Doctor of Philosophy / Biochemistry and Molecular Biophysics Interdepartmental Program / Gerald R. Reeck / Armet is a bifunctional protein that is apparently universally distributed among multicellular animal species, vertebrate and invertebrate alike. A member of the Unfolded Protein Response, (UPR) Armet promotes survival in cells that are under endoplasmic-reticulum (ER) stress. I have carried out biophysical studies on human Armet looking for compounds that bind to Armet and hence could reduce its anti-apoptotic function, thus potentially joining the growing class of pro-apoptotic drugs. Performed primarily with 1H-15N HSQC NMR, ligand studies showed that approximately 60 of the 158 residues are potentially involved with binding. The 60 residues are distributed throughout both domains and the linker suggesting multi-domain interaction with the ligand. Circular dichroism studies showed heat denaturation in a two-step unfolding process with independent unfolding of both domains of Armet with Tm values near 68°C and 83 C with the C-terminal domain unfolding first, as verified by 1H-15N HSQC NMR measurements. I also provide the first identification of UPR transcripts in pea aphids, Acyrthosiphon pisum, the genetic model among aphids. I measured transcript abundance with hope of finding future transcriptional targets for pest mitigation. I identified 74 putative pea aphid UPR components, and all but three of the components have higher transcript levels in aphids feeding on plants than those that fed on diets. This activated UPR state is attributed to the need for saliva proteins for plant feeding. Because aphids are agriculturally significant pests, and saliva is pivotal to their feeding on host plants, genes that encode saliva proteins may be targets for pest mitigation. Here I have sought the aphid’s saliva proteome by combining results obtained in several laboratories by proteomic and transcriptomic approaches on several aphid species. With these data I constructed a tentative saliva proteome for the pea aphid by compiling, collating, and annotating the data from several laboratories. I used RNA-seq to verify the transcripts in pea aphid salivary glands, thus expanding the proposed saliva proteome from approximately 50 components to around 130 components, I found that transcripts of saliva proteins are upregulated during plant feeding compared to diet feeding.

Orientador: Flavio Luis Schmidt / Dissertação (mestrado) - Universidade Estadual de Campinas. Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-10T19:44:29Z (GMT). No. of bitstreams: 1 Ferraz_MarianoBuenoMarcondes_M.pdf: 5652380 bytes, checksum: 42ae9a8aa05a8ec340cd75d6a1da2b75 (MD5) Previous issue date: 2008 / Resumo: A ervilha em lata é o segundo alimento em conserva mais consumido no Brasil. Praticamente todo o produto comercializado no país é processado a partir de grãos de ervilha seca, desidratada, comercializadas como commodities no mercado internacional. Existe uma necessidade de descrição detalhada de todo o processamento deste alimento para que exista a possibilidade de otimização dos processos envolvidos na produção desta conserva de baixa acidez. O objetivo deste trabalho foi descrever o comportamento de ervilhas (Pisum sativum L., variedade Flávia, cultivadas no estado do Rio Grande do Sul, safra 2004) desidratadas durante o processo de rehidratação, em relação a ganho de massa e volume. Foi determinada, também, a cinética de rehidratação deste alimento. Obtivemos também dados relacionados a textura destas ervilhas após o processamento térmico. Para este experimento, 100g de ervilhas foram hidratadas, em água destilada, à 05ºC, 25ºC, 45ºC, 65ºC e 85ºC, de modo a estabelecermos a cinética de hidratação das ervilhas, e determinar a temperatura ideal para hidratação. O modelo de Peleg apresentou melhor ajuste (R2 = 0,98) e a temperatura mais adequada foi de 25°C. Após o estudo da cinética e da definição da temperatura ideal para hidratação de ervilhas, foram hidratadas em soluções contendo NaCl 2%, CaCl2 0,5%, Ácido Cítrico 0,5% e EDTA 0,5%, além de todas as possíveis combinações entre os aditivos, assim como um grupo controle. Após a hidratação, as ervilhas foram envasadas em latas (73mm de de diâmetro x 85mm altura) e processadas a 121ºC por 15 minutos, resfriadas e avaliadas para determinação do ganho final de massa, volume e a variação da textura. Dentre os aditivos e suas combinações avaliadas, a solução de CaCl2 0,5% gerou o melhor conjunto de respostas, quanto aos parâmetros de ganho de massa e volume, além de uma melhor resposta em relação ao aumento da dureza. A influência deste aditivo foi avaliada em relação a aspectos sensoriais de cor, sabor, aparência geral e textura, além de atitude de compra. A textura foi o parâmetro que gerou maior divisão de opinião, com cerca de 50% dos consumidores aprovaram o aumento na dureza do produto, enquanto 50% dos consumidores desaprovaram o aumento da dureza. Em relação aos outros parâmetros, houve aprovação por 67% dos consumidores. Através dos resultados encontrados, foram estabelecidos alguns parâmetros que poderão ser utilizados na otimização de processos industriais de ervilha em conserva, diminuindo perdas econômicas, além da utilização racional de recursos hídricos, e do aumento na qualidade geral do produto que chega ao consumidor final / Abstract: Canned peas are the second most consumed canned good in Brazil. Pratically all of canned peas commercialized in this country is made from dried grains, marketed internationally as commodities. There is a need for a detailed description of the process as a whole, so the process could be optimized. The goal of this work was to describe the behaviour of dried peas (Pisum sativum, Flavia variety, grown is the state of Rio Grande do Sul, Brazil, harvested in 2004) during the rehydration process, in relation to quality parameters such as weight and volume gain, as well as the hydration kinetics of this vegetable. The texture of the peas was also evaluated. For this experiment, 100g of dried peas were rehydrated in distilled water at 05°C, 25°C, 45°C, 65°C and 85°C, se we could establish de rehydration kinetics, as well as the ideal rehydration temperature. This ideal temperature showed to be 25°C, and the rehydration kinetics fits the Peleg model. After detemining the ideal temperature and rehydration kinetics, 100g of dried peas were rehydrated in solutions containing 2%, CaCl2 0,5%, Ácido Cítrico 0,5% and EDTA 0,5%, and also every single combination between these additives, plus a control group, rehydrated with distilled water. After hydration, peas were canned (73mm diameter x 85mm height) and thermically processed at 121°C for 15 minutes, cooled at room temperature and evaluated for determination of final weight and volume gain, as well as texture. Among all the addtives and combinations tested, the CaCl2 0,5% solution showed the best results when all three parameters were analyzed. The influence of this additive was sensory evaluated regarding color, flavor, general appearance, and texture, as well as purchasing possibility. Texture was the parameter that divided consumers. 50% of the consumers approved the hardness of the peas, while 50% did not approved the hardness. Regarding the other parameters, most consumers approved the product. Through these results, is possible to establish some parameters that could be successfully used in the optimization of canned peas processing, minimizing economical losses and leading to a better use of water resources, as well as improving the quality of the final product / Mestrado / Mestre em Tecnologia de Alimentos

Pigeonpea (Cajanus cajan (L.) Millsp.) is a drought tolerant legume of the Fabaceae family in the order Fabales and the only cultivated species in the genus Cajanus. It is mainly cultivated in the semi-arid tropics of Asia and Oceania, Africa and America. In Malawi, one of the top producers of pigeonpea in Africa, it is grown by small scale farmers as a source of food and income and for soil improvement in intercropping systems. However, varietal contamination due to natural outcrossing causes significant yield losses for farmers. In this study, 48 polymorphic SSR markers were used to assess diversity in all pigeonpea varieties cultivated in Malawi with the aim of developing a genetic fingerprint to distinguish the released varieties. SSR alleles were separated by capillary electrophoresis on an ABI 3700 automated sequencer and allele sizes determined using GeneMapper 4.0 software. Allelic data was analysed with PowerMarker. A total of 212 alleles were revealed averaging 5.58 alleles per marker with a maximum number of 14 alleles produced by CCttc019 (Marker 40). Polymorphic information content (PIC) ranged from 0.03 to 0.89 with an average of 0.30. DARwin software was used to generate a neighbour-joining tree that displayed three major clusters with two sub clusters in Cluster I. The released varieties were scattered across all the clusters observed, indicating that they generally represent the genetic diversity available in Malawi, although it was observed that there is substantial variation that can still be exploited through further breeding. Screening of the allelic data associated with five popular pigeonpea varieties for which a DNA fingerprint was to be developed, revealed 6 markers – CCB1 (Marker 1), CCB7 (Marker 2), Ccac035 (Marker 7), CCttc003 (Marker 15), Ccac026 (Marker 37) and CCttc019 (Marker 40)– which gave unique allelic profiles for each of the five varieties. With further tests needed for its robustness, this genetic fingerprint can be used for seed certification to ensure only genetically pure seeds are delivered to Malawi farmers. / Agriculture and  Animal Health / M. Sc. (Agriculture)

No description available.

The bending and spring back behaviors of DP780 and DP980 were investigated using experiments, analytical models, and PEA. An PEA study using 3D shells was first performed of the work by Queener and DeAngelis (1968) and demonstrated that the 3D shell element behavior in bending and springback, is similar to that from simple bending theory. Experimental and FE studies were then performed using DP780 and DP980 steels under simple and general bending conditions. Simple bending conditions were studied in V -die bending. General bending was studied for DP780 using a commercial bending machine. The PEA examined the effect of element formulation and material hardening assumptions on springback, bending stresses, and the residual stress distributions after springback. Corresponding simple and general bending analytical models were also compared. The simple bending model was from Queener and DeAngelis ( 1968), and the general bending model was from Tan et al. ( 1995), modified for Ludwig hardening. Overall, the PEA predicted the springback magnitude in the order; 2D continuum >3D continuum> 3D shells, and kinematic hardening> mixed hardening> isotropic hardening. In the V -die bending study the 3D shell PEA, using a calibrated mixed hardening assumption, produced the most accurate results. The PEA using pure isotropic hardening demonstrated that bending and springback behaviors for both steels were consistent with that described by simple bending theory. The behaviors demonstrated by the FEA using mixed or pure kinematic hardening were not. In the general bending study, the 3D continuum element FEA using pure kinematic hardening was the most accurate. The 3D continuum element FEA captured the bending stress interaction with the hardening assumption as well as thinning deformation, in agreement with the analytical model and thinning measured experimentally. 3D Shell elements could not capture these behaviors and significantly under-predicted springback under the pure isotropic hardening assumption. / Thesis / Master of Applied Science (MASc)

Pea seeds were tempered at moisture contents of 12.0&amp / #61617 / 0.1, 13.0&amp / #61617 / 0.1, 14.0&amp / #61617 / 0.1 and 15.0&amp / #61617 / 0.3%. The seeds with different moisture contents were then milled and fractioned according to the particle size of 53, 106, 212, 425 and 850 &amp / #956 / m. Tempering the pea seeds (12.0&amp / #61617 / 0.1, 13.0&amp / #61617 / 0.1, 14.0&amp / #61617 / 0.1 and 15.0&amp / #61617 / 0.3%) did not significantly affect the mass and protein fraction in comparison with the pea seeds that are not tempered (11.45&amp / #61617 / 0.05%). For the production of pea protein isolate, aqueous-solvent extraction method was used. The protein was extracted with an alkali solution from the ground pea-seeds and precipitated from the extract by bringing the pH down to isoelectric point (pH=4.5). The precipitated protein was separated from the supernatant by centrifugation. The effects of extraction parameters on the yield of extraction such as pH, particle size, temperature, solvent to solid ratio, and salt were studied. The maximum yields were obtained at these conditions / pH: 12.0 for the alkalinity of the extraction medium, 53 &amp / #956 / m for the particle size, 40&amp / #61616 / C for the extraction temperature, 5.0 for the solvent to solid ratio and 0.0 M for the saline concentration. At these extraction conditions, the maximum protein recovery was 72.75% resulting in a product containing 93.29% protein on a dry basis.

The primary objectives of this study were to develop an effective reactive extrusion process to produce granular phosphorylated pea starches with enhanced enzyme resistance, and examine the effects of bulk phosphorylation conditions on the morphology, physicochemical and functional properties of extruded pea starch phosphates. Two types of commercially available pea starches (NutriPea and Meelunie) were chosen as the research subjects in this study with differing native resistance. A number of methods including optical microscopy, SEM, ICP-OES, Englyst method, DSC and rapid visco analysis (RVA) were used to characterize the morphology and properties of extruded pea starches. The effects of feed formulations and extrusion conditions on phosphorus incorporation and Englyst digestion profiles were examined systemically. The results showed that phosphorus content and digestion profiles were highly dependent on the feed moisture. Enzyme resistance did not positively correlate with phosphorus content for extruded pea starch phosphates in contrast to their counterparts prepared by conventional aqueous slurry. This was because extrusion processing can markedly increase the susceptibility of pea starch granules to enzymatic digestion. Lower feed moisture content (40%) gave lower phosphorus content, significantly lower RDS content, and higher SDS and/or RS content. Bulk phosphorylation in the extruder resulted in decreased RS2 content but increased RS4 content. Screw geometry with excessive mixing index was not desirable in terms of producing resistant starch. High screw speeds (150rpm and 200rpm) and low feed rate (1.02kg/h) brought about higher yields of SDS and RS in spite of lower phosphorus incorporation. Reactive extrusion of pea starches under optimized conditions achieved a significant but moderate increase either in RS content (from 18.67% to 22.57% for NutriPea) or in SDS content (from 37.18% to 42.23% for Meelunie) compared to their native counterparts. However, RS and SDS content could not be improved simultaneously at least based on these pea starches and the reactive extrusion processes in this study. The optical and SEM micrographs confirmed that the granule integrity was largely retained after optimized reactive extrusion process. DSC thermograms found no significant correlation between gelatinization characteristics and Englyst digestion profiles. Evidenced by RVA pasting profiles, NutriPea pea starch phosphates exhibited enhanced thermal and shear stability in comparison to their native counterpart. A novel foaming injection technology of cross-linking reagents solution was pioneeringly introduced to uniformly coat all starch particles at the lowest moisture level possible during the continuous production of granular NutriPea pea starch phosphates. Yet, the resulting phosphorus incorporation was much lower than expected and would require further studies. / Thesis / Master of Applied Science (MASc) / The Canadian food industry is increasingly interested in the potential to probe new avenues to produce enzyme-resistant food starches from pulses starches. Although extrusion cooking is commonly used for manufacturing cereals, snacks and other food products, no research has been reported on using an extruder to rapidly produce resistant pulse starches for functional food ingredients. This study aimed to develop an effective reactive extrusion process to produce phosphate cross-linked pea starches with enhanced enzyme resistance (i.e., increased slowly digestible starch (SDS) and resistant starch (RS) content ) based on an examination of the effects of reaction conditions on the properties of extrusion products. Two types of commercially available pea starches, NutriPea and Meelunie, were chosen as subjects of the research. The cross-linked pea starches under optimized conditions achieved a significant but moderate increase either in RS content (for NutriPea) or in SDS content (for Meelunie) compared to their native counterparts. However, RS and SDS content could not be improved simultaneously at least based on these pea starches and the reactive extrusion processes in this study.

Cajanus cajan (L.) Millsp. (Pigeonpea) belongs to the Leguminosae genus Cajanus which is composed of 34 species. Pigeonpea is the only cultivated member of the genus, while the remaining species are wild relatives belonging mainly to the secondary gene pool. DNA sequence data from the nuclear ITS region and the chloroplast trnL-F spacer were utilized to investigate the phylogenetic relationships between Cajanus and five other allied genera in the subtribe Cajaninae. This study revealed the non-monophyly of Cajanus and Rhynchosia and supported the monophyly of Eriosema and Flemingia, but more sampling ,especially from the large genera of Rhynchosia and Eriosema, is recommend to adequately test the hypothesis of generic monophyly. The phylogenetic relationships within the genus Cajanus resolved Cajanus scarabaeoides (L.) Thouars as the most basal species in the Cajanus clade. The study also utilized Single Nucleotide Polymorphism (SNP) markers derived from low copy orthologous genes and genotyped using the high throughput SNP-OPA Illumina golden gate assay. The aim was to understand phylogenetic and domestication history, genetic structure, patterns of genetic diversity, gene flow and historical hybridization between Cajanus cajan (pigeonpea) and wild relatives. The neighbor-joining tree resolved well-supported clusters, which reflect the distinctiveness of species and congruence with their geographical origin. It supported the ITS based phylogeny and resolved C. scarabaeoides as basal to the Cajanus clade. The phylogenetic signal and genetic signatures revealed insights into the domestication history of pigeonpea. Our results supported Cajanus cajanifolius as the presumed progenitor of pigeonpea and we speculate that for pigeonpea there was a single major domestication event in India. Genetic admixture and historical hybridization were evident between pigeonpea and wild relatives. Abundant allelic variation and genetic diversity was found in the wild relatives, with the exception of wild species from Australia, as compared to the domesticated pigeonpea. There was a reduction of about 75% in genetic polymorphism in domesticated pigeonpea as compared to the wild relatives, indicating a severe “domestication bottleneck” during pigeonpea domestication. We discovered SNP markers associated with disease resistance (NBS-LRR) loci. The SNPs were mined in a comparison of BAC-end sequences (BES) of C. cajan and amplicons of the wild species, C. scarabaeoides. A total of ~3000 SNPs were identified from 304 BES. These SNPs could potentially be used in constructing a genetic map and for marker assisted breeding.

Dans ce travail de thèse, nous avons étudié le transport du fer dans la plante, en nous focalisant sur la dernière étape de circulation : le remplissage de la graine. Il nous a paru important de comprendre 1- comment le fer arrive à la graine, 2- la manière dont il est délivré à l'embryon en développement, 3- sous quelles formes et 4- où et comment il est stocké dans l'embryon. Nous avons choisi de nous appuyer sur deux espèces modèles: Pisum sativum pour les approches de biochimie et Arabidopsis thaliana pour les approches de génétique. Le pois a été choisi car ses graines sont de grande taille et ont la particularité d'accumuler un albumen liquide qui sert de soutien nutritionnel pour l'embryon. Le point de départ de ce projet est l'étude de la spéciation du fer, c'est-à-dire l'identification des ligands du fer, dans cet albumen. Sur la base de la connaissance des complexes de fer circulant jusqu'à la graine, nous avons proposé de nouvelles fonctions physiologiques. La grande taille des cellules de pois a permis d'étudier la localisation du fer et sa spéciation in situ, à l'échelle subcellulaire. Chez Arabidopsis, nous avons étudié le rôle de deux gènes candidats, potentiellement impliqués dans le transport du fer vers la graine. / In this work, we studied iron transport in plants and we focused on the last circulation step: the seed filling. It seemt important to understand 1- how iron is arriving to the seeds, 2- the way it is delivered to developping embryos, 3- in which form and 4- where and how it is stored in embryos.To adress these questions, we used two model species: Pisum sativum for biochemical approaches and Arabidopsis thaliana for genetic approaches. Pea was choosen because of the large size of its seeds, and their characteristic to accumulate a liquid endosperm that feed embryos.The project started with the study of iron speciation, meaning the identification of iron ligands, in this endosperm. On the basis of the knowledge of the iron complexes arriving to seeds, we highlighted new physiological functions. The large size of pea cells also allowed us to study iron localization and in situ speciation, at subcellular level. In Arabidopis, we studied the role of two candidate genes putatively involved in iron transport to seeds.

A Neurobiologia Vegetal é um recente ramo das ciências vegetais que objetiva esclarecer os complexos padrões de comportamento vegetal, no que se refere à percepção, processamento, armazenamento e transmissão de sinais na planta e entre plantas. A detecção de vizinhos, é uma capacidade que implica em auto reconhecimento, uma vez que um organismo só terá sucesso em interações competitivas se for capaz de auto/não-auto discriminação. Assim, objetivou-se com este trabalho verificar se raízes de ervilha (Pisum sativum) cv. Mikado apresentam crescimento diferenciado quando na presença de raízes da mesma planta, e de raízes de outras plantas, mas pertencentes ao mesmo genótipo, para que se possa averiguar sua capacidade de auto/não-auto discriminação. Além disso, avaliou-se também o crescimento da parte aérea para observar em que grau a presença de plantas vizinhas pode influenciar o desenvolvimento vegetativo de plantas de ervilha. Quatro dias após a germinação, plântulas de Pisum sativum cv. Mikado tiveram a raiz principal cortada 5 mm abaixo do hipocótilo. Passados sete dias, foram retiradas as raízes secundárias, deixando-se apenas duas raízes, de igual tamanho, por planta (split-root). Plantas com duas raízes iguais foram replantadas, com cada vaso contendo duas raízes da mesma planta (tratamento Auto) ou duas raízes de plantas diferentes (Tratamento Não-auto). Os vasos foram agrupados em tríades. O experimento foi mantido em estufa incubadora sob condições de temperatura e fotoperíodo controladas e após 18 dias foram feitas avaliações do crescimento da parte aérea e das raízes, através das medições de: altura da planta (cm), peso fresco de parte aérea e de raiz (g), peso seco de parte aérea e de raiz (g), área foliar (cm2), área radicular (cm2), comprimento total de raiz (cm) e diâmetro médio de raiz (cm). A análise dos dados considerando os valores médios de cada tríade revelou não haver diferença significativa entre os tratamentos Auto e Não-auto com relação ao crescimento de parte aérea. No que se refere ao crescimento da raiz, com exceção do diâmetro médio, as demais variáveis diferiram significativamente, sendo que as plantas pertencentes ao tratamento Auto apresentaram valores de peso seco, área superficial e comprimento total 36,71%, 27,84% e 23,18%, respectivamente, maiores do que as plantas do tratamento Não-auto. Ou seja, as plantas que não estavam sob competição apresentaram maior crescimento de raiz. No entanto, quando se observou o comportamento das plantas entre si, em cada tríade, verificou-se, no tratamento não-auto, diferenças visíveis de crescimento tanto em parte aérea como na raiz entre as três plantas que constituía cada tríade. Verificou-se também que a raiz de uma mesma planta cresceu diferentemente de acordo com a identidade da raiz vizinha. Enquanto que no tratamento auto as três plantas que constituíam uma tríade tinham aproximadamente o mesmo tamanho de parte aérea e raiz. Assim, podemos afirmar que o crescimento das plantas no tratamento não-auto foi influenciado pelas interações entre as raízes e mais que isto, foi dependente da identidade da raiz vizinha implicando em auto/não-auto discriminação e reconhecimento parental. / The Plant Neurobiology is a recent branch of plant science that aims to clarify the complex patterns of behavior vegetable, with respect to perception, processing, storage and transmission of signals in plant and between plants. The detection of neighbors, is a capacity that involves self-recognition and an individual will only be successful in competitive interactions if it is capable of self/non-self discrimination. Thus, the objective was to determine whether roots of pea (Pisum sativum) cv. Mikado grow differently in the presence of the same plant roots, and roots of other plants, but within the same genotype, so that we can determine its capacity for self/non-self discrimination. In addition, we assessed also the growth of the shoot to see to what degree the presence of neighboring plants can influence the vegetative growth of pea plants. Five days from germination, seedlings of Pisum sativum cv. Mikado had the seminal root severed 5 mm below the hypocotyl. After seven days, all but two of these roots were removed, leaving only two roots of equal size per plant (split-root). Plants with two equal roots were replanted, with each pot containing two roots of the same plant (treatment self) or two roots of different plants (Treatment non-self). Pots were grouped in triplets. The experiment was kept in an incubator camera under controlled conditions of temperature and photoperiod and after 18 days were evaluated for growth of shoots and roots. It was measure plant height (cm), fresh weight of shoot and root (g), dry weight of shoot and root (g), leaf area (cm2), root area (cm2), total length of root (cm) and average root diameter (cm). The analysis of data considering the average values of each triplets showed no significant difference between treatments self and non-self in relation to the growth of shoots. With respect to root growth, except for the diameter, the other variables differed significantly, and plants belonging to treatment self had values of dry weight, surface area and total length of 36.71%, 27.84 % and 23.18%, respectively, higher than the treatment plants non-self. That is, plants that were not under competition had higher root growth. However, when we observe the behavior of plants in each triplet, it was found that the treatment non-self, the plants had sizes of shoot and root differ. It was also found that the root of the same plant grew differently depending on the identity of neighboring roots. While in treatment self, the three plants that constituted a triplet had, approximately, the same size of shoot and root. Thus, we can say that the growth of plants to treatment non-self was influenced by the interactions between roots and more that this was dependent on the identity of neighboring roots implying self/non-self discrimination and kin recognition.