Thesis (Ph. D.)--Oregon State University, 2007. / Printout. Includes bibliographical references (leaves 104-116). Also available on the World Wide Web.

Thesis ( Ph.D.)--University of Delaware, 2005 . / Principal faculty advisor: D. Janine Sherrier, Dept. of Plant and Soil Sciences. Includes bibliographical references.

Thesis (M.Sc. (Biochemistry, Microbiology & Biotechnology)) - Rhodes University, 2007.

The production, processing and marketing of sustainable and affordable food involve complex phenomena that affect the lives of millions of people worldwide. Due to the rapid growth of the world's population, the provision of food is a significant challenge for the agrifood industry and policy makers, as this is strictly interlinked with climate change and public health interventions. The overall aim of this research was to contribute to delivering nutritious food to feed an increasing unhealthy population. High-protein crops that can be grown sustainably in high latitude countries, including Scotland, could provide a healthy alternative to partially replace our dependency on unsustainable protein-rich foodstuffs. These include meat, the production of which is responsible for a substantial share of food-related environmental pressures. For this reason, green pea, lupin, fava bean, hemp and buckwheat were selected and analysed for their macro- and micro- nutrient content, as well as their phytochemical profile and compared to a red meat- and wheat-based meal in a human intervention trial. The crops studied were high in protein (ranging from 20 to 43% in buckwheat and lupin, respectively) and fibre (up to 25% in hemp) and also found to contain a diverse range of phenolic compounds, considered to participate in the prevention of diet-related disorders. As fava bean contained relatively high amounts of protein (approx. 22% w/w), protein fractions were isolated and further investigated to understand the contribution of the phytochemical components in terms of protein functionality and oxidative stability. Since fava bean protein isolates showed promising food applications, they were used to develop meat patties. The addition of fava bean proteins significantly decreased lipid and protein oxidation of the processed products. The results of this research could encourage a higher consumption of plant-based products, which would be favourable from both a health and environmental perspective.

Improving protein quality and identifying superior inbreds and hybrids are significant challenges in commercial maize breeding programs. These two problems were addressed in separate studies on inbreds and hybrids from a complete diallel cross of 12 elite proprietary inbred lines of maize evaluated in field trials in two locations for two years. One of the inbreds (WI) was a novel source of high quality protein obtained from Wilson Seeds, Inc. in Harlan, Iowa. In the first study, diallel analyses were used to study combining ability and types of gene action important in the inheritance of protein content, grain yield, grain moisture at harvest, time to silk, kernel hardness, and density. General combining ability (GCA) and specific combining ability (SCA) effects were highly significant for all traits indicating presence of both additive and non-additive effects, respectively. Reciprocal effects (REe), often assumed to be absent in maize diallel studies, were significant for grain yield and protein concentration, suggesting that choice of female parent may be important for these traits. Ratios expressing the relative importance of GCA and SCA indicated that protein concentration is controlled primarily by additive gene action. In the second study, restriction fragment length polymorphism (RFLP) data were obtained for the 12 inbreds using 42 genomic clones each with four restriction enzymes. Modified Roger's distances were calculated and used in cluster analyses for heterotic grouping of the inbreds. Two measures of level of heterozygosity and hybrid value were evaluated as means of predicting Fl performance of hybrids in the complete diallel set of hybrids and in groups of hybrids representing crosses between and within heterotic groups. Results from this study confirm those of previous investigations with respect to prediction of hybrid performance when comparable groupings of crosses between related and unrelated lines were evaluated. This study further indicates that RFLPs may also be useful for prediction of hybrid performance in situations typical of early generations of many maize breeding programs where recombinant inbreds are testcrossed to a common tester inbred. / Ph. D.

A fifteen day balance study was conducted to test the value of two diets providing 24 and 16 gm. of plant-protein daily and to approach the approximate minimum protein requirement using plant proteins for a 23-month old Chinese girl weighing 12 kg. The diets were supplemented to bring nutrients to the NRC recommended allowances except for protein and calories. Essential amino acids were planned to approximate the FAO provisional pattern. Nitrogen determinations were made on food, urine and feces. Food composites for each diet were analyzed for nine of the essential amino acids using a Beckman/Spince Model 120 Amino Acid Analyzer. Tryptophan was determined by microbiological assay. Nitrogen in the diets averaged 3.96 and 2.69 gm. respectively for the two protein levels of 24 and 16 gm. daily with retention of 0.33 gm. daily for all periods, or 0.48 gm. and 0.44 gm. for the test periods. The essential amino acids as analyzed represent 59 per cent and 34 per eent of the total protein in the moderate and low protein diets. The analyzed amino acid patterns were as follows: tryptophan 1.0 and 1.0; threonine 2.8 and 2.2; isoleucine 5.8 and 3.5; leucine 9.4 and 5.5; lysine 3.1 and 3.7; methionine 1.2 and 0.8; phenylalanine 6.1 and 3.6; valine 6.7 and 3.9; arginine 4.4 and 4.5; and histidine 1.3 and 2.0. A positive nitrogen retention of better than 0.4 gm. daily (0.033 gm./kg.) was obtained with daily nitrogen intake of 0.33 and 0.22 gm. per kg. of body weight for this 23-month old girl with approximately 90 and 84 calories per kg. per day. / Master of Science

Parkash Amarender. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 158-172). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Table of contents --- p.ii / Abstract --- p.xi / 撮要 --- p.xiv / List of Abbreviations --- p.xvi / List of Tables --- p.xvii / List of Figures --- p.xix / Chapter CHAPTER 1. --- INTRODUCTION / Chapter 1.1 --- Ribosome-inactivating proteins (RIPs) --- p.3 / Chapter 1.2 --- General Properties of RIPs --- p.5 / Chapter 1.2.1 --- Structure --- p.5 / Chapter 1.2.1.1 --- Type I and Type II RIPs --- p.5 / Chapter 1.2.1.2 --- Small RIPs --- p.10 / Chapter 1.2.2 --- Distribution --- p.12 / Chapter 1.2.3 --- Physicochemical properties --- p.15 / Chapter 1.3 --- Enzymatic activities of RIPs --- p.17 / Chapter 1.3.1 --- N-glycosidase activity --- p.17 / Chapter 1.3.2 --- Polynucleotide:adenosine glycosidase activity --- p.21 / Chapter 1.3.3 --- Ribonuclease (RNase) activity --- p.24 / Chapter 1.3.4 --- Deoxyribonucleolytic (DNase) activity --- p.25 / Chapter 1.3.5 --- Multiple depurination --- p.26 / Chapter 1.3.6 --- Inhibition of protein synthesis --- p.27 / Chapter 1.4 --- Biological activities of RIPs --- p.29 / Chapter 1.4.1 --- Interaction of ribosome-inactivating proteins with cells --- p.29 / Chapter 1.4.1.1 --- Internalization of type 1 ribosome-inactivating proteins --- p.29 / Chapter 1.4.1.2 --- Internalization of type 2 ribosome-inactivating proteins --- p.32 / Chapter 1.4.2 --- Effects on laboratory animals --- p.33 / Chapter 1.4.3 --- Immunosuppressive activity --- p.33 / Chapter 1.4.4 --- Abortifacient activity --- p.34 / Chapter 1.4.5 --- Antiviral activity --- p.35 / Chapter 1.5 --- Physiological roles of RIPs --- p.37 / Chapter 1.6 --- Applications of RIPs --- p.39 / Chapter 1.6.1 --- Possible uses in experimental and clinical medicine --- p.39 / Chapter 1.6.1.1 --- Anti-tumor therapy --- p.40 / Chapter 1.6.1.2 --- Immune disorders --- p.42 / Chapter 1.6.1.3 --- Neuroscience research --- p.43 / Chapter 1.6.2 --- Applications in agriculture --- p.44 / Chapter 1.7 --- Arginine/Glutamate Rich Polypeptides (AGRPs) --- p.46 / Chapter 1.8 --- Objectives of the present study --- p.48 / Chapter 1.8.1 --- Rationale of the study --- p.48 / Chapter 1.8.2 --- Outline of the thesis --- p.50 / Chapter Chapter 2 --- Materials and methods / Chapter 2.1 --- Introduction --- p.52 / Chapter 2.2 --- Materials and methods --- p.54 / Chapter 2.2.1 --- Materials --- p.54 / Chapter 2.2.2 --- Preparation of crude extract --- p.55 / Chapter 2.2.3 --- Purification of proteins --- p.55 / Chapter 2.2.4 --- Molecular weight determination with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) --- p.61 / Chapter 2.2.5 --- Protein determination --- p.64 / Chapter 2.2.6 --- N-terminal amino acid sequence --- p.64 / Chapter 2.2.7 --- Preparation of rabbit reticulocyte lysate --- p.65 / Chapter 2.2.8 --- Assay for cell-free protein synthesis- inhibiting activity --- p.65 / Chapter 2.2.9 --- Assay for N-glycosidase activity --- p.66 / Chapter 2.2.10 --- Assay for ribonuclease activity --- p.70 / Chapter 2.2.11 --- Assay for antifungal activity --- p.71 / Chapter 2.2.12 --- Assay for dehydrogenase activity --- p.71 / Chapter Chapter 3 --- Purification and characterization of proteins from their respective sources. / Chapter 3.1. --- Purification and Characterization of Hispidin from Hairy melon (Benincasa hispida var. chieh-qua) / Chapter 3.1.1. --- Introduction --- p.73 / Chapter 3.1.2. --- Results --- p.76 / Chapter 3.1.2.1. --- Purification --- p.78 / Chapter 3.1.2.2. --- Molecular weight determination --- p.84 / Chapter 3.1.2.3. --- N-terminal amino acid sequence --- p.85 / Chapter 3.1.2.4. --- Assay for cell-free protein synthesis-inhibiting activity --- p.86 / Chapter 3.1.2.5. --- Assay for N-glycosidase activity --- p.87 / Chapter 3.1.2.6. --- Assay for ribonuclease activity --- p.88 / Chapter 3.1.2.7. --- Assay for dihydrodiol dehydrogenase activity --- p.88 / Chapter 3.1.2.8. --- Assay for antifungal activity --- p.89 / Chapter 3.1.2.9. --- "Assessment of purity, yield and activity" --- p.91 / Chapter 3.1.3. --- Discussion --- p.92 / Chapter 3.2. --- Purification and Characterization of Momorchin from Dried Bitter Gourd (Momordica charantia) Seeds / Chapter 3.2.1. --- Introduction --- p.95 / Chapter 3.2.2. --- Results --- p.99 / Chapter 3.2.2.1. --- Purification --- p.100 / Chapter 3.2.2.2. --- Molecular weight determination --- p.103 / Chapter 3.2.2.3. --- N-terminal amino acid sequence --- p.104 / Chapter 3.2.2.4. --- Assay for cell-free protein synthesis- inhibiting activity --- p.105 / Chapter 3.2.2.5. --- Assay for ribonuclease activity --- p.105 / Chapter 3.2.2.6. --- Assay for N-glycosidase activity --- p.106 / Chapter 3.2.2.7. --- "Assessment of purity, yield and activity" --- p.107 / Chapter 3.2.3. --- Discussion --- p.108 / Chapter 3.3.3. --- Purification and Characterization of Luffacylin from Sponge Gourd (Luffa cylindrica) / Chapter 3.3.1. --- Introduction --- p.110 / Chapter 3.3.2. --- Results --- p.113 / Chapter 3.3.2.1. --- Purification --- p.115 / Chapter 3.3.2.2. --- Molecular weight determination --- p.119 / Chapter 3.3.2.3. --- N-terminal amino acid sequencing --- p.120 / Chapter 3.3.2.4. --- Assay for cell-free protein synthesis- inhibiting activity --- p.121 / Chapter 3.3.2.5. --- Assay for ribonuclease activity --- p.121 / Chapter 3.3.2.6. --- Assay for N-glycosidase activity --- p.122 / Chapter 3.3.2.7. --- Assay for antifungal activity --- p.123 / Chapter 3.3.2.8. --- "Assessment of purity, activity and yield" --- p.124 / Chapter 3.3.3. --- Discussion --- p.125 / Chapter 3.4. --- Purification and Characterization of α and β Benincasin from fresh Winter Melon {Benincasa hispida var. dong-gua) Seeds / Chapter 3.4.1. --- Introduction --- p.127 / Chapter 3.4.2. --- Results --- p.129 / Chapter 3.4.2.1. --- Purification --- p.130 / Chapter 3.4.2.2. --- Molecular weight determination --- p.135 / Chapter 3.4.2.3. --- N-terminal amino acid sequence --- p.136 / Chapter 3.4.2.4. --- Assay for cell-free protein synthesis- inhibiting activity --- p.137 / Chapter 3.4.2.5. --- Assay for ribonuclease activity --- p.137 / Chapter 3.4.2.6. --- Assay for antifungal activity --- p.138 / Chapter 3.4.2.7. --- "Assessment of purity, activity and yield" --- p.140 / Chapter 3.4.3. --- Discussion --- p.141 / Chapter 3.5. --- Purification and characterization of Moschins from Pumpkin (Cucurbita moschata) Seeds / Chapter 3.5.1. --- Introduction --- p.143 / Chapter 3.5.2. --- Results --- p.145 / Chapter 3.5.2.1. --- Purification --- p.146 / Chapter 3.5.2.2. --- Molecular weight determination --- p.149 / Chapter 3.5.2.3. --- N-terminal amino acid sequence --- p.150 / Chapter 3.5.2.4. --- Assay for cell-free protein synthesis- inhibiting activity --- p.151 / Chapter 3.5.2.5. --- Assay for ribonuclease activity --- p.151 / Chapter 3.5.2.6. --- "Assessment of purity, activity and yield" --- p.152 / Chapter 3.5.3. --- Discussion --- p.153 / Chapter Chapter 4 --- General Discussion and Conclusion --- p.154 / References --- p.158

Lectins and hemagglutinins are carbohydrate binding proteins present in a diversity of organisms including humans, vertebrate and invertebrate animals, plants, fungi, and bacteria. They are usually the abundant storage proteins in leguminous plants. They display a host of biological activities such as antitumor, antifungal, antiviral, insecticidal, and antibacterial activities. / The biological properties of isolated proteins, including hemagglutinating, antifungal, anti-tumor and HIV-1 reverse transcriptase inhibitory activities, were examined. Their biochemical and biological properties were compared with other purified proteins. / The seeds contain an abundance of proteins, some of which are storage proteins but may play a role of protection from pathogenic microbes and phytophagous insects. Antifungal peptides/proteins, antiviral proteins, ribosome-inactivating proteins, proteinase inhibitors, chitinases, proteinases, and defensins, are some examples of the myriad of seed proteins. The aforementioned proteins are collectively called plant defense proteins in view of their antipathogenic activities. These antifungal proteins exhibit a wide range of molecular masses and amino acid sequences. / Two lectins with potentially exploitable activities were purified from Capparis spinosa seeds and Hibiscus mutabilis seeds, respectively. A hemagglutinin was isolated from Phaselous vulgaris , cultivar "French bean 35", and detailed apoptotic pathway in breast cancer cells, MCF-7 cells, was investigated. A novel dimeric beta-lactoglobulin-like antifungal protein and an antifungal amidase were purified from Passiflora edilus seeds and Peltophorum pterocarpum, respectively. / Lam, Sze Kwan. / Adviser: Tsi Bun Ng. / Source: Dissertation Abstracts International, Volume: 72-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 188-204). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

We have studied the effects of exogenous salt on whole plant and suspension culture cells of the halophytic tomato Lycopersicon pennellii. Under low salt conditions (2.9 dS/M) plants showed enhanced (halophytic) growth (107% of control). At moderate (7.5 dS/M) and high (18.5 dS/M) salt levels, salt stress reduced growth to about 78% and 40% of control respectively. Salt-induced changes in root mRNAs were analyzed via two-dimensional PAGE of cell free translation (CFT) products. We have identified 14 proteins whose levels were enhanced by exogenous salt. One of these proteins was unique to low salt induced halophytic growth. This system allowed for discrimination between proteins up-regulated at all salt levels and those up-regulated only during salt stress induced growth reduction. Ten proteins were identified whose levels were reduced by exogenous salt. Once again, one could identify a subset of proteins whose levels were reduced only under salt stressed conditions. Proteins identified in this study are candidates for roles in growth maintaining stress adaptive metabolism in L.pennellii. These data underscore the complexity of the genetic control of salt metabolism in higher plants. The effects of exogenous salt on protein synthesis and accumulation were studied in suspension cultures of L.pennellii. Two salt levels were applied to the cells. Under low salt conditions (LS, 10 mM), L.pennellii cells showed enhanced (halophytic) growth. Under high salt conditions (HS, 50 mM), the cells showed reduced (salt-stressed) growth. Changes in proteins with time were analyzed by a combination of cell free translation, in vivo labeling and total accumulated protein. In vivo labeling studies showed that the pattern of steady state protein synthesis was disrupted shortly after addition of salt. High salt induced greater disruption in the pattern. Over time, the steady state levels of most proteins shifted back towards those of the unstressed-control. However, the level of several proteins remained altered. Analysis of proteins whose levels increased with exogenous salt showed differences in the response patterns that may allow for discrimination between proteins involved in growth maintaining and stress shock responses.

O produto do gene thi1 de Arabidopsis thaliana está provavelmente envolvido na biossíntese de tiamina (vitamina B1) e na proteção do DNA organelar contra danos. Estudos sobre a biossíntese da tiamina em plantas sugerem uma localização plastidial para este mecanismo, o que está de acordo com a existência de um peptídeo de trânsito cloroplástico (TP) na região N-terminal de THI1. Por outro lado, em leveduras a tiamina é sintetizada em mitocôndrias. Interessantemente, o cDNA de thi1 de A. thaliana complementa uma cepa de levedura com disrupção no gene homólogo thi4. A análise da seqüência de aminoácidos de THI1 revelou a presença de uma região capaz de formar uma estrutura do tipo a-hélice anfifílica, freqüentemente encontrada em preseqüências mitocondriais, localizada logo após o peptídeo de trânsito cloroplástico. O papel desta região na localização da proteína THI1 nas mitocôndrias foi comprovado a partir de ensaios envolvendo construções de genes quiméricos (contendo ou não a putativa seqüência de direcionamento mitocondrial) e um gene repórter (uidA). Estas construções foram introduzidas em plantas de tabaco e a localização da atividade GUS foi determinada nas frações subcelulares das plantas transgênicas. Análise direta da presença de THI1 nas mitocôndrias e cloroplastos de Arabidopsis foi realizada via imunolocalização. Também foram fornecidas evidências que as duas isoformas organelares são codificadas por um único transcrito nuclear. Experimentos de transcrição/tradução in vitro indicaram a ocorrência de dois produtos da tradução a partir de códons de iníciação em fase de leitura. Mutações sítio-específicas na seqüência de thi1 acoplados à experimentos usando a proteína fluorescente verde (GFP) mostraram que a tradução no primeiro AUG determina a localização da proteína nos cloroplastos, enquanto que a tradução no segundo AUG é responsável pelo endereçamento da proteína às mitocôndrias. A análise do contexto para início da tradução revelou que a região em torno do primeiro AUG é mais favorável para a tradução do mRNA de thi1. Além disso, observou-se a presença de uma forte estrutura em "grampo de cabelo" próximo ao segundo códon AUG, indicando um contexto subótimo capaz de interferir na tradução. Estas observações confirmaram os dados obtidos a partir da tradução in vitro na qual a iniciação se dá preferencialmente no primeiro AUG o que pode sugerir uma maior necessidade da proteína nos plastídeos. / Arabidopsis thaliana thi1 gene product is probably involved in both thiamine biosynthesis as well as protection of organellar DNA from damage. Studies of thiamine biosynthesis in plants suggest a plastid location for the pathway, which is in agreement with the predicted THI1 N-terminal chloroplastic transit peptide (TP). On the other hand, thiamine is synthesized in mitochondria in yeast cells. Interestingly, A. thaliana thi1 cDNA complements a yeast strain disrupted for the homologous thi4 gene. Analysis of THI1 amino acid sequence revealed the presence of a putative amphiphilic a-helix, which is typical for mitochondrial presequences, located downstream of the chloroplast transit peptide. The role of this sequence on mitochondrial import has been shown by chimeric gene constructs (carrying or not the putative mitochondrial presequence) and the uidA reporter gene. These constructions have been introduced into tobacco plants and the GUS activity has been measured in subcellular fractions of transgenic plants. Direct analysis of THIp in mitochondria and chloroplasts has been done via ImmunoGold labelling experiments. Additional evidence suggested that the two organellar isoforms were encoded by a single nuclear transcript. In vitro transcription/translation experiments revealed the presence of two translational products by a differential usage of two in-frame translational start codons. Coupling site-specific mutations on THI1 encoding sequence with green fluorescent protein (GFP) gene fusions showed that translation initiation in the first AUG directs translocation of THI1 to plastids. However, when translation initiates from the second AUG THI1 is addressed to mitochondria. Analysis of the translation efficiency of thi1 mRNA revealed that the best context for translation initiation is present at the first AUG. In addition, it has been shown a suboptimal context at the second AUG and a strong stem-and-loop structure which is likely to slow translation. These observation confirm the in vitro translation data in which translation occurs preferentially in the first AUG, what could suggest a higher requirement of the protein in plastids.