Background: Bowel preparation is essential for quality colonoscopy. Although most bowel preparation regimens recommend dietary restriction for 24 to 48 hours before the procedure, the evidence for this is poor. Objectives: To establish whether dietary restriction during bowel preparation improves the quality of bowel preparation. Methods: A prospective single blind, randomised controlled pilot study. The dietary restriction (DR) group was instructed not to ingest high fibre foods for 48 hours prior to the use of a polyethylene glycol (PEG) bowel preparation. The non-dietary restriction (NDR) group was not given any dietary modification, but received instructions for the use of the PEG-based preparation solution. On the day of colonoscopy, the quality of the bowel effluent was assessed, and additional preparation given as necessary. The primary endpoint was quality of bowel cleansing using the Harefield Cleansing Scale during colonoscopy. The secondary endpoint was the need for additional bowel preparation and quantity of additional bowel preparation given prior to endoscopy. Data were analysed on an intention to treat basis. Results: Twenty-three participants were randomised to the intervention group and thirty-four to the control group. Patient demographics were similar in both groups. Dietary restriction did not influence the success rate of bowel preparation: 97% successful bowel preparation in the DR group, vs 91% successful bowel preparation in the NDR group (p=0.559). Additional bowel preparation requirement were similar in both groups: 35% in DR group vs 39% in NDR group (p=0.768). Mean amount of additional bowel preparation required was similar: 560 ml in the DR group vs 460 ml in the NDR group (p=0.633). Conclusion: The quality of bowel preparation was comparable in patients with and without dietary restrictions prior to colonoscopy. Non-restrictive diets prior to bowel preparation should be considered to increase compliance. The sample size of this pilot study prohibited definite statistical conclusions but demonstrated this to be a reasonable methodology for a larger study.

The eukaryotic ribosome is a large molecular machine consisting of ~80 ribosomal proteins and 4 rRNAs. The 40S and 60S ribosomal subunits are assembled in the nucleolus by ~200 helper proteins then shipped into the cytoplasm or to the endoplasmic reticulum where protein translation takes place. Eventually ribosomes are removed from the cytoplasm and recycled through ribophagy, however, very is little is known about ribosomal protein exchange after assembly but before ribophagy. Using kinetic turnover measurements of ribosomal proteins and rRNA in vivo we determined ribosomal protein replacement rates are diverse suggesting ribosomal components may be replaced without destruction of the entire ribosome. Measurements from ad libitum fed and dietary restricted mice provide strong evidence that RPL10 exchanges rates are dramatically different between AL and DR while synthesis and degradation do not change. RPL10 turnover can be described using a two-pool kinetic model, which may be applied to many ribosomal proteins.

In this dissertation, I will review the history and general strategies for performing kinetic proteomics. I will then demonstrate that I have published an open source, user-friendly program for other scientists to use to perform kinetic proteomics data analysis, as well as publishing a novel discovery of key ribosomal subunits being replaced within the lifetime of the ribosome, which was discovered through use of kinetic proteomics. Finally, I will discuss work that is ongoing to improve my software tool for use in human subjects, and work being done to combine kinetic proteomics with other global analysis methods to make novel biological discoveries. Proteins are constantly synthesized and destroyed to ensure sufficient functioning proteins to meet cellular needs, a process called protein turnover. Synthesis and degradation are carefully balanced over time to ensure that average protein concentrations do not change drastically. The status quo of the cell, or protein homeostasis, is required for the health of the organism. If protein homeostasis breaks down, serious diseases, such as Alzheimers, can result when proteins aggregate instead of being degraded properly. Because protein turnover is the means to maintain protein homeostasis while keeping sufficient functioning proteins, measuring protein turnover is critical to understanding biological processes and disease states. Measuring protein turnover rates on a broad scale is possible using a method called kinetic proteomics, and the improvement of kinetic proteomics is where I have focused the work for this dissertation.

No description available.

No description available.

O desenvolvimento de uma resposta imune adequada é um processo extremamente importante para a manutenção da homeostase do organismo. Uma série de processos são desencadeados a partir do primeiro contato com micro-organismos patógenos até a efetivação da resposta imune de memória. Todos esses processos envolvem a participação e a complexa atuação de mediadores como as citocinas inflamatórias e também citocinas regulatórias, que exercerão efeitos controlando o processo inflamatório. Diversos mecanismos moleculares, subjacentes à resposta inflamatória, ainda não estão totalmente compreendidos, como por exemplo o controle da expressão de genes inflamatórios exercido pela IL-10. Os processos envolvidos na resposta inflamatória são mantidos às custas do consumo de nutrientes, dentre eles podemos destacar o aminoácido glutamina, que atua em nível molecular, fornecendo nitrogênio para a formação do material genético e fonte energética para determinadas células do sistema imunológico como os macrófagos. Portanto, neste trabalho, investigamos os efeitos da IL-10 na modificação de nucleossomos, evidenciando o papel dessa citocina em regular a expressão de genes inflamatórios em macrófagos. Avaliamos também a função da glutamina, modulando a expressão de RNAm de citocinas inflamatórias e regulatórias dessas células. E por último, desenvolvemos um modelo de restrição alimentar em camundongos, nos quais avaliamos os efeitos desse modelo considerando-se alguns aspectos hematológicos e estudamos as alterações na resposta inflamatória em células esplênicas e do peritônio, bem como avaliamos a suplementação de glutamina in vitro na produção das citocinas (IL-12, TNF-alfa, IL-10) e a expressão do fator de transcrição NFkB. Os resultados compilados mostraram que a IL-10 leva a uma rápida redução da acetilação de nucleossomos, modulando a arquitetura da cromatina de genes inflamatórios como a IL-12. A glutamina modula a expressão de citocinas inflamatórias, regulando positivamente a expressão de IL-10 e Interferon beta. E a restrição alimentar induz a redução de citocinas proinflamatórias (IL-12 e TNF-α), influenciadas pelo aumento da produção de IL-10 e finalmente a suplementação com glutamina não interfere nesses parâmetros nas células peritoneiais e esplênicas do grupo submetido à restrição alimentar. Conclusão: a IL-10 modula a expressão gênica através da modificação de nucleossomos em macrófagos derivados da medula; a glutamina modula a expressão de IL-10 inibindo a resposta inflamatória, e a restrição alimentar modula alguns aspectos hematológicos e possui propriedades anti-inflamatórias. / The development of an appropriate immune response is an important process to the organism\'s homeostatic maintenance. A series of processes are triggered upon the very first contact with pathogens, up to the immunological memory establishment. These processes implicate in the participation of complex mediators, such as inflammatory and regulatory cytokines that will control the inflammatory process. Some mechanisms underlying the inflammatory response are not totally understood, the control of inflammatory genes exerted by IL-10 is an example. The processes involved in the inflammatory response are kept with nutrients expense, among these nutrients we can highlight the amino acid glutamine. It acts in a molecular level, supplying nitrogen to genetic material formation and as an energy supply for immune cells such as macrophages. Thus, we investigated the IL-10 effects on nucleosome modifications evidencing this cytokine role regulating inflammatory genes expression in macrophages. We also evaluated glutamine functions modulating inflammatory and regulatory cytokines mRNA expression on these cells. Ultimately, we developed a dietary restriction animal model where we evaluated it\'s effects on selected haematological aspects, analyzing the alteration in the inflammatory response of splenic and peritoneal cells. We also evaluated in vitro glutamine supplementation assessing cytokines production (IL-12, TNF-α, and IL-10) and the expression of NFkB transcription factor. The compiled results a expressive reduction in nucleosome acetylation modifying the chromatin architecture of inflammatory genes such as IL-12 and IL-6. Glutamine modulates inflammatory cytokines gene expression upregulating the expression of IL-10 and interferon beta. The dietary restriction reduces proinflammatory cytokines production (IL-12 and TNF-α), these results are influenced by the upregulated IL-10 production, glutamine supplementation have no effect on these parameters in the dietary restriction group. In conclusion, we can infer that IL-10 modulates gene expression trough nucleosome modification in bone marrow derived macrophages, glutamine has a potential effect on IL-10 expression, inhibiting the inflammatory response and dietary restriction modifies hematological parameters, presenting anti-inflammatory properties.

Dietary restriction (DR), or the reduction of food consumption without malnutrition, is the most conserved method of preventing or reversing age associated diseases. It is also the most conserved method of increasing lifespan, across model organisms. We developed a robust liquid DR method for C. elegans, to investigate requirements for stress defense and NAD\(^+\)-associated mechanisms in mediating DR induced longevity. We found that DR lifespan extension depended upon stress defense regulators that act downstream of TORC1 and growth pathways, as well as SIR-2.1/SIRT1 and the NAD\(^+\) salvage pathway enzyme PNC-1. Surprisingly, PNC-1 was not required for improvement in two measures of healthspan, or the period of life spent free from disease. This suggests that the genetic regulation of DR effects can be uncoupled from one another, and that increased healthspan does not always indicate increased lifespan.

Dietary restriction (DR), is a reduction in food intake, either through overall calorie or specific macronutrient intake, while avoiding malnutrition. DR has been consistently shown to increase longevity and protect against age related diseases. Although originally thought to be the result of a reduction in caloric intake, recent evidence suggests that the ratio of macronutrients, particularly that of protein : non-protein energy, also plays a role. The broad range of species in which DR is known to be effective, suggests an evolutionary conserved mechanism. However, the suggestion of a strong model species bias and a potential sex bias have led some to question the ubiquity of responses to DR. Here, I address the following questions: (i) How consistent is the effect of DR on reproduction? (ii) How does varying macronutrient intake effect both growth and body composition in three-spine sticklebacks (Gasterosteus aculeatus)? (iii) What is the effect of dietary macronutrient intake on lifespan and reproduction in the three-spine stickleback? and (iv) How does changing macronutrient intake impact fitness related traits, such as condition and performance in three-spine sticklebacks? Through use of a systematic review and meta-analysis, I show that the effect of DR on reproduction is evolutionarily conserved, though the effect is stronger in model species. However, when accounting for all significant moderators there is no evidence of the suggested sex differences in the effect of DR. I show that body composition is predicted by dietary lipid intake, with sticklebacks targeting a lower ratio of protein : fat within the body, potentially via metabolism and excretion of protein. These results hint at a link between conversion and excretion of protein and survival costs associated with high protein diets. I show that mortality risk is reduced at balanced protein : lipid intakes in males and generally at low protein : lipid intakes for females. However, the effect in females is not consistent throughout life. I further show that reproduction is maximised on high protein : lipid intakes for both sexes. These results suggest a macronutrient mediated trade-off between lifespan and reproduction in male three-spine sticklebacks. Finally I show a positive effect of lipid intake on male condition (a possible indicator of overall health) hinting at a relationship between lipid intake, adiposity, health and lifespan in male three-spine sticklebacks.

O desenvolvimento de uma resposta imune adequada é um processo extremamente importante para a manutenção da homeostase do organismo. Uma série de processos são desencadeados a partir do primeiro contato com micro-organismos patógenos até a efetivação da resposta imune de memória. Todos esses processos envolvem a participação e a complexa atuação de mediadores como as citocinas inflamatórias e também citocinas regulatórias, que exercerão efeitos controlando o processo inflamatório. Diversos mecanismos moleculares, subjacentes à resposta inflamatória, ainda não estão totalmente compreendidos, como por exemplo o controle da expressão de genes inflamatórios exercido pela IL-10. Os processos envolvidos na resposta inflamatória são mantidos às custas do consumo de nutrientes, dentre eles podemos destacar o aminoácido glutamina, que atua em nível molecular, fornecendo nitrogênio para a formação do material genético e fonte energética para determinadas células do sistema imunológico como os macrófagos. Portanto, neste trabalho, investigamos os efeitos da IL-10 na modificação de nucleossomos, evidenciando o papel dessa citocina em regular a expressão de genes inflamatórios em macrófagos. Avaliamos também a função da glutamina, modulando a expressão de RNAm de citocinas inflamatórias e regulatórias dessas células. E por último, desenvolvemos um modelo de restrição alimentar em camundongos, nos quais avaliamos os efeitos desse modelo considerando-se alguns aspectos hematológicos e estudamos as alterações na resposta inflamatória em células esplênicas e do peritônio, bem como avaliamos a suplementação de glutamina in vitro na produção das citocinas (IL-12, TNF-alfa, IL-10) e a expressão do fator de transcrição NFkB. Os resultados compilados mostraram que a IL-10 leva a uma rápida redução da acetilação de nucleossomos, modulando a arquitetura da cromatina de genes inflamatórios como a IL-12. A glutamina modula a expressão de citocinas inflamatórias, regulando positivamente a expressão de IL-10 e Interferon beta. E a restrição alimentar induz a redução de citocinas proinflamatórias (IL-12 e TNF-α), influenciadas pelo aumento da produção de IL-10 e finalmente a suplementação com glutamina não interfere nesses parâmetros nas células peritoneiais e esplênicas do grupo submetido à restrição alimentar. Conclusão: a IL-10 modula a expressão gênica através da modificação de nucleossomos em macrófagos derivados da medula; a glutamina modula a expressão de IL-10 inibindo a resposta inflamatória, e a restrição alimentar modula alguns aspectos hematológicos e possui propriedades anti-inflamatórias. / The development of an appropriate immune response is an important process to the organism\'s homeostatic maintenance. A series of processes are triggered upon the very first contact with pathogens, up to the immunological memory establishment. These processes implicate in the participation of complex mediators, such as inflammatory and regulatory cytokines that will control the inflammatory process. Some mechanisms underlying the inflammatory response are not totally understood, the control of inflammatory genes exerted by IL-10 is an example. The processes involved in the inflammatory response are kept with nutrients expense, among these nutrients we can highlight the amino acid glutamine. It acts in a molecular level, supplying nitrogen to genetic material formation and as an energy supply for immune cells such as macrophages. Thus, we investigated the IL-10 effects on nucleosome modifications evidencing this cytokine role regulating inflammatory genes expression in macrophages. We also evaluated glutamine functions modulating inflammatory and regulatory cytokines mRNA expression on these cells. Ultimately, we developed a dietary restriction animal model where we evaluated it\'s effects on selected haematological aspects, analyzing the alteration in the inflammatory response of splenic and peritoneal cells. We also evaluated in vitro glutamine supplementation assessing cytokines production (IL-12, TNF-α, and IL-10) and the expression of NFkB transcription factor. The compiled results a expressive reduction in nucleosome acetylation modifying the chromatin architecture of inflammatory genes such as IL-12 and IL-6. Glutamine modulates inflammatory cytokines gene expression upregulating the expression of IL-10 and interferon beta. The dietary restriction reduces proinflammatory cytokines production (IL-12 and TNF-α), these results are influenced by the upregulated IL-10 production, glutamine supplementation have no effect on these parameters in the dietary restriction group. In conclusion, we can infer that IL-10 modulates gene expression trough nucleosome modification in bone marrow derived macrophages, glutamine has a potential effect on IL-10 expression, inhibiting the inflammatory response and dietary restriction modifies hematological parameters, presenting anti-inflammatory properties.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22358号 / 医博第4599号 / 新制||医||1042(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 長船 健二, 教授 岩田 想, 教授 富樫 かおり / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM