Aktuell gibt es für Selen (Se) keine Bedarfsempfehlungen für das Damwild (Dama dama) in Gehegehaltung. Diese Studie soll die typische Se-Aufnahme bei Gehegehaltung von Damwild ermitteln und klären, welche Parameter sich eignen, um die Se-Versorgung des Damwildes zu reflektieren. Dazu wurden 19 Damhirsche in zwei Gruppen unter identischen Bedingungen gehalten. Die Tiere ernährten sich von dem natürlichen Grasaufwuchs und Mischfutter (0,15 mg/kg TS bzw. 1,07 - 1,91 mg/kg TS). In Blut, Plasma und die Organen wurden der Se-Gehalt, die Aktivität der Se-abhängigen Glutathionperoxidase (GPx) sowie teilweise die Gesamt-GPx-Aktivität (gesGPx), die Aktivität der Glutathion-S-Transferase (GST) und die Expression verschiedener GPx analysiert. Durch die Se-Supplementierung wurden ein signifikanter Anstieg des Plasma-Se in der Versuchsgruppe und ein moderater Unterschied der Vollblut-Se-Konzentration sowie der Vollblut-GPx-Aktivität zwischen der Kontroll- und der Versuchsgruppe beobachtet (p = 0,08). Außerdem wurde in allen Organen der Versuchsgruppe ein höherer Se-Gehalt als in der Kontrollgruppe festgestellt. In der Hierarchie der untersuchten Organe ist die Niere am höchsten angeordnet, absteigend folgen der Herz- und Skelettmuskel, die Milz und die Leber. Eine Se-Aufnahme von 0,05 - 0,08 mg/kg TS führt beim Damwild nicht zur Ausprägung von Se-Mangelsymptomen und stellt daher eine ausreichende Se-Versorgung dar. Die Empfehlungen zur Se-Versorgung für Damwild sind somit nicht von Hauswiederkäuern zu übernehmen. Im Plasma und im Vollblut scheinen Se-Konzentrationen von 28 - 64 µg/l und 81 - 200 µg/l für eine ausreichende Se-Versorgung zu sprechen, in der Leber Se-Konzentrationen von 270 - 663 µg/kg TS.:Inhaltsverzeichnis Inhaltsverzeichnis I Abbildungsverzeichnis V Tabellenverzeichnis VI Verzeichnis der Anhangstabellen VIII Abkürzungsverzeichnis IX 1. Einleitung 1 2. Literaturübersicht 2 2.1 Se als chemisches Element 2 2.2 Geschichte des Se und seiner Proteine 3 2.3 Se-Gehalte in Boden, Pflanzen, Nahrungs- und Futtermitteln 4 2.3.1 Se-Gehalte im Boden 4 2.3.2 Se-Gehalte in Pflanzen 5 2.3.3 Se-Gehalte in Nahrungsmitteln 7 2.3.4 Se-Gehalte in Futtermitteln 8 2.4 Se im Stoffwechsel 9 2.4.1 Resorption 9 2.4.2 Transport, Metabolismus und Speicherung 10 2.4.2.1 Transport 10 2.4.2.3 Speicherung 11 2.4.3 Versorgung über Plazenta und Milch 12 2.4.4 Exkretion 14 2.5 Biologische Funktionen des Se 16 2.5.1 SeP 16 2.5.2 Funktionen 18 2.5.2.1 Spezielle Funktionen der GPx 18 2.5.2.2 Weitere Funktionen der Selenoenzyme 20 2.6 Damwild (Dama dama) 21 2.6.1 Systematische und historische Einordnung des Damwildes 21 2.6.2 Physiologie und Ernährung des Damwildes 22 2.6.3 Se-Status bei Cerviden 23 2.7 Se-Bedarf 24 2.8 Se- und Enzymwerte im Organismus 25 2.8.1 Se-Gehalte im Blut 25 2.8.2 Se-Gehalte in verschiedenen Organen 28 2.9 Se und Erkrankungen 30 2.9.1 Se-Mangel assoziierte Erkrankungen 30 2.9.2 Se-Toxizität 31 2.9.2.1 Die akute Se-Intoxikation 32 2.9.2.2 Die subakute Se-Intoxikation 33 2.9.2.3 Die chronische Se-Intoxikation 33 3 Tiere, Material und Methoden 35 3.1 Versuchsziel 35 3.2 Tiere 35 3.3 Haltung 35 3.4 Fütterung und Supplementierung 35 3.5 Versuchsablauf 37 3.6 Probenentnahmen 38 3.6.1 Blutproben 38 3.6.2 Organ- und Gewebeproben 38 3.6.3 Wiegen 40 3.6.4 Futterproben 41 3.7 Versuchsparameter 43 3.8 Analytische Methoden 43 3.8.1 Futteranalyse 43 3.8.1.1 TS 43 3.8.1.2 Rohasche (Ra) 44 3.8.1.3 Organische Substanz (oS) 44 3.8.1.4 Rohprotein (Rp) 44 3.8.1.5 Rohfett (Rfe) 44 3.8.1.6 Rohfaser (Rfa) 44 3.8.1.7 N-freie Extraktstoffe (NfE) 45 3.8.1.8 Spurenelemente: Se, Cu, Zn 45 3.8.2 Vollblut-, Plasma-, Organ- und Gewebeanalyse 45 3.8.2.1 Histologie der Skelettmuskulatur 45 3.8.2.2 Se-Gehalt 46 3.8.2.3 TS-Gehalt 47 3.8.2.4 GPx-Aktivität 47 3.8.2.5 Proteingehalt 48 3.8.2.6 Hämoglobingehalt 49 3.8.2.7 GPx-mRNA-Expression 49 3.8.2.8 α -Glutathion-S-Transferase-Aktivität (GST) 53 3.9 Statistische Auswertung 54 4. Ergebnisse 55 4.1 KM der Tiere und Gewichte der Schlachtkörperhälften 55 4.1.1 KM der Tiere zu Versuchsbeginn 55 4.1.2 Gewichte der Schlachtkörperhälften zu Versuchende 55 4.2 Histologie der Skelettmuskulatur 56 4.3 Se-Gehalte in Plasma, Vollblut und Organen 58 4.3.1 Se-Gehalte in Plasma und Vollblut 58 4.3.2 Se-Gehalte in verschiedenen Organen 59 4.4 Se-abhängige und -unabhängige Enzyme 60 4.4.1 GPx-Aktivitäten in Plasma und Vollblut 60 4.4.2 GPx-Aktivität in verschiedenen Organen 62 4.4.3 GPx-mRNA-Expression 63 4.4.4 α-GST-Aktivität 64 5. Diskussion 67 5.1 Kritik der Methoden 67 5.1.1 Se-Supplementierung der Tiere 67 5.1.1.1 Futteraufnahme 67 5.1.1.2 Höhe der Se-Supplementierung 67 5.1.1.3 Dauer der Se-Supplementierung 68 5.1.1.4 Art der Se-Supplementierung 68 5.1.2 Probengewinnung 69 5.1.3 Untersuchungsparameter 69 5.1.4 Vitamin E 69 5.2 Diskussion der Versuchsergebnisse 70 5.2.1 Einschätzung der Se-Versorgung vor Se-Supplementierung 70 5.2.2 Einschätzung der Se-Versorgung nach unterschiedlicher Se-Supplementierung 72 6. Zusammenfassung 81 7. Summary 83 8. Literaturverzeichnis 85 9. Anhang……………………………………………………………………..110 Danksagung 117

Call number: LD2668 .T4 1952 M3 / Master of Science

Selenium (Se) is a contaminant of potential concern in aquatic systems due to its efficient incorporation into food webs, potential for bioaccumulation at higher trophic levels, and role as a developmental toxicant in oviparous vertebrates. While the presence of embryonic/larval deformities due to in ovo Se exposure is considered the most sensitive toxicological endpoint, elevated levels of dietary Se have also been associated with alterations to bioenergetic and hormonal status of adult female fishes, which consequently could lead to diminished fitness and impaired reproduction. Adverse reproductive effects in fishes have been the primary focus of Se research thus far, while studies focusing on Se toxicity in amphibians in any regard are severely lacking. The US EPA has recently proposed a new set of criteria for the protection of freshwater aquatic life with regards to acceptable Se tissue threshold levels; however, these values were generated based on effects observed in fishes with negligible existent data on amphibians to assist in this process. Thus, the overall goal of this thesis research was to characterize the reproductive and developmental effects of elevated dietary Se exposure in Xenopus laevis, in order to provide a foundation for amphibian related Se research that may assist in establishing effective regulatory guidelines that protect this highly vulnerable and ecologically valuable taxon. The research presented in this thesis was performed as one large generational bioassay with the analysis of experimental variables divided into three sections in order to evaluate the effects of elevated in ovo Se exposure via maternal transfer on early and late stages of larval development in addition to the overall fitness of adult X. laevis females after a dietary exposure. Adult X. laevis females were fed a diet augmented with L-selenomethionine (SeMet) for 68 days after which they were bred with untreated males. The resultant embryos were incubated up to 5 days post fertilization (dpf) to determine fertilization success, hatchability, mortality and frequency/severity of malformations. Subsamples of 5 dpf tadpoles were selected and raised to completion of metamorphosis for evaluation of mortality, growth and maturation rate. In addition, tissue and blood samples as well as morphometric indices were collected from X. laevis females, upon completion of the exposure period and subsequent breeding, to ascertain Se tissue distribution, triglyceride and glycogen levels, cortisol concentrations and the overall health status of SeMet-treated females. Within the data gathered throughout this research, a foundation of knowledge characterizing Se toxicity in amphibians was established along with the development of an early life stage toxicity threshold for the frequency of teratogenic abnormalities in X. laevis. The bioenergetic and stress status in addition to the overall body condition of adult females after a 68 day dietary exposure showed no significant differences among treatment groups. The concentrations of Se measured in the ovary, egg, liver and muscle samples increased with female dietary Se levels with strong positive relationships between egg Se concentrations and the other three tissues being illustrated. Elevated in ovo Se exposure had no biologically significant effect on fertilization success, hatchability or mortality within the first 5 dpf; however, the frequency and severity of morphological abnormalities was significantly greater in tadpoles from the highest dose group, with eye lens abnormalities most prominently observed. Late stage larval survival and growth was unaffected by in ovo Se exposure; however, the distribution of developmental stages observed at the set time point when 50% of tadpoles completed metamorphosis showed a larger portion of tadpoles at earlier stages of development in the highest dose group despite no overall change in time to metamorphosis. The results of this thesis research in its entirety suggest that amphibians, as represented by X. laevis, are potentially more tolerant to elevated in ovo and dietary Se exposures than other oviparous vertebrates studied to date; however, without sufficient data for comparison it is unknown whether X. laevis is a tolerant, average or sensitive species among amphibians.

Selenium is an important nutritional element that is required in a minute amount for maintenance of proper health in both humans and animals. Many biochemical processes in human and animal depends on Se and selenoprotein function, and various studies have suggested that Se supplementation can improve fundamental immune function in both humans and animals. Se status in the UK is very low; therefore, there is a need to enhance selenium intake through diet. One way of doing that is through the introduction of selenium to farmed animals. In this Ph.D. study, we fed rainbow trout with diet containing different spiked concentrations (0.8 - 8.9 μg g-1) of Se for 14 weeks. Fish were sampled every two weeks and liver, kidney, muscle, gills and whole blood were collected and processed. The first phase of this Ph.D. study focused on selenium distribution and biotransformation in tissues of rainbow trout. Three methods were developed and used to determine total selenium concentration; species' distributions in tissues and Se peptide sequence to determine the possible incorporation of Se into proteins respectively. In the first method total selenium concentration was determined using ICP-MS and the second and third methods using HPLC-ICP-MS/ESI-MS. Total selenium concentrations in trout tissues were determined, and the highest selenium concentrations were found in liver followed by kidney, gills, and muscle. SeMet and SeCys were found to be the major species in all tissues followed by inorganic and other unknown species. To determine the possibility of Se incorporation in to protein, peptide de novo sequencing was carried out. Few selenoprotein were identified using automated de novo sequence and database search. Se species distributions in tissues of beached Pilot whales were studied. Several low molecular weights Se species were identified with selenite pre-dominating other species in most of the adult whales tissues analysed.

Background: Peripartum cardiomyopathy (PPCM) is an incompletely understood disease that causes significant morbidity and mortality in many parts of the world, including Northern Nigeria. The aims of this Thesis were: [1] to determine if selenium deficiency, serum ceruloplasmin and traditional birth practices are risk factors for PPCM, in Kano, Nigeria; [2] to describe the one year survival and left ventricular reverse remodeling (LVRR) in a group of patients with PPCM from three referral hospitals in Kano, Nigeria; [3] to identify potential electrocardiographic (ECG) predictors of PPCM; and [4] to assess right ventricular systolic dysfunction (RVSD) and remodelling in a cohort of PPCM patients in Kano, Nigeria. Materials and Methods: The studies were carried out in 3 referral hospitals in Kano, Nigeria. Study 1: This was a case-control study. Critically low serum selenium concentration was defined as <70μg/L. Study 2: This was a longitudinal study. LVRR was defined as absolute increase in LV ejection fraction (LVEF) by ≥10.0% and decrease in LV end-diastolic dimension indexed to body surface area (LVEDDi) ≤33.0 mm/m2, while recovered LV systolic function as LVEF ≥55%, at 12 months follow-up. Study 3: This was a case-control study. Logistic regression models and a risk score were developed to determine ECG predictors of PPCM. Study 4: This was a longitudinal study and patients were followed up for 12 months. RVSD was defined as the presence of either tricuspid annular plane systolic excursion (TAPSE) <16mm or peak systolic wave (S’) tissue Doppler velocity of RV free wall <10cm/s. Recovery of RV systolic function was defined as an improvement of reduced TAPSE to ≥16mm or S’ to ≥10cm/s, without falling to reduced levels again, during follow-up. Results: Study 1: Total of 39 PPCM patients and 50 controls were consecutively recruited after satisfying the inclusion criteria. Mean serum selenium in patients (61.7±14.9μg/L) was significantly lower than in controls (118.4±45.6μg/L) (p<0.001). The prevalence of serum selenium <70μg/L was significantly higher among patients (76.9%) than controls (22.0%) (p<0.001). The mean ceruloplasmin and prevalence of socio-economic indices, multiparity, pregnancy-induced hypertension, obesity and twin pregnancy were not different between the groups (p>0.05). Logistic regression showed that rural residency significantly increased the odds for serum selenium <70μg/L by 2.773 fold (p=0.037). Study 2: A total of 33 patients were followed-up. Of the 17 survivors at 12 months, 8 patients (47.1%) satisfied the criteria for LVRR, of whom 5 (29.4%) had recovered LV systolic function, but LVRR was not predicted by any variable in the regression models. The prevalence of normal LV diastolic function increased from 11.1% at baseline to 35.3% at twelve months (p=0.02). At one year follow-up, 41.4% of patients had died (two thirds of them within the first 6 months), but mortality wasn’t predicted by any variable including LVRR. Study 3: A total of 54 PPCM and 77 controls were studied. A rise in heart rate by 1 beat/minute increased the odds of PPCM by 6.4% (p=0.001), while presence of ST-T-wave changes increased the odds of PPCM by 12.06 fold (p<0.001). In patients, QRS duration modestly correlated (r=0.4; p<0.003) with LV dimensions and end-systolic volume index (LVESVI), and was responsible for 19.9% of the variability of the latter (R2 = 0.199; p=0.003). A risk score of ≥2 had a sensitivity of 85.2%, specificity of 64.9%, negative predictive value of 86.2% and area under the curve of 83.8% (p<0.0001) for potentially predicting PPCM. Study 4: A total of 45 patients were studied. RV systolic function recovery occurred in a total of 8 patients (8/45; 17.8%), of whom 6 (75.0%) recovered in 6 months after diagnosis. The prevalence of RVSD fell from 71.1% at baseline to 36.4% at 6 months (p=0.007) and 18.8% at one year (p=0.0008 vs baseline; p=0.41 vs 6 month). Although 83.3% of the deceased had RVSD, it didn’t predict mortality in the regression models (p>0.05). Conclusion: These studies have shown that selenium deficiency seems to be a risk factor for PPCM in Kano, Nigeria, related to rural residency. However, serum ceruloplasmin, customary birth practices and some other characteristics were not associated with PPCM in the study area. They have also shown that PPCM patients had modest LVRR but high mortality at one year. In addition, using the ECG risk score could help to streamline the diagnosis of PPCM with significant accuracy, prior to confirmatory investigations in postpartum women. Finally, RVSD and reverse remodelling were common in Nigerians with PPCM, in whom the first 6 months after diagnosis seem to be critical for RV recovery and survival. / Summary

Epidemiological evidence indicates that selenium supplementation may increase risk for ocular hypertension and glaucoma. The purpose of this project was to determine the effects of selenium on the conventional "trabecular" aqueous outflow pathway, a likely site of pathology for glaucoma. Human trabecular meshwork (HTM) cells and human umbilical vein endothelial cells (HUVECs) were treated with selenium (MSeA) at or near physiologically relevant concentrations. Selenium uptake by cells was monitored using mass spectrometry. While detectible changes in intracellular selenium were observed after exposure to 1-10 uM MSeA for 24 hours, the majority remained in the conditioned medium. The high concentrations of extracellular selenium we observed raised the possibility that selenium has an extracellular target.To investigate the role of selenium in extracellular matrix turnover, I examined alterations in protein secretion and intracellular signaling. MSeA treatment (5-10 uM) led to a significant decrease in the secretion of matrix metalloproteinase -2 and its inhibitor after 6-24 hours and to a dose-dependent decrease in kinase signaling. Later, I investigated the possibility that integrins are an extracellular target of selenium by monitoring morphological changes in HTM cells and by treating them with divalent cations. MSeA stimulated morphological changes consistent with a decrease in integrin function. These occurred before (less than 3 hours) alterations in protein secretion and intracellular signaling (3-6 hours). Zinc treatment prevented MSeA-mediated alterations in protein secretion and changes in cell-matrix adhesion.Finally markers of HTM cell homeostasis were examined. MSeA treatment (5 uM) led to a 60% decrease in protein synthesis after 3 hours and a 60% reduction in protein secretion, without causing significant alterations in cell viability and total ATP. To assess the physiological relevance of my results, anterior segments were perfused with MSeA to determine its effects on aqueous outflow facility. Preliminary results suggest that MSeA leads to a decrease in outflow facility.The combination of MSeA-induced decreases in several indicators of HTM cell homeostasis (without adversely effects on cell viability at physiologically relevant doses) and decreases in outflow facility provide a possible mechanism for selenium-associated ocular hypertension.

Introduction: It has been reported that the principal source of exposure for humans to inorganic arsenic (As) comes from drinking water. It is known that selenium (Se) competes with the reductive metabolism and methylation of As and Se compete for the availability of glutathione. The overarching goal of this dissertation research is to assess relationships between arsenic intake from water and other fluids with urinary arsenic output and then to assess how urinary arsenic output is modified by selenium exposure. Methods: Households in the Binational Arsenic Exposure Survey (BAsES) were selected for their varying groundwater arsenic concentrations. A first morning urine void and water samples from all household drinking sources were collected for As quantification. Relationships were examined between various urinary arsenic biomarkers and estimated arsenic exposures. The association between urinary arsenic biomarkers and dietary intake and urinary output of selenium was also evaluated. Results: Arizonans reported consuming 18.5 mL/kg-day of water and 34.3 mL/kg-day from all fluids. In contrast, participants from Mexico reported 3.5 mL/kg-day of water and 12.3 mL/kg-day from all fluids. Median urinary inorganic As concentration among Arizona participants (ranging from 1.2 to 2.0 µg/L) was lower than among participants from Mexico (range 2.5 to 6.2 µg/L). Estimated arsenic intake from drinking water was associated with urinary total arsenic concentration (p<0.001), urinary inorganic arsenic concentration (p<0.001), and urinary sum of species (p<0.001). Urinary arsenic concentrations increased between 7% and 12% for each one percent increase in arsenic consumed from drinking water. No statistically significant relationships were seen between urinary methylated arsenic biomarkers with either dietary intake of selenium or the urinary selenium concentration. Conclusion: Water was the primary contributor to total fluid intake among Arizonans while Mexico participants primarily consumed carbonated beverages. Arsenic intake from water was significantly associated with urinary arsenic output; however, the concentration of arsenic consumed explained a small fraction of urinary arsenic levels. While selenium can biologically interact with arsenic in the liver, no relationship between urinary arsenic biomarkers were identified with either dietary intake of selenium or urinary output of selenium.

Liver glutathione peroxidase activity is affected by changes in selenium (Se) status. To investigate the effect of Se status on GSH-Px protein we prepared antibodies against rat liver GSH-Px and used them in an ELISA. The immunoreactivity of the anti-GSH-Px antibodies against GSH-Px was both tissue and species specific. When rats were depleted of Se, liver GSH-Px activity decreased exponentially to zero with a half-life of 2.8 d. Liver GSH-Px protein also decreased exponentially, but not to zero, with a longer half-life of 5.2 d. Dietary repletion of Se-deficient rats with 0.5 mg Se/kg diet increased GSH-Px protein and activity after 1 d. After 14 d of repletion the levels of GSH-Px protein and activity had plateaued at the levels present in Se-adequate rats. When Se-deficient rats were injected with 15 or 60 ug Se, only rats injected with 60 ug Se and killed 24 h later showed an increase in GSH-Px protein and activity. These results suggest that when Se is limiting, GSH-Px protein and GSH-Px activity are coordinately regulated by the available Se, but in Se-adequacy homeostatic processes control the level of GSH-Px.

The effects of dietary methionine (Met) on the utilization of selenium (Se) from stored tissue Se and dietary selenomethionine (SeMet) for glutathione peroxidase (GSH-Px) synthesis were studied in male rats. Plasma, liver and muscle Se significantly increased when rats were fed 0.5 mg Se/kg diet as SeMet in a Met-deficient diet for 21 d, whereas tissue GSH-Px activities decreased 43-50% during the SeMet supplementation period, suggesting that Se is deposited as SeMet in general body proteins. By calculation, a significant lower percentage of Se was associated with GSH-Px in Met-deficient as compared to Met-supplemented rats. Dietary Met supplementation increased the incorporation of 75Se from 75SeMet into specific rat selenoproteins in addition to liver GSH-Px. Overall, these results suggest that intact SeMet is preferentially incorporated non-specifically into general body proteins in Met-deficient rats, whereas with supplemental Met, more SeMet is degraded and the released Se used for specific selenoprotein synthesis. (Abstract shortened with permission of author.)

為滿足人類對微量元素硒的需求，在本研究中，我們進一步完善優化利用少硒化肥強化水稻的生物農業性狀，並且全面地檢測了富硒稻米中硒的生物有效性和生物利用度。首先，我們發現低濃度的亞硒酸鈉（2毫克/升）在提高水稻幼苗生長方面有顯著的成效。通過抽穗後葉面噴施亞硒酸鈉生產富硒稻米及調控低量的亞硒酸鈉（10.5克硒/公頃）能顯著增加水稻籽粒中硒含量高達51倍；同時，水稻產量也上調了1.24倍。此外，通過硒形態分析、體外胃腸消化和抗氧化實驗來評估，在富硒稻米中，硒的主要富集形態是硒代蛋氨酸；同時，富硒稻米具有明顯較高的抗氧化生物活性。這種富硒大米在人類補硒方面具有巨大潛力。 / 硒對植物生長的作用有兩方面，既有有利作用又有毒副作用。水稻種植應用低濃度的亞硒酸鈉能促進生長，而較高濃度的亞硒酸鈉則抑制生長。為詳細解釋這種兩面性影響機制，我們應用二維凝膠電泳（2-DE）結合基質輔助鐳射解吸離子化-飛行時間質譜（MALDI-TOF/TOF MS）進行蛋白質組學研究。將硒處理組與對照組水稻幼苗之間的凝膠圖像進行比較，確定了莖葉和根中分別有66和97個差異表達的蛋白質。基因聚類分析顯示，水稻的中心代謝，光合作用和氧化還原平衡高度受硒處理影響。低硒處理（2和6毫克/升亞硒酸鈉）啟動抗氧化系統，增強光合作用和初級代謝。而較高的硒處理（10毫克/升的亞硒酸鈉）則抑制光合作用和初級代謝。此項研究在未來生產富硒水稻方面具有指導性意義。 / 為了更好地瞭解水稻穀粒中硒富集的生物機制，我們應用2-DE結合MALDI-TOF/TOF MS及1-DE結合傅裏葉變換離子迴旋共振質譜（FTMS）進行了水稻穀粒的蛋白質組學研究。這項研究提供了最全面的稻米穀粒蛋白質表達圖譜。通過硒處理和對照之間的比較，62和250個差異表達的蛋白質分別被雙電離飛行時間質譜和傅裏葉變換質譜所鑒定。通過基因功能分類，在成熟的稻穀中，硫代謝，碳代謝，細胞的氧化還原調控，和種子的營養儲存過程中涉及的蛋白質受到硒富集的高度影響。此外，有6個蛋白被檢測具有含硒氨基酸片斷，這是高等植物中含硒蛋白的首次鑒定。 / 富硒水稻的基因工程能提高人類的補硒預期。因此，為獲得可以應用於基因工程改造的合適的水稻基因，我們通過在經典模型植物擬南芥中過表達水稻O-乙醯絲氨酸硫解酶（OASTL）的基因，包括在胞漿中表達的OASTLA基因、在質體中表的OASTLB基因和線粒體中表達的OASTLC基因，用以研究這些基因在轉基因植株中對硒富集的影響。在不同硒濃度處理下，與野生型植物相比，此三個基因均表達顯著提高轉基因植物中的硒含量。即時定量反轉錄PCR分析結果顯示，由於過表達的水稻OASTL基因，硒同化的整個代謝途徑被啟動，尤其是與半胱氨酸和蛋氨酸合成有關的基因被啟動，這可能就是引起更多的硒富集在轉基因植物裏的原因。此外，過表達水稻OASTL基因也啟動穀胱甘肽還原酶，這可能增強富硒轉基因植物的抗氧化系統從而提高抗逆性並增加產量。因此，OASTL基因在基因工程生產富硒稻米方面具有重要潛在價值。 / To fulfill the natural human needs of selenium (Se), I further improved the agronomic biofortification of rice (Oryza sativa) with less Se fertilizers and comprehensively evaluated Se bioaccessibility and bioavailability in the Se-enriched rice. Se-enriched rice grains were prepared by foliar application of selenite after rice heading. As compared with control, low amount of sodium selenite (10.5 g Se/ha) significantly increased Se content in rice grains by up to 51 times; at the same time, rice yield was also up-regulated by up to 1.24 times. Furthermore, by Se speciation analysis, in vitro gastrointestinal digestion and antioxidant assays, the Se-enriched rice grains contain readily absorbable selenomethionine as the major Se species and have significantly higher antioxidant bioactivities. This Se-enriched rice has enormous potential for Se supplementation in humans. / Se shows both beneficial and toxic effects on plant growth. Treatments with lower concentrations of sodium selenite enhanced the growth of rice seedlings, whereas higher concentrations of sodium selenite repressed seedling growth. To reveal the regulatory mechanisms underlying these effects, a comparative proteomics study combining 2-dimensional gel electrophoresis (2-DE) and matrix assisted laser desorption ionization (MALDI)-tandem time of flight (TOF/TOF) mass spectrometry (MS) were performed. By comparison of gel images between Se treatments and control, 66 and 97 differentially expressed proteins were identified in shoot and root, respectively. Gene Ontology and Clustering analysis reveal primary metabolism, photosynthesis and redox homeostasis are the most highly affected biological processes by Se treatments. Lower Se treatments (2 and 6 mg/L sodium selenite) activated antioxidative system, enhanced photosynthesis and primary metabolism. However, higher Se treatment (10 mg/L sodium selenite) damaged photosynthesis apparatus, inhibited photosynthesis and primary metabolism. This study provided novel insights into Se response in rice at the proteome level, which are expected to be highly useful for dissecting the Se response pathways in higher plants and for producing of Se enriched rice cultivars in the future. / To better understand the regulatory mechanism under Se accumulation in rice grains, a comparative proteomics study using 2-DE coupled MALDI-TOF/TOF MS and 1-dimensional gel electrophoresis (1-DE) coupled liquid chromatography (LC) - Fourier transform-ion cyclotron resonance (FT-ICR) MS were carried out. By comparison of Se treatments and control, 62 and 250 differentially expressed proteins were identified by 2-DE/MALDI-TOF/TOF MS and 1-DE/LC-FT-ICR MS, respectively. By gene functional classification, proteins involved in the processes of sulfur metabolism, carbon metabolism, cell redox regulation, and seed nutritional storage were the most highly affected by Se accumulation in mature rice grains. In addition, there were 6 proteins identified to contain fragments of selenoamino acid modification, which was the first identification of selenoproteins in higher plants. / Genetic engineering of Se-enriched rice will have important implications for human health in Se deficient regions. Therefore, to acquire appropriate rice genes as candidates for bioengineering of Se-enriched rice cultivars, I overexpressed three of the rice O-Acetylserine(thiol)lyase (OASTL) genes encoding cytosolic OASTLA, plastic OASTLB and mitochondrial OASTLC, individually in the model plant Arabidopsis (Arabidopsis thaliana) to characterize the effects of Se accumulation in transgenic plants. The results showed that compared to the wild type plants, overexpression of all these genes significantly increased Se content in transgenic plants under treatments of different selenite concentrations. By real-time RT-PCR analysis, I found that the whole metabolic pathway of selenite assimilation was activated by overexpressing rice OASTL genes, especially the genes involved in cysteine and methionine biosynthesis, which may give rise to more Se accumulation in the transgenics. In addition, overexpression of rice OASTL genes also activated the antioxidative system by activating the glutathione reductase, which may be responsible for the increased biomass of Se-enriched transgenic plants. Therefore, OASTL genes could be good candidate for the future genetic engineering of Se-enriched rice. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Wang, Yudong. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 149-159). / Electronic reproduction. 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Available via World Wide Web. / Abstract also in Chinese. / Declaration of Originality --- p.i / Acknowledgments --- p.ii / Abstract --- p.iii / 摘要 (Abstract in Chinese) --- p.v / List of Abbreviations --- p.vii / List of Figures --- p.x / List of Tables --- p.xi / Chapter Chapter 1: --- Introduction --- p.1 / Chapter 1.1. --- Implications of Se for human health and its metabolism in plants --- p.1 / Chapter 1.2. --- Systemic study of Se metabolism and regulation in rice --- p.3 / Chapter 1.3. --- Candidate genes for genetic engineering of Se-enriched rice --- p.4 / Chapter 1.4. --- Objectives of this project --- p.7 / Chapter Chapter 2: --- Generation of selenium-enriched rice with enhanced grain yield, selenium content and bioavailability through fertilization with selenite --- p.9 / Chapter 2.1. --- Introduction --- p.9 / Chapter 2.2. --- Materials and methods --- p.12 / Chapter 2.2.1. --- Reagents --- p.12 / Chapter 2.2.2. --- Plant materials and growth conditions --- p.12 / Chapter 2.2.3. --- Se speciation analysis --- p.14 / Chapter 2.2.4. --- Antioxidant assays --- p.16 / Chapter 2.2.5. --- Data analysis --- p.18 / Chapter 2.3. --- Results and discussion --- p.18 / Chapter 2.3.1. --- Effects of fertilization of selenite on the growth and Se content of rice seedlings --- p.18 / Chapter 2.3.2. --- Effects of fertilization of selenite on the antioxidant activity of rice seedlings --- p.19 / Chapter 2.3.3. --- Effect of fertilization of selenite on Se content in rice products --- p.20 / Chapter 2.3.4. --- Effect of fertilization of selenite on rice yield --- p.21 / Chapter 2.3.5. --- Analysis of Se bioaccessibility in Se-enriched rice grains --- p.22 / Chapter 2.3.6. --- Analysis of Se bioavailability in Se-enriched rice grains --- p.23 / Chapter 2.4. --- Conclusion --- p.24 / Chapter Chapter 3: --- Proteomics analysis reveals multiple regulatory mechanisms in response to selenium in rice --- p.37 / Chapter 3.1. --- Introduction --- p.37 / Chapter 3.2. --- Materials and methods --- p.39 / Chapter 3.2.1. --- Plant materials and growth conditions --- p.39 / Chapter 3.2.2. --- Physiological measurements --- p.40 / Chapter 3.2.3. --- Total protein extraction --- p.40 / Chapter 3.2.4. --- 2-DE separation, gel staining and image analysis --- p.40 / Chapter 3.2.5. --- Trypsin digestion, mass spectrometry and protein identification --- p.41 / Chapter 3.2.6. --- Protein functional classification and hierarchical cluster analysis --- p.43 / Chapter 3.2.7. --- Statistical analysis --- p.43 / Chapter 3.3. --- Results and discussion --- p.43 / Chapter 3.3.1. --- Effects of Se on rice seedlings --- p.43 / Chapter 3.3.2. --- Effects of Se on shoot and root proteomes of rice seedlings --- p.44 / Chapter 3.3.3. --- Gene ontology analysis of Se-responsive proteins --- p.46 / Chapter 3.3.4. --- Clustering analysis revealed the dynamics of functional protein groups under Se treatment --- p.47 / Chapter 3.3.5. --- Se treatment induced redox and stress related proteins --- p.48 / Chapter 3.3.6. --- Se-responsive proteins preferentially associated with primary metabolism and photosynthesis --- p.50 / Chapter 3.3.7. --- Post translational modifications involved in plant Se-response --- p.52 / Chapter 3.4. --- Conclusion --- p.53 / Chapter Chapter 4: --- Comparative proteomics analysis of selenium responses in selenium-enriched rice grains --- p.79 / Chapter 4.1. --- Introduction --- p.79 / Chapter 4.2. --- Materials and methods --- p.82 / Chapter 4.2.1. --- Plant materials and growth conditions --- p.82 / Chapter 4.2.2. --- Total protein extraction --- p.83 / Chapter 4.2.3. --- 2-DE separation, gel staining and image analysis --- p.83 / Chapter 4.2.4. --- Trypsin digestion, mass spectrometry and protein identification --- p.84 / Chapter 4.2.5. --- Preparative SDS-PAGE separation and trypsin digestion --- p.85 / Chapter 4.2.6. --- NanoLC-FT-ICR MS and protein identification --- p.86 / Chapter 4.2.7. --- Label-free quantitation of identified proteins --- p.87 / Chapter 4.2.8. --- Functional classification of Se-responsive proteins --- p.87 / Chapter 4.3. --- Results and discussion --- p.88 / Chapter 4.3.1. --- Effects of foliar application of selenite in rice grain production --- p.88 / Chapter 4.3.2. --- 2-DE/MALDI-TOF/TOF MS analysis of Se-enriched rice grains --- p.89 / Chapter 4.3.3. --- Label-free 1-DE/LC-FT-ICR-MS analysis of Se-enriched rice grains --- p.89 / Chapter 4.3.4. --- Gene ontology analysis of rice grain proteome and Se-responsive proteins --- p.90 / Chapter 4.3.5. --- Sulfur metabolism were highly repressed in Se-enriched rice --- p.91 / Chapter 4.3.6. --- Proteins involved in redox regulation were induced in Se-enriched rice --- p.92 / Chapter 4.3.7. --- Se-responsive proteins are preferentially associated with carbon metabolism --- p.93 / Chapter 4.3.8. --- Proteins involved in seed nutritional storage --- p.95 / Chapter 4.4. --- Conclusion --- p.97 / Chapter Chapter 5: --- Overexpressing rice O-Acetylserine(thiol)lyase Genes Enhances Selenium Accumulation in Arabidopsis --- p.123 / Chapter 5.1. --- Introduction --- p.123 / Chapter 5.2. --- Materials and methods --- p.126 / Chapter 5.2.1. --- DNA constructs --- p.126 / Chapter 5.2.2. --- Transient gene expression and subcellular localization --- p.127 / Chapter 5.2.3. --- Arabidopsis plant transformation and growth --- p.127 / Chapter 5.2.4. --- Selenium treatment and physiological measurements --- p.127 / Chapter 5.2.5. --- Total Se content assay --- p.127 / Chapter 5.2.6. --- RT-PCR analysis --- p.128 / Chapter 5.3. --- Results and discussion --- p.128 / Chapter 5.3.1. --- Phenotypes of OASTL-transgenic Arabidopsis --- p.128 / Chapter 5.3.2. --- Se accumulated in OASTL-transgenic Arabidopsis under Se treatment --- p.129 / Chapter 5.3.3. --- Overexpression of rice OASTL genes activated Se assimilation pathways --- p.130 / Chapter 5.3.4. --- ATSAT genes were highly expressed in OASTL-transgenics --- p.132 / Chapter 5.3.5. --- Overexpression of rice OASTL genes activated the antioxidative system --- p.133 / Chapter 5.3.6. --- Methionine synthesis was enhanced in OASTL-transgenics --- p.134 / Chapter 5.4. --- Conclusion --- p.134 / Chapter Chapter 6: --- Conclusion --- p.146 / References --- p.149 / Chapter Appendix I: --- Publications --- p.160