Dissertations / Theses on the topic 'Functional Characterization'

In order to guarantee cell survival, and the transmission of the correct genetic information to their offspring, organisms had to develop molecular pathways to deal with damaged genetic material and mechanisms to fix the damage. During their life, organisms are exposed to enormous amounts of DNA damage due both to mutagenic agents and normal environmental conditions. For instance, hydrolysis causes the release of up to 10000 purine bases from the DNA of a single cell per day. Therefore, DNA Damage Response (DDR) pathways comprise a variety of mechanisms including control and arrest of cell growth, tolerance of datpaged DNA, and DNA repair pathways. In this thesis, I focused on Wemer helicase interacting protein 1 (WRNIPl), and its role in the regulation of DDR. WRNIPl contains a single UBZ domain, which is also found in a group of proteins that are involved in DNA damage tolerance. Moreover WRNIP 1 is post-translationally modified with polyubiquitin chains and polyubiquitinylation is selectively induced after exposure to UV irradiation, suggesting that WRNIPl may have a role in the DNA damage response. Despite this evidence, the specific function of WRNIP 1 in mammalian cells remains unclear. To address this point, I have used different cell based viability assays and I have verified that the level of expression of WRNIP 1 negatively correlates with cell survival upon DNA damage. Significantly, down-regulation of WRNIPl results in a faster release from cell cycle check points, which is one of the main cellular responses to DNA damage. I have also found that these phenotypes are correlated with a WRNIP1-dependent negative regulation of DNA repair. Importantly, I have demonstrated that the effect of WRNIPl on these processes is dependent on the activity of the AAA + ATPase domain. Furthermore, my results on WRNIPl ubiquitinylation revealed that lysine 274 is specifically ubiquitinylated upon UV irradiation. Importantly, lysine 274 is a key residue for the Activity of the AAA+ ATPase, indicating that the ATPase activity of WRNIPl is impaired as part of the cellular response to UV damage. Taken together, these results suggest that WRNIPl functions as negative regulator of DNA repair, and that this function is specifically inhibited through ubiquitinylation when cells face UV-induced DNA damage.

Symbiotic nitrogen fixation occurs in plants harboring nitrogen-fixing bacteria within the plant tissue. The most widely studied association is between the legumes and rhizobia. In this relationship the plant (legumes) provides the bacteria (rhizobia) with reduced carbon derived from photosynthesis in exchange for reduced atmospheric nitrogen. This allows the plant to survive in soil, which is low in available of nitrogen. Rhizobia infect and enter plant root and reside in organs known as nodules. In the nodules the bacteria fix atmospheric nitrogen. The association between the legume, Medicago truncatula and the bacteria Sinorhizobium meliloti, has been studied in detail. Medicago mutants that have defects in nodulation help us understand the process of nitrogen fixation better. One such mutant is the Mtnip-1. Mtnip-1 plants respond to S. meliloti by producing abnormal nodules in which numerous aberrant infection threads are produced, with very rare rhizobial release into host plant cells. The mutant plant Mtnip-1 has an abnormal defense-like response in root nodules as well as defects in lateral root development. Three alleles of the Mtnip/latd mutants, Mtnip-1, Mtlatd and Mtnip-3 show different degrees of severity in their phenotype. Phylogenetic analysis showed that MtNIP/LATD encodes a protein belonging to the NRT1(PTR) family of nitrate, peptide, dicarboxylate and phytohprmone transporters. Experiments with Mtnip/latd mutants demonstrats a defective nitrate response associated with low (250 μM) external nitrate concentration rather than high (5 mM) nitrate concentration. This suggests that the mutants have defective nitrate transport. To test if MtNIP/LATD was a nitrate transporter, Xenopus laevis oocytes and Arabidopsis thaliana mutant plants Atchl1-5, defective in a major nitrate transporter AtNRT1.1(CHL1), were used as surrogate expression systems. Heterologous expression of MtNIP/LATD in X. laevis oocytes and Atchl1-5 mutant plants conferred on them the ability to take up nitrate from external media with high affinity, thus demonstrating that MtNIP/LATD was a high affinity nitrate transporter. Km for MtNIP/LATD was determined to be approximately160 μM in the X. laevis system and 113 μM in the Arabidopsis Atchl1-5 mutant lines thus supporting the previous observation of MtNIP/LATD being a high affinity nitrate transporter. X. laevis expressing the mutant Mtnip-1 and Mtlatd, were unable to transport nitrate. However X. laevis oocytes, expressing the less severe mutant allele Mtnip-3 were able to transport nitrate suggesting another role of the Mtnip/latd besides high affinity nitrate transport. Experimental evidence suggested that MtNIP/LATD might transport another substrate beside nitrate. MtNIP/LATD levels are regulated by phytohormones. Experiments performed with ABA (abscisic acid), IAA (indole acetic acid) and histidine as substrates in X. laevis system show that the MtNIP/LATD mRNA injected oocytes efflux IAA but do not transport histidine or ABA. When wild type A17 and mutant Mtnip-1 and Mtnip-3 plants, grown in the presence of different sources of nitrogen were screened in herbicide chlorate, a structural analog of nitrate, the A17 and Mtnip-3 mutant showed levels of susceptibility that was different from mutant Mtnip-1 lines. Evidence suggested that the amount of chlorate transported into the plants were regulated by the C:N status of the A17 and Mtnip-3 plants. This regulation was missing in the Mtnip-1 lines thus suggesting a sensor function of MtNIP/LATD gene.

Thesis (Ph.D.)--Biomedical Engineering, Georgia Institute of Technology, 2007.
Committee Chair: Hu, Xiaoping; Committee Member: Brummer, Marijn; Committee Member: Butera, Robert; Committee Member: Oshinski, John; Committee Member: Sathian, Krish.

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

Nowadays, material, energy and information technologies are three pillar industries. The materials that have close relation with our life have also been the foundation for the development of energy and information technologies. As the new member of the material family, functional molecular materials have become increasingly important for many applications, for which the design and characterization by the theoretical modeling have played the vital role. In this thesis, three different categories of functional molecular materials, the endohedral fullerenes, the fullerene derivatives and the self-assembled monolayers (SAMs), have been studied by means of first principles methods. The non-metal endohedral fullerene N@C60 is a special endohedral fullerene that is believed to be relevant to the construction of future quantum computer. The energy landscape inside the N@C60 has been carefully explored by density functional theory (DFT) calculations. The most energy favorable potential energysurfaces (PESs) for the N atom to move within the cavity have been identified. The effect of the charging on the PESs has also been examined. It is found that the inclusion of dispersion force is essential in determining the equilibriumstructure of N@C60. Furthermore, the performance of several commonly useddensity functionals with or without dispersion correction has been verified for ten different endohedral fullerenes A@C60 with the atom A being either reactive nonmetal or nobel gases elements. It shows that the inclusion of the dispersion forcedoes provide better description for the binding energy (BE), however, none ofthem could correctly describe the energy landscape inside all the ten endohedral fullerenes exclusively. It thus calls for the further improvement of current density functionals for weak interacting systems. Soft X-ray spectroscopy is a powerful tool for studying the chemical and electronic structures of functional molecular materials. Theoretical calculations have been proven to be extremely useful for providing correct assignments for spectraof large systems. In this thesis, we have performed first principles simulations forthe near-edge X-ray absorption fine structure (NEXAFS) and X-ray photoelectron spectra (XPS) of fullerene derivatives and aminothiolates SAMs. Our calculatedspectra can accurately reproduce experimental results available for all the systemsunder investigations, and identify the species or structures that are responsible for those unexpected spectral features observed in experiments. We have suggested a modified building block (MBB) approach that allows to calculate NEXAFS spectraof a large number of fullerene derivatives with very small computational cost, and resolved the long standing puzzle around the experimental XPS and NEXAFS spectra of SAMs with aminothiolates.

QC 20120523

Group A Streptococcus is a Gram-positive human pathogen able to colonize both upper respiratory tract and skin. GAS is responsible for several acute diseases and autoimmune sequelae that account for half a million deaths worldwide every year (Cunningham et al., 2000). As other bacteria, GAS infections requires the capacity of the pathogen to adhere to host tissues and to form cell aggregates. The ability to persist in distinct host niches like the throat and the skin and to trigger infections is associated with the expression of different GAS virulence factors. GAS pili has been described as important virulence factors encoded by different FCT-operon regions. Based on this information, we decided to study the possible effect of environmental conditions that could regulate the pili expression. In this study we reported the influence of pH environment variations in biofilm formation for strains pertaining to a panel of different GAS FCT-types. The biofilm formation was promoted, excepted in the FCT-1 strains, by a changing in pH from physiological to acidic condition of growth in in vitro biofilm assay. By analyzing the possible association between biofilm formation and pH dependence, we have found that in FCT-2 and FCT-3 strains, the biofilm is promoted by pH reduction leading to an increase of pili expression. These data confirmed a direct link between pH dependent pilus expression and biofilm formation in GAS. As pili are a multi component structure we decided to investigate the functional role of one of its subunits, the AP-1 protein. AP-1 is highly conserved through the different FCT-types and suggests a possible essential role for the pili function. We focused our attention on the AP-1 protein encoded by the FCT-1 strains (M6). In particular this AP-1 protein contains the von Willebrand Factor A (VWFA) domain, which share an homology with the human VWFA domain that has been reported to be involved in adhesion process. We have demonstrated that the AP-1 protein binds to human epithelial cells by its VWFA domain, whereas the biofilm formation is mediated by the N-terminal region of AP-1 protein. Moreover, analyzing the importance of AP-1 in in vivo experiments we found a major capacity of tissue dissemination for the wild-type strain compared to the isogenic AP-1 deletion mutant. Pili have been also reported as potential vaccine candidates against Gram positive bacteria. For these reason we decided to investigate the relationship between cross reaction of sera raised against different GAS and GBS pilin subunits and the presence of a conserved Cna_B domain, in different pilin components. Our idea was to investigate if, using pilus conserved domains, a broad coverage vaccine against streptococcal infection could be possible.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2014.
Cataloged from PDF version of thesis.
Includes bibliographical references (pages 101-106).
Despite being responsible for 90% of cancer mortality, metastasis is not well understood. This thesis is focused on the circulation step of the metastatic cascade, examining three types of mobilized tumor cells: circulating tumor cells (CTCs), intraoperatively shed tumor cells, and malignant pleural effusions (MPE). We investigated the functional behavior of mobilized tumor cells in order to explain the discrepancy between the number of tumor cells in circulation and the number overt metastases. The first part of this thesis examines the functional behavior of CTCs isolated from the peripheral blood of metastatic castration-resistant prostate cancer patients. Individual CTCs were compartmentalized using arrays of nanowells to enable clonal comparison and mapping of heterogeneity. The viability, invasiveness and secretory profiles of CTCs were measured. Only a subset of CTCs was found to possess malignant traits indicative of metastatic potential. These CTCs were resistant to anoikis, were invasive or secreted proteolytic enzymes. The second part of this thesis determines the presence of intraoperatively shed tumor cells using blood samples withdrawn from the pulmonary vein after pulmonary lobectomy procedures. Previous studies did not distinguish tumor cells from normal epithelial cells specifically or sensitively. Single-cell genetic approaches were used to compare the genotype of isolated single cells to matched tumor cells and normal adjacent tissue, thereby confirming the malignancy of shed epithelial cells. The third and last part of the thesis delineates the tumorigenic population with surface markers using MPEs. A total of 35 surface antigens were screened from four categories: 1) cancer stem cell 2) epithelial-mesenchymal transition 3) metastatic signature and 4) tyrosine kinase receptors. Surface antigen CD24 was found to be specifically and abundantly expressed in MPE, and was required for the colonization of the lung. In conclusion, metastatic inefficiency is due to the presence of inactive cells and cellular heterogeneity. Inactive cells are either normal epithelial cells or apoptotic tumor cells. Cellular heterogeneity may arise from differences in surface marker expression or functional states. Therefore, only a subset of mobilized tumor cells can give rise to metastases, and therapeutic strategies should be focused on this subset.
by Xiaosai Yao.
Ph. D.

De nombreuses études ont récemment fait émerger le rôle majeur de la régulation des programmes transcriptionnels par des mécanismes épigénétique. Parmi les complexes qui entrainent des modifications post-traductionnelles des extrémités N-terminales des histones, les complexes ayant une activité histone acétyl-transférase (HAT) sont des acteurs majeurs des voies d’activation de la transcription. GCN5 fut la première protéine contenant une activité HAT à être identifiée. Des études détaillées ont permis d’établir la composition de ces deux complexes. De plus, Il a été montré que ces deux complexes contenant GCN5 (CCG) sont capables d’activer la transcription in vitro. Cependant, le rôle in vivo de ces complexes ainsi que son intégration dans les réseaux de régulation transcriptionelle est mal compris. Des anticorps de qualité ont été obtenus pour les différentes sous unités des complexes qui ont permis leur utilisation dans deux approches différentes : (i) Identification de facteurs interagissant avec les CCG. Les CCG (ATAC ou SAGA) ainsi que leurs interactants potentiels ont été purifiés. Nous avons notamment montré que ATAC interagissait dans des contextes cellulaires spécifiques avec un autre co-activateur, transcriptionnel nommé Mediateur. Puis nous avons pu montrer que ce complexe formé des deux entités ATAC-Mediateur avait un rôle important dans la régulation de la transcription certains ARN non codants. (ii) Identification des sites de fixation des CCG a l’échelle du génome. Nous avons utilisé la technique de chromatin immuno-précipitation (ChIP) couplé au séquençage direct à haut débit des fragments purifiés (ChIP-seq) pour identifier le catalogue exhaustif des sites de fixation des CCG. Grace a la comparaison des sites de fixation de ATAC avec la structure de la chromatine, nous avons pu montrer que ATAC avait un double rôle, en participant à l’activation de promoteurs proximaux mais aussi d’éléments de régulation distants (enhancers) des gènes
In higher eukaryotes, RNA Polymerase II transcription is a central cellular process allowing the spatio-temporal expression of a particular set of genes contained in a given genome. Accurately regulated transcription is a prerequisite for many key aspects of the life of an organism. Thus, transcription is tightly controlled by multiple regulatory layers. GCN5 is a member of the Histone Acetyl Transferase (HAT) family that are part of the chromatin modifier complexes. HATs deposit acetyl groups on histone tails that is believed to favour chromatin opening and positively influence transcription initiation. GCN5 is contained in two different muti-subunit macromolecular complexes named ATAC (Ada-Two-A-Containing) and SAGA (Spt-Ada-Gcn5-Acetyl-transferase) that modulate its functional specificity. While the composition and the in vitro activity of these two complexes were intensively studied, little is known about the function of these complexes in vivo. During my thesis, I aimed to better characterise the mode of action of GCN5 containing complexes (GCC) by using recent technology developments. First through the systematic analysis of the in vivo interacting partners of SAGA and ATAC by sensitive mass spectrometry, I identified a stable functional interaction between ATAC and the Mediator, another coactivator complex. I could show that this particular complex is recruited to regulate a set of ncRNA genes. Second, I analysed genome wide binding maps of ATAC produced in different cell types by chromatin immunoprecipitation coupled with high throughput sequencing (ChIP-seq I could show that ATAC binds both active promoter and active enhancer elements. Moreover, I demonstrate that while the ATAC binding at promoters is generally invariant across cell lines, the binding at enhancer is highly variable. This suggests that cell specific programs controlled by ATAC are mainly regulated at the enhancer level

Mestrado em Engenharia e Ciência dos Materiais
Pretendeu-se com o presente trabalho preparar e caracterizar dois tipos de argamassas funcionais: argamassas estruturais e argamassas decorativas obtidas a partir da incorporação de nanotubos de carbono e compostos fotocrómicos respectivamente. Quanto às argamassas estruturais (SCM), utilizaram-se nanotubos de carbono (superficialmente modificados, ou não) como material de reforço. Neste contexto foram estudados: (i) os limites de concentração, (ii) diferentes métodos de dispersão e, (iii) estratégias preparativas que minimizam o problema da hidrofobicidade dos nanotubos quando adicionados à argamassa. Os materiais foram caracterizados por testes de solubilidade, resistência à compressão, resistência à tracção, resistência a flexão, módulo de elasticidade, capilaridade e microscopia electrónica de varrimento (SEM). Os resultados mais promissores foram obtidos após modificação superficial dos CNT, ou recorrendo à dispersão numa suspensão de TiO2. As propriedades mecânicas das argamassas resultantes foram superiores à amostra padrão no mas as suas propriedades físicas foram mantidas similares. Tendo em conta a natureza aplicada deste projecto foi ainda feita uma breve avaliação económica sobre a utilização de CNT na indústria de argamassas. Essa avaliação apontou para a necessidade de novos estudos semelhantes ao aqui apresentado afim de minimizar as quantidades de CNTs a usar. Em relação às argamassas decorativas (DCM) o uso de compostos fotocrómicos foi explorado. Nesse sentido, estudou-se o efeito da adição de: (i) haletos de prata, (ii) lentes fotocrómicas moídas, (iii) trióxido de tunsténio em pó e, (iv) compostos de trióxido de tungsténio e óxido de titânio. Os materiais foram caracterizados por colorimetria. Foram obtidos resultados positivos para algumas das amostras estudadas nomeadamente as que envolveram o uso de trióxido de tungsténio e compostos de trióxido de tungsténio e óxido de titânio, as quais após exposição à luz solar revelaram uma súbtil variação na tonalidade ou indício de alteração da cor. Por fim foi ainda estudado o efeito da adição de CNT a argamassas decorativas no sentido de avaliar a possibilidade de utilizar CNTs com vista a uma melhoria das propriedades mecânicas de DCM. Tal como no caso das SCM a modificação superficial dos CNT revelou-se necessária. Deste modo obteve-se um conjunto de materiais com propriedades inovadoras, nomeadamente ópticas e estruturais, podendo dar origem a argamassas com possíveis mudanças de cor quando expostas à luz solar e argamassas com elevada resistência mecânica respectivamente.
The present work aims at developing two different types of functional mortars: (a) Structural cement mortars (SCM) and (b) decorative mortars (DCM). In the case of SCM, multiwalled carbon nanotubes (MWCNT) were used for reinforcement and the effect of different sample preparation variables on the mechanical properties of the ensuing composites was studied. This include: (i) concentration of MWCNT, (ii) surface modification of MWCNT and (iii) the dispersion method. The materials were characterised by solubility tests, bend resistance, strength resistance, and compressive strength, modulus of elasticity, capillarity and Scanning Electron Microscopy (SEM). The most promising results were obtained using surface modified CNTs or dispersing them in a TiO2 suspension. The ensuing cement mortars had improved properties when compared to the standard cement mortar and the physical properties were similar. In view of the applied research nature of this project a brief economical analysis was carried out. Such analysis points that further studies are required in order to minimise the amount of CNT to be used. As regards DCM the addition of photochromic materials such as silver halides, tungsten oxide and of a tungsten oxide + titanium oxide composite was explored in order to yield a light responsive material which could change its colour or at least shade depending on the intensity of light. The materials obtained were characterised by colorimetry. The results obtained for samples prepared using tungsten trioxide and composites of tungsten trioxide and titanium oxide were encouraging. Upon sunlight exposure either a subtle colour shade or colour change was observed. Finally, addition of CNT to DCM was also studied aiming at increasing the mechanical properties of the decorative mortar. As in the case of SCM surface modification of CNT proved to be required.

Smith-Magenis Syndrome (SMS; OMIM #182290) is a multiple congenital abnormality and intellectual disability (ID) disorder caused by either an interstitial deletion of the 17p11.2 region containing the retinoic acid induced-1 (RAI1) gene or a mutation of the RAI1 gene. Individuals diagnosed with SMS typically present characteristics such as ID, self-injurious behavior, sleep disturbance, ocular and otolaryngological abnormalities, craniofacial and skeletal abnormalities, neurological and behavioral abnormalities, as well as other systemic defects and manifestations. Previous work by Vyas in 2009 showed temporal expression of rai1 in zebrafish embryos as early as 9 hpf. We hypothesize that there is maternal rai1 expression as early as zero hours post fertilization in wild type embryos. Using end-point PCR, we found that in fact there is maternal rai1 expression is detectable as early as 2 hours post fertilization (hpf) in wild type zebrafish embryos. Furthermore, we quantified rai1 expression using qPCR and found that rai1 expression declines significantly after 6 hpf. We hypothesize that a down regulation of rai1 or loss of rai1 will lead to morphological phenotypes, especially if that loss of rai1 function occurs during the earliest stages of zebrafish embryogenesis. Using a rai1morpholino oligonucleotide (MO), we found a loss of rai1 expression did not induce a morphological phenotype in in wild type embryos; furthermore, we also found that a loss of maternal rai1 expression did not induce a morphological phenotype as well. Utilizing a mutant rai1 zebrafish line, we found that both rai1 +/fh370 progeny nor rai1 fh370/fh370 progeny exhibited a morphological phenotype and that downstream targets such as bdnf were not affected by a reduction or complete loss of rai1. Prior research has shown that retinoic acid (RA) can induce rai1 expression. We hypothesize that RA can induce expression of rai1 during zebrafish embryogenesis. Using wild type fish and a rai1 in situ hybridization probe, we found that RA treatment at 25 hpf induced expression of rai1. The construction of a rai1 overexpression vector used for overexpression studies was started. Further development of GFP expression vector and zebrafish rai1 antibody are needed to determine if the morpholino is reducing rai1 protein expression.

CB1 cannabinoid receptors are G-protein-coupled receptors that mediate the central nervous system (CNS) effects of marijuana and endocannabinoids. Recently, cannabinoid receptor interacting protein 1a (CRIP1a) was discovered as a novel protein that binds to the CB1 receptor C-terminus and inhibits CB1 receptor activity without affecting CB1 expression. This thesis investigated the functions of CRIP1a by characterizing the first CRIP1a knockout (KO) mouse line. The absence of CRIP1a was confirmed in KO mice using quantitative PCR and immunoblotting. I hypothesized that CRIP1a KO mice would exhibit enhanced CB1 receptor-mediated G-protein activity in the CNS, as well as cannabimemetic phenotypes and enhanced sensitivity to cannabinoid agonists in vivo. Results showed increased CB1 agonist-stimulated G-protein activity in the amygdala of CRIP1a KO relative to WT mice, but not in cerebellum, hippocampus or spinal cord. CB1 receptor levels did not differ between genotypes in in any region examined. Interestingly, CRIP1a KO mice exhibited an anxiolytic-like phenotype and decreased nociceptive sensitivity in vivo, but did not differ from WT mice in tests of motor activity or coordination. Surprisingly, sensitivity to agonist-induced antinociception, hypothermia, catalepsy or motor incoordination did not differ between genotypes. Our findings suggest that CRIP1a could play a selective role in modulation of anxiety by endocannabinoids, and this action could be mediated through the amygdala. Thus, CRIP1a might serve as a future pharmacological target for studying and treating anxiety disorders.

Signaling scaffolds are critical for the correct spatial organization of enzymes within the ERK1/2 signaling pathway and proper transmission of intracellular information. However, mechanisms that control molecular dynamics within scaffolding complexes, as well as biological activities regulated by the specific assemblies, remain unclear. The scaffold protein Shoc2 is critical for transmission of the ERK1/2 pathway signals. Shoc2 accelerates ERK1/2 signaling by integrating Ras and RAF-1 enzymes into a multi-protein complex. Germ-line mutations in shoc2 cause Noonan-like RASopathy, a disorder with a wide spectrum of developmental deficiencies. However, the physiological role of Shoc2, the nature of ERK1/2 signals transduced through this complex or mechanisms regulating the function of Shoc2 remain largely unknown. My dissertation addresses the mechanisms by which Shoc2 accelerates ERK1/2 signal transmission and the biological outputs of the Shoc2-guided signals. To delineate Shoc2-mediated ERK1/2 signals, I have utilized a vertebrate zebrafish model. I demonstrated that loss of Shoc2 protein expression leads to early embryonic lethality resulting from a significant reduction in the number of circulating erythropoietic and myelopoietic blood cells, underdeveloped neurocranial and pharyngeal cartilages, and a profound delay in calcification of bone structures. Together, this data demonstrates that the Shoc2 scaffolding module transmits ERK1/2 signals in neural crest development and blood cell differentiation. This dissertation also addresses the mechanistic basis of how allosteric ubiquitination of Shoc2 and RAF-1 is controlled. I have characterized a molecular interaction of Shoc2 with its previously unknown binding partner Valosin-Containing Protein (VCP/p97). These studies demonstrated that hexametric ATPase VCP modulates ubiquitination of Shoc2 and RAF-1 through the remodeling of the scaffolding complex in a spatial-restricted manner. Experiments utilizing fluorescence microscopy and biochemical methods show that VCP/p97 sequesters the E3 ligase HUWE1 from the Shoc2 module, thereby altering the ubiquitination of Shoc2 and RAF-1 as well as the amplitude of ERK1/2 signals. These studies also show that the levels of Shoc2 ubiquitination and ERK1/2 phosphorylation are imbalanced in fibroblasts isolated from Inclusion Body Myopathy with Paget’s disease of bone and Frontotemporal Dementia (IBMPFD) patients harboring VCP germline mutations. This data also suggests that ERK1/2 pathway deregulation is part of IBMPFD pathogenesis. In summary, these studies make a significant advance in our understanding of the mechanisms by which the Shoc2 scaffold regulates specificity and the dynamics of the ERK1/2 signaling networks. They also make important insights into our understanding of biological activities and targets of Shoc2-mediated ERK1/2 signals at the early stages of embryonic development and disease.

Background: Colorectal cancer is now one of the most common causes of death in Australia, with an estimated 1486 new cases in the country in 2010, accounting 12.7% of all cancer deaths (ACIM, 2014). In addition to its significance in Australia, it is one of the most common global health concerns. At present colorectal cancer is the third most common cancer worldwide, which cost more than 600,000 lives every year. Most of the colorectal cancer is diagnosed at a late stage but if it is diagnosed at an early stage, the five-year survival rate exceeds in 90% cases. This is the reason there is a need to find out biomarker for early detection and the exact underlying cause for designing a better treatment for colorectal cancer. GAEC1 (Gene amplified in esophageal cancer 1) showed a series of amplifications and deletions in oesophageal cancer. The gene is located at 7q22.1. GAEC1 has tumorigenic potential approximately equal to the Ras gene family and overexpression of this gene played a pivotal role in the cancer transformation of oesophageal squamous cell carcinoma. GAEC1 has higher amplification in colorectal adenocarcinoma tissues when compared to non-cancer colorectal tissues. In this study, we focused on finding out the oncogenic properties of GAEC1, correlation with clinical and pathological features and its underlying mechanism in colorectal cancer initiation and progression. Materials and method: Human colon cancer cell lines (SW480, SW48, HCT116 cells) and non-neoplastic colonic epithelium cell (FHC cells) were purchased from American Type Culture Collection (ATCC). SW480, SW48 and HCT116 cell lines were maintained in Dulbecco's Modified Eagle Medium (DMEM) (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum at 37 ℃ in 5% CO2. FHC cells were maintained in DMEM: F-12 (1:1) with 10% fetal bovine serum with containing an extra 10 mM N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES) (Thermo Fisher Scientific) (for a final concentration of 25 mM), 10 ng/ml cholera toxin, 0.005 mg/ml insulin, 0.005 mg/ml transferrin, 100 ng/ml hydrocortisone. Fresh frozen human colorectal cancer tissues and adjacent non-cancer tissues were collected with no selection bias. Expression levels of mRNA and protein were measured by real-time PCR and western blot analysis respectively. Immunocytochemistry, immunohistochemistry and immunofluorescence assay were used to identify the localization of GAEC1 protein in colon cancer cells and colon cancer tissues. Flow cytometry was used for the detection of apoptotic cells and cell cycle alteration. Co-immunoprecipitation followed by mass spectrometry analysis was used to identify the protein-protein interaction. Severe combined immunodeficiency (SCID) mice were used for tumour xenograft experiment. Results: We found differential expression of GAEC1 protein and mRNA in different pathological stages of colon cancer cells (SW480-Stage II, SW48-Stage III and HCT116-Stage IV) when compared to non-neoplastic colon cells (FHC cells). GAEC1 protein was predominantly expressed in the cytoplasm of colon cancer cells (SW480, SW48, and HCT116) and the nucleus of non-neoplastic colon epithelial cells (FHC). The transient knockdown of GAEC1 using siRNA induced apoptosis in SW480 and SW48 cells, which was associated with G2/M phase arrest and decreased expression of Bcl-2 and K-ras proteins and increased expression of p53. In addition, down-regulation of GAEC1 significantly inhibited cell proliferation, reduced migration capacity and decreased clonogenic potentiality of colon cancer cells (SW480 and SW48 cells). Furthermore, a xenotransplantation model showed that stable knockdown of GAEC1 using shRNA constructs in colon cancer cells entirely suppressed xenograft tumour growth in mice. Approximately 52.5% of patients with colorectal cancers showed high expression of GAEC1 mRNA whereas 47.5% exhibited low expression compared to their matched non-neoplastic tissues. Similarly, ~ 66% (53/80) of colorectal cancer tissues showed high GAEC1 protein expression (positive staining), while the remaining colorectal cancer cases were noted with no GAEC1 protein (negative) expression. GAEC1 protein was predominantly located in the cytoplasm and showed low to no expression in normal colon tissues. High expression of GAEC1 mRNA was predominantly seen among patients below 60 years compared to those patients over 60 years of age (78%, versus 44%, p=0.008). Patients with synchronous colorectal adenocarcinomas mostly exhibited with low expression of GAEC1 mRNA. On the other hand, compared to poorly differentiated colorectal carcinomas (grade III), patients with well and moderately differentiated colorectal carcinomas (grade I+II) colorectal cancers showed a high expression of GAEC1 mRNA. Similarly, high GAEC1 mRNA expression was frequently noted among patients presented without any pre-neoplastic adenomas in their colorectal cancer tissues compared to patients with an adenoma in their colorectal cancer tissues. By co-immunoprecipitation followed by mass spectrometry analysis 31 interacting protein was identified. The interaction between GAEC1 and four proteins (HIGD1A, Rhotekin, Granulin and eIF3J) was further confirmed. Western blot analysis detected reduced expression of these proteins following stable knockdown of GAEC1 in colon cancer cells. GEAC1 endogenously interacts with p53 in SW480 and SW48 colon cancer cells. In this study, we have noted that overexpression of GAEC1 increased cell proliferation, migration, and reduced apoptosis in colon cancer cells. Also, these cells showed cell cycle arrest at the synthetic phase, activation of Bcl-2, K-ras, pAKT proteins as well as inhibition of p53, PUMA, p21 and BAX proteins. Furthermore, silencing of GAEC1 reduces the nuclear import of MDM2 and increase the expression of p53 in the nucleus suggesting that GAEC1 expression is essential for interaction of p53-MDM2 and nuclear translocation of MDM2 in colon cancer cells. Conclusion: In summary, the expression analysis, in vitro and in vivo data indicated that GAEC1 is differentially expressed in cancer cells and act as an oncogene in colon cancer progression. The high expression of GAEC1 mRNA/protein, as well as its correlation with multiple clinical and pathological characteristics in patients with colorectal carcinoma, strongly, suggests that GAEC1 is a key regulator in the initiation of colorectal carcinogenesis. In addition, the protein-protein interaction with a number of proteins and the effect of GAEC1 modulation on the expression of interacting proteins indicates the potential role of GAEC1 in the signalling pathway of colon cancer pathogenesis.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Medicine
Griffith Health
Full Text

Single amino acid variants (SAVs) are one of the main causes of Mendelian disorders, and play an important role in the development of many complex diseases. At the same time, they are the most common kind of variation affecting coding DNA, without generally presenting any damaging effect. With the advent of next generation sequencing technologies, the detection of these variants in patients and the general population is easier than ever, but the characterization of the functional effects of each variant remains an open challenge. It is our objective in this work to tackle this problem by developing machine learning based in silico SAVs pathology predictors. Having the PMut classic predictor as a starting point, we have rethought the entire supervised learning pipeline, elaborating new training sets, features and classifiers. PMut2017 is the first result of these efforts, a new general-purpose predictor based on SwissVar and trained on 12 different conservation scores. Its performance, evaluated bothby cross-validation and different blind tests, was in line with the best predictors published to date. Continuing our efforts in search for more accurate predictors, especially for those cases were general predictors tend to fail, we developed PMut-S, a suite of 215 protein-specific predictors. Similar to PMut in nature, Pmut-S introduced the use of co-evolution conservation features and balanced training sets, and showed improved performance, specially for those proteins that were more commonly misclassified by PMut. Comparing PMut-S to other specific predictors we proved that it is possible to train specific predictors using a unique automated pipeline and match the results of most gene specific predictors released to date. The implementation of the machine learning pipeline of both PMut and PMut-S was released as an open source Python module: PyMut, which bundles functions implementing the features computation and selection, classifier training and evaluation, plots drawing, among others. Their predictions were also made available in a rich web portal, which includes a precomputed repository with analyses of more than 700 million variants on over 100,000 human proteins, together with relevant contextual information such as 3D visualizationsof protein structures, links to databases, functional annotations, and more.
Les mutacions puntuals d’aminoàcids són la principal causa de moltes malalties mendelianes, i juguen un paper important en el desenvolupament de moltes malalties complexes. Alhora, són el tipus de variant més comuna que afecta l’ADN codificant de proteïnes, sense provocar, en general, cap efecte advers. Amb l’adveniment de la seqüenciació de nova generació, la detecció d’aquestes variants en pacients i en la població general és més fàcil que mai, però la caracterització dels efectes funcionals de cada variant segueix sent un repte. El nostre objectiu en aquest treball és abordar aquest problema desenvolupant predictors de patologia in silico basats en l’aprenentatge automàtic. Prenent el predictor clàssic PMut com a punt de partida, hem repensat tot el procés d’aprenentatge supervisat, elaborant nous conjunts d’entrenament, descriptors i classificadors. PMut2017 és el primer resultat d’aquests esforços, un nou predictor basat en SwissVar i entrenat amb 12 mètriques de conservació de seqüència. La seva precisió, mesurada mitjançant validació creuada i amb tests cecs, s’ha mostrar en línia amb els millors predictors publicats a dia d’avui. Continuant els nostres esforços en la cerca de predictors més acurats, hem desenvolupat PMut-S, un conjunt de 215 predictors específics per cada proteïna. Similar a PMut en la seva concepció, PMut-S introdueix l’ús de descriptors basats en la coevolució i conjunts d’entrenament balancejats, millorant el rendiment de PMut2017 en 0.1 punts del coeficient de correlació de Matthews. Comparant PMut-S a d’altres predictors específics hem provat que és possible entrenar predictors específics seguint un únic procediment automatitzat i assolir uns resultats tan bon com els de la majoria de predictors específics publicats. La implementació del procediment d’aprenentatge automàtic tant de PMut com de PMut-S ha sigut publicat com a un mòdul de Python de codi obert: PyMut, el qual inclou les funcions que implementen el càlcul dels descriptors i la seva selecció, l’entrenament i avaluació dels classificadors, el dibuix de diverses gràfiques... Les prediccions també estan disponibles en un portal web que inclou un repositori precalculat amb els anàlisis de més de 700 milions de variants en més de 100 mil proteïnes humanes, junt a rellevant informació de context com visualitzacions 3D de les proteïnes, enllaços a bases de dades, anotacions funcionals i molt més.

The focus of this work was to functionally characterize the CD300e receptor expressed in human monocytes and myeloid dendritic cells and investigate the implications that receptor engagement has on their biology. We provide evidence formally supporting that CD300e functions as an activating receptor capable of regulating the innate immune response by triggering various pro- inflammatory functions including intracellular calcium mobilization, superoxide anion production, pro-inflammatory cytokine release and up-regulation of co-stimulatory molecules in myeloid cells. We also report that ligation of CD300e on the surface of monocytes results in their differentiation to functional MΦ2-like macrophages by an autocrine mechanism that involves M-CSF and its receptor (CD115).
L'objectiu d'aquest treball ha estat caracteritzar funcionalment el receptor CD300e expressat en monòcits i cèl·lules dendrítiques mieloides humanes, així com investigar les implicacions que l'activació d'aquest receptor pot tenir en la seva biologia. Demostrem formalment que el receptor CD300e funciona com un receptor activador capaç de regular la resposta immune innata activant diverses funcions proinflamatòries, incloent la mobilització de calci intracel·lular, la producció d'anió superòxid, la secreció de citocines proinflamatòries i la inducció de molècules coestimuladores en cèl·lules mieloides. També descrivim que l'activació del receptor CD300e a la superfície dels monòcits provoca la seva diferenciació cap a macròfags funcionals del tipus MΦ2 gràcies a un mecanisme autocrí que funciona a través del M-CSF i el seu receptor (CD115).

The JAK/STAT pathway is a non-receptor tyrosine kinase signaling pathway that is well conserved and highly re-utilized in many mammalian and Drosophila developmental processes. Compared to dozens of ligands and receptors in mammalian JAK/STAT, Drosophila JAK/STAT pathway is simpler with one receptor and three ligands, Upd, Upd2 and Upd3, which have similar amino acid sequences. Previous literature shows that upd and upd2 exhibit the same dynamic striped expression pattern in embryos and have semi-redundant functions during embryogenesis. Do Upd and Upd3 also have redundant functions? To answer this question, the functions of Upd3 in Drosophila development were investigated in this dissertation. In addition, the coordinate expression mechanism of upd and upd3 in eye discs was also analyzed. To study the functions of Upd3 in development, the expression pattern of upd3 was examined and detected in larval eye discs, wing discs, haltere discs, lymph glands and adult ovaries with in situ hybridization to upd3 mRNA and an upd3 reporter line. Consistent with the expression pattern, the loss of function mutants of upd3 exhibit small eyes, outstretched wings, downward extended halteres and reduced circulating blood cell concentration, demonstrating the roles of Upd3 in these tissues’ development. However, functions of Upd3 in other aspects of immune response were not detected. To investigate the mechanism of the coordinate expression of upd and upd3, the genetic and molecular relationship of upd, upd3 and os was dissected. The os alleles, oso, oss and os1, are a group of classical alleles which display outstretched wings, small eyes, or both, respectively. The genetic complementation tests of upd, upd3 and os showed that both upd and upd3 failed to complement os while upd complemented upd3, suggesting functions of both upd and upd3 are affected in os alleles. Consistent with the genetic tests, the expression of upd and upd3 in eye discs is lost in os allele. Molecularly, putative enhancer regions are deleted at the 5’ end of upd3 in os alleles. Hence, a transcriptional co-regulation model of upd and upd3 is proposed in which upd and upd3 share a common cis-regulatory region, lesions of which cause the os phenotype.

The ring opening polymerization of D,L-lactide (DLLA) using multifunctional hydroxyl-terminated initiators and catalyst/coinitiator systems based on Sn(Oct)2 afforded the preparation of star-shaped, poly(D,L-lactide)s (PDLLA)s of controlled molar mass, narrow molar mass distributions, and well-defined chain end functionality. Various modifications of star-shaped PDLLA resulted in macromolecules with tailored functionalities for biomedical applications. Star-shaped PDLLAs were modified to contain photoreactive methacrylate end groups and subsequent photo-crosslinking was performed. Photo-crosslinked networks based on methacrylated star-shaped PDLLAs exhibited thermal properties and mechanical performance that were superior to current approved clinical adhesives. In addition, the thermal and mechanical properties of the networks were strongly dependent on the composition and molar mass of the star-shaped PDLLA precursors. Tensile strengths in the range of 8-21 MPa were obtained while the Youngâ s modulus increased from 12 to 354 MPa and were higher for networks based on urethane containing polymers. Star-shaped PDLLAs bearing complementary adenine and thymine terminal units were also prepared. The hydrogen bonding associations between complementary PDLLA macromolecules depended strongly on molar mass and hence, the concentration of multiple hydrogen bonding units. 1H NMR spectroscopy confirmed the formation of hydrogen-bonded complexes with a 1:1 optimal stoichiometry and an association constant of 84 M-1. The hydrogen-bonded complexes also exhibited significantly higher solution viscosities than non-blended polymer solutions of similar molar mass and concentration. Thermoreversible associations of PDLLA-based complementary polymers were observed in the melt phase and the melt viscosity of a blended complex was consistently an order of magnitude higher than non-functionalized star-shaped PDLLA of similar molar mass. Furthermore, melt electrospinning of the hydrogen-bonded complexes successfully resulted in fibers of significantly larger diameter (9.8 ± 2.0 µm) compared to the individual precursors (PDLLA-A = 4.0 ± 0.6 µm and PDLLA-T = 4.4 ± 1.0 µm). These results suggested that thermoreversibility, as well as the strength of the hydrogen bonding interactions between the end groups of the tailored star-shaped PDLLA-based supramolecular polymers controlled the fiber diameter in the melt electrospinning process. Highly ordered microporous honeycomb structures were developed on photo-functional star-shaped PDLLA surfaces. The pore dimensions were dependent on polymer solution concentration, polymer molar mass and relative humidity. The combination of self-organizing and cross-linking techniques resulted in free-standing, PDLLA membranes with high chemical stability as well as higher mechanical strength for further material patterning. Amikacin, an antibiotic commonly used for treating infections was successfully encapsulated in star-shaped PDLLA fibers that were electrospun from solution. Preliminary results suggested that molecular architecture influenced the encapsulation of the antibiotic and subsequent drug release profile.
Ph. D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Materials Science and Engineering, May, 2020
Cataloged from the official PDF of thesis.
Includes bibliographical references (pages 161-179).
In solutions, the dynamic behavior of soft materials is often critical to their function. In biological materials such as proteins and peptides, the edict that 'structure dictates function' has been supplanted in recent decades by recognition that features like intrinsic disorder, conformational distribution, and solvent dynamics often play a part which is equally fundamental to the binding and reactivity of these materials. The same revelation holds for many other functional soft materials, including abiotic peptides and self-assembling materials, where function is controlled by the dynamic behavior of both the compound and the substrate. In this work, I elucidate the role of dynamics in several significant functional polyamides by the synthesis and characterization of samples spin-labeled for electron paramagnetic resonance (EPR) spectroscopy. By this approach, I developed insight into several soft-materials systems, including abiotic peptide tags, combinatorially selected for bioconjugation; fibronectin mimetic peptides, designed for therapeutic purposes, biomaterials and drug delivery; and finally, novel, self-assembling polyamide materials designed for water purification and energy conservation.
by William Robin Lindemann.
Ph. D.
Ph.D. Massachusetts Institute of Technology, Department of Materials Science and Engineering

Pour s’adapter à leur environnement, les plantes ont acquis la capacité de produire une grande quantité de métabolites spécialisés à partir d’un nombre limité de structures de base. Parmi les modifications apportées à ces structures de base, la réaction de glycosylation permet d’augmenter la solubilité du composé, de réduire sa toxicité et de contribuer à une meilleure stabilité de certaines molécules ayant pour conséquence la modification de leur transport et/ou de leur stockage. Les UDP-glycosyltransférases (UGT) forment une vaste famille de glycosyltransférases chez les plantes. Elles regroupent des enzymes glycosylant principalement des hormones et des phénylpropanoides en utilisant l’UDP-sucre comme donneur de sucre. L’objectif de ce travail consiste à contribuer à la caractérisation fonctionnelle de la famille des UGT72 chez le peuplier afin d’identifier le rôle de ses membres dans les processus développementaux liés aux arbres et dans leurs réponses au stress. Plusieurs membres de cette famille ont déjà été caractérisés chez d’autres espèces comme A. thaliana, M. truncatula et C. sinensis, mettant en évidence la capacité de certaines UGT72s à glycosyler les monolignols, une implication dans le processus de lignification, un rôle dans des processus de défense contre les pathogènes ou encore une fonction de détoxification de certains polluants. Parmi les 8 UGT72s identifiées chez le peuplier, nous avons montré qu’in vitro UGT72AZ2 glycosyle l’acide férulique et l’acide sinapique, UGT72B37 le p-coumaraldéhyde, le coniféraldéhyde, le sinapaldéhyde, le coniferyl alcool et le sinapyl alcool, UGT72B39 le coniféryl alcool et UGT72A2 la naringénine. Tous les membres de la famille UGT72 sont exprimés dans les tissus vasculaires, suggérant un rôle dans le développement vasculaire. La surexpression de UGT72AZ1 ou UGT72AZ2 provoque l’accumulation de glucosides de monolignols (respectivement coniférine et syringine ou coniférine seulement), sans toutefois affecter la quantité totale de lignine. Concernant la localisation subcellulaire, excepté pour UGT72A2, les UGT72s du peuplier sont localisées dans le réticulum endoplasmique et le noyau, suggérant respectivement, un rôle dans la régulation de la voie des phénylpropanoides et dans la maintenance de l’ADN. UGT72A2 se démarque des autres membres de cette famille, car elle est localisée dans les chloroplastes et dans des vésicules associées aux chloroplastes, suggérant un rôle dans la régulation des phénylpropanoides dans le chloroplaste et/ou dans la maintenance du chloroplaste. En appui de ces hypothèses, nous avons constaté que la photosynthèse est affectée dans les lignées sous-exprimant UGT72A2, provoquant un jaunissement des feuilles. De plus, les feuilles de lignées sous-exprimant UGT72A2 développent un stress oxydatif associé à une réduction de l’accumulation des flavonoïdes et de l’activité des enzymes antioxydantes, suggérant un rôle de UGT72A2 dans l’homéostasie des formes réactives de l’oxygène (ROS).
In order to adapt to their environment, plants have developed the capacity to produce a diversified range of specialized metabolites by modifying a core set of molecules. Among those modifications, glycosylation allows to increase the solubility, to reduce the toxicity and to stabilize compounds in order to modify their transport and/or allow their storage. The UDP-glycosyltransferases (UGT) forming the largest glycosyltransferase superfamily in plants, combine enzymes which glycosylate mainly hormones and phenylpropanoids by using UDP-sugar as sugar donor. The purpose of this dissertation is to contribute to the functional characterization of the UGT72 family in poplar to unravel the role of its members in tree developmental processes and in stress response. Members of this family already characterized in other species (e.g. Arabidopsis thaliana, Medicago truncatula and Camellia sinensis) have been found to glycosylate monolignols and some of them have been associated with lignification, defence against pathogens and detoxification of pollutants. Among the 8 UGT72s identified in poplar, we have shown that UGT72AZ2 glycosylates in vitro ferulic acid and sinapic acid, UGT72B37 p-coumaraldehyde, coniferaldehyde, sinapaldehyde, coniferyl alcohol and sinapyl alcohol, UGT72B39 coniferyl alcohol and UGT72A2 naringenin. All the UGT72 members are expressed in vascular tissues suggesting a role in vascular development. The overexpression of UGT72AZ1 or UGT72AZ2 in poplar triggers the accumulation of monolignol glucosides (both coniferin and syringin or only coniferin, respectively) but has no impact on lignin content. With respect to the subcellular localization, except for UGT72A2, poplar UGT72s are localized in the endoplasmic reticulum and in the nucleus suggesting a possible role in the phenylpropanoid pathway regulation and in DNA maintenance, respectively. UGT72A2 stands out from the other poplar UGT72s by being localized in the chloroplast and chloroplast associated bodies, suggesting a role in the phenylpropanoid regulation in chloroplasts and/or in chloroplast maintenance. Moreover, supporting these hypotheses, photosynthesis was affected in lines downregulated for UGT72A2, as shown by a leaf yellowing phenotype and an oxidative stress in these lines as compared to the wild type. The flavonoid biosynthesis and the activity of enzymes involved into the reactive oxygen species (ROS) scavenging seem to be reduced by the downregulation of UGT72A2 suggesting a role of this UGT in the ROS homeostasis.
Doctorat en Sciences
info:eu-repo/semantics/nonPublished

Sortases have been known to be essential in Gram-positive bacteria for attaching proteins onto the peptidoglycan layer of the bacterium. Sortase A has been found to be useful as a “molecular stapler”, although; in vivo, the enzyme is responsible for attaching proteins to the peptidoglycan layer of Gram-positive bacteria. It accomplishes both of these tasks by joining two proteins together via an LPXTG sorting sequence. The enzyme has been proven to be very useful in attaching any two proteins together without worrying about recombinant techniques to generate the fusion protein. The problem with this enzyme is that the catalytic diad, which is composed of Cys-184 and His-120, has to be in a certain form that exists .2% of the time at pH 7.0. There is also a hydrolytic shunt that the enzyme can undergo instead of the productive transpeptidase reaction. These issues lead to groups attempting to place S.aureus SrtA through directed evolution in order to increase the catalytic efficiency of the enzyme. Although mutants have been generated that increase the catalytic efficiency 13-fold and 130-fold, the structural basis behind this increase is poorly understood. Using crystallography, we will attempt to discover the structural basis behind the rate enhancement as well as understand more about different species of SrtA. We also will attempt to kinetically characterize the S.aureus SrtA enzyme, its mutants, and different strains of SrtA. Thus far G.moribillorum SrtA has been crystallized and its structure shows that there is a distinction in the β6/β7 loop which has been implied to be important to catalysis. Furthermore, the pentaglycine kinetics shone some light on how the different mutants interact with the pentaglycine substrate of S.aureus SrtA.

The comprehensive identification of genetic alterations is critical to understanding the pathophysiology of cancer. Recent advances in sequencing technology have enabled the detailed description of cancer genomes. However, to translate these findings into a deeper understanding of cancer biology, analyzing the functional impact of cancer-associated genetic aberration is essential. Here I investigate how to accelerate the functional characterization of two classes of genetic alterations, point mutations and amplifications. The wide spectrum of point mutations that arise in cancer makes them challenging to study comprehensively. I have developed a scalable systematic method to experimentally infer the functional impact of cancer-associated gene variants. I performed pooled in vivo tumor formation assays and gene expression profiling using 474 mutant alleles curated from 5,338 human tumors. I identified 12 transforming alleles including two in genes (PIK3CB, POT1) that have not been previously shown to be tumorigenic. One rare KRAS allele, D33E, displayed tumorigenicity and constitutive activation of RAS effector pathways. By correlating gene expression changes induced upon expression of wild type and mutant alleles, I could infer the activity of specific alleles. These approaches enable the interrogation of cancer-associated alleles at scale and demonstrate that rare alleles may be functionally important. Frequently amplified regions in cancer often harbor oncogenic drivers. However, identifying the driver gene among many other amplified genes is challenging. In high-grade serous ovarian cancer (HGSOC), 1,825 genes are amplified across 63 amplicons. We employed systematic loss-of-function RNAi data to identify amplified genes that were essential in the ovarian lineage. We identified 50 amplified and essential genes and validated FRS2, an adaptor protein in FGFR pathway. FRS2-amplified cancer cell lines were dependent on FRS2 expression and FRS2 overexpression in immortalized cell lines was sufficient to promote anchorage independent growth and tumorigenesis in nude mice. This approach demonstrates that intersecting structural genomics with functional genomics can facilitate the discovery of driver genes in recurrently amplified regions. Collectively, the methods I present here provide a framework to study point mutations and amplifications to accelerate the interpretation of the cancer genome.
Medical Sciences

Dissertation (Ph.D.)--Worcester Polytechnic Institute.
Keywords: electrodeposition; chemical vapor deposition; AAO template-assisted nanofabrication; 1 D nanomateirals; inorganic nanomaterials; nanostructured electrode. Includes bibliographical references (leaves 102-103).

Pectin was isolated by aqueous extraction at pH 6.0 or 2.0 from okra (Abelmoschus esculentus L.) pods. An isolation protocol was designed to extract pectin and study the influence of the extraction pH on its chemical composition, macromolecular and functional properties. The extraction protocols resulted in the isolation of pectin of high purity as evidenced by their high total carbohydrate (70.0 – 82%) and low protein (4.3 – 6.3%) contents. Samples contained between 47-57% galacturonic acid, had broad molecular weight distributions, a low degree of methylation (40 and 25 %) and high degree of acetylation (52 and 38 %). Neutral sugar analysis showed that pectin extracted at pH 6.0 contained more neutral sugars, particularly, galactose, rhamnose and arabinose than that extracted at pH 2.0 indicating variations in fine structure. In addition, molecular parameters of the isolated pectins, such as intrinsic viscosity (2.8 – 4.4 dL g-1), critical concentration (0.15 – 0.45 dL g-1) and coil overlap parameter (0.66 –1.51), showed that extraction conditions resulted in pectin with different chain macromolecular characteristics. Following extraction, the functional properties of okra pectin were investigated in high and low moisture systems and also in colloidal dispersions. It has been shown that okra polysaccharides are non-gelling pectins and their inability to form ordered structures was attributed to the high degree of acetylation and branching of the side-chains. The pH sensitivity of okra pectins has been further demonstrated in high solid systems, where the mechanical relaxation of LM-pectin in the presence of co-solute has been altered by pH. It has been shown that high pH values result in extended chain conformation and early vitrification events. In contrast, viscoelastic functions of polyelectrolyte decreased and resulted in delayed vitrification events at low pH. The next step of present work was focused on potential utilization of okra polysaccharides in fabrication of oil-in-water emulsions for food and pharmaceutical applications. The emulsifying properties of crude okra extracts and okra isolates (rich in pectin) have been investigated under different conditions (e.g., oil volume fraction, biopolymer concentration, pH values, energy input methods) in order to produce fine emulsions with long-term stability. It has been shown that pH of extraction has a pronounced effect on the interfacial activity of both crude extract and pectin isolates. Extracts or isolates obtained at high pH demonstrated higher emulsifying capacity than those extracted at low pH. In general, okra pectin isolates were more efficient in emulsion stabilisation than crude extracts by producing emulsions of smaller droplet sizes. Moreover, emulsifying capacity of okra pectin was affected by the pH and stable emulsions were produced only at low pH values (pH 2.0 or 3.0). It has been shown that okra pectin-stabilized emulsions evolve under the effects of Ostwald ripening and coalescence during the long-term storage. The present work shows the potential of okra pectins as emulsifiers under acidic conditions and serves as the basis for the development of such systems in encapsulation technology of bioactive components.

Nuclear hormone receptors (NHRs) are transcription factors that regulate a wide variety of developmental and physiological processes. NHRs are targets of numerous drugs. However, due to limited knowledge on NHR specificity, many such drugs activate multiple biological pathways downstream of NHRs, leading to undesired side effects. To study NHR specificity in vivo, I used the model organism Caenorhabditis elegans. One C. elegans NHR is NHR-49, which regulates various aspects of lipid metabolism. Specifically, it activates genes involved in fatty acid desaturation and fatty acid β-oxidation by binding to a subunit of the Mediator multiprotein complex, MDT-15. Vice versa, NHR-49 represses genes involved in sphingolipid breakdown by heterodimerizing with another C. elegans NHR, NHR-66. Recently, three point mutations in nhr-49 were identified that promote fatty acid desaturation, but whether these alleles act specifically in this pathway or also affect other nhr-49 regulated processes is not clear. To test whether the mutated residues are linked to specific biological functions, I studied how they affect gene expression and protein-protein interactions by real time quantitative PCR and Yeast 2 Hybrid assays. I found that the three point mutations have different effects on nhr-49 dependent metabolic processes. While all three alleles broadly promoted nhr-49 dependent activation, only one allele affected nhr-49 dependent repression. This shows that the mutations and the corresponding amino acid residues have some association with specific nhr-49 dependent biological processes.
Medicine, Faculty of
Medical Genetics, Department of
Graduate

Macrophages can be polarized into M1 and M2 macrophages based on the composition of the milieu. Human macrophages have been poorly characterized. In this study, various macrophage subsets were generated by treating monocyte-derived macrophages (MDMs) with IFNγ (M1), IL-4 (M2a), LPS and IL-1β (M2b) or IL-10 (M2c) which were characterized with respect to their cell surface marker profile and functional profile in the context of cytokine production, susceptibility to HIV infection and apoptosis. Each polarization state demonstrated a unique cell surface marker profile and cytokine profile. In addition M1 macrophages were shown to produce IFNγ post TLR stimulation. Moreover, M1 macrophages were highly sensitive to apoptosis following Smac mimetic treatment. Furthermore, M2a and M2c macrophages were resistant to apoptosis, induced by PI3K blockage and IAPs degradation respectively, and at the same time supported productive HIV infection unlike the other macrophage subsets. These findings might lead to better understanding of HIV reservoir formation and be used to develop therapies to eradicate it.

Adult skeletal muscles can regenerate after injury due to the presence of satellite cells, a quiescent population of myogenic progenitor cells characterized by expressing the transcription factor Pax7. Once activated, satellite cells repair the muscle damage and replenish the stem cell niche due to the coordinated function of several transcription factors including Pax7 and the myogenic regulatory factors (MRFs). MRFs are skeletal muscle-specific transcription factors that can convert non-muscle cells into the myogenic lineage. MRFs are known to cooperate with other transcription factors in regulating the complex transcriptional network driving myogenic differentiation of muscle progenitors. The Six4 transcription factor emerges as a strong candidate for cooperating with MRFs. Six4 is expressed in skeletal muscles; the lack of a muscle development phenotype in Six4-null mice has been attributed to compensation by other Six family members. However, this did not exclude a critical role for Six4 during muscle development as Six1;Six4 double mutant mice show a more severe muscle phenotype than Six1 mutant mice. Nevertheless, the role of Six4 during adult muscle regeneration has never been addressed. I combined a partial loss-of-function of Six4 with high-throughput approaches to address the role of Six4 during adult skeletal muscle regeneration. I observed an important function of Six4 during muscle regeneration in vivo and in in vitro cell models. Using RNA interference assays against Six4 in tibialis anterior muscle regeneration after cardiotoxin-induced muscle damage, I observed for the first time that Six4 plays a role in proper muscle regeneration. The ability of the MRF MyoD, a central regulator of skeletal myogenesis, to convert a non-muscle cell model into the myogenic lineage was impaired with attenuated Six4 expression. I employed genome-wide approaches by combining ChIP-sequencing with gene expression profiling and identified a set of muscle genes coordinately regulated by both Six4 and MyoD. Throughout the genome, the cooperation between Six4 and MyoD was associated with binding of the H3K27me3 demethylase Utx and depletion of the H3K27me3 repressive chromatin mark. Together, these results reveal an important role for Six4 during adult muscle regeneration, and suggest a widespread mechanism of cooperation between Six4 and MyoD that correlates with modifying the epigenetic landscape of the regulatory regions of a large set of genes needed for efficient myogenesis.

碩士
國防醫學院
微生物及免疫學研究所
93
英文摘要 H-REV107 gene family belongs to the type Ⅱ tumor suppressor genes, which includes H-REV107, RIG1, HRASLS, HRASLS2 and HRLP5. Proteins of this gene family contain the N-terminal NC domain and the C terminal hydrophobic transmembrane domain. Presence of the C terminal transmembrane domain is essential for functions of the proteins. The H-REV107 family proteins exhibit activities to suppress cell growth and to induce cellular apoptosis, and/or differentiation. The anti-tumor activities of the protein family may be mediated through suppressing of Ras-mediated signal pathways. Although displays high sequence homology with H-REV107 family proteins, functions of the HRAS-like suppressor 2 (HRASLS2) have not be investigated. This study analyzed expression profiles and biological functions of the human HRASLS2. The HRASLS2 was expressed at high levels in normal tissues of the colon, small intestine, stomach, kidney and trachea. However, it was expressed at low levels in breast (MCF-7) and colorectal (HCT116) cancer cell lines. Expression vectors that synthesize HRASLS2 fusion proteins were constructed by subcloning of the HRASLS2 cDNA amplified from the SW480 colorectal cancer cells. Successful expression of HRASLS2 fusion proteins was detected in transfected HtTA cells by Western bloting and immunocytochemical staining. The full length HRASLS2-DsRed fusion protein predominantly expressed on the Golgi apparatus but not the plasma membrane nor the nucleus. Expression of the HRASLS2-myc fusion protein in MCF-7 breast cancer cells resulted in suppression of colony formation. Also, transient HRASLS2 transfection induced the release of lactate dehydrogenase and chromatin condensation in concentration and time dependent mannars in HtTA cervical cancer cells. The carboxyl-terminal truncated HRASLS2 fusion proteins expressed as the diffuse pattern in cytoplasm and had no effect on colony formation and the release of lactate dehydrogenase. In conclusion, HRASLS2 protein exhibited growth suppressive and proapoptotic activities on cancer cells. The carboxyl-domain was indispensable for endomembrane localization and biological functions of the HRASLS2 protein.

碩士
國立臺灣大學
微生物學研究所
94
Telomere is replicated by telomerase in most eucaryotes. Most yeast cells that lack telomerase enter into senescence and eventually die. However, a few cells bypass the senescence phenotype and survive. These survivors elongate their telomeres by the alternative recombination pathway. The transcriptional level of YPL014W is increased in telomerase-independent alternative lengthening of telomeres (ALT) pathway, which is used by type II survivor cells. Previous study showed that Ypl014w forms a complex with Cdc28 and Cln3(Uetz et al., 2000), indicating that Ypl014w may involve in cell cycle regulation. Study the function of Ypl014w may help us to find the relationship between telomere elongation pathway and cell cycle regulation. In my study, YPL014W knockout has no influence on cell cycle regulation and the phosphorylation status of Cdc28-Cln3 substrate Whi5. I also find that Ypl014w is a phosphorylated protein, but CLN3 knockout and temperature sensitive mutant of CDC28 have no influence on its phosphorylation status. I did not find the redundant gene of YPL014W with synthetic lethal. I can not confirm the previous study that Ypl014w interacts with Cdc28 and Cln3 in yeast two hybrid. I did not find other proteins that interact with Ypl014w in yeast-two-hybrid screen and IP analysis. According to sequence analysis, YPL014W expression may be controlled by Mcm1, a transcription factor that regulates many cell cycle regulated genes. I showed that Mcm1 binds to the promoter of YPL014W with CHIP and found the two ECB elements that Mcm1 binds.

碩士
國立臺灣大學
微生物學研究所
92
Telomeres is replicated by telomerase in budding yeast. Most cells that lack telomerase enter into senescence and eventually die. However, a few cells bypass the senescence phenotype and survive. These survivors elongate their telomeres by the alternative recombination pathway. The transcriptional level of YPL014W is increased during this elongation process. YPL014W associates with two cell cycle proteins, Cln3 and Cdc28. Here we analyzed the growth characteristics of the ypl014w△ mutant and cln3△ypl014w△ double mutant. In addition, we also investigated whether Ypl014w affects on the glucose regulation of Cln3, Cln3-Cdc28 kinase activity. Analysis of cell morphology and cell cycle revealed that the ypl014w△ mutant accumulates in the G2 phase of the cell cycle, wherever the cln3△ypl014w△ double mutant accumulates in the G1 phase. These data suggest that Ypl014w may involve in cell cycle regulation through Cln3. In the assay of glucose regulation of YPL014W, we found that glucose affects the transcription of YPL014W and Ypl014w has no influence on the glucose regulation of CLN3.Our findings suggest that Ypl014w may involve in cell cycle and glucose regulation.