.1% [36] vs. 20.7% [42]). Conclusions: The majority of RUNX1 mutations clustered in the RUNT domain are VOUS. This study demonstrates that both truncations and RUNT domain mutations harbor molecular signatures that reflect similar oncogenic mechanisms. These molecular signatures are also present when filtering exclusively by VOUS in the RUNT domain. Citation Format: Frank J. Scarpa, Madhuri Paul, Rachel Daringer, Sally Agersborg, Vincent A. Funari, Forrest J. Blocker. Molecular profiling of the RUNX1 RUNT domain in myeloid disorders [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2281.

The Core Binding Factor is a heterodimeric transcription factor complex in vertebrates that is composed of a DNA binding α-subunit and a non-DNA binding β-subunit. The α-subunit is encoded by members of the Runt Domain family of proteins and the β-subunit is encoded by the CBFβ gene. In Drosophila, two genes encoding α-subunits, runt and lozenge, and two genes encoding β-subunits, Big brother and Brother, have been previously identified. Here, a sensitized genetic screen was used to isolate mutant alleles of the Big brother gene. Expression studies show that Big brother is a nuclear protein that co-localizes with both Lozenge and Runt in the eye imaginal disc. The nuclear localization and stability of Big brother protein is mediated through the formation of heterodimeric complexes between Big brother and either Lozenge or Runt. Big brother functions with Lozenge during cell fate specification in the eye, and is also required for the development of the embryonic PNS. ds-RNA-mediated genetic interference experiments show that Brother and Big brother are redundant and function together with Runt during segmentation of the embryo. These studies highlight a mechanism for transcriptional control by a Runt Domain protein and a redundant pair of partners in the specification of cell fate during development.

Abstract AML1/Runx1 is a frequent target of human leukemia–associated gene aberration and encodes a transcription factor with nonredundant biologic functions in initial development of definitive hematopoiesis, T-cell development, and steady-state platelet production. AML1/Runx1 and 2 closely related family genes, AML2/Runx3 and AML3/Runx2/Cbfa1, present in mammals, comprise the Runt-domain transcription factor family. Although they have similar structural and biochemical properties, gene-targeting experiments have identified distinct biologic roles. To directly determine the presence of functional overlap among runt-related transcription factor (Runx) family molecules, we replaced the C-terminal portion of acute myeloid leukemia 1 (AML1) with that derived from its family members, which are variable in contrast to conserved Runt domain, using the gene knock-in method. We found that C-terminal portions of either AML2 or AML3 could functionally replace that of AML1 for myeloid development in culture and within the entire mouse. However, while AML2 substituted for AML1 could effectively rescue lymphoid lineages, AML3 could not, resulting in a smaller thymus and lymphoid deficiency in peripheral blood. Substitution by the C-terminal portion of AML3 also led to high infantile mortality and growth retardation, suggesting that AML1 has as yet unidentified effects on these phenotypes. Thus, the C-terminal portions of Runx family members have both similar and distinct biologic functions.

Runx proteins are bifunctional transcription factors that both repress and activate transcription in animal cells. Typically, Runx proteins work in concert with other transcriptional regulators, including co-activators and co-repressors to mediate their biological effects. In Drosophila melanogaster the archetypal Runx protein, Runt, functions in numerous processes including segmentation, neurogenesis and sex determination. During primary sex determination Runt acts as one of four X-linked signal element (XSE) proteins that direct female-specific activation of the establishment promoter (Pe) of the master regulatory gene Sex-lethal (Sxl). Successful activation of SxlPe requires that the XSE proteins overcome the repressive effects of maternally deposited Groucho (Gro), a potent co-repressor of the Gro/TLE family. Runx proteins, including Runt, contain a C-terminal peptide, VWRPY, known to bind to Gro/TLE proteins to mediate transcriptional repression. We show that Runt’s VWRPY co-repressor-interaction domain is needed for Runt to activate SxlPe. Deletion of the Gro-interaction domain eliminates Runt-ability to activate SxlPe, whereas replacement with a higher affinity, VWRPW, sequence promotes Runt-mediated transcription. This suggests that Runt may activate SxlPe by antagonizing Gro function, a conclusion consistent with earlier findings that Runt is needed for Sxl expression only in embryonic regions with high Gro activity. Surprisingly we found that Runt is not required for the initial activation of SxlPe. Instead, Runt is needed to keep SxlPe active during the subsequent period of high-level Sxl transcription suggesting that Runt helps amplify the difference between female and male XSE signals by counter-repressing Gro in female, but not in male, embryos.

Abstract AML1/ETO is the chimeric fusion protein resulting from the t(8;21) found in AML of the M2 subtype. It contains the N-terminal 177 amino acids of RUNX1 and virtually all (575aa) of ETO. The RUNX1 component includes the Runt domain, which mediates both DNA binding and heterodimerization with CBFβ, but lacks the more C-terminal sequences required for transactivation. AML1/ETO occupies RUNX target genes in vivo and is associated with a repressive chromatin structure characterized by reduced levels of acetylated histone H3. AML1/ETO is thought to repress transcription by recruiting a SMRT (N-CoR)/Sin3A/HDAC complex to chromatin via sequences in ETO. ETO is the human homologue of the Drosophila Nervy protein and shares 4 regions of homology with Nervy called Nervy Homology Regions (NHR) 1–4. Deletion studies have shown that three of the AML1/ETO domains essential for its repressive function are the Runt domain, NHR2, and NHR4. The NHR2 domain is a hydrophobic heptad repeat that mediates oligomerization of AML1/ETO, interaction with ETO family members, and also with mSin3A and HDACs. We recently solved an x-ray structure of the NHR2 domain and found it to be an alpha-helical tetramer. Based on this structure we have introduced amino acid substitutions into the NHR2 domain that disrupt tetramer formation but not AML1/ETO stability. These mutations impair the ability of AML1/ETO to inhibit the differentiation of GR-1+/Mac-1+ cells following retroviral transduction into primary mouse bone marrow cells, and also inhibit the serial replating ability of AML1/ETO expressing bone marrow cells in vitro. We additionally show that mutations reported by Amann et al. (Mol Cell Biol. 21, 6470, 2001) to disrupt mSin3A binding to NHR2 do not affect the biological activity of AML1/ETO in vitro. We also introduced mutations in the Runt domain of AML1/ETO that disrupt CBFβ binding by defined amounts (40-fold, 200-fold, 500-fold), and demonstrated that CBFβ binding by AML1/ETO is essential for its dominant negative activity. The latter results suggest that small molecules designed to selectively impair heterodimerization of AML1/ETO with CBFβ could potentially block AML1/ETO’s dominant negative activity.

RUNX2 belongs to the RUNT domain family of transcription factors of which three have been identified in humans (RUNX1, RUNX2 and RUNX3). RUNX proteins are vital for metazoan development and participate in the regulation of cellular differentiation and cell cycle progression (Coffman, 2003). RUNX2 is required for proper bone formation by driving the differentiation of osteoblasts from mesenchymal progenitors during development (Ducy et al, 1997; Komori et al, 1997; Otto et al, 1997). RUNX2 is also vital for chondrocyte maturation by promoting the differentiation of chondrocytes to the hypertrophic phenotype (Enomoto et al, 2000). The consequences of completely disrupting the RUNX2 locus in mice provided compelling and conclusive evidence for the biological importance of RUNX2 where knockout mice died shortly after birth with a complete lack of bone formation (Komori et al, 1997; Otto et al, 1997). A further indication of the requisite role of RUNX2 in skeletal development was the discovery that RUNX2 haploinsufficiency in humans and mice caused the skeletal syndrome Cleidocranial Dysplasia (CCD) (Mundlos et al, 1997; Lee et al, 1997). A unique feature of RUNX2 is the consecutive polyglutamine and polyalanine tracts (Q/A domain). Mutations causing CCD have been observed in the Q/A domain of RUNX2 (Mundlos et al, 1997). The Q/A domain is an essential part of RUNX2 and participates in transactivation function (Thirunavukkarasu et al, 1998). Previous genotyping studies conducted in our laboratory identified several rare RUNX2 Q/A variants in addition to a frequently occurring 18 base pair deletion of the polyalanine tract termed the 11Ala allele. Analysis of serum parameters in 78 Osteoarthritis patients revealed the 11Ala allele was associated with significantly decreased osteocalcin. Furthermore, analysis of 11Ala allele frequencies within a Geelong Osteoporosis Study (GOS) fracture cohort and an appropriate age matched control group revealed the 11Ala allele was significantly overrepresented in fracture cases indicating an association with increased fracture risk. To further investigate the 11Ala allele and rare Q/A variants, 747 DNA samples from the Southeast Queensland bone study were genotyped using PCR and PAGE. The experiment served two purposes: 1) to detect additional rare Q/A variants to enrich the population of already identified mutants and 2) have an independent assessment of the effect of the 11Ala allele on fracture to either support or refute our previous observation which indicated the 11Ala allele was associated with an increased risk of fracture in the GOS. From the 747 samples genotyped, 665 were WT, 76 were heterozygous for the 11Ala allele, 5 were homozygous for the 11Ala allele and 1 was heterozygous for a rare 21 bp deletion of the polyglutamine tract. Chi-square analysis of RUNX2 genotype distributions within fracture and non-fracture groups in the Southeast Queensland bone study revealed that individuals that carried at least one copy of the 11Ala allele were enriched in the fracture group (p = 0.16, OR = 1.712). The OR of 1.712 was of similar magnitude to the OR observed in the GOS case-control investigation (OR = 1.9) providing support for the original study. Monte-Carlo simulations were used to combine the results from the GOS and the Southeast Queensland bone study. The simulations were conducted with 10000 iterations and demonstrated that the maximum probability of obtaining both study results by chance was less than 5 times in two hundred (p < 0.025) suggesting that the 11Ala allele of RUNX2 was associated with an increased fracture risk. The second element of the research involved the analysis of rare RUNX2 Q/A variants identified from multiple epidemiological studies of bone. Q/A repeat variants were derived from four populations: the GOS, an Aberdeen cohort, CAIFOS and a Sydney twin study. Collectively, a total of 20 rare glutamine and one alanine variants were identified from 4361 subjects. All RUNX2 Q/A variants were heterozygous for a mutant allele and a wild type allele. Analysis of incident fracture during a five year follow up period in the CAIFOS revealed that Q-variants (n = 8) were significantly more likely to have fractured compared to non-carriers (p = 0.026, OR 4.932 95% CI 1.2 to 20.1). Bone density data as measured by quantitative ultrasound was available for CAIFOS. Analysis of BUA and SOS Z-scores revealed that Q-repeat variants had significantly lower BUA (p = 0.031, mean Z-score of -0.79) and a trend for lower SOS (p = 0.190, mean Z-score of -0.69). BMD data was available for all four populations. To normalize the data across the four studies, FN BMD data was converted into Z-scores and the effect of the Q/A variants on BMD was analysed using a one sample approach. The analysis revealed Q/A variants had significantly lower FN BMD (p = 0.0003) presenting with a 0.65 SD decrease. Quantitative transactivation analysis was conducted on RUNX2 proteins harbouring rare glutamine mutations and the 11Ala allele. RUNX2 proteins containing a glutamine deletion (16Q), a glutamine insertion (30Q) and the 11Ala allele were overexpressed in NIH3T3 and HEK293 cells and their ability to transactivate a known target promoter was assessed. The 16Q and 30Q had significantly decreased reporter activity compared to WT in NIH3T3 cells (p = 0.002 and 0.016, for 16Q and 30Q, respectively). In contrast 11Ala RUNX2 did not show significantly different promoter activation potential (p = 0.54). Similar results were obtained in HEK293 cells where both the 16Q and 30Q RUNX2 displayed decreased reporter activity (p=0.007 and 0.066 for 16Q and 30Q respectively) whereas the 11Ala allele had no material effect on RUNX2 function (p = 0.20). The RUNX2 gene target reporter assay provided evidence to suggest that variation within the glutamine tract of RUNX2 was capable of altering the ability of RUNX2 to activate a known target promoter. In contrast, the 11Ala allele showed no variation in RUNX2 activity. The third feature of the research served the purpose of identifying potential RUNX2 gene targets with particular emphasis on discovering genes cooperatively regulated by RUNX2 and the powerful bone promoting agent BMP2. The experiment was conducted by creating stably transfected NIH3T3 cells lines overexpressing RUNX2 or BMP2 or both RUNX2 and BMP2. Microarray analysis revealed very few genes were differentially regulated between standard NIH3T3 cells and cells overexpressing RUNX2. The results were confirmed via RT-PCR analysis which demonstrated that the known RUNX2 gene targets Osteocalcin and Matrix Metalloproteinase-13 were modestly induced 2.5 fold (p = 0.00017) and 2.1 fold (p = 0.002) respectively in addition to identifying only two genes (IGF-II and SCYA11) that were differentially regulated greater than 10 fold. IGF-II and SYCA11 were significantly down-regulated 27.6 fold (p = 1.95 x 10-6) and 10.1 fold (p = 0.0002) respectively. The results provided support for the notion that RUNX2 on its own was not sufficient for optimal gene expression and required the presence of additional factors. To discover genes cooperatively regulated by RUNX2 and BMP2, microarray gene expression analysis was performed on standard NIH3T3 cells and NIH3T3 cells stably transfected with both RUNX2 and BMP2. Comparison of the gene expression profiles revealed the presence of a large number of differentially regulated genes. Four genes EHOX, CCL9, CSF2 and OSF-1 were chosen to be further characterized via RT-PCR. Sequential RT-PCR analysis on cDNA derived from control cells and cells stably transfected with either RUNX2, BMP2 or both RUNX2/BMP2 revealed that EHOX and CSF2 were cooperatively induced by RUNX2 and BMP2 whereas CCL9 and OSF-1 were suppressed by BMP2. The overexpression of both RUNX2 and BMP2 in NIH3T3 fibroblasts provided a powerful model upon which to discover potential RUNX2 gene targets and also identify genes synergistically regulated by BMP2 and RUNX2. The fourth element of the research investigated the role of RUNX2 in the ascorbic acid mediated induction of MMP-13 mRNA. The study was carried out using NIH3T3 cell lines stably transfected with BMP2, RUNX2 and both BMP2 and RUNX2. The cell lines were grown to confluence and subsequently cultured for a further 12 days in standard media or in media supplemented with AA. RT-PCR analysis was used to assess MMP-13 mRNA expression. The RT-PCR results demonstrated that AA was not sufficient for inducing MMP-13 mRNA in NIH3T3 cells. In contrast RUNX2 significantly induced MMP-13 levels 85 fold in the absence of AA (p = 0.0055) and upregulated MMP-13 mRNA levels 254 fold in the presence of AA (p = 0.0017). The results demonstrated that RUNX2 was essential for the AA mediated induction of MMP-13 mRNA in NIH3T3 cells. The effect of BMP2 on MMP-13 expression was also investigated. BMP2 induced MMP-13 mRNA transcripts a modest 3.8 fold in the presence of AA (p = 0.0027). When both RUNX2 and BMP2 were overexpressed in the presence of AA, MMP-13 mRNA levels were induced a massive 4026 fold (p = 8.7 x 10-4) compared to control cells. The investigation revealed that RUNX2 was an essential factor for the AA mediated induction of MMP-13 and that RUNX2 and BMP2 functionally cooperated to regulate MMP-13 mRNA levels.

RUNX2 belongs to the RUNT domain family of transcription factors of which three have been identified in humans (RUNX1, RUNX2 and RUNX3). RUNX proteins are vital for metazoan development and participate in the regulation of cellular differentiation and cell cycle progression (Coffman, 2003). RUNX2 is required for proper bone formation by driving the differentiation of osteoblasts from mesenchymal progenitors during development (Ducy et al, 1997; Komori et al, 1997; Otto et al, 1997). RUNX2 is also vital for chondrocyte maturation by promoting the differentiation of chondrocytes to the hypertrophic phenotype (Enomoto et al, 2000). The consequences of completely disrupting the RUNX2 locus in mice provided compelling and conclusive evidence for the biological importance of RUNX2 where knockout mice died shortly after birth with a complete lack of bone formation (Komori et al, 1997; Otto et al, 1997). A further indication of the requisite role of RUNX2 in skeletal development was the discovery that RUNX2 haploinsufficiency in humans and mice caused the skeletal syndrome Cleidocranial Dysplasia (CCD) (Mundlos et al, 1997; Lee et al, 1997). A unique feature of RUNX2 is the consecutive polyglutamine and polyalanine tracts (Q/A domain). Mutations causing CCD have been observed in the Q/A domain of RUNX2 (Mundlos et al, 1997). The Q/A domain is an essential part of RUNX2 and participates in transactivation function (Thirunavukkarasu et al, 1998). Previous genotyping studies conducted in our laboratory identified several rare RUNX2 Q/A variants in addition to a frequently occurring 18 base pair deletion of the polyalanine tract termed the 11Ala allele. Analysis of serum parameters in 78 Osteoarthritis patients revealed the 11Ala allele was associated with significantly decreased osteocalcin. Furthermore, analysis of 11Ala allele frequencies within a Geelong Osteoporosis Study (GOS) fracture cohort and an appropriate age matched control group revealed the 11Ala allele was significantly overrepresented in fracture cases indicating an association with increased fracture risk. To further investigate the 11Ala allele and rare Q/A variants, 747 DNA samples from the Southeast Queensland bone study were genotyped using PCR and PAGE. The experiment served two purposes: 1) to detect additional rare Q/A variants to enrich the population of already identified mutants and 2) have an independent assessment of the effect of the 11Ala allele on fracture to either support or refute our previous observation which indicated the 11Ala allele was associated with an increased risk of fracture in the GOS. From the 747 samples genotyped, 665 were WT, 76 were heterozygous for the 11Ala allele, 5 were homozygous for the 11Ala allele and 1 was heterozygous for a rare 21 bp deletion of the polyglutamine tract. Chi-square analysis of RUNX2 genotype distributions within fracture and non-fracture groups in the Southeast Queensland bone study revealed that individuals that carried at least one copy of the 11Ala allele were enriched in the fracture group (p = 0.16, OR = 1.712). The OR of 1.712 was of similar magnitude to the OR observed in the GOS case-control investigation (OR = 1.9) providing support for the original study. Monte-Carlo simulations were used to combine the results from the GOS and the Southeast Queensland bone study. The simulations were conducted with 10000 iterations and demonstrated that the maximum probability of obtaining both study results by chance was less than 5 times in two hundred (p < 0.025) suggesting that the 11Ala allele of RUNX2 was associated with an increased fracture risk. The second element of the research involved the analysis of rare RUNX2 Q/A variants identified from multiple epidemiological studies of bone. Q/A repeat variants were derived from four populations: the GOS, an Aberdeen cohort, CAIFOS and a Sydney twin study. Collectively, a total of 20 rare glutamine and one alanine variants were identified from 4361 subjects. All RUNX2 Q/A variants were heterozygous for a mutant allele and a wild type allele. Analysis of incident fracture during a five year follow up period in the CAIFOS revealed that Q-variants (n = 8) were significantly more likely to have fractured compared to non-carriers (p = 0.026, OR 4.932 95% CI 1.2 to 20.1). Bone density data as measured by quantitative ultrasound was available for CAIFOS. Analysis of BUA and SOS Z-scores revealed that Q-repeat variants had significantly lower BUA (p = 0.031, mean Z-score of -0.79) and a trend for lower SOS (p = 0.190, mean Z-score of -0.69). BMD data was available for all four populations. To normalize the data across the four studies, FN BMD data was converted into Z-scores and the effect of the Q/A variants on BMD was analysed using a one sample approach. The analysis revealed Q/A variants had significantly lower FN BMD (p = 0.0003) presenting with a 0.65 SD decrease. Quantitative transactivation analysis was conducted on RUNX2 proteins harbouring rare glutamine mutations and the 11Ala allele. RUNX2 proteins containing a glutamine deletion (16Q), a glutamine insertion (30Q) and the 11Ala allele were overexpressed in NIH3T3 and HEK293 cells and their ability to transactivate a known target promoter was assessed. The 16Q and 30Q had significantly decreased reporter activity compared to WT in NIH3T3 cells (p = 0.002 and 0.016, for 16Q and 30Q, respectively). In contrast 11Ala RUNX2 did not show significantly different promoter activation potential (p = 0.54). Similar results were obtained in HEK293 cells where both the 16Q and 30Q RUNX2 displayed decreased reporter activity (p=0.007 and 0.066 for 16Q and 30Q respectively) whereas the 11Ala allele had no material effect on RUNX2 function (p = 0.20). The RUNX2 gene target reporter assay provided evidence to suggest that variation within the glutamine tract of RUNX2 was capable of altering the ability of RUNX2 to activate a known target promoter. In contrast, the 11Ala allele showed no variation in RUNX2 activity. The third feature of the research served the purpose of identifying potential RUNX2 gene targets with particular emphasis on discovering genes cooperatively regulated by RUNX2 and the powerful bone promoting agent BMP2. The experiment was conducted by creating stably transfected NIH3T3 cells lines overexpressing RUNX2 or BMP2 or both RUNX2 and BMP2. Microarray analysis revealed very few genes were differentially regulated between standard NIH3T3 cells and cells overexpressing RUNX2. The results were confirmed via RT-PCR analysis which demonstrated that the known RUNX2 gene targets Osteocalcin and Matrix Metalloproteinase-13 were modestly induced 2.5 fold (p = 0.00017) and 2.1 fold (p = 0.002) respectively in addition to identifying only two genes (IGF-II and SCYA11) that were differentially regulated greater than 10 fold. IGF-II and SYCA11 were significantly down-regulated 27.6 fold (p = 1.95 x 10-6) and 10.1 fold (p = 0.0002) respectively. The results provided support for the notion that RUNX2 on its own was not sufficient for optimal gene expression and required the presence of additional factors. To discover genes cooperatively regulated by RUNX2 and BMP2, microarray gene expression analysis was performed on standard NIH3T3 cells and NIH3T3 cells stably transfected with both RUNX2 and BMP2. Comparison of the gene expression profiles revealed the presence of a large number of differentially regulated genes. Four genes EHOX, CCL9, CSF2 and OSF-1 were chosen to be further characterized via RT-PCR. Sequential RT-PCR analysis on cDNA derived from control cells and cells stably transfected with either RUNX2, BMP2 or both RUNX2/BMP2 revealed that EHOX and CSF2 were cooperatively induced by RUNX2 and BMP2 whereas CCL9 and OSF-1 were suppressed by BMP2. The overexpression of both RUNX2 and BMP2 in NIH3T3 fibroblasts provided a powerful model upon which to discover potential RUNX2 gene targets and also identify genes synergistically regulated by BMP2 and RUNX2. The fourth element of the research investigated the role of RUNX2 in the ascorbic acid mediated induction of MMP-13 mRNA. The study was carried out using NIH3T3 cell lines stably transfected with BMP2, RUNX2 and both BMP2 and RUNX2. The cell lines were grown to confluence and subsequently cultured for a further 12 days in standard media or in media supplemented with AA. RT-PCR analysis was used to assess MMP-13 mRNA expression. The RT-PCR results demonstrated that AA was not sufficient for inducing MMP-13 mRNA in NIH3T3 cells. In contrast RUNX2 significantly induced MMP-13 levels 85 fold in the absence of AA (p = 0.0055) and upregulated MMP-13 mRNA levels 254 fold in the presence of AA (p = 0.0017). The results demonstrated that RUNX2 was essential for the AA mediated induction of MMP-13 mRNA in NIH3T3 cells. The effect of BMP2 on MMP-13 expression was also investigated. BMP2 induced MMP-13 mRNA transcripts a modest 3.8 fold in the presence of AA (p = 0.0027). When both RUNX2 and BMP2 were overexpressed in the presence of AA, MMP-13 mRNA levels were induced a massive 4026 fold (p = 8.7 x 10-4) compared to control cells. The investigation revealed that RUNX2 was an essential factor for the AA mediated induction of MMP-13 and that RUNX2 and BMP2 functionally cooperated to regulate MMP-13 mRNA levels.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Medical Science
Faculty of Health
Full Text

AbstractLifted (family-based) static analysis by abstract interpretation is capable of analyzing all variants of a program family simultaneously, in a single run without generating any of the variants explicitly. The elements of the underlying lifted analysis domain are tuples, which maintain one property per variant. Still, explicit property enumeration in tuples, one by one for all variants, immediately yields combinatorial explosion. This is particularly apparent in the case of program families that, apart from Boolean features, contain also numerical features with large domains, thus giving rise to astronomical configuration spaces.The key for an efficient lifted analysis is a proper handling of variability-specific constructs of the language (e.g., feature-based runtime tests and $$\texttt {\#if}$$ # if directives). In this work, we introduce a new symbolic representation of the lifted abstract domain that can efficiently analyze program families with numerical features. This makes sharing between property elements corresponding to different variants explicitly possible. The elements of the new lifted domain are constraint-based decision trees, where decision nodes are labeled with linear constraints defined over numerical features and the leaf nodes belong to an existing single-program analysis domain. To illustrate the potential of this representation, we have implemented an experimental lifted static analyzer, called SPLNum$$^2$$ 2 Analyzer, for inferring invariants of C programs. An empirical evaluation on BusyBox and on benchmarks from SV-COMP yields promising preliminary results indicating that our decision trees-based approach is effective and outperforms the baseline tuple-based approach.

This study analyzes the relationship between the elderly dependency ratio and income inequality over the period 1972-2019 in countries such as the USA, Japan, the UK, France, Germany, Canada, and Italy, which rank top in the population aging, using the Fourier-Shin cointegration test. According to the results, the rise in the elderly dependency ratio of all countries included in the analysis, except for France, has a positive impact on income inequality. The result implying that the rise in the elderly dependency ratio increases the income inequality and renders some policy recommendations possible. Accordingly, the provision of adequate childcare programs and family aids can result in greater labor force participation in the short- and long-run. In addition, a pension system can be developed to lower the elderly dependency ratio, more money can be saved for the retirement period, and working domains can be developed for the post-retirement period.

In this chapter, I focus on a writer who got her start writing poetry and prose for the cheap popular press: Frances Brown. Like Cook, Brown was working class, but her impoverished upbringing as the daughter of a postmaster in a remote Ulster village placed her on a lower rung of the social ladder than Cook with fewer resources at her disposal. She lost her sight to smallpox when she was eighteen months old and learned about the world by listening to her siblings’ lessons and having family members read aloud to her. Once she began writing, her sister Rebecca served as her amanuensis. She began her career contributing poetry to the Irish Penny Journal, later published her work in the Penny Magazine, and developed an extensive career contributing poetry and prose to Chambers’s Edinburgh Journal. Her careful navigation of the market for popular literature reveals the importance of cheap media formats (with differing levels of copyright protection) in a fashioning a writing career. Even though Brown’s work was often repurposed by scissors-and-paste journalists as if it were free content within the public domain, she was successful in establishing a celebrity identity and publishing her work in book form.

Whether you are breaking prairie sod in the nineteenth century or raising a family and scrambling to make ends meet in the twenty-first, it is hard to get worked up over abstract possibilities. There is too much that needs doing, right here, right now. Even knowing the odds, people still live in earthquake zones, hurricane alleys, and the unprotected floodplains of mighty rivers. The warm embrace of a thirsty aridland city is not so different. Generally speaking, it is hard for any of us to get seriously concerned about what might happen until it does happen. That’s why the politics of climate change are so difficult. The measurements and observations that convince scientists about the warming of Earth are invisible to the rest of us. We fail to sense them at the scale of our personal lives. And believing in the verdicts of computer models about what might happen twenty or forty years in the future, well, that is tantamount to a leap of faith, and most people don’t ordinarily jump that far. Believing in the growth of cities can be difficult, too. Beginning in 2007, the domino of subprime mortgage defaults knocked over the domino of overleveraged investment banks, which toppled a wobbly world credit system, which upended industries around the globe and ushered in the Great Recession. 1 The home-building industries of growth-crazy cities like Las Vegas and Phoenix collapsed virtually overnight. Suburbs from Florida to California became ghost towns where wind-driven litter piled up in doorways and weeds grew higher than the sills of boarded-up windows. Some analysts predicted the emergence of a new generation of suburban slums and the death of gas-guzzling, car-dependent, long-commute suburban lifestyles. 2 Indeed, in the long run, considering the implications of peak oil and peak water and the likelihood of more severe climate reckonings than we’ve yet seen, such a demise seems likely—though maybe not quite yet.

The spin test is a standard industrial practice employed for the qualification of rotor blades and disks. The expected results are the modal properties of blades and assemblages at different rotation velocities. If a significant dynamic coupling among the blades exists, global vibration modes appear, reflecting into a set of closely spaced natural frequencies for each mode family. In case of perfectly-tuned bladed disks, the circumferential structure of the mode shapes is known and can be exploited during the identification process so that traditional single-dof models may be applied. On the contrary, the mode irregularities produced by mistuning prevents the use of single-dof models requiring the development of more sophisticated approaches. In this work, we propose a multi-dof identification technique organized as follow: 1) the FRF of the bladed disk in the neighborhood of a resonance crossing is identified by the wavelet transform of the measured response; 2) the modal parameters of the system are estimated using a mixed stochastic-deterministic subspace algorithm formulated in the frequency domain. The procedure is validated using a realistic numerical simulation.

For the past 25 years, heuristic search has been used to solve domain-independent probabilistic planning problems, but with heuristics that determinise the problem and ignore precious probabilistic information. In this paper, we present a generalization of the operator-counting family of heuristics to Stochastic Shortest Path problems (SSPs) that is able to represent the probability of the actions outcomes. Our experiments show that the equivalent of the net change heuristic in this generalized framework obtains significant run time and coverage improvements over other state-of-the-art heuristics in different planners.

A numerical–computational procedure is described to determine a multi-dimensional functional or an operator for the representation of the computational results of a numerical transport code. The procedure is called Numerical Transport Code Functionalization (NTCF). Numerical transport codes represent a family of engineering software to solve, for example, heat conduction problems in solids using ANSYS; heat and moisture transport problems in porous media using NUFT; or laminar or turbulent flow and transport problems using FLUENT, a computational fluid dynamic (CFD) model. The NTCF procedure is developed to determine a model for the representation of the code for a variety of input functions. Couple solution of multiphysics problems often require repeated, iterative calculations for the same model domain and with the same code, but with different boundary condition values. The NTCF technique allows for reducing the number of runs with the original numerical code to the number of runs necessary for NTCF model identification. The NTCF procedure is applied for the solution of coupled heat and moisture transport problems at Yucca Mountain, Nevada. The NTCF method and the supporting software is a key element of MULTIFLUX, a coupled thermohydrologic-ventilation model and software. Numerical tests as well as applications for Yucca Mountain, Nevada are presented using both linear and nonlinear NTCF models. The performance of the NTCF method is demonstrated both in accuracy and modeling acceleration.

Industrial scale reactor system components are frequently characterized by a complex geometry that requires a large number of nodes in a computational grid to resolve significant flow features and by reaction and additional complex physics, such as spray vaporization, that greatly increase the number of partial differential equations (PDEs) to solve. On a fast serial workstation, simulations of these systems can take several days, making use of the model for extensive parametric and optimization studies impractical. A parallel multiphase reacting flow code was developed from a family of related serial codes using the industry standard Message-Passing Interface (MPI) and domain decomposition in the primary flow direction. The parallel computational fluid dynamics (CFD) code can now be run on a cluster of networked computers. This capability makes detailed simulation of many industrial reactors feasible without incurring the large increase in cost of moving to simulation on massively parallel computing platforms. The required computers and network are often already present in organizations and idle at night. The parallel CFD code has been applied to studies of transient heat transfer to monolith catalyst substrates and to the design of inlet chambers that evenly distribute a high speed hot air inlet stream over the monolith inlet plane. These studies are part of a program to investigate the feasibility of rapid start fuel reformers. The use of fuel reformers in light duty fuel cell powered vehicles requires a reformer start up time of less than 60 seconds. Parallelization issues and parallel performance are presented with examples of reformer component flow and comparison with experimental data from monolith heat up studies.

The new godunov-type high order method via flux-reconstruction, are capable of unifiying several popular methods including the discontinuous Galerkin, staggered-grid multi-domain method, or the spectral difference/spectral volume methods into a single family. Compared to discontinuous galerkin method, this new method retain the arbitrary order and compact properties but written in differential form and without mass matrix. The flux-reconstrcution method using general unstructured 2D/3D mesh including triangles, quadrilaterals, tetrahedrons, pyramids, prisms and hexahedrons are implemented. Compared to discontinuous galerkin method, FR is differential form without any quandature, so it runs faster and is easier to implement on GPU to achieve higher speed up ratio. The present paper investigates three different solving methods for this new method in order to cut down the hugh computational cost and memory requirement for fully implicit methods. Explicit multi-stage Runge-Kutta, nonlinear Lower-Upper Symmetry Gauss-Seidel (LU-SGS) and generalized minimal residual (GMRES) with matrix-free preconditioning methods are implemented and all these three methods use p-multigrid to smooth low-frequency errors on lower order. The results of 2D steady-state external/internal viscous flows are presented and the convergence properties for different solving method are compared. Efficiency and robustness are improved through symmetric Gauss-Seidel (SGS) iterations as preconditioning, a remarkable feature of the present GMRES+SGS method is that the storage of the full Jacobian matrix can be eliminated and only its diagonal stored. A laplacian artificial viscosity is tested to capture shock in one element and performs well for this new high order method, an L2 projection method is used to improve the perfomance of aliasing error in conservative form. The one equation Spalart-Allmaras turbulence model is unsed to solve the Raynolds average navier-stokes equations. MPI is used for parallel simulations for ralatively complex 3D internal flow when using high order method. Compared to other high order methods, results in this paper show that Flux-Reconstruction methods with efficient solving method performs well for 2D/3D general unstructured mesh in wide range of reynorlds number, and is also potential for “real geometry” simulations.