Accurate DNA replication is vital for maintaining genomic stability. Consequently, the machinery required to drive this process is designed to ensure the meticulous maintenance of information. However, random misincorporation of errors reduce the fidelity of the DNA and lead to pre-mature aging and age-related disorders such as cancer and neurodegenerative diseases. Some of the incorporated errors are the result of the error prone DNA polymerase alpha (Pol a), which initiates synthesis on both the leading and lagging strand. Lagging strand synthesis acquires an increased number of polymerase a tracks because of the number of Okazaki fragments synthesized per round of the cell cycle (~50 million in mammalian cells). The accumulation of these errors invariably reduces the fidelity of the genome. Previous work has shown that these pol a tracks can be removed by two redundant pathways referred to as the short and long flap pathway. The long flap pathway utilizes a complex network of proteins to remove more of the misincorporated nucleotides than the short flap pathway which mediates the removal of shorter flaps. Lysine acetylation has been reported to modulate the function of the nucleases implicated in flap processing. The cleavage activity of the long flap pathway nuclease, Dna2, is stimulated by lysine acetylation while conversely lysine acetylation of the short flap pathway nuclease, FEN1, inhibits its activity. The major protein players implicated during Okazaki fragment processing (OFP) are known, however, the choice of the processing pathway and its regulation by lysine acetylation of its main players is yet unknown. This dissertation identifies three main findings: 1) <i>Saccharomyces cerevisiae</i> helicase, petite integration frequency (Pif1) is lysine acetylated by Esa1 and deacetylated by Rpd3 regulating its viability and biochemical properties including helicase, binding and ATPase activity ii) the single stranded DNA binding protein, human replication protein A (RPA) is modified by p300 and this modification stimulates its primary binding function and iii) lysine acetylated human RPA directs OFP towards the long flap pathway even for a subset of short flaps.

The study deals with interactions of polyelectrolytes polystyrene sulfonate and hyaluronic acid with nitrogenic amphiphilic substances, represented by lysine and albumine. To study the interactions pH-metry, conductance, viscositic and turbidity measurement, DLS and reometry were used. All mixtures of different concentrations were measured and the data were compered with data obtained from measurement of samples with amphiphilic sumstances without polyelectrolytes. Observed interactions occured in the aminoacid concentrations between 0 to 20 mmoldm-3, then the PSS interaction groups were fully bonded by lysine and no more interactions were recognized. The same behaviour were observed in albumine solutions with concentration under 2 gdm-3.

Dietary supplementation of nicotinamide adenine dinucleotide (NAD+) precursors has been suggested as a treatment for non-alcoholic fatty liver disease and obesity. In the liver, NAD+ is primarily generated by nicotinamide phosphoribosyltransferase (NAMPT), and hepatic levels of NAMPT and NAD+ have been reported to be dependent on age and body composition. The aim of the present study was to identify time course-dependent changes in hepatic NAD content and NAD+ salvage capacity in mice challenged with a high-fat diet (HFD). We fed 7-week-old C57BL/6JBomTac male mice either regular chow or a 60% HFD for 6, 12, 24, and 48 weeks, and we evaluated time course-dependent changes in whole body metabolism, liver steatosis, and abundance of hepatic NAD-associated metabolites and enzymes. Mice fed a 60% HFD rapidly accumulated fat and hepatic triglycerides with associated changes in respiratory exchange ratio (RER) and a disruption of the circadian feeding pattern. The HFD did not alter hepatic NAD+ levels, but caused a decrease in NADP+ and NADPH levels. Decreased NADP+ content was not accompanied by alterations in NAD kinase (NADK) abundance in HFD-fed mice, but NADK levels increased with age regardless of diet. NAMPT protein abundance did not change with age or diet. HFD consumption caused a severe decrease in protein lysine malonylation after six weeks, which persisted throughout the experiment. This decrease was not associated with changes in SIRT5 abundance. In conclusion, hepatic NAD+ salvage capacity is resistant to long-term HFD feeding, and hepatic lipid accumulation does not compromise the hepatic NAD+ pool in HFD-challenged C57BL/6JBomTac male mice.

Under environmental stress, such as glucose deprivation, cells form stress granules - the accumulation of cytoplasmic aggregates of repressed translational initiation complexes, proteins, and stalled mRNAs. Recent research implicates stress granules in various diseases, such as neurodegenerative disease, but the exact regulators responsible for the assembly and disassembly of stress granules are unknown. Studies detect post-translational modifications on core stress granule proteins. One modification is lysine acetylation, in which a substrate is regulated by a lysine acetyltransferase (KAT) and lysine deacetylase (KDAC). My project deciphers the impact of lysine acetylation on an essential protein found in stress granules, poly(A) binding protein (Pab1) in Saccharomyces cerevisiae. In this work, I demonstrated that acetylation mimic of Pab1-K131 reduces stress granule formation upon glucose deprivation, and other stressors such as ethanol, raffinose, and vanillin. A potential KDAC that might be facilitating this role is Rpd3. Further, electromobility shift assay studies suggest that acetylation mimic of Pab1-K131 negatively impacts poly(A) RNA binding. This work will be useful when exploring therapeutic options when combating diseases linked to stress granules.

<p> Cell encapsulation aims at the delivery of a therapeutic protein to a patient from transplanted cells. Conventional approaches involve immune-isolating cell lines that have been genetically modified to express a therapeutic protein, in alginate-based microcapsules. The long-term success of this approach hinges on the structural stability of the microcapsules, as well as their ability to maintain an environment suitable for the long-term survival of encapsulated cells. The most commonly studied type of microcapsule is the alginate-poly-Llysine-alginate (APA) microcapsule. However, the main concern with AP A microcapsules is the Joss of structural integrity during long-term implantation due to the exchange of calcium ions with other physiological ions, as well as the loss of the polyelectrolyte overcoats. </p> <p> In order to increase the structural stability of the microcapsules, we developed and characterized a number of synthetic polyelectrolytes that undergo phase separation upon complexation, and which are capable of forming covalent cross-links. These reactive polyelectrolytes are designed to take the place of poly-L-lysine and the outer alginate layer. We also explored combining cross-linkable synthetic polyanions with sodium alginate to strengthen the Ca Alginate core, by forming a core cross-linked network extending throughout the microcapsules. The polyelectrolyte complexes, encapsulation processes and microcapsule properties were studied in detail using extensive characterization techniques, including collaborative work on cell viability and host-immune response. </p> <p> Overall, this thesis describes a novel approach and prom1smg materials for cell encapsulations that offer enhanced microcapsule resistance to chemical and mechanical stresses, while preserving the desired biocompatibility. These materials may ultimately be useful for clinical immunosuppressive therapies. </p> / Thesis / Doctor of Philosophy (PhD)

No description available.

Fifty twelve-month female Sprague-Dawley rats were used to study a lysine requirement for tissue maintenance. Animals were randomly assigned into five groups with dietary lysine levels ranging from 0.097 to 0.317 percent and fed for 60 days. Liver composition and carcass composition were determined, and a lysine requirement was also predicted. Results showed that the group fed 0.317 percent lysine had significantly increased liver fat content and decreased protein content. Neither liver moisture content nor total liver nitrogen content was related to dietary lysine levels. There was no significant finding on the analysis of carcass composition. The data indicated that the mature rat had a requirement for lysine lower than 0.097 percent in the diet. It was suggested that either adequate lysine was provided by wheat gluten in the diet, or the mature rat did not require lysine in order to maintain tissue level of protein. In future studies, it was necessary to use a diet with lysine levels lower than 0.097 percent to determine the minimum lysine requirement, and a concurrent baseline group for comparison with the treatment animals. / M.S.

L'utilisation de gluten de maïs (GM) jaune dans les moulées d'aquaculture est une pratique de plus en plus courante. En plus d'être très digeste, le GM est faible en phosphore et contient plus de 60% de protéines digestes. Le GM jaune est très concentré en pigments caroténoïdes xanthophylles jaunes. TI a cependant été démontré que ces pigments ont un pouvoir colorant indésirable sur la chair des salmonidés. Le GM est aussi pauvre en lysine et les régimes alimentaires contenant un haut niveau de GM peuvent montrer une légère déficience en lysine, ce qui limite la déposition protéique chez les poissons. Quatre régimes alimentaires (36% de protéines digestibles, 19 MJ kg-1 d'énergie digestible, 50 ppm d' astaxanthine) ont été formulés pour contenir 12% de farine de poisson et 30% de GM blanc ou jaune. Un régime contenant 36% de farine de poisson et aucun GM a également été utilisé. Les moulés ont été distribuées à des truites arc-en-ciel (poids initial = 119 g) élevés à 12° C pendant 140 jours. Le premier objectif de cette étude était d'étudier l'effet des pigments xanthophylles sur la déposition des pigments dans la chair de la truite arc-en-ciel. Le second objectif consistait à étudier l'effet de différents niveaux de lysine dans la moulée sur les performances et la pigmentation de la truite arc-en-ciel.

Lysine acetylation is a widespread protein post-translational modification that influences diverse cellular processes. An association between acetylation of histone N-terminal tails and transcriptional activation has been recognised since the 1960s. However, it has only become apparent since 2000 that many of the effects of histone acetylation are mediated by proteins that bind to acetyl-lysine through a specialised acetyl-lysine recognition domain, the bromodomain. Small-molecule inhibitors of bromodomain-histone interactions can greatly assist studies into the functions of bromodomain-containing proteins, and show promise as treatments for several diseases, including cancers. Herein I describe the discovery and development of a novel chemical series of bromodomain-binding ligands containing the 3,5-dimethyisoxazole moiety. This heterocycle acts as an acetyl-lysine bioisostere, mimicking key interactions formed between acetyl-lysine and the bromodomain. Optimised compounds show sub-micromolar affinities for bromodomains of the BET family, a class of transcriptional co-regulators. Crystallographic and structure-activity relationship studies shed light on the structural requirements for potent and selective BET ligands. Furthermore, the compounds show cellular effects consistent with BET bromodomain inhibition: cytotoxicity studies in a range of cell lines, including the NCI-60 human tumour cell line screen, reveal differential activity, with leukaemias showing particular sensitivity. 3,5-Dimethylisoxazole-containing compounds were also shown to downregulate known BET target genes. Further studies investigated the effect of modifying or replacing the methyl groups of 3,5-dimethylisoxazole on BET bromodomain affinity, which indicated that the 3-methyl group is necessary for affinity. Finally, three novel isoxazole-containing amino acids were synthesised and incorporated into histone peptides as potential bromodomain-binding, non-hydrolysable, acetyl-lysine mimics. These amino acids might be useful in uncovering the function of individual acetylated lysine residues. The identification of methyl-isoxazoles as acetyl-lysine-mimetic bromodomain ligands represents a significant advance in our understanding of structure-activity relationships for these important proteins. The confirmed cellular activity of these compounds will enable their use in future biological studies.

A semente de milho representa uma importante fonte de proteínas para alimentação humana e de animais monogástricos. Porém, como membros da família dos cereais não apresentam proteínas com um balanço nutricional adequado, devido principalmente ao baixo conteúdo de lisina. As proteínas de reserva da semente de milho são classificadas como fração não-zeína (albumina, globulina e glutelina) e zeína. Mutantes de endosperma de milho, como o2 apresentam quantidades maiores de lisina na semente. Porém, muitos mutantes considerados "alta lisina" não foram ainda caracterizados bioquimicamente. Uma série de mutantes, opaco (o1, o2, o5, o7, o10, o11 e o13) e floury (fl1 e fl2), foram estudadas para determinar as quantidades de proteínas de reserva, o perfil electroforético das proteínas e o conteúdo de LYS na semente, endosperma e embrião. Foi observado que os mutantes apresentaram redução no conteúdo de zeína e aumentos da fração não-zeína com variações dependendo do mutante, do background genético e do tecido analisado. A análise da semente determinou aumentos principalmente da fração albumina e globulina nos mutantes, exceto para o5 com aumentos apenas da fração glutelina. No endosperma foi observado aumento principalmente de albumina em o2, o7 e o5; e globulina em fl2, o10, o11 e o13. No embrião foram registrados os níveis maiores de albumina e globulina da semente, porém a quantidade de proteínas de reserva foi similar entre os genótipos. As quantidades de lisina presentes nas frações protéicas foram sempre maiores nos mutantes, porém para o10, o11 e o13 diferenças significativas, foram observadas principalmente para LYS na fração glutelina. O perfil SDS-PAGE revelou a presença de numerosas bandas protéicas variando entre 100kDa e 10kDa, sendo que a fração não-zeína revelou maior heterogeneidade no número de bandas. Bandas protéicas de maior intensidade foram observadas nos mutantes. Análise 2D-PAGE de proteínas de reserva do endosperma de mutantes o1, o2, fl1 e fl2, revelou padrões similares de distribuição de proteínas, aumentos de intensidade de spots protéicos e a presença de spots restrita ao perfil dos mutantes. Os resultados sugerem que os mutantes opacos e floury avaliados apresentam quantidades maiores de LYS na semente quando comparados aos genótipos selvagens que lhes deram origem. A futura análise dos spots que apresentaram alterações altamente significativas permitirá uma maior compreensão dos efeitos específicos dessas mutações sobre a regulação da biossíntese das proteínas de reserva e do acúmulo de lisina no grão. / The maize seed is an important source of proteins for humans and monogastric animals. However, such as all cereals, maize storage proteins is nutritionally poor mainly due to the low content of lysine. Maize seed storage proteins can be classified as non-zeins (albumins, globulins and glutelins) and zein. Some storage proteins mutants such as the o2 mutants exhibit higher contents of lysine. However, many of these high-lysine mutants have not yet been biochemically characterized. The opaque mutants o1, o2, o5, o7, o10, o11, o13 and floury mutants fl1 and fl2 were analyzed for storage proteins contents, eletrophoretic profile and lysine content in the seeds, endosperm, and embryo. It was observed that the mutants exhibited reduction in the zein fraction and increase in the non-zein fraction which varied according to the mutant, genetic background and tissue analyzed. The whole seed analysis revealed increases mainly in albumins and globulins, with the exception of the o5 mutants which exhibited a higher increase in the glutelin fraction. In the endosperm it was observed increase in the albumin fraction in o2, o7 and o5 and in the globulin fraction in fl2, o10 and o13. Higher concentrations of albumin and globulin were observed in the embryo, but with similar concentrations of total proteins among the mutants. The lysine content was higher always higher in the storage proteins of the mutants, however, in o10, o11 and o13 significant differences for lysine content were observed mainly in the glutelin fraction. SDSPAGE analysis revealed the presence of several band varying from 10kDa to 100kDa, with a higher heterogeneity among the non-zein fraction. Bands with higher intensity have been observed in the mutants. 2D-PAGE analysis revealed that the o1, o2, fl1 and fl2 mutants exhibited similar band patterns with increases in similar spots and mutant specific bands. The results suggest that the opaque and floury mutants exhibited higher lysine content when compared to their respective wild-type counterparts. Future analysis of the protein spots that exhibited significant variations will allow a better understanding of the specific effects of each mutation on the regulation of storage protein synthesis and lysine accumulation in the seeds.