Rozprawy doktorskie na temat „Proteins in human nutrition”

Thesis (MNutr)--Stellenbosch University, 2014.
ENGLISH ABSTRACT: Introduction and purpose The aim of the study was to determine the renal handling of a once-off bolus dietary protein load in patients treated for nephroblastoma. Patients who have been managed for nephroblastoma always have suboptimal amounts of kidney tissue as a result of their medical management which includes nephrectomies, chemotherapy and or radiotherapy. Little data are available indicating the extent of renal impairment expected in such patients as a result of their disease and management. The study was to determine whether the use of regular screening tests such as serum urea, creatinine and urine microalbumin, in conjunction with a dietary protein load could help detect early progressive deterioration of kidney function in nephroblastoma patients. Methodology The study was a quantitative non-randomised intervention study in which patients served as their own control before and after a protein load. Thirty-four participants were included in the study. Each participant was provided with a supplemental protein drink providing 2 g/kg body weight of protein. Serum creatinine, urea and urine microalbumin were assessed at baseline and four hours after the intervention. These pre- and post intervention biochemical values were then analysed together with descriptive data relating to the participants, such as age, stage of nephroblastoma, aspects of medical management and the period of time since they had been treated for nephroblastoma, and statistical relationships were assessed. Data were collected from May 2010 to November 2010. Results Descriptive statistics indicated that the mean [± Standard deviation (SD)] age of the population was 92 (± 55) months, the mean age at diagnosis was 41 (± 27) months and the mean age from the diagnosis to the time of the study was 51 (± 53) months. There was a statistically significant increase (p = 0.00) in serum urea post intervention; however, no significant difference was noted between pre- and post intervention serum creatinine and urine microalbumin values. The stages of nephroblastoma failed to show a statistical correlation with the response to the dietary protein bolus load in terms of the difference in pre- and post intervention biochemical data. No statistical correlation was found between post-pubescence and response to the protein load. Similiarly, no statistical correlation could be demonstrated for a longer period between the diagnosis and the time of this study, on the one hand, and the prevalence of high values in the biochemical data, on the other. Conclusion The study was unable to demonstrate statistically that participants managed for nephroblastoma had poor renal handling of a once-off dietary protein load in terms of the objectives specified. The study had limitations including a small population with even smaller subgroups of participants, therefore results of the study need to be interpreted in context to the size of the population.
AFRIKAANSE OPSOMMING: Doel Die doel van die studie was om die renale hantering van ’n eenmalige bolus dieetproteïenlading by pasiënte wat vir nefroblastoom behandel word, te bepaal. Pasiënte wat vir nefroblastoom behandel word, het altyd ‘n subopitmale hoeveelheid nierweefsel as gevolg van hulle mediese behandeling wat nefrektomies, chemoterapie en / of radioterapie insluit. Min data is beskikbaar omtrent die omvang van die nierbelemmering wat in sulke pasiënte verwag word as gevolg van hulle siekte en behandeling. Die studie is uitgevoer om te bepaal of die gebruik van gereelde siftingstoetse soos serum-ureum, kreatinien en mikroalbuminurie, in samewerking met ‘n dieetproteïenlading, kan help om vroeë progressiewe agteruitgang van nierfunksie in nefroblastoom pasiënte, op te spoor. Metodologie Die studie was ‘n kwantitatiewe nie-ewekansige intervensie studie waar pasiënte as hul eie kontrole gedien het voor en na ‘n proteïenlading. Altesaam 34 deelnemers is by die studie betrek. Elke deelnemer het ’n proteïenaanvullingsdrankie ontvang wat 2 gram proteïen per kilogram liggaamsgewig voorsien het. Serumkreatinien, serum-ureum en mikro-albuminurie is op die basislyn sowel as vier uur na die intervensie gemeet. Hierdie biochemiese waardes voor en na die intervensie is daarna saam met beskrywende data van die deelnemers – soos ouderdom, stadium van nefroblastoom, aspekte van mediese behandeling en tydsverloop sedert behandeling vir nefroblastoom – ontleed. Statistiese verwantskappe is vervolgens beoordeel. Data is vanaf Mei 2010 tot November 2010 ingesamel. Resultate Beskrywende statistieke het op ’n gemiddelde [± Standaard afwyking (SA)] populasie-ouderdom van 92 (± 55) maande, ’n gemiddelde diagnose-ouderdom van 41(± 27) maande en ’n gemiddelde ouderdom van 51(± 53) maande vanaf diagnose tot en met die studie gedui. Ná die intervensie is ’n statisties beduidende toename (p = 0.00) in serum-ureum opgemerk, hoewel daar geen beduidende verskil in serumkreatinien en mikro-albuminurie waardes, voor en na behandeling, was nie. Biochemiese data voor en na die intervensie het geen statistiese verwantskap tussen die stadium van nefroblastoom en die reaksie op die dieetproteïenlading getoon nie. Boonop is geen statistiese verwantskap opgemerk tussen post-pubesensie en die reaksie op die proteïenlading, of tussen ’n langer tydsverloop tussen die diagnose en die studie en die voorkoms van hoë waardes in die biochemiese data nie. Gevolgtrekking Wat die studie-doelwitte betref, kon die navorsing nie statisties bewys dat deelnemers wat vir nefroblastoom behandel word, swak renale hantering van ’n eenmalige dieetproteïenlading toon nie. Die beperkinge van die studie sluit ‘n klein populasie met selfs kleiner subgroepe in; die resultate van die studie moet derhalwe in die konteks van die grootte van die populasie, geÏnterpreteer word.

Humans obtain approximately 70% of their dietary protein from plant sources on a global basis. In developing countries, vegetable protein intake is higher than in developed countries (Lusas and Rhee, 1986). Cereals, pulses and oilseeds are not only very important plant foods in the human diet, but also the main components of feeds for livestock, which can be considered as source of dairy products and meat for humans (Lasztity and Hidvegi, 1983). Cereals contribute the major dietary source of carbohydrates, and a substantive source of protein, vitamins, and minerals. Oilseeds are one of the main sources of lipid, and pulses supply protein and / or lipid. In many countries, including developed and developing countries, wheat products are consumed as a major component of the diet. Wheat flour is one of the most important foods in many countries in the world. Wheat grain contains 6-20% protein, 63-77% starch, approximately 2% fat, 2.0-2.7% crude fiber and 1.4-2.0% ash, depending in part on variety and class, and on environmental conditions during growth (Pomeranz, 1988). In many Asian countries, rice is the main cereal in the diet.

Protein co-precipitates were prepared from whey powder and soybean flour using various extraction and co-precipitation techniques. The effect of extraction and co-precipitation on co-precipitate yield was investigated. Native and sodium dodecyl sulfate polyacrylamide gel electrophoresis (Native-PAGE, SDS-PAGE) and light compound microscopy (LCM) were used to study the structure of the co-precipitates. The rheological and gelation properties of the co-precipitates were determined. Highest yield (45%) for NaOH/Isoelectric Point IEP-Heating-Cooling, co-precipitate was obtained using the following conditions of extraction; extraction temperature, 40°C; temperature of precipitation 95°C, and pH of precipitation was 4.5. The yield of co-precipitates was affected by chelating agents and pH of precipitation and temperature of precipitation. Native-PAGE showed that 2 new protein bands result from the interactions between whey and soybean proteins during preparation of the co-precipitate. SDS-PAGE showed that the new proteins dissociated to give the protein subunits of whey and soybean proteins. LCM results showed differences in microscopic structure between the whey and soybean protein precipitates and the protein co-precipitates. Gels were characterized by measurement of water holding capacity (WHC), gelation start temperature (GST) and denaturation start temperature (DST) and gel strength (GS). Gels (16%) from a protein co-precipitate Mixed Powder MP:NaOH/IEP-Cooling had higher WHC and GS than gels from whey protein precipitate, soybean protein precipitate and protein co-precipitates Mixed Extract ME:NaOH/IEP-Cooling and co-precipitates MP: and ME:NaOH/IEP-Heating-Cooling. The DST of protein co-precipitates was dependent on protein concentration and pH, while GST was relatively dependent on protein concentration.

The effect of three sources of protein: soy, dairy, and meat protein, and two levels of calcium on zinc, iron, copper, calcium, and magnesium retention in young adult men was determined in a 30-day metabolic balance study. The study was divided into a twenty-day baseline period, a thirty day controlled feeding period from which all the balance data was collected, and a twenty day follow-up period. During the controlled feeding period, twenty-four subjects were randomly assigned to one of three dietary treatment groups which differed in respect to protein source. The dairy treatment group was fed a diet in which 70% of the dietary protein was derived from dairy products; the soy treatment group was fed a diet in which 67% of the dietary protein was derived from soy products; and the meat treatment group was given a diet in which 70% of the dietary protein was provided by animal meat products. To test the effect of calcium level on mineral retention, the controlled feeding period was divided into two periods: Period I, in which the subjects consumed moderate levels of calcium (mean= 1206.77 + 193.29 mg/day) and Period II, in which the subjects consumed high levels of calcium (mean 2134.51 + 164.63 mg/day).
Master of Science

Our objective was to investigate the mode of interaction between selected food proteins and phenolic compounds. Bovine serum albumin (BSA), bovine beta-lactoglobulin, and soybean glycinin were used with the following phenolic compounds; 3,4,5-trihydroxybenzoic acid (gallic acid), 3,4-dihydroxy cinnamic acid (caffeic acid), p -hydroxycinnamic acid (courmaric acid), and 5,7-dihydroxy 4-methoxy isoflavone (biochanin A). The interaction was investigated using incubation temperatures of 35°, 45° and 55°C at pH 5, 7 and 9. Native and SDS-polyacrylamide gel electrophoresis (PAGE), differential scanning calorimetry (DSC), and Fourier transform infrared (FTIR) spectroscopy were used to identify protein-phenol interactions. Certain phenolic compounds combined with BSA and prevented protein aggregation. In general, the thermal stability of the proteins increased as a result of interaction with phenolic compounds; the most pronounced effect was observed with beta-lactoglobulin in the presence of gallic acid at pH 7. The interaction of the phenols with the proteins resulted in changes in protein secondary structure. (Abstract shortened by UMI.)

The high rate of malnutrition (180/1000 live births) in Sierra Leone (U N Demographic Year Book, 1985) has become a major concern to the government, development agencies, medical and nutritional personnel. This study was designed to determine whether there were any significant differences between the nutritional status of the children women who participated in an FAO project and those who did not and was conducted in six villages in the Koinadugu District of Sierra Leone.By referring to a list of local protein rich foods, three-day recall of food frequency was done to estimate the protein intake. Anthropometric measures were collected and body mass index was determined. All measures were compared with standards for African-American population.The weight-for-age was significantly greater for the the 50th percentile than that of the non-project children (NPC). Protein intake less than the average of 57 grams, were seen among older children with lower weight-for -height percentile, suggesting that total food intake was inadequate for the older children. This researcher believes that the FAO project should continue but that nutrition education should be an important component of the program.
Department of Home Economics

Thesis (M.S.)--West Virginia University, 2007.
Title from document title page. Document formatted into pages; contains vii, 44 p. : ill. Includes abstract. Includes bibliographical references (p. 30-33).

[Truncated abstract] Obesity is now a major public health problem worldwide. More than half the Australian population is now overweight. This is an important public health concern primarily because of the impact of overweight and obesity on risk of diabetes, hypertension and cardiovascular disease. Many strategies have been proposed to fight the obesity epidemic. One possible strategy involves understanding of the role of dietary components in the control of food intake. In this regard, dietary protein and fibre appear to be the most satiating nutrients. Foods enriched in protein, replacing energy from carbohydrate, or dietary fibre can increase satiety and reduce energy intake in the short-term. Longer-term trials suggest benefits of increasing protein or fibre intake on weight loss and features of the metabolic syndrome. The effects of dietary approaches which increase both protein and fibre at the expense of refined carbohydrate are uncertain. A practical approach to increasing both protein and fibre content of processed foods is to incorporate high protein and fibre ingredients into high carbohydrate foods. Lupin kernel flour is a novel food ingredient derived from the endosperm of lupin. It contains 40 to 45% protein, 25 to 30% fibre, and negligible sugar and starch. Lupin kernel flour can be incorporated into refined carbohydrate rich foods such as bread to increase protein and fibre content at the expense of refined carbohydrate. ... Body weight was measured every 2 weeks throughout the 16 week intervention, and these data were analysed to determine whether there was any between group difference in the rate of change in weight over 16 weeks. Over 16 weeks, lupin bread compared to white bread resulted in a significant increase in protein (13.7 (2.3, 25.0) g/d) and fibre (12.5 (8.8, 16.2) g/d) intakes, and a decrease in carbohydrate intake (-19.9 (-45.2, 5.5) g/d). There was a significant difference between groups in the rate of weight change over the 16 weeks (P=0.05). However, at 16 weeks there was no significant effect on body weight (-0.4 (-1.3, 0.6) kg), fat mass (-0.5 (-1.2, 0.2) kg) or fat free mass (0.2 (-0.5, 0.8) kg). Plasma adiponectin and leptin were not altered. Mean 24 hour systolic blood pressure (-2.4 (-3.4, -1.3) mm Hg) and pulse pressure (-3.1 (-3.9, -2.3) mm Hg) were lower for lupin relative to white bread, but diastolic blood pressure was not significantly different between groups. Apart from a lower HDL cholesterol for lupin relative to white bread (-0.09 (-0.17, -0.01) mmol/L), there were no significant differences in other blood lipids and glucose and insulin concentrations. Interpretation of the results was not influenced after adjustment for potential confounding factors. These studies assessed effects of bread enriched in lupin kernel flour relative to white bread, resulting in a higher protein and fibre intake and lower refined carbohydrate intake. This increased satiety and reduced energy intake acutely, but did not significantly influence body weight over 16 weeks. Systolic blood pressure and pulse pressure were significantly reduced. There were no significant improvements in blood lipids or glucose and insulin concentrations. Therefore, increasing protein and fibre intake at the expense of refined carbohydrate using lupin kernel flour may benefit satiety and blood pressure. Longer-term trials incorporating weight loss may be needed to observe benefits on body weight.

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As folhas de mandioca constituem boa fonte de nutrientes o que possibilita sua aplicação como ingrediente para as indústrias alimentícias. Apesar dos elevados teores de proteína, fibras, minerais e vitaminas, as folhas de mandioca contêm cianeto, substância tóxica que impede o seu consumo na forma in natura. No Brasil, as folhas de mandioca têm sido pouco exploradas para fins alimentícios, principalmente pela falta de conhecimento de seus aspectos nutricionais e da viabilidade econômica de sua exploração comercial. No processamento das folhas de mandioca para a obtenção da farinha, produto com ampla potencialidade de utilização como insumo para as indústrias alimentícias, é necessário observar não somente a redução do teor de cianeto, mas também, os aspectos nutricionais e reológicos desta. A extrusão termoplástica vem sendo explorada nos últimos anos no desenvolvimento de diversos produtos alimentícios. O objetivo principal deste trabalho foi avaliar a utilização das folhas de mandioca visando sua possível valorização, como matéria-prima para produtos alimentícios extrusados. O experimento foi conduzido em 3 fases complementares, de acordo com os seguintes objetivos específicos: Experimento I: produzir e caracterizar a farinha de folhas de mandioca quanto à composição química e propriedades de pasta. Experimento II: avaliar os efeitos de parâmetros de extrusão e porcentagem de farinha de folhas de mandioca sobre as propriedades físicas, reológicas e térmicas de farinhas instantâneas. Experimento III: avaliar os efeitos dos parâmetros variáveis do processo de extrusão e composição da matéria-prima sobre a qualidade de massas alimentícias instantâneas. O processo de extrusão dos Experimentos II e III foi realizado em extrusor mono-rosca, em delineamento central composto rotacional para quatro fatores: porcentagem de farinha de folhas em mistura...
Cassava leaves are good source of nutrients which enables its application as an ingredient for the food. Despite high levels of protein, fiber, minerals and vitamins, cassava leaves contain cyanide, a toxic substance that prevents their consumption in natura. In Brazil, cassava leaves have yet been little exploited for food, especially the lack of knowledge of their nutritional value and also the economic viability of commercial exploitation. Processing of cassava leaves to obtain the flour, a product with broad potential for use as raw material for the food, it is necessary to observe not only the reduction of cyanide content, but also the nutritional and rheological aspects of this. The thermoplastic extrusion has been explored in recent years in the development of various food products. Aiming at the enhancement of cassava leaves as raw material for food products, this study was conducted in three complementary experiments which had the following objectives: to produce and characterize cassava leaves flour for chemical composition and total cyanide content (Experiment I); evaluate the effects of extrusion parameters, as well as, the percentage of cassava leaves flour on the physical, rheological and thermal properties of instant flour (Experiment II); evaluate the effects of extrusion process variables and the composition of the raw material on the quality of instant pasta (Experiment III). The extrusion process of the Experiments II and III was performed in single-screw extruder in central composite design for four factors: percentage of cassava leaves flour mixed with cassava starch, moisture of mixtures, extrusion temperature and screw speed. The results obtained in the characterization of cassava leaves flour showed the presence of 23.00 g.100g-1 protein, 30.68 g.100g-1 fiber, 10.23 g.100g-1 ash, 56.6 mg.100g-1 vitamin C, 38.00 mg.100g-1 β-caroten and 0.30 mg.100g-1 total cyanide. In the analy ...

Orientador: Magali Leonel
Banca: Silvio José Bicudo
Banca: Regina Marta Evangelista
Banca: Mariana Schmidt Rechsteiner
Banca: Silene Bruder Silveira Sarmento
Resumo: As folhas de mandioca constituem boa fonte de nutrientes o que possibilita sua aplicação como ingrediente para as indústrias alimentícias. Apesar dos elevados teores de proteína, fibras, minerais e vitaminas, as folhas de mandioca contêm cianeto, substância tóxica que impede o seu consumo na forma in natura. No Brasil, as folhas de mandioca têm sido pouco exploradas para fins alimentícios, principalmente pela falta de conhecimento de seus aspectos nutricionais e da viabilidade econômica de sua exploração comercial. No processamento das folhas de mandioca para a obtenção da farinha, produto com ampla potencialidade de utilização como insumo para as indústrias alimentícias, é necessário observar não somente a redução do teor de cianeto, mas também, os aspectos nutricionais e reológicos desta. A extrusão termoplástica vem sendo explorada nos últimos anos no desenvolvimento de diversos produtos alimentícios. O objetivo principal deste trabalho foi avaliar a utilização das folhas de mandioca visando sua possível valorização, como matéria-prima para produtos alimentícios extrusados. O experimento foi conduzido em 3 fases complementares, de acordo com os seguintes objetivos específicos: Experimento I: produzir e caracterizar a farinha de folhas de mandioca quanto à composição química e propriedades de pasta. Experimento II: avaliar os efeitos de parâmetros de extrusão e porcentagem de farinha de folhas de mandioca sobre as propriedades físicas, reológicas e térmicas de farinhas instantâneas. Experimento III: avaliar os efeitos dos parâmetros variáveis do processo de extrusão e composição da matéria-prima sobre a qualidade de massas alimentícias instantâneas. O processo de extrusão dos Experimentos II e III foi realizado em extrusor mono-rosca, em delineamento central composto rotacional para quatro fatores: porcentagem de farinha de folhas em mistura...
Abstract: Cassava leaves are good source of nutrients which enables its application as an ingredient for the food. Despite high levels of protein, fiber, minerals and vitamins, cassava leaves contain cyanide, a toxic substance that prevents their consumption in natura. In Brazil, cassava leaves have yet been little exploited for food, especially the lack of knowledge of their nutritional value and also the economic viability of commercial exploitation. Processing of cassava leaves to obtain the flour, a product with broad potential for use as raw material for the food, it is necessary to observe not only the reduction of cyanide content, but also the nutritional and rheological aspects of this. The thermoplastic extrusion has been explored in recent years in the development of various food products. Aiming at the enhancement of cassava leaves as raw material for food products, this study was conducted in three complementary experiments which had the following objectives: to produce and characterize cassava leaves flour for chemical composition and total cyanide content (Experiment I); evaluate the effects of extrusion parameters, as well as, the percentage of cassava leaves flour on the physical, rheological and thermal properties of instant flour (Experiment II); evaluate the effects of extrusion process variables and the composition of the raw material on the quality of instant pasta (Experiment III). The extrusion process of the Experiments II and III was performed in single-screw extruder in central composite design for four factors: percentage of cassava leaves flour mixed with cassava starch, moisture of mixtures, extrusion temperature and screw speed. The results obtained in the characterization of cassava leaves flour showed the presence of 23.00 g.100g-1 protein, 30.68 g.100g-1 fiber, 10.23 g.100g-1 ash, 56.6 mg.100g-1 vitamin C, 38.00 mg.100g-1 β-caroten and 0.30 mg.100g-1 total cyanide. In the analy ...
Doutor

Thesis (M.S.)--East Tennessee State University, 2004.
Title from electronic submission form. ETSU ETD database URN: etd-0329104-095404. Includes bibliographical references. Also available via Internet at the UMI web site.

The function of homologous DNA recombination in human mitochondria has been a topic of ongoing debate for many years, with implications for fields ranging from DNA repair and mitochondrial disease to population genetics. While genetic and biochemical evidence supports the presence of a mitochondrial recombination activity, the purpose for this activity and the proteins involved have remained elusive. The work presented in this thesis was designed to evaluate the mitochondrial localization of the major recombinase protein in human cells, Rad51, as well as determine what function it plays in the maintenance of mitochondrial DNA (mtDNA) copy number that is critical for production of chemical energy through aerobic respiration. The combination of subcellular fractionation with immunoblotting and immunoprecipitation approaches used in this study clearly demonstrates that Rad51 is a bona fide mitochondrial protein that localizes to the matrix compartment following oxidative stress, where it physically interacts with mtDNA. Rad51 was found to be critical for mtDNA copy number maintenance under stress conditions. This requirement for Rad51 was found to be completely dependent on ongoing mtDNA replication, as treatment with the DNA polymerase gamma (Pol ϒ) inhibitor, ddC, suppresses both recruitment of Rad51 to the mitochondria following the addition of stress, as well as the mtDNA degradation observed when Rad51 has been depleted from the cell. The data presented here support a model in which oxidative stress induces a three-part response: (1) The recruitment of repair factors including Rad51 to the mitochondrial matrix, (2) the activation of mtDNA degradation systems to eliminate extensively or persistently damaged mtDNA, and (3) the increase in mtDNA replication in order to maintain copy number. The stress-induced decrease in mtDNA copy number observed when Rad51 is depleted is likely the result of failure to stabilize or repair replication forks that encounter blocking lesions resulting in further damaged to the mtDNA and its eventual degradation.

Thesis (M.S.)--West Virginia University, 2009.
Title from document title page. Document formatted into pages; contains vii, 42 p. : ill. Includes abstract. Includes bibliographical references (p. 29-35).

Human health is a complex and wide-ranging subject far beyond nutrition and physical exercise. Still, these factors have a huge impact on global health by their ability to prevent diseases and thus promote health. Thus, to identify health risks and benefits, it is necessary to reveal the underlying mechanisms of nutrition and exercise, which in many cases follows a complex chain of events. As a consequence, current health research is generating massive amounts of data from anthropometric parameters, genes, proteins, small molecules (metabolites) et cetera, with the intent to understand these mechanisms. For the study of health responses, especially related to physical exercise and nutrition, alterations in small molecules (metabolites) are in most cases immediate and located close to the phenotypic level and could therefore provide early signs of metabolic imbalances. Since there are roughly as many different responses to exercise and nutrients as there are humans, this quest is highly multifaceted and will benefit from an interpretation of treatment effects on a general as well as on an individual level. This thesis involves the application of chemometric methods to the study of global metabolic reactions, i.e. metabolomics, in a strategy coined predictive metabolomics. Via the application of predictive metabolomics an extensive hypothesis-free biological interpretation has been carried out of metabolite patterns in blood, acquired using gas chromatography-mass spectrometry (GC-MS), related to physical exercise, nutrition and diet, all in the context of human health. In addition, the chemometrics methodology have computational benefits concerning the extraction of relevant information from information-rich data as well as for interpreting general treatment effects and individual responses, as exemplified throughout this work. Health concerns all lifestages, thus this thesis presents a strategic framework in combination with comprehensive interpretations of metabolite patterns throughout life. This includes a broad range of human studies revealing metabolic patterns related to the impact of physical exercise, macronutrient modulation and different fitness status in young healthy males, short and long term dietary treatments in overweight post menopausal women as well as metabolic responses related to probiotics treatment and early development in infants. As a result, the studies included in the thesis have revealed metabolic patterns potentially indicative of an anti-catabolic response to macronutrients in the early recovery phase following exercise. Moreover, moderate differences in the metabolome associated with cardiorespiratory fitness level were detected, which could be linked to variation in the inflammatory and antioxidaive defense system. This work also highlighted mechanistic information that could be connected to dietary related weight loss in overweight and obese postmenopausal women in relation to short as well as long term dietary effects based on different macronutrient compositions. Finally, alterations were observed in metabolic profiles in relation to probiotics treatment in the second half of infancy, suggesting possible health benefits of probiotics supplementation at an early age.
Embargo until 2012-06-01

The purpose of this research was to evaluate whether serum prealbumin levels would serve as a predictor of hospital length of stay for elderly orthopedic patients who underwent hip replacement surgery. The study consisted of a set of 54 patients admitted to a hospital in Bristol, Tennessee. Patients with depleted prealbumin levels, low to low/normal prealbumin levels, or normal prealbumin levels were analyzed. Data collected from a retrospective chart review included: age, length of stay, serum glucose, sodium, potassium, hematocrit, hemoglobin, BUN, creatinine, WBC, prealbumin, and post operative diet consumption. Data were analyzed using analysis of variance for treatment effects. Because of the limited size of the data set, probabilities approaching p<0.10 were considered and levels of p<0.05 were considered significant. The research failed to show a significant relationship between prealbumin levels at admission and length of patient stay during post-operative recovery.

La perte de masse et de force musculaires liée à l'âge, ou sarcopénie, pourrait être partiellementexpliquée par un défaut de récupération de masse musculaire après des épisodes générateursd'atrophie musculaire. Ainsi, les périodes d'immobilisation qui augmentent avec l'âge (alitement,convalescence, fracture) pourraient être suivies d'une absence de récupération musculaire etcontribuer à la fonte musculaire au cours du vieillissement. Les causes de ce défaut de récupérationimpliquent notamment un déséquilibre du taux de renouvellement protéique et du taux derenouvellement cellulaire. L'objectif de cette thèse a donc été de mettre en évidence les mécanismesresponsables de l'atrophie musculaire chez le rat âgé au cours de l'immobilisation et ceux quiseraient défaillants afin de déceler les mécanismes à cibler pour favoriser la récupérationmusculaire.Des rats âgés ont été immobilisés pendant 8 jours par plâtrage unilatéral de la patte arrière, puislaissés en récupération pendant 40 jours après le déplâtrage. Nous avons montré que chez cesanimaux nourris avec un régime contenant 13% de caséine, l'immobilisation entraîne une atrophiedes muscles immobilisés mais, contrairement au rat adulte, le rat âgé ne récupère jamais la massemusculaire perdue. L'atrophie des muscles immobilisés peut être expliquée par 1/ une augmentationde l'apoptose et de la protéolyse ubiquitine-protéasome-dépendante musculaires, 2/ une diminutionde la régénération des cellules musculaires et 3/ une diminution de la protéosynthèse musculaire àl'état nourri. Tous ces phénomènes pourraient résulter de la présence d'un fort stress oxydant etd'une importante inflammation intramusculaire. Tous ces paramètres sont normalisés dès 10 jours derécupération, ce qui permet de stopper l'atrophie mais ne permet pas d'initier la phase derécupération musculaire. Nous avons donc testé l'effet de différentes supplémentationsnutritionnelles au cours de la période de récupération afin de favoriser un gain de masse musculairepost immobilisation. Des supplémentations en leucine (acide aminé bien connu pour stimuler laprotéosynthèse et inhiber la protéolyse) ont ainsi été réalisées. Chez les rats supplémentés, uneamélioration de la synthèse protéique et une normalisation plus précoce des activités protéolytiquesdu protéasome ont été observées. Cependant cette amélioration du métabolisme protéique ne s'estpas traduite par un gain de masse musculaire. Par contre, la modulation qualitative et quantitative desapports en protéines a pu permettre d'obtenir une récupération significative de masse musculaire :ainsi des régimes contenant 13% de lactosérum et des régimes hyper-protéinés ont permis de gagner50% de la masse perdue et ce, dès 20 jours de récupération.Nos résultats montrent que l'immobilisation chez le rat âgé aggrave la sarcopénie. Une fortealtération du métabolisme protéique permet d'expliquer la perte de muscle et la seule normalisationde la protéolyse et de la protéosynthèse permet d'expliquer l'absence de récupération musculaire.Nous avons montré que la modulation des apports en protéines au cours de la phase de récupérationpouvait permettre un gain de protéines.

Background Modern neonatal care has improved the survival rate of extremely preterm infants. These infants are at high risk of malnutrition and growth failure during 3-4 months of hospital care. The objectives of this study was to investigate nutritional intakes during hospitalization and explore associations between nutritional intakes, postnatal growth and retinopathy of prematurity (ROP). Perioperative nutrition in infants undergoing surgery for patent ductus arteriosus (PDA) was also investigated. Methods This is a population-based study of Swedish extremely preterm infants (<27 weeks) born during 2004-2007 (n=602). Detailed data on nutritional supply and anthropometric measurements during hospitalization were retrospectively retrieved from hospital records. Comprehensive data on cohort characteristics, neonatal morbidity and infant mortality were obtained from the Extremely Preterm Infants in Sweden Study (EXPRESS). Results During the first 70 days of life, intakes of energy, protein and several micronutrients, with the exception of iron and some vitamins, were less than estimated requirements, and infants showed severe postnatal growth failure. Energy and protein intake predicted growth in all anthropometric outcomes even when adjusting for severity of illness, and fat intake was positively associated with head growth. Low folate intake was positively correlated with poor weight and length gain while high iron intake, mainly explained by blood transfusions, was negatively associated with poor length gain. Furthermore, a low energy intake was associated with severe ROP (stage 3-5). An increased energy intake of 10 kcal/kg/d was associated with 24% decrease in severe ROP (p=0.01). During the first month, 99% of the infants were exclusively fed human milk. Infants who underwent surgery for PDA (n=140) were malnourished, with energy and macronutrient intakes below minimum estimated requirements before, during and after surgery. Conclusions The severe postnatal growth failure observed in Swedish extremely preterm infants may be prevented by improved intakes of energy, protein, fat and folate and a reduction of the number of blood transfusions. Human milk is the main enteral food source and analyses of human milk macronutrient contents facilitates individualized fortification. Provision of adequate energy intakes during the first four weeks of life may be an effective way to reduce the risk of severe ROP. Perioperative nutrition in infants undergoing PDA surgery needs to be improved. The study results have important implications for nutritional regimens, postnatal growth and health outcome in this new generation of survivors.

Cu, Zn superoxide dismutase (SOD1) is a dimeric, β-sandwich, metalloenzyme responsible for the dismutation of superoxide. Mutations covering nearly 50% of the amino acid sequence of SOD1 have been found to acquire a toxic gain-of-function leading to amyotrophic lateral sclerosis. A hallmark of this disease is the presence of insoluble aggregates containing SOD1 found in the brain and spinal cord. While it is unclear how these aggregates or smaller, precursor oligomeric species may be the source of the toxicity, mutations leading to increased populations of unstable, partially folded species along the folding pathway of SOD1 may be responsible for seeding and propagating aggregation. In an effort to determine the responsible species, we have systematically characterized the stability and folding kinetics of five well studied ALS variants: A4V, L38V, G93A, L106V and S134N. The effect of the amino acid substitutions was determined on a variety of different constructs characterizing the various post-translational maturation steps of SOD1: folding, disulfide bond formation and Zn binding. Zn was found to bind progressively tighter along the folding pathway of SOD1, minimizing populations of monomeric species. In contrast, ALS variants were found to have the greatest perturbation in the equilibrium populations of the folded and unfolded state for the most immature, disulfide-reduced metal-free SOD1. In this species, at physiological temperature, four out of five ALS variants were >50% unfolded. Finally the energetic barriers in the folding and unfolding reaction were studied to investigate the unusually slow folding of SOD1. These results reveal that both unfolding and refolding are dominated by enthalpic barriers which may be explained by the desolvation of the chain and provide insights into the role of sequence in governing the folding pathway and rate.

Sarcopenia is a growing health problem in this country as more Americans are living well into old age. It has been thought that a higher protein intake may be related to greater lean muscle mass as well as greater muscular strength and physical functioning. The purpose of this research was to examine protein intake and its relationship to lean body mass and physical functioning in a nationally representative sample of U.S. adults. This research used cross-sectional data from the NHANES 2003-2004 to examine the relationship between protein intake, as an average from two 24h recalls, and dual X-ray absorptiometry (DXA) lean body mass measures. Additionally, these lean body mass measures were examined as predictive of Physical Limitation Score (PLS), an index created from self-reported difficulty in performing basic activities of daily living. Protein intake was examined as total grams of intake, a percentage of total daily energy and as a percent contributed by animal foods. The lean body mass measures used in this analysis included: total lean mass, appendicular lean mass, muscle mass index [lean mass / height (m2)], appendicular lean mass [appendicular lean mass / height (m2)] and percent lean body mass. Questions used to create the self-reported index of functioning, the Physical Limitation Score (PLS), included difficulty in independently: walking ¼ mile, walking up ten stairs without resting, lifting and carrying 10lbs and standing from an armless straight chair. It was found that protein intake significantly, positively predicted lean body mass in select age-gender groups, while more often in men than in women. Total grams of protein intake positively predicted total and appendicular muscle mass index in men ages 19-50 (p-value <0.05), appendicular muscle mass index in men ages 51-70 (p-value 0.038) and percent lean mass in men 71+ years (p-value 0.026). Protein as a percent of energy was a significant, positive predictor of appendicular lean mass in men 19-50 (p-value 0.048), muscle mass index in women 19-50 (p-value 0.007), appendicular muscle mass index in women 19-50 (p-value 0.024) and percent lean mass in men 71+ years (p-value 0.019). Protein as a percent of energy was a significant negative predictor of percent lean mass in older women 71+ years (p-value 0.046). Protein as a percent contributed by animal foods was not a significant predictor of lean mass in any age-gender group. It was also found that Physical Limitation Score (PLS) was surprisingly positively predicted by total and appendicular lean and total and appendicular muscle mass index in nearly all age-gender groups (p-value <0.05) or at least moderately, positively predicted by these (p-value <0.10), meaning that having a greater amount of lean mass predicted greater physical limitation. The only exception was percent lean mass, which was a significant negative predictor of PLS in men and women 60-70 years (p-value <0.05). In this case, a higher percent lean mass was associated with less physical limitation. Additional follow-up analyses revealed that total body fat mass (kg) and percent body fat were significant positive predictors of PLS in most age-gender groups (p-value <0.05), indicating that higher amounts of body fat predicted greater self-reported limitation. Protein expressed as total grams per day or as a percent of total daily energy were generally significant or marginally significant predictors of lean mass in men of all ages but not often in women while protein as a percent contributed by animal foods was not found to be a significant predictor in any age-gender group. Total lean mass, total fat mass and total percent body fat were generally significant positive predictors of physical limitation, calling into question the functional implications of body composition in an older population. Results suggest that in older adults, excess body fat may be a stronger predictor of physical limitation than low lean muscle mass.

CAPES; CNPQ
A Spirulina platensis é reconhecida como uma fonte não convencional de proteínas, em função da sua constituição favorável deste nutriente (46 a 63%), possuindo concentração superior a das carnes e da soja. Além disso, apresenta potencial como matéria-prima para a produção de bioetanol, podendo acumular entre 8,0 e 14,0% de carboidratos. A fim de abranger o conceito de Biorrefinarias Integradas, o objetivo deste trabalho consistiu em avaliar a extração conjunta de proteínas e carboidratos da biomassa de Spirulina platensis utilizando tratamento ultrassônico e agitação em meio alcalino, e a posterior produção e caracterização do concentrado proteico. Na primeira etapa do trabalho, aplicou-se uma estratégia sequencial de planejamento experimental (Planejamento Fatorial Fracionário (PFF) seguido de Delineamentos Compostos Centrais Rotacionais (DCCR)) para seleção e maximização das variáveis com influência significativa sobre o processo de extração. Com as condições de extração otimizadas, foi possível atingir recuperação final de 75,85% e de 41,54% de proteínas e carboidratos, respectivamente. Na segunda etapa do trabalho foi realizada a precipitação de proteínas, para a separação da fase líquida contendo os carboidratos e obtenção do concentrado proteico, o qual foi caracterizado quimicamente e de acordo com sua funcionalidade tecnológica. O concentrado proteico apresentou coloração verde azulada com 75,97% de proteínas (b.s.), concentrações apreciáveis de aminoácidos, sendo o que o triptofano apresentou o maior escore químico (1,71) e o aminoácido limitante foi a histidina; na análise da estrutura secundária das proteínas, as conformações mais abundantes foram β-folha e α-hélice. Na etapa de avaliação da funcionalidade tecnológica observou-se que o pH apresentou influência nas propriedades de capacidade de absorção de água, capacidade de formação e estabilidade de espuma e emulsão, e capacidade de formação de gel, o que pode ser justificado pela solubilidade desta proteína, que é mínima em pH 3,0 e máxima em 9,0. A concentração de concentrado proteico também interferiu no desempenho destas propriedades; melhores resultados foram obtidos em maiores níveis de concentração, exceto para a capacidade de absorção de água e de óleo. Desta forma foi possível determinar que as proteínas de Spirulina platensis podem contribuir na formulação de alimentos, possuindo características eficazes de formação de emulsões, espumas ou géis, bem como pode ser utilizada como fonte suplementar de proteínas.
Spirulina platensis is considered an unconventional source of protein, because its avorably constitution on this component (46 to 63%), which is higher than the meat and soy. Furthermore, it has potential as a feedstock for bioethanol production and can accumulate between 8.0 to 14.0% of carbohydrate. In order to cover the concept of Integrated Biorefineries, the aim of this study was to evaluate the combined extraction of proteins and carbohydrates from Spirulina platensis biomass using sonication and agitation, under alkaline conditions, and the subsequent production and characterization of protein concentrate. The first stage of this work consisted of applying a sequential strategy of experimental design (Fractional Factorial Design FFD) and Central Composite Rotatable Design (CCRD)) by selecting and maximizing variables with significant influence on the protein and carbohydrates extraction. With the extraction conditions established, a final yield of 75.85% and 41.54% from protein and carbohydrate, respectively, was reached. In the second step, the protein concentrate obtained by precipitation was submitted to chemical and echnological functionality analyzes. The protein concentrate showed blue-green color with 75.97% of proteins (dry weight), appreciable concentrations of amino acids, where tryptophan had the highest chemical score (1.71) and the limiting amino acid was histidine; the secondary structure of proteins showed that the most abundant conformations present were β-sheet and α-helice. At the step of echnological functionality evaluation it was observed that the pH influenced on the properties of water absorption capacity, foaming and emulsion capacity and stability, and gelation capacity; it can be justified by the solubility of this protein which is minimal at pH 3.0 and maximum at 9.0. The level of addition of protein concentrate also interfered on the performance of these properties; better results have been obtained at higher concentrations levels, except for water and oil absorption capacity. Thus, it was confirmed that the Spirulina platensis proteins may contribute in different ormulations of foods, having effective characteristics to form emulsions, foams or gels, and can be used as a supplemental source of protein.

n-3 polyunsaturated fatty acids (n-3 PUFA) have been postulated to improve the insulin resistance associated with type 2 diabetes since the 1960s when observational studies in the Alaskan Inuit noted a reduced prevalence of type 2 diabetes when this population consumed a traditional diet. These findings were supported by animal studies but results of human intervention studies have been variable with most showing no change in glucose metabolism. More recent studies in growing farm animals suggested that muscle membrane phospholipids required to be enriched to a minimum of 14% n-3 PUFA in order for a change in insulin sensitivity to occur. This study sought to establish the effect of long-term (9 month) high-dose (3g/day) supplement of the n-3 PUFA eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on insulin sensitivity of glucose and protein metabolism. Thirty-three subjects with impaired glucose regulation underwent hyperinsulinaemic-euglycaemic-euaminoacidaemic clamps pre- and postintervention of n-3 PUFA or a control (maize) oil. A second cohort who all received n-3 PUFA supplementation underwent pre- and post-intervention muscle biopsies. Secondary outcomes included an assessment of inflammatory status and determining whether erythrocyte membrane phospholipid could act as a surrogate for muscle membrane phospholipid. In the clamp cohort, there were no changes in glucose metabolism postintervention; however, there was an increase in insulin-stimulated protein metabolism following the fish oil intervention. In the biopsy cohort, no subject achieved 14% PUFA enrichment in muscle membrane phospholipids; however, all subjects who received n-3 PUFA supplementation did achieve a minimum of 14% enrichment of n-3 PUFA in erythrocyte membrane phospholipid. In agreement with the majority of the literature, n-3 PUFA did not affect glucose metabolism. Insulin-stimulated protein metabolism was improved supporting the findings of another recent human study. These changes in protein metabolism may reduce the sarcopenia associated with aging, potentially delaying the progression of frailty.

Weight gain due to poor diet and lack of exercise is responsible for over 300,000 deaths each year (U.S. Department of Health and Human Services, 2010). Obese adults have an increased risk for serious health conditions including high blood pressure and cholesterol, type 2 diabetes, coronary heart disease, stroke, gallbladder disease, osteoarthritis, sleep apnea, respiratory problems, and certain cancers (National Cancer Institute, 2012). Participation in exercise can help control weight, strengthen muscles and bones, and reduce the incidence of cardiac events, stroke, hypertension, type 2 diabetes, colon and breast cancers, osteoporotic fractures, gallbladder disease, obesity, depression, anxiety, and delay mortality (ACSM, 2009). The purpose of this study was to determine the effectiveness of exercise participation during weight loss on a high protein-low carbohydrate diet plan during a 12-week span in females aged 15 to 25 years. Specifically, this research study was a comparison of markers of health such as weight, fat mass, percent body fat, and fat-free mass in females who consistently exercised during the diet (Exercisers) from those who did not participate in consistent exercise (Non- Exercisers). The population in this study was selected due to the transition from high school to college being a critical period because it is associated with many identity choices and lifestyle changes that can lead to weight gain (Anderson, Shapiro, & Lundgren, 2003). The data indicate participation in regular exercise, while consuming a high protein-low carbohydrate diet plan, increases the loss of body weight, fat mass, and percent body fat when compared to participating in the diet plan alone. There was no significant difference in fat-free mass reduction between the groups. One implication for practice is recommending moderate to vigorous exercise for a minimum of 30 minutes at a time, totaling a minimum of 150 minutes per week, for females trying to achieve weight loss. Based from the results of this research study, in order to achieve a greater amount of body weight, fat mass, and percent body fat reduction one should consider incorporating exercise participation and high protein-low carbohydrate dieting into their weight loss plan.

CAPES
O glúten é uma proteína advinda do trigo e subdivide-se em duas frações: gliadina e glutenina. A doença celíaca é uma intolerância ao glúten, que causa inflamação à mucosa do intestino, dificultando a absorção de nutrientes causando diarreia crônica, flatulência, anemia, entre outros problemas. Em função da má absorção, o celíaco necessita consumir alimentos fortificados para auxiliar no aporte nutricional. A maca peruana (Lepidium meyenii) é uma raíz originária do Peru e possui altos teores de cálcio (150 mg/100 g de farinha) e ferro (16,6 mg de ferro/100 g de farinha) e pode ser uma opção para o enriquecimento nutricional dos pães sem glúten. A farinha das sementes de niger (Guizotia abyssinica) e alpiste (Phalaris canariensis) também podem ser uma ótima alternativa para aumentar o valor nutricional de pães sem glúten. O presente estudo teve como objetivo elaborar pães sem glúten enriquecidos com farinhas de maca peruana, alpiste e niger. Para tanto foram utilizados dois planejamentos de misturas para compor o melhor pão (um planejamento para atingir melhor proporção de maca peruana, fécula de batata, polvilhos doce e azedo e outro planejamento para obter melhor pão enriquecido com farinhas de niger e alpiste, a partir da utilização da melhor formulação com maca peruana). Os planejamentos de misturas com pseudocomponentes serviram para investigar as melhores respostas nos quesitos volume específico, perfil de textura e análise sensorial dos pães enriquecidos com maca, alpiste e niger. Para os pães enriquecidos com maca, a formulação que melhor atendeu aspectos nutricionais, reológicos (volume específico e perfil de textura) e o fator desejabilidade foi a formulação 4 (25% de maca; 50% de fécula de batata e 25% de polvilho doce). Para os pães enriquecidos com niger e alpiste, a formulação que melhor atendeu aspectos nutricionais, sensoriais, fator de desejabilidade e parâmetros reológicos (volume específico e perfil de textura) foi a formulação 9 (compondo 16% de niger, 67% de alpiste e 16% de arroz). Assim, podese comprovar que a maca peruana, as sementes de niger e alpiste podem ser uma alternativa viável no enriquecimento de farinhas para pães sem glúten.
Gluten is a wheat protein and is subdivided into two fractions: gliadin and glutenin. Celiac disease is an intolerance to gluten, which causes inflammation of the intestinal mucosa, making it difficult to absorb nutrients, causing chronic diarrhea, flatulence, anemia, among other problems. Due to malabsorption, the celiac needs to consume fortified foods to aid in nutritional intake. Maca (Lepidium meyenii) is a native root from Peru and has high levels of calcium (150 mg / 100 g flour) and iron (16.6 mg iron / 100 g flour) and may be an option for nutritional enrichment of gluten-free breads. The flour from niger seeds (Guizotia abyssinica) and canary seed (Phalaris canariensis) can also be used to increase the nutritional value of gluten-free breads. The present study had as objective to elaborate gluten-free breads enriched with flours of maca, canary and niger. In order to do this, two statistical modeling of mixtures were used to compose the bread improvement (a planning to get the best proportion between maca, potato starch, sweet and sour manioc and another planning to obtain a better bread enriched with niger and canary flour, using as base the best formulation with maca). The statistical modeling of mixtures with pseudo components served to investigate the best answers in the specific volume, texture profile and sensory analysis of the enriched breads with maca, canary seed and niger. In maca enriched breads, the formulation with best nutritional and rheological factors and desirability requirements was formulation 4 (25% maca, 50% potato starch and 25% sweet manioc). In breads enriched with niger and canary seed, the formulation with best results in nutritional, sensory, desirability and rheological requirements was formulation 9 (16% niger, 67% canary seed and 16% rice flour). Thus, it could be proven that maca, niger and canary seed flours, can be a viable alternative in flour enrichment for gluten-free breads.

Postprandial urea production in subjects with insulin dependent diabetes mellitus (IDDM) on conventional insulin therapy is normal when the previous diet is high in protein, but there is an incomplete adaptive reduction in urea production following protein restriction. To evaluate the nutritional implications of restricted protein intake in human diabetes mellitus, it is first necessary to establish a reliable method to measure changes in urea production and amino acid catabolism in response to changes in dietary protein intake. We therefore tested (1) the accuracy of the urea production rate (Ra) to depict changes in urea production, (2) whether sulfate production can be accurately depicted using tracer or nontracer approaches, after establishing the use of electrospray tandem mass spectrometry to measure sulfate concentrations and 34SO4 enrichments following administration of the stable isotope tracer sodium [34S]sulfate, (3) the reproducibility of urea and sulfate measurements following a test meal low in protein (0.25 g/kg) in subjects previously adapted to high (1.5 g/kg.d) and low (0.3 g/kg.d) protein intakes, and compared the metabolic fate of [ 15N]alanine added to the test meal with that of [15N] Spirulina platensis, a 15N-labeled intact protein, and (4) whether we could identify the differences in postprandial urea and sulfate productions between normal subjects and persons with IDDM receiving conventional insulin therapy previously adapted to high protein intake, when the test meal was limiting in protein. Under basal conditions, steady state urea Ra is an accurate measure of urea production. Following changes in urea production, both the tracer and nontracer methods seriously underestimated total urea Ra. The tracer method overestimated sulfate production by 20%, but the nontracer method provided an accurate measure of sulfate production and, hence, sulfur amino acid catabolism. Postprandial changes in urea and sulfate productions following normal ada

Conoscenze preesistenti: Le transglutaminasi (TGase) sono una classe di enzimi ampiamente diffusa tra gli organismi procarioti ed eucarioti. Gli enzimi di questa famiglia catalizzano modifiche post-traduzionali in molte proteine attraverso reazioni di trasferimento dell’acile, reazioni di deaminazione e di crosslinking (polimerizzazione) tra residui peptidici di lisina (accettore di acile) e glutammina (donatore di acile) intra- o inter-catena proteica. A causa della sua facilità di espressione e di purificazione, l’unica TGase ampiamente usata per le applicazioni industriali è la TGase microbica estratta da Streptomyces mobaraensis (MTGase). Oggigiorno la MTGase è disponibile in commercio ed è ampiamente usata nell’industria dei biopolimeri, in cosmetica, per applicazioni cliniche, nell’industria tessile della lana e soprattutto nell’industria alimentare. La sua abilità di catalizzare legami crociati in molti substrati proteici differenti è sempre più usata non solo per la produzione di salsicce, prosciutti e formaggi ma, molto recentemente, anche per la detossificazione della farina, come possibile terapia alternativa alla dieta senza glutine. Ne consegue che oggigiorno le applicazioni industriali della MTGase stiano aumentando, coinvolgendo sempre più settori e producendo una ricerca scientifica su questo argomento sempre più fervente, allo scopo di tentare di rispondere a specifiche esigenze industriali, come l’implementazione di sistemi di purificazione della MTGase più efficienti, la ricerca di fonti alternative di transglutaminasi microbica, e di fonti sicure di enzimi ricombinanti. Scopo del progetto di dottorato: lo scopo principale del progetto è l’identificazione di nuove forme di transglutaminasi microbica che possano diventare un’alternativa a quella attualmente in uso. È stata eseguita un’analisi approfondita delle sequenze note allo scopo di ottenere una classificazione delle TGase microbiche attraverso la loro similarità a forme note. Per selezionare le migliori candidate che possano essere forme attive in appropriate condizioni, le sequenze selezionate sono state soggette di modellamento molecolare e simulazioni molecolari. Per testare l’attività enzimatica, sono stati effettuati dei saggi sperimentali su una nuova forma trovata ed un’ulteriore nuova forma è stata espressa. Risultati: il presente lavoro propone in primo luogo un’analisi, ad oggi assente, dell’ampio panorama delle transglutaminasi microbiche, sviluppando la prima classificazione delle TGase microbiche basata sulle loro caratteristiche di sequenza e sulle loro specifiche strutture secondarie predette. Al fine di classificare ed analizzare le caratteristiche strutturali di tutte le sequenze annotate come aventi un TGase core, sono state utilizzate tecniche computazionali che coinvolgono analisi di sequenza, studi comparativi, costruzione di alberi filogenetici, modellamento per omologia e simulazioni di dinamica molecolare. Tramite questo approccio, è stata effettuata una classificazione preliminare di queste sequenze dividendole in cinque gruppi principali. Ogni gruppo è stato studiato dal punto di vista delle sequenze per analizzare la presenza di motifs specifici. Per tre di questi cinque gruppi, sono state studiate anche le strutture secondarie e, da questa analisi, sono state rilevate caratteristiche specifiche per ogni gruppo. Inoltre, due nuove forme di TGase microbica (mTGase) sono state studiate in dettaglio: K. albida mTGase e l’ipotetica mTGase da SaNDy (organismo non rivelato per possibilità di brevetto). Per la prima, in comparazione con la MTGase, sono state effettuate analisi della tasca relativa al sito attivo e simulazioni di dinamica molecolare. Per la seconda, invece, sono state utilizzate tecniche sperimentali per purificare l’ipotetico enzima al fine di testarne l’attività su substrati alimentari. Saggi sperimentali su entrambe le proteine sono ancora in corso, al fine di trovare le migliori condizioni di attività enzimatica e i migliori substrati di reazione. Le simulazioni di dinamica molecolare eseguite sulla mTGase di K. albida hanno suggerito alcune spiegazioni alla maggiore specificità di questo enzima rispetto alla MTGase, dimostrata sperimentalmente da Steffan e colleghi, ed alcune indicazioni per variare le condizioni di attività usate per testarla. Inoltre, l’analisi dei substrati ha permesso di trovare nuovi possibili substrati, sui quali l’enzima potrebbe essere impiegato ai fini della riduzione delle allergenicità. D’altro canto, l’enzima estratto da SaNDy, mostrando una più alta somiglianza con la MTGase, potrebbe essere meno selettivo della mTGase da K. albida nei confronti di specifici substrati, pertanto potrebbe essere possibile una sua applicazione anche su substrati gliadinici, tuttavia, per provare ciò, sono necessari ulteriori esperimenti. Note: il presente lavoro di dottorato è stato principalmente svolto presso il Laboratorio di Bioinformatica del CNR di Avellino sotto la supervisione del Dr. Facchiano, tuttavia, tutte le simulazioni di dinamica molecolare sono state eseguite presso il Dipartimento di Biochimica dell’Università di Zurigo, nel laboratorio di biologia strutturale e computazionale sotto la supervisione del Prof. A. Caflisch e del suo gruppo di ricerca (periodo di formazione all’estero obbligatorio). I saggi di attività sperimentale sul substrato gliadinico sono stati effettuati dal laboratorio di spettrometria di massa CeSMA-ProBio presso il CNR di Avellino; e l’ipotetica mTGase da SaNDy è stata invece clonata, espressa e purificata durante la collaborazione con il laboratorio di Molecular Sensing presso il CNR of Avellino.
Background: transglutaminases (TGase) are a class of enzymes widely spread in eukaryotic and prokaryotic organisms. Enzymes of this family catalyze post-translational modifications in many proteins by acyl transfer reactions, deamidation and crosslinking (polymerisation) between protein intra- or inter-chain glutamine (acyl donor) and lysine (acyl acceptor) peptide residues. Due to its facility of expression and purification, the only TGase enzyme widely used for industrial applications is the microbial TGase extracted from Streptomyces mobaraensis (MTGase). Nowadays the MTGase is commercially available and widely used in biopolymers industry, in cosmetics, in clinical applications, in wool textiles, and above all in the food processing industry. Its ability to catalyze crosslinks on many different protein substrates is increasingly used not only for sausage, ham and cheese production but, very recently, also for flour detoxification, as a possible alternative therapy to the gluten free diet. It follows that nowadays the industrial applications of MTGase have increased, covering more and more fields producing a very active scientific research about this topic aimed at attempt to meet specific industrial needs, as the implementation of more efficient system for MTGase production, the research of alternative sources of microbial TGase, and safe source of recombinant enzymes. Aims of the doctorate project: the main aim of the project is the identification of novel forms of microbial TGases that could become an alternative to that in use. A depth screening of known sequences has been performed, with the aim of obtaining a classification of microbial TGases for their similarity to known forms. To select the best candidates to be active forms under appropriate conditions, molecular modelling and molecular simulations have been performed on selected sequences. To test the enzymatic activity, experimental assays have been performed with a novel form, and another novel form has been expressed. Results: the present work proposes at first an analysis, lacking so far, of the wide microbial transglutaminase world, developing the first classification of the microbial TGase based on their sequence features and their specific predicted secondary structures. In order to classify and analyze the structural features of all the sequences annotated as having a TGase core computational techniques involving sequence analyses, comparative studies, building of phylogenetic trees, homology models and molecular dynamic simulations have been used. From this approach, a preliminary classification of these sequences was done by dividing them in five main groups. Each group has been investigated from the sequence point of view to analyze the presence of specific motifs. For three of this five groups, also the secondary structures have been investigated and, from this analysis, features specific for each group have been detected. Moreover, two novel forms of microbial TGase (mTGase) have been investigated in the detail: K. albida mTGase and the hypothetical mTGase from SaNDy (organism not disclosed for patent opportunity). Molecular dynamics simulations and active site pocket analyses have been performed for the first, in comparison with MTGase. For the second, instead, experimental technique has been used to purify the hypothetical enzyme in order to test it on food related substrates. Experimental assays on both the proteins are still ongoing, to find the best enzymatic activity conditions and the best substrates of reaction. The molecular dynamic simulations performed on K. albida mTGase have suggested some explanations to the higher specificity of this enzyme than MTGase, experimentally demonstrated by Steffen et colleague, and several indications to change the activity conditions used to test it. Moreover, the substrates screening has allowed to find novel possible substrates, on which this enzyme could be employed for the allergenicity reduction. On the other hand, the enzyme extracted from SaNDy, showing a higher similarity with MTGase, could be less selective than K. albida mTGase for specific substrates, so it could be possible its application also on the gliadin substrate, but to prove it further experiments are necessary. Note: the present PhD work has been mainly performed in the Bioinformatics Laboratory at the CNR of Avellino under Dr. Facchiano’s supervision, however all the MD simulations have been performed at the Biochemistry Department of the University of Zurich, in the computational and structural biology laboratory under the supervision of Prof. A. Caflisch and his research group (compulsory abroad training period). Experimental activity assays on gliadin substrate have been performed by the spectrometry mass CeSMA-ProBio lab at the CNR of Avellino; and the hypothetical mTGase from SaNDy was instead cloned, expressed and purified in collaboration with the Laboratory for Molecular Sensing at the CNR of Avellino.

Bacteria are one of the largest causes of human disease, with millions of deaths every year attributed to bacterial infections, and they have become more difficult to tackle with the widespread emergence of antibiotic resistance. In this thesis, I describe my studies that pursued two approaches: one focus was on using antimicrobial histones as an alternative to treatment for antibiotic resistant bacteria; in another approach the recombinant version of an eggshell cuticle protein was expressed and purified for testing against food-safety pathogens. One major pathogen that is contributing to this challenge of antibiotic resistance is Staphylococcus aureus. The methicillin-resistant strain of S. aureus leads to increased hospital stays and increased mortality in patients. The impact of such pathogens is worsened when bacteria form surface-attached aggregates known as biofilms. Development of new approaches to eradicate antibiotic- resistant biofilms will benefit human health. This study looked at an alternative method to eradicate bacteria compared to traditional antibiotics. Histones with antimicrobial activity were extracted from chicken blood and tested against methicillin-sensitive and methicillin-resistant strains of Staphylococcus aureus biofilm (MSSA and MRSA). The histone mixture completely eradicated both strains in biofilm form at relatively low concentrations. In addition, the histone mixture also displayed fast kill kinetics against planktonic forms of the two strains. Finally, the interaction of the histone mixture with the bacterial membrane in MRSA biofilms was observed by scanning electron microscopy (SEM). Bacteria treated with the histone mixture showed clear morphological changes, including pore formation and cell collapse. Therefore, the histone mixture purified from chicken red blood cells could prove to be a good alternative to traditional antibiotics for protection against antibiotic-resistant strains of bacteria in their planktonic and biofilm forms. Reduction of food-borne illness is another important aspect in the promotion of human health. A significant contributor to food-borne illness is contaminated table eggs. The unfertilized egg can be contaminated by a variety of pathogens including Salmonella spp. and Bacillus spp. The egg is protected by the eggshell which is traversed by respiratory pores that are normally covered by a cuticle plug to restrict pathogen entry. This cuticle consists of several proteins including ovocaxlyin-32 (OCX-32). OCX-32 has a large number of naturally occurring haplotypes due non-synonymous single nucleotide polymorphisms (SNPs). In this study, the goal was to express five of the most common haplotypes of OCX-32 in Escherichia coli and purify the recombinant protein for assay of its antimicrobial activity. Five constructs that contain the cDNA of common OCX-32 haplotypes (A, B, C, D, and O) with a histidine tag at the C-terminus were generated. The constructs were subcloned into pGEX4T-1 vector which encodes Glutathione-S-transferase (GST) upstream of the multiple cloning site. My study developed methods to optimize the expression conditions, and to increase the solubility of the recombinant protein. Various expression strains of E. coli and solubility buffers were tested. In addition, the construct was subcloned into a plasmid containing the small ubiquitin-like modifier (SUMO) fusion tag; the solubility of the new SUMO-OCX-32 haplotype A recombinant fusion protein was evaluated. The best results were obtained by slow dialysis refolding of denatured SUMO-OCX-32 fusion protein. This recombinant protein showed almost complete solubility with minimal precipitation and was tested against the egg-related pathogen, Bacillus cereus. Unfortunately, the SUMO-OCX-32 recombinant protein did not inhibit growth of B. cereus. In my studies reported in this thesis, two very different approaches were taken. A histone mixture was isolated from an abundant starting material, which proved to be highly effective and promising in the eradication of S. aureus biofilms at relatively low concentrations. Alternatively, expression of a soluble recombinant protein for functional activity assay was very challenging and required the optimization of a number of methods to prepare soluble protein for testing. One of the methods tested proved effective in obtaining large amounts of soluble protein. However, further developmental work will be essential to determine if this approach is a viable strategy in acquiring functional protein.

Mast cells form an integral part of both innate and adaptive immunity; they help to orchestrate the inflammatory immune response through the release of a variety of inflammatory mediators. Adverse reaction to allergens can lead to activation of mast cells, causing degranulation and release of a range of pro-inflammatory mediators contributing to the onset of allergy. The most studied activation pathway in the adaptive immune response of mast cells is through the Immunoglobulin E (IgE) cell surface receptor FceRI. Crosslinking of FceRI leads to degranulation and de novo synthesis of mediators. Every eukaryotic cell undergoes constitutive secretion. Alongside this general process, cells such as neuronal endocrine and immune cells, including mast cells, perform regulated secretion. This enables the cell to rapidly release mediators stored in secretory granules upon stimulation by a particular extracellular ligand. Mediators released fall into two categories; pre-formed, contained within these secretory granules; monoamines such as histamine as well as many proteases, and de novo synthesized that are released through the constitutive secretory pathway, including prostaglandins, leukotrienes, cytokines and chemokines. Elucidating the mechanisms of mast cell mediator release is imperative for understanding many disease processes; however, knowledge of the precise mechanisms by which mast cell exocytosis is controlled remains elusive. The aim of this study was to identify and characterise Soluble NSF attachment protein receptor (SNARE) proteins involved in the release of inflammatory mediators in human mast cells. Using LAD 2 human mast cells and primary human lung mast cells (HLMCS), expression of a variety of syntaxins and Vesicle associated membrane proteins (VAMPs), as well as the ubiquitously expressed SNAP-23 were found. To study the roles of individual VAMPs in exocytosis a novel technique utilising pH sensitive pHluorins was developed. Using VAMPs tagged with pHluorins, the cellular distribution of VAMP-3 and VAMP-8 containing vesicles and their behaviour upon IgE stimulation in live cells was monitored. In unstimulated cells, VAMP- 3 and 8 were found to have distinct cellular distributions. Upon IgE stimulation both VAMP-3 and VAMP-8 containing vesicles translocated to the membrane and underwent membrane fusion, consistent with roles in exocytosis. However, their responses showed distinct time courses and calcium dependences. Importantly the VAMP-3 vesicle pool could be selectively targeted with a botulinum neurotoxin serotype B (BoNT)/B LC construct and in doing so inhibited the release of IL-6. The findings in this study support the notion that distinct vesicle pools, defined in part by expression of VAMP-3 and VAMP-8, regulate the release of inflammatory mediators from mast cells and that BoNTs might provide a novel means of targeting the release of chronic inflammatory mediators from mast cells for treatment of chronic inflammatory diseases.

This research investigates the protein interactions that occur when soy protein is added to milk and subjected to renneting or heating. Milk was fortified with 20% soy protein and enzymic coagulation studied at 35°C at various pH's and CaCl2 levels. The first part deals with the interaction between milk and soy proteins during rennet-induced milk coagulation. The first goal was to determine how soy proteins affected milk coagulation. The effects of native versus heat-denatured soy proteins on rennet coagulation time and curd firmness were compared. lmmunogold labeling along with transmission electron microscopy was used to identify and localfze soy proteins in coagulated milk. Partitioning of ß-conglycinin and glycinin, the two main soy protein fractions, between cheese and whey was determined by electrophoresis. Soy proteins affected milk coagulation to the greatest extent at pH 6.6. Both heat-denatured and native soy proteins increased rennet coagulation time. Only heat-denatured soy proteins affected final curd firmness. Most of ß-conglycinin was lost in whey, whereas glycinin was retained in curd. Soy proteins existed in the curd as aggregates that were less electron dense than casein micelles. At pH 6.6, heat-denatured soy proteins were fibrous and adhered to the surfaces of casein micelle, preventing direct micelle-micelle contact. This would delay aggregation rate and decrease curd firmness by decreasing the number and strength of links between casein micelles. Native soy proteins did not bind to the casein micelles but rather were physically trapped within curd. Their effect of delaying aggregation is thought to be a function of their binding of calcium. Adding CaCl2 or lowering the pH to 6.3 or 6.0 helped restore coagulation properties. The second goal was to determine what heat-induced interaction occurs between milk and soy proteins, specifically between κ-casein and glycinin. Both κ-casein and glycinin are heat labile and form insoluble aggregates when heated. When glycinin and κ-casein were heated together, some acidic polypeptides of glycinin crosslinked with κ-casein via disulfide linkages. However, when disulfide linkage was prevented by adding ß-mercaptoethanol , non-covalent interactions between κ-casein and both acidic and basic polypeptides of glycinin occurred that prevented the heat precipitation of glycinin. This non-covalent interaction between glycinin polypeptides and κ-casein may explain why the heat-treated soy proteins became attached to the surfaces of casein micelles during rennet coagulation of milk.

Three experiments were conducted to study the sensitivity of the pH-stat (in vitro) method in the prediction of true digestibility (TD), as measured by amount of base added, of plant proteins, either alone or in the presence of specific additives (nitrogen-free mixture, vitamin mixture and/or mineral mixture) as part of a complete diet of plant proteins that had been subjected to various levels and forms of heating. The in vitro TD values were then compared with TD values obtained in. vivo (Wistar rats). In experiment 1, the effect of temperature (dry-heating at 80, 100, 120, 150, 180 and 240° C or autoclaving at 121° C) and time (30, 60, 120 and 240 minutes) of heat application on in vitro base consumption (BC) was measured in 3 grains (wheat, barley and sorghum) and whole defatted soybeans. The largest increase in BC measured by the pH-stat method was that of soybeans in response to 30 minutes of autoclaving. Dry heating had various effects on the BC by soybeans, depending upon temperature and time of application, but none of the treatments was as beneficial as autoclaving. Mild, dry-heating of grains at 80-120° C improved BC slightly. The improvement was most marked for wheat. Both dry-heating of grain at temperatures above 120° C and autoclaving reduced the BC significantly for all durations. In experiment 2, the effect of inclusion of non-protein dietary components (minerals, vitamins and a nitrogen-free mixture, singly and in combination) on in. vitro BC measured by the pH-stat method of wheat and fat-extracted soybeans (both proteins in the raw and autoclaved forms) was monitored. For the wheat treatments, the inclusion of a mineral mixture significantly (p<0.001>) increased digestibility. This effect was greatest with autoclaved wheat. It was concluded that, in general, the presence of minerals increased the rate of hydrolysis. With raw soybeans, the distinction between treatments was less well-defined. The treatments containing vitamin or nitrogen-free and mineral combination mixtures were digested to a significantly greater extent than the raw soybeans alone. With autoclaved soybeans, additives had no effect. This lack of response to additives may have been due to the rather large amount of base required by the autoclaved soybean protein alone. In experiment 3, a series of rat-feeding trials were conducted in conjunction with in. vitro digestions. Diets were fed to groups of Wistar rats to determine TD, Biological Value (BV), and Net Protein Utilization (NPU) in vivo. Although BV was measured it was not relevant for this work. Concurrently, the same diets were tested for in. vitro TD by the pH-stat method. Specific regression equations were developed for each protein-type tested, after it was determined that a much lower correlation coefficient was obtained when one general equation was utilized. The newly-developed equations followed the format y = a + bx, where y = TD (as a part of one), a = the y-intercept, b = slope of the function and x = ml 0.10N NaOH added during the 10-minute digestion. Regression equations, correlation coefficients (r) and standard errors for each regression (s) between in. vitro and in vivo true digestibility of proteins were as follows; Soybean, soybean (autoclaved), soybean/wheat combinations (n = 6) r = 0.93 TD = 0.7868 + 0.2175x s = 0.018 Sorghum (raw, autoclaved, 90° C, 120° C, 180° C dry-heated, steamed) (n = 6) r = 0.92 TD = 0.4575 + 1.8841x a = 0.058 Alfalfa pellets/hay in combination with either wheat or barley (n = 13) r = 0.91 TD = 0.3446 + 1.0356x s = 0.046Alfalfa hay and barley combinations (n = 5) r = 0.96 TD = 0.2360 + 1.3194x s = 0.048 Grains (19 barleys, 10 triticales, 6 sorghums, and 2 wheats) (n = 37) r = 0.74 TD = 0.7419 + 0.4759x s = 0.044 In general, it can be stated that the pH-stat method is a useful method for screening proteins for the effect of various treatments on digestibility. Damage due to abnormally severe processing conditions (i.e. heating) is readily detected by the pH-stat technique as indicated by a decrease in the amount of base consumed during enzymatic hydrolysis.
Land and Food Systems, Faculty of
Graduate

This thesis examined aspects of production and utilization of acetate in humans via measurements of plasma concentrations in different circumstances with particular attention to changes in diabetes. Circulating plasma acetate was measured by a modified acetate kinase-based enzymatic spectrophotometric method with adequate sensitivity and specificity for levels encountered in human plasma. Fasting plasma acetate was increased in diabetics and correlated with glucose and indices of glucose disposal. Levels increased further when they were fed different high- fibre diets. The rise in acetate levels after lactulose ingestion correlated with changing breath hydrogen excretion in subjects with suspected malabsorption. Plasma acetate levels increased during fat infusion, and conversely, fell with suppression of fatty acid levels during euglycaemic clamping. Insulin appeared to promote acetate production from glucose by enhancing glycolysis and acetyl CoA availability, although its activity in reducing lipolysis had an opposite effect. The hepatic formation of acetate from ethanol did not appear influenced by prior chlorpropamide intake. Glucose tolerance was unaffected by a 150mmol/hr acetate load, but acetate tolerance was impaired when glucose was simultaneously available. Adipose tissue lipolysis was suppressed during acetate infusions as evident from reduced levels of glycerol and non-esterifled fatty acids. Blood 'ketone body' levels were increased, suggesting direct conversion from acetate. Possibly as a result, fat oxidation assessed from gaseous exchange, was reduced with infused acetate. Acetate utilization was impaired in diabetic patients from higher fasting plasma levels and slower metabolic clearance. The defect in diabetes was probably due to both over-production and under-utilization, and could be related to the enhanced lipolysis, hyperglycaemia and a reportedly reduced hepatic activity of acetyl CoA synthetase. It was concluded that acetate is derived from both colonic fermentation and endogenous catabolism of glucose and fatty acids and appears rapidly metabolisable in humans. Some areas of further interest in human acetate metabolism were highlighted.