The SRC gene encodes pp60c-Src, a 60 kDa non-receptor tyrosine kinase that is frequently activated and/or overexpressed in many cancers including colon cancer. In a subset of colon cancer cell lines, it has been shown, that the overexpression of c-Src can be explained, in part, by the transcriptional activation of the SRC gene. As a result, the general goal of this thesis was to further characterize how SRC is transcriptionally regulated in human cancer cell lines. Two highly dissimilar promoters, the housekeeping-like SRC1A promoter, as well as the HIF-1Ñ regulated tissue-specific SRC1Ñ promoter, regulate SRC expression. hnRNP K and the Sp family of factors regulate the SRC1A promoter; however, the true impact of Sp3 on SRC1A activity was not understood. In this thesis, a comprehensive analysis of the effect of Sp3 on SRC1A activity was performed. Physiologically, Sp3 exists as four translational isoforms that, in part, dictate the activation potential of Sp3. In general, the longer forms of Sp3 were modest transcriptional activators of the SRC1A promoter whereas the shorter forms were unable to activate the SRC1A promoter. An analysis of all Sp3 isoforms identified that the shorter Sp3 isoforms could be converted into transcriptional activators of SRC1A if the SUMOylation of a critical lysine residue within the inhibitory domain was prevented. Conversely, SUMOylation of the same isoform had little effect on the activation potential of the longer Sp3 isoforms at the SRC1A promoter. These results suggest that transcriptional activation by Sp3 is promoter context-, isoform- and modification-dependent.<p>SRC is transcriptionally repressed by histone deacetylase inhibitors (HDIs) and despite unsuccessful studies attempting to identify HDI-responsive elements within the SRC promoter regions none could be identified. This finding also suggests that histone deacetylases (HDACs) may be required for SRC expression. Historically, it was believed that HDIs act at the histone level to alter chromatin dynamics through the inactivation of HDACs to result in histone hyperacetylation and increased transcriptional activation. As such, a systematic investigation of the changes in histone H3 and H4 acetylation status at the transcriptionally repressed SRC promoter regions and the transcriptionally activated p21WAF1 promoter region was performed. The p21WAF1 promoter was used as control in this study as p21WAF1 is a classical example of a gene transcriptionally activated by HDIs. Interestingly, similar changes in histone acetylation at the p21WAF1 promoter and both SRC promoter regions were observed. Upon closer examination of acetylation changes at discreet histone residues, it was observed that in the rare case that a particular residue was differentially acetylated upon treatment at the promoter regions analyzed, the SRC1Ñ and p21WAF1 promoter regions demonstrated more similar changes in acetylation as compared to SRC1A. Taken together, these results suggest that histone acetylation status is not an accurate indicator of transcriptional activity following HDI treatment. To further investigate HDI-mediated SRC repression, RNA Pol. II occupancy at the promoter and regions downstream of the promoter were assessed. Despite the continued occupancy of RNA Pol. II at the promoter regions, RNA Pol. II was lost from the 3¡¦ UTR upon treatment with HDIs. These findings suggest that RNA Pol. II . may be sequestered at the promoter regions upon treatment with HDIs possibly as a result of impeded transcription initiation and/or elongation. Further analysis of the phosphorylation status of RNA Pol. II identified that transcriptional initiation was indeed occurring despite HDI treatment; however, productive transcriptional elongation could not be confirmed thus suggesting a role for abrogated elongation in HDI mediated SRC repression. Complimentary analysis of the effects of HDACs on SRC expression suggested that while class I HDACs abrogated SRC expression, class II HDACs were required for the maintenance of SRC transcript levels in a promoter-independent fashion. Together, these results provide the basis for a model whereby HDIs repress SRC transcriptional expression through the inhibition of class II HDAC activity to eventually result in curtailed SRC transcriptional elongation.

Post translational modifications in histone proteins are transmissible changes that are not coded for in the DNA sequence itself but have a significant affect in the control of gene expression. Eukaryotic transcription is a regulated process, and acetylation plays a major role in this regulation. Deranged equilibrium of histone acetylation can lead to alteration in chromatin structure and transcriptional dysregulation of genes that are involved in the control of proliferation, cell-cycle progression, differentiation and or apoptosis. Evidence shows that high glucose conditions mimicking diabetes can increase histone acetylation and augment the inflammatory gene expression. Recent advances also highlight the involvement of altered histone acetylation in gastrointestinal carcinogenesis or hyperacetylation in amelioration of experimental colitis. However, the role of histone acetylation under obesity conditions is not yet known. Therefore in the present study, western blot analysis in the liver of Zucker obese versus lean rats was performed to determine the pattern and level of H3 and H4 acetylation (both in nuclear and homogenate fractions) at specific lysine (K) in pathological state of hepatic steatosis The same technique was also applied in the liver of obese rats fed higher amounts of vitamin B6 (OH) versus those fed normal amounts of vitamin B6 (ON) to assess if hyper-acetylation can be a protective response to hepatic steatosis. In both experimental models, it was also of interest to elucidate the expression of anti- and pro- apoptotic factor Bcl-2 and Bax in respect to histone acetylation. It was observed that, in liver homogenate fractions in control animals (LC/OC), there was a higher level of histone H3 acetylation at (K9, K14) and H4 acetylation at K5 in the obese animals. In contrast, the nuclear level of H3 and H4 acetylation at the same lysine residues was considerably higher in the lean and lower in the obese animals. Obese animals contained lower liver preneoplastic lesions as well as liver weight as a result of higher amounts of vitamin B6, had significantly higher H3 acetylation at K9 and K14 and H4 acetylation at K5, in both homogenate and nuclear fractions. However, histone acetylation was not detected for histone H4 at lysine 12 (K12) in either control group (LC/OC) or obese with different B6 diet group (OH/ON). Nevertheless, global histone H3 and H4 acetylation in both homogenate and nuclear fractions, was slightly higher in the lean rats and obese rats fed higher amounts of B6. By using the western blot technique, the level of anti- and pro- apoptotic Bcl-2 and Bax were also evaluated. The moderately higher level expression of anti-apoptotic Bcl2 protein was found in lean animals, whereas the expression of pro-apoptotic Bax was significantly higher in obese animals. Furthermore, anti-apoptotic Bcl2 protein expression was slightly higher in the obese rats fed normal amounts of B6 diet; but, pro-apoptotic Bax was higher in the obese rats fed higher amounts of vitamin B6. This is the first study which shows that hyperacetylation of histones in liver nuclei can be correlated with amelioration of hepatic steatotis. Histone acetylation and B6 rich diet might be involved in the regulation of biological availability of key apoptotic proteins, which, in turn, can possibly modify the severity of the disease.

Post translational modifications in histone proteins are transmissible changes that are not coded for in the DNA sequence itself but have a significant affect in the control of gene expression. Eukaryotic transcription is a regulated process, and acetylation plays a major role in this regulation. Deranged equilibrium of histone acetylation can lead to alteration in chromatin structure and transcriptional dysregulation of genes that are involved in the control of proliferation, cell-cycle progression, differentiation and or apoptosis. Evidence shows that high glucose conditions mimicking diabetes can increase histone acetylation and augment the inflammatory gene expression. Recent advances also highlight the involvement of altered histone acetylation in gastrointestinal carcinogenesis or hyperacetylation in amelioration of experimental colitis. However, the role of histone acetylation under obesity conditions is not yet known. Therefore in the present study, western blot analysis in the liver of Zucker obese versus lean rats was performed to determine the pattern and level of H3 and H4 acetylation (both in nuclear and homogenate fractions) at specific lysine (K) in pathological state of hepatic steatosis The same technique was also applied in the liver of obese rats fed higher amounts of vitamin B6 (OH) versus those fed normal amounts of vitamin B6 (ON) to assess if hyper-acetylation can be a protective response to hepatic steatosis. In both experimental models, it was also of interest to elucidate the expression of anti- and pro- apoptotic factor Bcl-2 and Bax in respect to histone acetylation. It was observed that, in liver homogenate fractions in control animals (LC/OC), there was a higher level of histone H3 acetylation at (K9, K14) and H4 acetylation at K5 in the obese animals. In contrast, the nuclear level of H3 and H4 acetylation at the same lysine residues was considerably higher in the lean and lower in the obese animals. Obese animals contained lower liver preneoplastic lesions as well as liver weight as a result of higher amounts of vitamin B6, had significantly higher H3 acetylation at K9 and K14 and H4 acetylation at K5, in both homogenate and nuclear fractions. However, histone acetylation was not detected for histone H4 at lysine 12 (K12) in either control group (LC/OC) or obese with different B6 diet group (OH/ON). Nevertheless, global histone H3 and H4 acetylation in both homogenate and nuclear fractions, was slightly higher in the lean rats and obese rats fed higher amounts of B6. By using the western blot technique, the level of anti- and pro- apoptotic Bcl-2 and Bax were also evaluated. The moderately higher level expression of anti-apoptotic Bcl2 protein was found in lean animals, whereas the expression of pro-apoptotic Bax was significantly higher in obese animals. Furthermore, anti-apoptotic Bcl2 protein expression was slightly higher in the obese rats fed normal amounts of B6 diet; but, pro-apoptotic Bax was higher in the obese rats fed higher amounts of vitamin B6. This is the first study which shows that hyperacetylation of histones in liver nuclei can be correlated with amelioration of hepatic steatotis. Histone acetylation and B6 rich diet might be involved in the regulation of biological availability of key apoptotic proteins, which, in turn, can possibly modify the severity of the disease.

The SRC gene encodes pp60c-Src, a 60 kDa non-receptor tyrosine kinase that is frequently activated and/or overexpressed in many cancers including colon cancer. In a subset of colon cancer cell lines, it has been shown, that the overexpression of c-Src can be explained, in part, by the transcriptional activation of the SRC gene. As a result, the general goal of this thesis was to further characterize how SRC is transcriptionally regulated in human cancer cell lines. Two highly dissimilar promoters, the housekeeping-like SRC1A promoter, as well as the HIF-1Ñ regulated tissue-specific SRC1Ñ promoter, regulate SRC expression. hnRNP K and the Sp family of factors regulate the SRC1A promoter; however, the true impact of Sp3 on SRC1A activity was not understood. In this thesis, a comprehensive analysis of the effect of Sp3 on SRC1A activity was performed. Physiologically, Sp3 exists as four translational isoforms that, in part, dictate the activation potential of Sp3. In general, the longer forms of Sp3 were modest transcriptional activators of the SRC1A promoter whereas the shorter forms were unable to activate the SRC1A promoter. An analysis of all Sp3 isoforms identified that the shorter Sp3 isoforms could be converted into transcriptional activators of SRC1A if the SUMOylation of a critical lysine residue within the inhibitory domain was prevented. Conversely, SUMOylation of the same isoform had little effect on the activation potential of the longer Sp3 isoforms at the SRC1A promoter. These results suggest that transcriptional activation by Sp3 is promoter context-, isoform- and modification-dependent.<p>SRC is transcriptionally repressed by histone deacetylase inhibitors (HDIs) and despite unsuccessful studies attempting to identify HDI-responsive elements within the SRC promoter regions none could be identified. This finding also suggests that histone deacetylases (HDACs) may be required for SRC expression. Historically, it was believed that HDIs act at the histone level to alter chromatin dynamics through the inactivation of HDACs to result in histone hyperacetylation and increased transcriptional activation. As such, a systematic investigation of the changes in histone H3 and H4 acetylation status at the transcriptionally repressed SRC promoter regions and the transcriptionally activated p21WAF1 promoter region was performed. The p21WAF1 promoter was used as control in this study as p21WAF1 is a classical example of a gene transcriptionally activated by HDIs. Interestingly, similar changes in histone acetylation at the p21WAF1 promoter and both SRC promoter regions were observed. Upon closer examination of acetylation changes at discreet histone residues, it was observed that in the rare case that a particular residue was differentially acetylated upon treatment at the promoter regions analyzed, the SRC1Ñ and p21WAF1 promoter regions demonstrated more similar changes in acetylation as compared to SRC1A. Taken together, these results suggest that histone acetylation status is not an accurate indicator of transcriptional activity following HDI treatment. To further investigate HDI-mediated SRC repression, RNA Pol. II occupancy at the promoter and regions downstream of the promoter were assessed. Despite the continued occupancy of RNA Pol. II at the promoter regions, RNA Pol. II was lost from the 3¡¦ UTR upon treatment with HDIs. These findings suggest that RNA Pol. II . may be sequestered at the promoter regions upon treatment with HDIs possibly as a result of impeded transcription initiation and/or elongation. Further analysis of the phosphorylation status of RNA Pol. II identified that transcriptional initiation was indeed occurring despite HDI treatment; however, productive transcriptional elongation could not be confirmed thus suggesting a role for abrogated elongation in HDI mediated SRC repression. Complimentary analysis of the effects of HDACs on SRC expression suggested that while class I HDACs abrogated SRC expression, class II HDACs were required for the maintenance of SRC transcript levels in a promoter-independent fashion. Together, these results provide the basis for a model whereby HDIs repress SRC transcriptional expression through the inhibition of class II HDAC activity to eventually result in curtailed SRC transcriptional elongation.

Histone deacetylases (HDACs) are a family of enzymes whose functions have been overwhelmingly associated with gene expression and chromatin dynamics by modifying the histone tails. In recent years, intensive studies have demonstrated that many non-histone proteins also could serve as substrates for HDACs. And their functions and activities have been found to be regulated by posttranslational acetylation on the ε-amino group of lysines. Here, we report that two DNA repair factors including NBS1 (Nijmegen breakage syndrome 1) and ATDC (Ataxia-Telangiectasia Group D Complementing) are acetylated proteins. SIRT1 could maintain NBS1 in a hypoacetylated state, which is required for ionizing radiation-induced NBS1 Ser343 phosphorylation. And by modulating the acetylation of ATDC, HDAC9 could prevent ATDC-p53 complex formation, promoting IR-induced cell death. These data suggest HDACs play much wider roles in cells in addition to their transcriptional repression function.

No description available.

No description available.

<p>NatA binds inositol hexakisphosphate and other ligands, and exhibits conformational flexibility dependent on the ligand bound.</p> / Dissertation

No description available.

Diese Arbeit besteht aus drei Teilen. Im ersten Teil wurde Holz mit verschiedenen, kommerziell erhältlichen Kieselsolen behandelt, die unterschiedliche pH-Werte und Oberflächenmodifikationen aufwiesen. Basische Kieselsole vermochten nicht in das Holz einzudringen, da ihr pH-Wert während des Eindringens absinkt und es zur Ausfällung des Kieselsols im Holz kommt. Neutrale und saure Kieselsole hingegen konnten problemlos in das Holz eingebracht werden. Eines der sauren Kieselsole, welches mittels Aluminumoxychlorid kationisch modifiziert war, reduzierte die Wasseraufnahme und den pilzlichen Abbau durch die Braunfäule Coniophora puteana (Kiefer) und die Weißfäule Trametes versicolor (Buche). Im Bläuetest zeigte sich ein verminderter Befall durch Aureobasidium pullulans, allerdings kein kompletter Schutz gegen diesen Pilz. Auch die kleinsten verfügbaren Partikelgrößen für Kieselsole ergaben keinerlei Zunahme des Zellwandvolumens (chemische Quellung, Bulking), was darauf hinweist, dass eine Eindringung in die Zellwand nicht stattfand. Es erscheint daher nicht möglich, Kieselsole in die Zellwand einzubringen und die Dimensionsstabilität des Holzes zu verbessern. Da Kieselsol lediglich in die Lumen der Holzzellen eingebracht werden kann, kann die Behandlung nicht als wirkliche Holzmodifizierung angesehen werden. Wegen der vielversprechenden Ergebnisse in den Wasseraufnahmeversuchen und den Pilztests wurde mit dem kationischen Kieselsol behandeltes Holz thermogravimetrisch und in einem Brandtest untersucht. Im thermogravimetrischen Test zeigte sich eine leicht verminderte Pyrolysetemperatur (eine übliche Wirkung von Feuerschutzmitteln), die Holzkohlemenge war jedoch nicht erhöht. Dies zeigt, dass die Menge an brennbaren Gasen, die während der Pyrolyse freiwerden, durch das Kieselsol nicht vermindert wurde. Auch zeigte die resultierende Holzkohle gleiche Oxidationseigenschaften wie die Holzkohle der Kontrollen. Im Brandtest wurden die Branddauer, die Brandgeschwindigkeit und der Gewichtsverlust vermindert. Das Nachglühen der Holzkohle wurde komplett unterbunden. Alle diese Effekte waren jedoch relativ klein verglichen mit den Effekten eines kommerziell erhältlichen Feuerschutzsalzes, welches ebenfalls als Referenzbehandlung getestet wurde. Im zweiten Teil der Arbeit wurden acetoxyfunktionelles Silan und verschiedene Polydimethylsiloxane (PDMS) mit Acetanhydrid kombiniert, um Holz zu acetylieren. Die PDMS hatten die folgenden Funktionalitäten: Amino, Acetoxy, Hydroxy und nicht-funktionell. Die beste Hydrophobierung des acetylierten Holzes wurde durch die Kombination mit acetoxyfunktionellem PDMS erreicht, welches anschließend in verschiedenen Konzentrationen getestet wurde. Eine Konzentration von 1% in Acetanhydrid zeigte bereits eine maximale Hydrophobierung, welches darauf schließen lässt, dass die inneren Oberflächen des Holzes mit dem PDMS belegt und hydrophobiert wurden. Die Pilzresistenz des behandelten Holzes wurde durch die Kombination mit dem PDMS nicht beeinflusst. Bei Wasserlagerung zeigte sich eine leichte Überquellung des Holzes, welches mit der Kombination von Acetanhydrid und PDMS acetyliert worden war. Untersuchungen der Biegefestigkeit und Bruchschlagarbeit ergaben jedoch keinen Einfluss. Im dritten Teil der Arbeit wurden wasserbasierte Emulsionen von funktionellen PDMS zur Imprägnierung von Holz eingesetzt. Es wurde untersucht, ob Resistenz gegen pilzlichen Abbau und Hydrophobierung wie auch erhöhte Dimensionsstabilität mit dieser Behandlung erreicht werden kann. Die α-ω-gebundenen Funktionalitäten der PDMS waren: Amino, Carboxy, Epoxy und Carbobetain. Die stärkste Hydrophobierung wurde mit dem carbobetain-funktionellen PDMS erreicht, allerdings ergab diese Behandlung keine verbesserte Pilzresistenz gegenüber einem Abbau durch Coniophora puteana und Trametes versicolor. In dieser Hinsicht die beste Wirkung zeigte die Behandlung mit carboxy-funktionellem PDMS. Dieses Material verminderte jedoch die Wasseraufnahmerate nur ungenügend und wurde außerdem stark ausgewaschen. Daher wurden in der Folge amino-funktionelles und carboxy-funktionelles PDMS kombiniert, um durch eine Salzbildung der beiden Funktionalitäten eine verbesserte Fixierung des carboxy-funktionellen Siloxans zu erreichen. Die Kombination zeigte bei einem Überschuss an amino-funktionellem PDMS eine gute Fixierung, jedoch ansonsten keine Synergieeffekte. Die Dimensionsstabilität des Holzes wurde durch die Behandlungen nur sehr geringfügig verbessert. Hierfür müsste eine gute Eindringung der Chemikalien in die Zellwand erfolgen und ein dauerhaftes Bulking erzielt werden. Die Eindringung der Chemikalien in die Zellwand war jedoch in allen Fällen nur gering.