The aim of my study was to create a Drosophila line lacking the expression of all Sirtuin genes, check its developmental phenotype and characterise its response in stress conditions. The flies had bigger weight than controls, they had decreased fertility and fecundity and they developed more slowly. They showed a trend towards increased resistance to chill coma, but they did not show a significant difference in starvation or oxidative stress assay. Its effect on lifespan is being investigated.

Over 100 years ago, X-ray crystallography gave scientists a window to the atomic world with varied applications in biology, chemistry and physics among other subjects. Macromolecular crystallography is now considered an essential tool for solving the three-dimensional structure of proteins and understanding their physiological role at the atomic level. As crystal growth remains a bottleneck in crystallography, various other techniques are often employed to help understand the protein structure and function. These methods range from simple analysis of the protein sequence to experiments such as dynamic light scattering, isothermal titration calorimetry, activity assays, and analytical ultracentrifugation. The additional knowledge gained about proteins from these methods can then assist in the modification of the protein to facilitate its crystallisation. The structural biology of proteins belonging to two diverse families; Galectins and Sirtuins, both involved in the regulation of cancer, was studied in this thesis. Galectins are evolutionarily conserved and ubiquitously present animal lectins with a high affinity for -galactose containing oligosaccharides. To date, 15 mammalian galectins have been identified. Their involvement in cell–cell and cell–matrix interactions has highlighted their importance in signal transduction and other intracellular processes. Human Galectin-7 (hGal-7) is a 16 kDa prototype galectin which is involved in the stimulation and development of cancer. The crystal structure of native hGal-7 and its complex with galactose and lactose have been reported. In this study, cross-linking of hGal-7 by glycodendrons and the resulting clustering and lattice formation have been studied. For this purpose, the high resolution X-ray structures of hGal-7 in complex with carbohydrate-based multivalent dendrons have been elucidated and analysed. Also discussed in this thesis are preliminary binding affinity results obtained using isothermal calorimetry. Supramolecular assembly formation was also assessed using dynamic light scattering. These experiments reveal how multivalent glycodendendrons interact with and form cross-links with hGal-7 molecules. Understanding how these dendrimeric compounds interact with hGal-7 would help in the design of new tools to investigate the recognition of multivalent carbohydrates by lectins and their resulting role in aggregation processes in tumour embolisation and survival. Sirtuins are NAD+-dependent deacylases that are involved in the regulation of diverse biological functions such as ageing, metabolism and stress resistance, in normal cellular physiology. Their role in ageing and ageing-related diseases, including cancer and neurodegenerative diseases among others, has received much attention and sirtuins have been extensively studied to help in extension of human lifespan. Seven members of the sirtuin family (SIRT1-7) are known, which are diversely sub-localised in the cell. A myriad of questions regarding their deacylation activity, their interplay and their role in various diseases still remain unanswered. In this study, structural biology techniques have been used to understand the role of SIRT1, SIRT2 and SIRT7. The cloning, expression, purification and crystallisation of these sirtuins are presented in this thesis. Various supporting techniques used to confirm the identity and activity of the proteins are also discussed. A brief discussion of the methods that can be employed to overcome various barriers in structural biology is also presented in this thesis. Elucidating the structure of full length sirtuins would help in the development of highly selective modulators of sirtuins to aid in the understanding of their role in ageing and ageing-related diseases.

Accumulation of intracellular damage by reactive oxygen species accelerates biological aging, leading to the development of age-related lung diseases such as idiopathic pulmonary fibrosis (IPF). Mitochondrial dysfunction and mitochondria-related oxidative stress has been implicated in the pathogenesis of many age-related diseases. Selective autophagic degradation of mitochondria (mitophagy) is critical to maintain a proper pool of the organelle and preserve cellular energy homeostasis. Oxidative stress resulting from age-dependent defects in the quality of proteins and degradation of mitochondria promotes alveolar epithelial cell damage potentiating lung injury. Our research found diminished autophagy corresponding with elevated levels of oxidized proteins and lipofuscin in response to lung injury in old and middle-aged mice compared to younger animals. More importantly, older mice exposed to lung injury are characterized by deficient mitophagic responses. The pro-fibrotic cytokine transforming growth factor beta 1 (TGFβ1) plays a pivotal role in driving fibroblast-to-myofibroblast differentiation (FMD), an important feature of pulmonary fibrosis. TGFβ1-mediated FMD is characterized by reduced autophagy flux, altered mitophagy and defects in mitochondrial function. In accordance, PINK1 expression is reduced in the aging murine lung and biopsies from IPF patients compared to controls. "nOur research also revealed a decline in mitochondrial protein deacetylase sirtuin 3 (SIRT3) expression in the lungs of aging mice. Low levels of SIRT3 transcripts were observed in two different animal models of pulmonary fibrosis. SIRT3 expression was reduced in fibrotic regions of lung tissues from patients with fibrotic diseases. We demonstrated that down-regulation of SIRT3 by TGFβ1 promotes acetylation of major oxidative stress response regulators, such as superoxide dismutase 2 (SOD2) and isocitrate dehydrogenase 2 (IDH2), and that resveratrol induced SIRT3 expression and ameliorated acetylation changes induced by TGFβ1. Knockdown of SIRT3 expression by siRNA exacerbated TGFβ1-induced FMD. By contrast, promotion of SIRT3 expression attenuated the effect of TGFβ1 on myofibroblast differentiation. Finally, SIRT3-deficient mice were more susceptible to pulmonary fibrosis in response to bleomycin and had increased collagen deposition compared to control mice. Collectively, our research indicates that an age-related decline in autophagy, SIRT3 expression, and mitochondrial homeostasis may contribute to the promotion and/or perpetuation of pulmonary fibrosis. / Meredith L Sosulski

Chen, Juan. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 124-154). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Both pronuclei of the zygote undergo epigenetic changes after fertilization, which determines the quality of the zygote and successful early mammalian embryonic development. Shortly after fertilization epigenetic asymmetry among the pronuclei of the zygote is evident, while the paternal pronucleus undergoes active DNA demethylation, the DNA of the maternal pronucleus remains methylated. The male pronucleus in addition undergoes histone acetylation, whereas the histones of the female pronucleus remain methylated. Asymmetry of pronuclei and their epigenetic status predicts successful reprogramming of the genome, and thus the success of embryonic development. For the successful development of the embryo is therefore required correct formation of both of these pronuclei of the zygote and this formation of pronuclei is regulated by post-translational histone modifications called histone code. It was hypothesized that the histone code is regulated by the activity of NADP+ - dependent histone deacetylases, sirtuins. In the experiment were used fully grown in vitro maturated pig oocytes that were fertilized with pig spermatozoa in vitro. After isolation of zygotes cultured with addition of the activator sirtuin resveratrol was performed immunofluorescence analysis of acetylated and methylated histone H3 at lysine K9 of pronuclei of the zygotes. From the results of control group asymmetry between the pronuclei of the zygote is evident; wherein the male pronucleus exhibits higher acetylation intensity contrast female pronucleus exhibits higher methylation intensity. After adding resveratrol to all experimental groups female pronucleus showed a significant increase of the methylated histone H3 at lysine K9, and contrary to the male pronucleus significant decrease of acetylated histone H3 at lysine K9. Sirtuins are involved in the regulation of histone code in porcine zygote and it can be assumed that they also play a role during subsequent embryonic development, which is the subject of further study.

Recently, the increasing importance of reproductive biotechnologies rises. They provide us to get higher performance of livestock or to improve treatment in human medicine. It is neccessary to have a sufficient amount of developmentaly competent oocytes and further healthy liveable embryos for in vitro culture to supply a progress of reproductive technologies. Immediately after fertilization, pronucleus formation is a key moment for further embryonicdevelopment. Male and female pronuclei have their own pattern of histone code. For development of early embryo, it is neccessary to supply the correct pattern of histone code. NAD+-dependent histon deacetylases, sirtuins, are one of the mechanism which plays in regulation of histone code. These family contains seven isoforms, SIRT1-7. Based on current research, we decided for hypothesis that sirtuins are present in porcine fertilized oocytes and regulate the pronucleus formation. In this thesis, porcine COCs were culture in modificated culture medium and after 44 hr. maturation, only oocytes with extruded first polar body were chosen and used for further in vitro fertilization. Presumed zygotes were subsequently cultured with sirtuins inhibitors, nicotinamide or sirtinol. After 22 hr. of in vitro culture, zygotes were subjected by imunocytochemicaly localization of methylated and acetylated (on lysine K9) histone H3 and image analysis. Our results show that SIRT1 is localizated in porcine zygotes, especially in pronuclei. There are changes in acetylation and methylation H3K9 after sirtuin inhibition. Significant increase of H3K9 acetylation and decrese in H3K9 methylation are appeared. Sirtinol usage has confirmed that the changes are result of SIRT1 action. Role of SIRT1 in histone code regulation of pronucleus formation is still not enought described in porcine.

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Acetylation was previously thought to occur exclusively on histones, but recent high-throughput screens have identified thousands of non-histone substrates. Despite the identification of these sites, little is known about how these acetyltransferase enzymes target their substrates. Gcn5 is the catalytic acetyltransferase found within the highly conserved SAGA complex. Recently, a member of this complex, Ada2, was found to impact Gcn5 substrate selection. In the yeast model organism Saccharomyces cerevisiae, a synthetic substrate developed from a proposed Gcn5-specific consensus sequence is used to identify regulators of Gcn5 substrate selection. This work is the first to demonstrate that addition of a consensus sequence is enough to confer acetylation of a non-substrate. With this method, Ada3 was identified as a key regulator, and acetylome profiling identified novel targets for Gcn5 dependent acetylation specifically regulated by Ada3. This system could be adapted for other acetyltransferases to identify regulators of substrate selection.

Autophagy is a regulated and evolutionarily conserved catabolic process that serves to degrade superfluous or damaged organelles and recycle their biochemical components for use in energy production and other biosynthetic reactions. It is a crucial cellular response to various stresses including oxidative stress and starvation. Sirtuins are NAD+ dependent deacetylases that link transcriptional regulation to cellular energy homeostasis, DNA damage, ROS response, cell cycle control, apoptosis and autophagy. The role of autophagy in cancer is complex as autophagy exerts both cell protective and damaging functions depending on the circumstances. The purpose of this study is to investigate the role of individual Sirtuin family members in the regulation of gene expression of the known autophagy markers LC3 and Beclin 1 under DNA damage and oxidative stress. To investigate the regulation of autophagy, we followed the gene expression of autophagy genes LC3 and Beclin 1 under diverse stress conditions in human osteosarcoma cells (U2OS). The protein and mRNA levels of LC3-II and the formation of autophagosomes were increased in etoposide and rotenone treated cells. While LC3-II and LC3-1 protein expressions were increased in Sirt1 overexpressing cells, no significant change was observed in Beclin 1 protein level. However, inhibition of Sirt1 by siRNA did not affect the cellular levels of the autophagy markers suggesting the potential involvement of other Sirtuin family members in the regulation of autophagy. Elevated LC3 mRNA was observed in cells overexpressing any of the Sirtuins family members; however etoposide treatment selectively inhibited Sirt1 and Sirt2 dependent upregulation of LC3 mRNA. Induction of LC3-Luc reporter activity was observed in Sirt5 transfected cells, which was further increased by etoposide treatment. Sirt5 carrying mutation in its catalytic domain is not able to induce autophagy and Sirt5 mediating autophagy is under the control of NF-KB transcription factor. These results support the notion that Sirtuins are important regulators of autophagy and the function of each member is to differentially regulate DNA damage and oxidative stress responses.