>Magister Scientiae - MSc / Salvia hispanica L. is an annual pseudocereal food crop, locally known as chia that has the ability to grow in water stress environments. The importance of chia dates back to the pre-columbian era where it was consumed as staple food by the indigenous South Americans due to its high nutritional and medicinal benefits. A single chia plant produces two seed variants: white seed genotype (denoted as WSG) and black seed genotype (denoted as BSG). Chia seeds have been proven to have a huge potential as a healthy food source and contained various medicinal properties. However, these plants are still prone to environmental stress conditions such as salinity that is one of the major abiotic stresses that influence crop production and yield worldwide. Despite the nutritional impact of the chia seeds, limited information regarding their molecular responses to abiotic stress conditions are known. This study was divided into two distinct parts. Firstly, the study comparatively analysed the leaf proteomes of two chia genotypes using gelbased proteomic analysis coupled with mass spectrometry. Total soluble proteins were extracted from chia leaves and subjected to 2-D PAGE analysis. Proteins were visualized by CBB and identified by MALDI-TOF MS/MS. A total of 284 and 209 spots were detected in WSG and BSG, respectively. Using mass spectrometry, 36 differentially expressed protein spots were successfully identified based on their protein abundance using homology database searches. Interestingly, two defensive-related proteins (osmotin-like protein and the chalcone isomerase) were only present in WSG and absent in BSG. In light of previous information regarding the nutritional profiles (no significant difference) of these two genotypes, this study has shown that there are distinct molecular differences between these genotypes. Therefore, WSG will be used in further downstream analysis. The second part of this study focused on the influence of salt stress (imposed by 100 mM NaCl) on the leaf proteome of WSG. Using gel-based proteomic analysis, 61 differentially expressed proteins were identified and classified into nine functional categories. Most of the proteins identified in this study were upregulated by salt stress. Interesting to note, 12 proteins identified in this study were only present in response to salt stress but were absent in the control. These proteins include ATP-dependent zinc metalloprotease FTSH 2 (spot 48), HSP70 proteins (spots 46 and 47), superoxide dismutases (spots 10, 41 and 42) and an ascorbate peroxidase (spot 56). All these proteins are important antioxidants that play a significant role in scavenging reactive oxygen species (ROS). Previous studies have shown that these antioxidants play vital roles in stress tolerance. These proteins could serve as potential biomarkers that could be used to enhance salt stress tolerance in pseudocereals and cereal food crops. / National Research Foundation (NRF) and Agricultural Research Council

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22148号 / 医博第4539号 / 新制||医||1039(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 坂井 義治, 教授 小西 靖彦, 教授 滝田 順子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Pseudoexfoliation (PEX) syndrome is an age-related condition characterized by the production and accumulation of extracellular fibrillary material in the anterior segment of the eye. PEX predisposes for several pathological conditions, such as glaucoma and complications during and after cataract surgery. The pathogenesis of PEX is not yet fully understood. It is multifactorial with genetics and ageing as contributing factors. We aimed to study the proteome in aqueous humor (AH) in PEX in order to increase the knowledge about its pathophysiology. Therefore, we developed sampling techniques and evaluated separation methods necessary for analyzing small sample volumes. Other objectives were to study the lens capsule in eyes with PEX regarding small molecules, and to investigate the association between PEX and cataract surgery in a population-based 30-year follow-up study. Samples of AH from eyes with PEX and control eyes were collected during cataract surgery. In pooled, and individual samples, various liquid based separation techniques and high resolution mass spectrometry were utilized. For quantitation, various methods for labeling, and label free techniques were applied. Lens capsules were collected from some of the patients, and analysed by imaging mass spectrometry. A cohort of 1,471 elderly individuals underwent a comprehensive ophthalmological examination at baseline. Medical information was obtained by questionnaires, and from medical records. Incident cases of cataract surgery were identified by review of medical records. In the initial study, several techniques were explored for protein detection, and a number of proteins were identified as differentially expressed. In the individually labelled samples, changes in the proteome were observed. Eyes with PEX contained higher levels of proteins involved in inflammation, oxidative stress, and coagulation, suggesting that these mechanisms are involved in the pathogenesis in PEX. The levels of β/γ-crystallins were significantly increased in PEX, which is a novel finding. In the lens capsules from individuals with PEX, changes in the lipid composition was observed with time-of-flight secondary ion mass spectrometry. These changes remain to be elucidated. By multivariate analysis, lens opacities were the first, and PEX the second most important predictor for cataract surgery, the later accounting for a 2.38-fold increased risk for cataract surgery.