The use of traditional and natural medicines in primary healthcare or alternative therapy is on the increase. However, the safety and efficacy of these medicines have not yet been confirmed. Pharmacognosy, the study of the properties of drugs, potential drugs or drug substances of natural origin and the search for new drugs from natural resources, is therefore of extreme importance in today’s healthcare environment. Cotyledon orbiculata L. var. orbiculata (Haw.) DC., a succulent shrub that is widely distributed over the whole of southern Africa, is an example of a plant used in traditional medicine for its antiepileptic effects. Oxidative stress can either be the cause of, or be secondary to epilepsy pathogenesis. Lipid peroxidation causes the disruption of cell membranes which leads to cell destruction and, in the case of neurological disorders, neurodegeneration. Reactive species have also been found to influence neurotransmission by affecting neurotransmitter metabolism and functions. Reactive species can therefore be responsible for the development of convulsions. Conventional anti-epileptics have shown to exert neuroprotective effects but information or research regarding their ability to prevent epilepsy from becoming chronic does either not exist or is not promising. Antioxidants have potential in the treatment of epileptic seizures as well as the prevention of chronic epilepsy by preventing the effects that oxidative stress has on neurotransmitter metabolism and functions that cause alterations in neuronal excitability and seizure threshold, ultimately leading to epileptic foci. The aim of this study was to evaluate the potential of the bufadienolide orbicusides of C. orbiculata and analogues as anti-epileptic treatment through antioxidant activity. Initially the isolation of novel antioxidants from C. orbiculata leaf juice was attempted. The antioxidant activity of the concentrated juice and fractions resulting thereof were evaluated with two assays. The thiobarbituric acid (TBA) assay was used to measure the extent of lipid peroxidation and nitroblue tetrazolium (NBT) assay was used to measure superoxide scavenging activity in rat brain homogenate. The low concentrations of orbicusides prompted the determination of the activity of two commercial bufadienolides (bufalin and cinobufotalin) and two bufadienolide analogues, synthesised by the esterification of trans-androsterone and androstanolone, respectively, using coumalic acid, producing Compound 1 and Compound 2. The toxicity of the commercial bufadienolides and synthesised analogues were evaluated by using the MTT assay (a cell viability assay). C. orbiculata juice showed significant pro-oxidant activity in both assays. Bufalin showed significant pro-oxidant activity in the TBA assay. Cinobufotalin showed no significant activity. Compound 1 showed pro-oxidant activity in the TBA assay and Compound 2 showed slight antioxidant activity in the NBT assay. The commercial bufadienolides showed low cell viability, indicating significant toxicity. The synthesised analogues showed a significant reduction in toxicity (despite Compound 2 being moderately toxic) when compared to the toxicity of the commercial bufadienolides. The low concentrations of orbicusides in the plant material and the antioxidant assay results of the two commercial bufadienolides suggested that the orbicusides may not be involved in the antioxidant properties of C. orbiculata. However, the antioxidant activity of Compound 2 showed that altering the pyrone moiety of bufadienolides could possibly improve antioxidant activity. The reduced toxicity and slight antioxidant activity of the synthesised bufadienolide analogues motivates further investigation. / MSc (Pharmaceutical Chemistry), North-West University, Potchefstroom Campus, 2014

South Africa is a country of great diversity. Different climate zones and a host of different habitats make South Africa the perfect platform for rich floral diversity. This floral diversity lends itself to the study of natural products by discovering new natural drugs that can be used in the treatment of many illnesses. Studies into the antioxidant properties of plants that are used in traditional medicine are an important aspect of research to determine the rationale of the use of plants by traditional healers. Many neurodegenerative diseases, like epilepsy, Parkinson s and Alzheimer s diseases, are linked to oxidative stress. Antioxidants could play a major role as neuroprotective agents and could alter the progression of these diseases. Epilepsy is one of the world s most prevalent central nervous system disorders and affects more than seventy per one thousand children in South Africa. Most of these cases are people in rural areas of South Africa where communities rely on the use of traditional medicine. Cotyledon orbiculata L. var orbiculata (Haw.) DC. is widely used in traditional medicine to treat epilepsy and other central nervous system disorders. The need to screen these plants for activity and toxicity is very important to understand the complex mechanism of action in the treatment of patients. In this study the methanol extract and three different fractions of the methanol extract of Cotyledon orbiculata were used to test for antioxidant activity and toxicity towards neuroblastoma cells. The freeze dried leaves of Cotyledon orbiculata were extracted with methanol using a Soxhlet apparatus. The concentrated extracts were analysed using HPLC (high pressure liquid chromatography) and three major peaks were selected for isolation. Three assays were performed to assess the antioxidant activity and toxicity of the isolated compounds. The thiobarbituric acid assay (TBA) quantifies the extent of the inhibition of lipid peroxidation in rat brain homogenates by the isolated fractions. All of the samples were able to attenuate lipid peroxidation as seen from the results obtained from the TBA assay. The methanol extract showed the best attenuation of lipid peroxidation in the rat brain homogenate with fraction 1 and 2 showing greater attenuation of lipid peroxidation than fraction 3. The nitroblue tetrazolium assay (NBT) quantifies the ability of the fractions to scavenge superoxide radicals in a rat brain homogenate. All samples were able to scavenge superoxide radicals as indicated by the NBT assay. The methanol extract showed the best superoxide scavenging abilities in the assay whereas fraction 1 showed better scavenging abilities than fraction 2 and 3. The 3–(4,5–dimethylthiazol–2–yl)–2,5–diphenyltetrazolium bromide assay (MTT) indicates the toxicity of the fractions towards neuroblastoma cells. The methanol extract and fraction 2 in the highest concentration of 10 mg/ml were the only samples that showed toxicity towards neuroblastoma cells. The molecular structure of a compound from fraction 2 was determined by using nuclear magnetic resonance spectroscopy (NMR), infrared spectroscopy (IR), and mass spectroscopy (MS). This compound was identified as diethyl malate. Diethyl malate is an artefact that is generated in HPLC procedures in the presence of malic acid (which naturally occurs in the leaves of Cotyledon orbiculata) and ethanol. The methanol extract of Cotyledon orbiculata has high antioxidant activity and could be due to the presence of malic acid in the leaves of the plant. The rationale in the use of Cotyledon orbiculata in the treatment of epilepsy could not be determined due to the isolation of an artefact, diethyl malate, obtained from the fraction. Further research should include methods to prevent artefact formation and purification of the samples that are obtained. / Thesis (MSc (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2012.

South Africa is a country of great diversity. Different climate zones and a host of different habitats make South Africa the perfect platform for rich floral diversity. This floral diversity lends itself to the study of natural products by discovering new natural drugs that can be used in the treatment of many illnesses. Studies into the antioxidant properties of plants that are used in traditional medicine are an important aspect of research to determine the rationale of the use of plants by traditional healers. Many neurodegenerative diseases, like epilepsy, Parkinson s and Alzheimer s diseases, are linked to oxidative stress. Antioxidants could play a major role as neuroprotective agents and could alter the progression of these diseases. Epilepsy is one of the world s most prevalent central nervous system disorders and affects more than seventy per one thousand children in South Africa. Most of these cases are people in rural areas of South Africa where communities rely on the use of traditional medicine. Cotyledon orbiculata L. var orbiculata (Haw.) DC. is widely used in traditional medicine to treat epilepsy and other central nervous system disorders. The need to screen these plants for activity and toxicity is very important to understand the complex mechanism of action in the treatment of patients. In this study the methanol extract and three different fractions of the methanol extract of Cotyledon orbiculata were used to test for antioxidant activity and toxicity towards neuroblastoma cells. The freeze dried leaves of Cotyledon orbiculata were extracted with methanol using a Soxhlet apparatus. The concentrated extracts were analysed using HPLC (high pressure liquid chromatography) and three major peaks were selected for isolation. Three assays were performed to assess the antioxidant activity and toxicity of the isolated compounds. The thiobarbituric acid assay (TBA) quantifies the extent of the inhibition of lipid peroxidation in rat brain homogenates by the isolated fractions. All of the samples were able to attenuate lipid peroxidation as seen from the results obtained from the TBA assay. The methanol extract showed the best attenuation of lipid peroxidation in the rat brain homogenate with fraction 1 and 2 showing greater attenuation of lipid peroxidation than fraction 3. The nitroblue tetrazolium assay (NBT) quantifies the ability of the fractions to scavenge superoxide radicals in a rat brain homogenate. All samples were able to scavenge superoxide radicals as indicated by the NBT assay. The methanol extract showed the best superoxide scavenging abilities in the assay whereas fraction 1 showed better scavenging abilities than fraction 2 and 3. The 3–(4,5–dimethylthiazol–2–yl)–2,5–diphenyltetrazolium bromide assay (MTT) indicates the toxicity of the fractions towards neuroblastoma cells. The methanol extract and fraction 2 in the highest concentration of 10 mg/ml were the only samples that showed toxicity towards neuroblastoma cells. The molecular structure of a compound from fraction 2 was determined by using nuclear magnetic resonance spectroscopy (NMR), infrared spectroscopy (IR), and mass spectroscopy (MS). This compound was identified as diethyl malate. Diethyl malate is an artefact that is generated in HPLC procedures in the presence of malic acid (which naturally occurs in the leaves of Cotyledon orbiculata) and ethanol. The methanol extract of Cotyledon orbiculata has high antioxidant activity and could be due to the presence of malic acid in the leaves of the plant. The rationale in the use of Cotyledon orbiculata in the treatment of epilepsy could not be determined due to the isolation of an artefact, diethyl malate, obtained from the fraction. Further research should include methods to prevent artefact formation and purification of the samples that are obtained. / Thesis (MSc (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2012.

Le grain de maïs est composé de 3 compartiments : l’embryon et l’albumen issus de la double fécondation et l’enveloppe d’origine maternelle. Le développement du grain et l’accumulation de réserves demande l’établissement d’une communication étroite entre l’embryon et l’albumen pour coordonner leur développement respectif. Si, des régulateurs majeurs impliqués dans le développement de la graine d’Arabidopsis ont été décrits, ces connaissances restent parcellaires chez les céréales. Les objectifs de ma thèse consistaient d’une part à étudier le contrôle de la communication entre l’embryon et l’albumen et d’autre part la régulation du remplissage du grain de maïs. Par l’analyse de lignées transgéniques sous exprimant ZmZHOUPI (ZmZOU-RNAi), nous avons établi que ce facteur de transcription à domaine bHLH, bien que s’exprimant exclusivement dans l’albumen, affecte significativement le développement de l’embryon (taille de l’embryon, persistance du suspenseur). L’analyse de données RNAseq (grains sauvages versus grains ZmZOU-RNAi) a permis d’identifier des gènes cibles potentiels de ZmZOU. De plus, nous avons montré que 3 facteurs de transcription de type bHLH homologues d’INDUCER OF CBP EXPRESSION (ICE) forment un partenariat avec ZmZOU.D’autre part, nous avons étudié les homologues d'ABA INSENSITIVE3, FUSCA3 et LEAFY COTYLEDON2 (AFL) qui forment un réseau de facteurs de transcription, à domaine B3, régulant l’accumulation d’huile et de protéines de réserves dans l’embryon d’Arabidopsis. Grâce à des analyses phylogénétiques et d’expression, nous avons établi que chez le maïs le réseau AFL, constitué de 5 membres (ZmAFLs), est partiellement conservé. Par dosages et analyse d’expression, nous avons montré que ZmAFL4, en particulier, est impliqué dans le contrôle de la biosynthèse de l’amidon dans l’albumen. / Maize kernel is composed of three major compartments: an embryo and an endosperm both produced by double fertilization and the maternally derived seed coat. Seed development and reserves accumulation demands coordination and thus communication between embryo and endosperm allowing specific growth of each compartment. While major regulators involved in seed development have been already described in Arabidopsis, knowledge in cereals remains limited. My thesis purposes were to study on one hand the control of communication between embryo and endosperm and on the other hand regulation of maize kernel filling.By analysis of transgenic lines knock down ZmZHOUPI (ZmZou-RNAi), we showed that this bHLH domain transcription factor, exclusively expressed in endosperm, affect significantly embryo development, size of embryo proper and suspensor persistence. RNAseq data analyses let find putative direct targets of ZmZOU. Additionally, we identified ZmZOU partners, 3 bHLH domain transcription factor homologs of INDUCER OF CBP EXPRESSION (ICE).Furthermore, we studied homologs of three B3 domain transcription factors named ABA INSENSITIVE3, FUSCA3 et LEAFY COTYLEDON2 (AFL) which form a regulatory network governing oil and seed storage proteins accumulation in Arabidopsis embryo. By phylogenetic and expression analysis, we established that 5 genes (ZmAFLs) constitute in maize a partially conserved AFL network. Through dosage and expression analysis, we established that particularly ZmAFL4 is involved in starch biosynthesis regulation.

Na maioria das sementes a emergência da radícula caracteriza o término da germinação e marca o início do desenvolvimento da plântula. A atividade das hidrolases da parede celular durante a pré-emergência pode estar associada ao amolecimento do tecido que circunda o embrião, principalmente na região micropilar, onde ocorre a protrusão da radícula. A atividade dessas enzimas após a emergência é associada à degradação de reservas polissacarídicas da parede celular, mobilizadas para suprir a plântula de açúcares antes que se torne autotrófica. No presente trabalho foram investigadas várias hidrolases da parede celular no endosperma de Euphorbia heterophylla durante a germinação e o desenvolvimento inicial pós-germinativo da plântula. Foi também realizada a purificação parcial de endo-b-1,4-glucanases da semente dessa espécie. As atividades da xiloglucano endotransglicosilase (XET), endo-b-mananase e b-manosidase são maiores no período de pré-emergência da semente de E. heterophylla, comparando-se com o período de pós-emergência. Por outro lado, as atividades da b-galactosidase, b-glucosidase, a-xilosidase, b-xilosidase e das glucanases que hidrolisam CMC, xiloglucanos de Hymenaea courbaril ou de Copaifera langsdorffii, xilano, Avicel e liquenano são elevadas no período de pós-emergência. A atividade sobre a laminarina ocorre durante ambos os períodos, de pré- e pós-emergência da radícula. A atividade da poligalacturonase não foi detectada nessa semente. Os resultados sugerem que XET, endo-b-mananase e b-manosidase podem estar envolvidas no processo de germinação, enquanto que as demais enzimas podem estar relacionadas ao processo de mobilização de reservas da semente. Em E. heterophylla o embrião está encerrado num endosperma rico em lipídios e proteínas, cujos produtos da degradação são absorvidos pelos cotilédones que iniciam a expansão 24 horas após o início da embebição. É possível que a hidrólise da parede celular do endosperma diminua a resistência contra a expansão em área do cotilédone facilitando, ao mesmo tempo, a rápida difusão dos produtos da degradação para os cotilédones. A purificação parcial das endoglucanases foi realizada utilizando-se colunas de Sephacryl S-100-HR numa primeira etapa, resultando em 2 grupos com atividade sobre CMC denominados I e II. Em uma segunda etapa, as endoglucanases do grupo I foram sequencialmente purificadas em coluna DEAE-Sephadex e de CF11-Celulose (afinidade). Através da focalização isoelétrica em gel de poliacrilamida e técnicas de sobreposição em gel de ágar para a determinação de atividade, foram detectadas endoglucanases de pIs de aproximadamente 3,0, 3,3, 3,8, 4,4, 4,9, 5,4 e 5,7 e um outro grupo de enzimas cujos pIs variam entre 8,5 e 10,0. As enzimas de pIs 3,0, 3,3 e 3,8 hidrolisam CMC e xiloglucano de C. langsdorffii; as de pIs entre 8,5 e 10,0 hidrolisam CMC e o xiloglucano de H. courbaril, mas não hidrolisam o de C. langsdorffii; as enzimas de pIs 4,4, 4,9 e 5,7 hidrolisam somente CMC; a de pI 5,4 hidrolisa somente xiloglucano de H. courbaril. As endoglucanases do grupo II foram purificadas em coluna de CM-celulose, tendo sido detectada uma enzima de pI 3,2 que degrada CMC bem como xiloglucano de C. langsdorffii e de H. courbaril. Foi também detectada uma xiloglucanase de pI 4,2 que hidrolisa somente xiloglucano de H. courbaril e um grupo de isozimas cujos pIs variam entre 8,5 e 10,0, que hidrolisa somente CMC. As xiloglucanases têm sido investigadas em sementes cujo xiloglucano é armazenado no cotilédone. O presente trabalho é o primeiro a relatar a ocorrência de xiloglucanases em uma semente endospérmica. / In most seeds radicle protrusion characterizes the termination of germination and the beginning of seedling development. The activity of cell wall hydrolases during the pre-emergence stage may be related to the softening of the tissue which involves the embryo, notably in the micropylar region where the radicle protrusion occurs. The activity of these enzymes following radicle emergence is related to the reserve degradation which may be cell wall polysaccharides mobilized to supply the seedling with sugars before it becomes autotrophic. In the present work it was investigated several cell wall hydrolases from the endosperm of Euphorbia heterophylla during germination and initial seedling development of this species. A partial purification of endo-b-1,4-glucanase was also performed. The activities of xyloglucan endotransglycosylase (XET), endo-b-mannanase and b-manosidase in E. heterophylla seeds are higher over the pre-emergence when compared to the post-emergence period. On the other hand the activities of b-galactosidase, b-glucosidase, a-xylosidase, b-xylosidase and glucanases which hydrolise CMC, xyloglucans from Hymenaea courbaril and Copaifera langsdorffii, xylan, Avicel and liquenan are higher over the post-emergence period. Activity on laminarin occurs over both periods. Polygalacturonase activity has not been detected in this seed. These results suggest that XET, endo-b-mannanase and b-manosidase may be involved in the process of germination whereas the other enzymes may be more related to reserve mobilization. In E. heterophylla the endosperm contains high contents of lipids and proteins and the degradation products of these reserves are absorbed by the cotyledons which expansion begins 24 hours since the start of imbibition. Probably endosperm cell wall hydrolysis facilitates cotyledon expansion by lowering endosperm resistance and at the same time the diffusion of degradation products into cotyledons. Partial purification of endoglucanases was carried out on Sephacryl S-100-HR as a first step resulting 2 active fractions (I and II) on CMC. Fraction I was further purified on DEAE-Sephadex and CF11-Cellulose (affinity) columns. Polyacrylamide gel isoelectric focusing followed by gel-overlay assay technique indicated several endoglucanases with pIs around 3.0, 3.3, 3.8, 4.4, 4.9, 5.4 and 5.7 and another group between 8.5 and 10.0. The pI 3.0, 3.3 and 3.8 enzymes hydrolyse both CMC and xyloglucan from Copaifera langsdorffii; the group between 8.5 and 10.0 hydrolyse both CMC and xyloglucan from Hymenaea courbaril but not from C. langsdorffii; pIs 4.4, 4.9 and 5.7 enzymes hydrolyse only CMC; pI 5.4 enzyme hydrolyse only xyloglucan from H. courbaril. Fraction II was further purified on CM-celullose column. It was detected a pI 3.2 endoglucanase which degrades CMC and xyloglucan from both C. langsdorfii and H. courbaril, a pI 4.2 xyloglucanase which hydrolyses only xyloglucan from H. courbaril, and a group of isozymes (pI 8.5 to 10.0) which hydrolyses only CMC. Xyloglucanases have been investigated only in seeds that have stored xyloglucan in the cotyledon. The present work is the first one to report the occurrence of xyloglucanases in an endospermic seed.

Cloning of trees using somatic embryogenesis could have a major impact on tree breeding and commercial plantation forestry. To initiate somatic embryogenesis in Anatolian black pine (Pinus nigra Arnold. subspecies pallasiana), one-year old cones containing immature seeds were collected from eight trees located in METU campus, Ankara. Embryogenic tissues were derived from immature zygotic embryos excised from the seeds. The zygotic embryos at the time of collection were at the precotyledonary stage of development. For this study, Douglas-fir cotyledon revised medium (DCR) supplemented with 13.6&micro / M 2,4-D, 2.2&micro / M BAP, 0.5 g/L casein hydrolysate, 0.25 g/L L-glutamine and 3% sucrose was used. The media was solidified with 0.2% gelrite. Embryogenic tissue initiation was calculated for each genotype and collection date. Overall initiation frequencies were recorded as 0.92% for 2004 and 1.96% for 2005. Highest initiation frequency was calculated for 5-July 2005 sampling time (4.06). ANOVA revealed significant differences between trees and collection date for initiation frequencies. Also, ECL (Established cell lines) recorded after five subcultures. Overall, 0.38% and 0.62% of the initial explants were converted into ECLs for 2004 and 2005 respectively.

Na maioria das sementes a emergência da radícula caracteriza o término da germinação e marca o início do desenvolvimento da plântula. A atividade das hidrolases da parede celular durante a pré-emergência pode estar associada ao amolecimento do tecido que circunda o embrião, principalmente na região micropilar, onde ocorre a protrusão da radícula. A atividade dessas enzimas após a emergência é associada à degradação de reservas polissacarídicas da parede celular, mobilizadas para suprir a plântula de açúcares antes que se torne autotrófica. No presente trabalho foram investigadas várias hidrolases da parede celular no endosperma de Euphorbia heterophylla durante a germinação e o desenvolvimento inicial pós-germinativo da plântula. Foi também realizada a purificação parcial de endo-b-1,4-glucanases da semente dessa espécie. As atividades da xiloglucano endotransglicosilase (XET), endo-b-mananase e b-manosidase são maiores no período de pré-emergência da semente de E. heterophylla, comparando-se com o período de pós-emergência. Por outro lado, as atividades da b-galactosidase, b-glucosidase, a-xilosidase, b-xilosidase e das glucanases que hidrolisam CMC, xiloglucanos de Hymenaea courbaril ou de Copaifera langsdorffii, xilano, Avicel e liquenano são elevadas no período de pós-emergência. A atividade sobre a laminarina ocorre durante ambos os períodos, de pré- e pós-emergência da radícula. A atividade da poligalacturonase não foi detectada nessa semente. Os resultados sugerem que XET, endo-b-mananase e b-manosidase podem estar envolvidas no processo de germinação, enquanto que as demais enzimas podem estar relacionadas ao processo de mobilização de reservas da semente. Em E. heterophylla o embrião está encerrado num endosperma rico em lipídios e proteínas, cujos produtos da degradação são absorvidos pelos cotilédones que iniciam a expansão 24 horas após o início da embebição. É possível que a hidrólise da parede celular do endosperma diminua a resistência contra a expansão em área do cotilédone facilitando, ao mesmo tempo, a rápida difusão dos produtos da degradação para os cotilédones. A purificação parcial das endoglucanases foi realizada utilizando-se colunas de Sephacryl S-100-HR numa primeira etapa, resultando em 2 grupos com atividade sobre CMC denominados I e II. Em uma segunda etapa, as endoglucanases do grupo I foram sequencialmente purificadas em coluna DEAE-Sephadex e de CF11-Celulose (afinidade). Através da focalização isoelétrica em gel de poliacrilamida e técnicas de sobreposição em gel de ágar para a determinação de atividade, foram detectadas endoglucanases de pIs de aproximadamente 3,0, 3,3, 3,8, 4,4, 4,9, 5,4 e 5,7 e um outro grupo de enzimas cujos pIs variam entre 8,5 e 10,0. As enzimas de pIs 3,0, 3,3 e 3,8 hidrolisam CMC e xiloglucano de C. langsdorffii; as de pIs entre 8,5 e 10,0 hidrolisam CMC e o xiloglucano de H. courbaril, mas não hidrolisam o de C. langsdorffii; as enzimas de pIs 4,4, 4,9 e 5,7 hidrolisam somente CMC; a de pI 5,4 hidrolisa somente xiloglucano de H. courbaril. As endoglucanases do grupo II foram purificadas em coluna de CM-celulose, tendo sido detectada uma enzima de pI 3,2 que degrada CMC bem como xiloglucano de C. langsdorffii e de H. courbaril. Foi também detectada uma xiloglucanase de pI 4,2 que hidrolisa somente xiloglucano de H. courbaril e um grupo de isozimas cujos pIs variam entre 8,5 e 10,0, que hidrolisa somente CMC. As xiloglucanases têm sido investigadas em sementes cujo xiloglucano é armazenado no cotilédone. O presente trabalho é o primeiro a relatar a ocorrência de xiloglucanases em uma semente endospérmica. / In most seeds radicle protrusion characterizes the termination of germination and the beginning of seedling development. The activity of cell wall hydrolases during the pre-emergence stage may be related to the softening of the tissue which involves the embryo, notably in the micropylar region where the radicle protrusion occurs. The activity of these enzymes following radicle emergence is related to the reserve degradation which may be cell wall polysaccharides mobilized to supply the seedling with sugars before it becomes autotrophic. In the present work it was investigated several cell wall hydrolases from the endosperm of Euphorbia heterophylla during germination and initial seedling development of this species. A partial purification of endo-b-1,4-glucanase was also performed. The activities of xyloglucan endotransglycosylase (XET), endo-b-mannanase and b-manosidase in E. heterophylla seeds are higher over the pre-emergence when compared to the post-emergence period. On the other hand the activities of b-galactosidase, b-glucosidase, a-xylosidase, b-xylosidase and glucanases which hydrolise CMC, xyloglucans from Hymenaea courbaril and Copaifera langsdorffii, xylan, Avicel and liquenan are higher over the post-emergence period. Activity on laminarin occurs over both periods. Polygalacturonase activity has not been detected in this seed. These results suggest that XET, endo-b-mannanase and b-manosidase may be involved in the process of germination whereas the other enzymes may be more related to reserve mobilization. In E. heterophylla the endosperm contains high contents of lipids and proteins and the degradation products of these reserves are absorbed by the cotyledons which expansion begins 24 hours since the start of imbibition. Probably endosperm cell wall hydrolysis facilitates cotyledon expansion by lowering endosperm resistance and at the same time the diffusion of degradation products into cotyledons. Partial purification of endoglucanases was carried out on Sephacryl S-100-HR as a first step resulting 2 active fractions (I and II) on CMC. Fraction I was further purified on DEAE-Sephadex and CF11-Cellulose (affinity) columns. Polyacrylamide gel isoelectric focusing followed by gel-overlay assay technique indicated several endoglucanases with pIs around 3.0, 3.3, 3.8, 4.4, 4.9, 5.4 and 5.7 and another group between 8.5 and 10.0. The pI 3.0, 3.3 and 3.8 enzymes hydrolyse both CMC and xyloglucan from Copaifera langsdorffii; the group between 8.5 and 10.0 hydrolyse both CMC and xyloglucan from Hymenaea courbaril but not from C. langsdorffii; pIs 4.4, 4.9 and 5.7 enzymes hydrolyse only CMC; pI 5.4 enzyme hydrolyse only xyloglucan from H. courbaril. Fraction II was further purified on CM-celullose column. It was detected a pI 3.2 endoglucanase which degrades CMC and xyloglucan from both C. langsdorfii and H. courbaril, a pI 4.2 xyloglucanase which hydrolyses only xyloglucan from H. courbaril, and a group of isozymes (pI 8.5 to 10.0) which hydrolyses only CMC. Xyloglucanases have been investigated only in seeds that have stored xyloglucan in the cotyledon. The present work is the first one to report the occurrence of xyloglucanases in an endospermic seed.

Fundação de Amparo a Pesquisa do Estado de Minas Gerais / The purpose of this study is to evaluate the amount of transcripts from the Major Histocompatibility Complex (MHC) class I classical (BoLA-N *) and non-classical (BoLA-NC*), as well as of TNF-α (Tumor Necrosis Factor alpha) and TGF-β1 (Transforming Growth Factor beta 1) cytokines in bovine non-pregnant uterus, caruncles, cotyledons and blastocysts throughout the pregnancy period, in order to understand the immune and physiological mechanisms that occur in pregnancy in cows. The samples were analyzed by qPCR, the qualification (presence or absence) by conventional RT-PCR and the immune fluorescence with sheep monoclonal antibody against MHC class I. As positive control we used a sample of tissue from the bovine lymph node and/or spleen in all experimental tests. The amount of transcript BoLA-N* and BoLA-NC* was small (fold change 0 to 3) in blastocysts and cotyledons. Cotyledons transcription BoLA-NC* is greater than the transcription of BoLA-N*. Blastocysts transcription of BoLA-N* was higher than BoLA-NC*. Blastocysts transcribed more BoLA-NC2 than in cotyledons. Blastocysts not transcribed BoLA-NC4. Cotyledons transcribed BoLA-NC4 more than BoLA-NC2. The caruncles showed large amounts of transcripts (fold change 5 to 400), which were higher for pregnancy period of \"8-9\" month to BoLA-N* and one month for BoLA-NC*. Caruncles BoLA-NC2 transcription was higher than the BoLA-NC4. The uterus non pregnant had fewer transcripts BoLA-N* and BoLA-NC* than in caruncles. The uterus non pregnant no have transcripts of BoLA-NC2, but they had a small amount of transcripts of BoLA-NC4. Caruncles had large amounts of transcripts (fold change 50 to 500) of BoLA-NC2 with pregnancy age greater than one month; those are seemed to protect the fetus from rejection. TNF-α appears in greater amount in late gestation (8 and 9 months), showing the beginning of an inflammatory reaction, necessary to the adequate removal of the fetus and the placenta during delivery. Since TGF-β1 has a peak of expression in the sixth and seventh months of gestation, just when the fetus has its greatest growth, demonstrating a probable involvement of this cytokine in this phenomenon. / Com o intuito de entender os mecanismos imunofisiológicos da gestação em vacas esta tese teve como objetivo avaliar a quantidade de transcritos do Complexo de Histocompatibilidade Principal (MHC) de classe I clássico (BoLA-N*) e não clássico (BoLA-NC*), bem como das citocinas TNF-α (Fator de Necrose Tumoral alfa) e TGF-β1 (Fator de Crescimento Transformador beta 1) em úteros não gestantes, carúnculas, blastocistos e cotilédones bovino durante todo o período gestacional. Utilizou-se a quantificação por qPCR, a qualificação (presença ou ausência) via RT-PCR convencional e a imunofluorescência com anticorpo primário monoclonal MHC de classe I ovino. Como controle positivo foi utilizado uma amostra de tecido de linfonodo e/ou baço bovino em todos os ensaios experimentais. A quantidade de transcrito de BoLA de classe I clássico e não clássico foi pequena (fold change variando de 0 a 3) nos blastocistos e cotilédones. Nos cotilédones a transcrição de BoLA-NC* foi maior do que a transcrição de BoLA-N*. Nos blastocistos houve maior transcrição de BoLA-N* do que de BoLA-NC*. BoLA-NC2 transcreveu mais nos blastocisto do que nos cotilédones. BoLA-NC4 não transcreveu nos blastocistos. Os cotilédones transcreveram mais BoLA-NC4 do que BoLA-NC2. As carúnculas apresentaram grandes quantidades de transcritos (fold change variando de 5 a 400), que foram maiores nos períodos gestacionais de 8 a 9 para BoLA_N* e até 1 mês para BoLA-NC*. A transcrição de BoLA-NC2 foi maior do que a de BoLA-NC4 nas carúncula. O útero não gestante teve menor quantidade de transcritos de BoLA-N* e BoLA-NC* do que nas carúnculas. Não há transcrição de BoLA-NC2 no útero não gestante, mas há uma pequena quantidade de transcritos de BoLA-NC4 neste tecido. A presença de grandes quantidades de transcritos (fold change variando de 50 a 500) de BoLA-NC2 nas carúnculas, com idade gestacional maior do que um mês, parece proteger o feto da rejeição. Nas carúnculas parece existir um pico de transcrição de TGF-β1 no sexto e sétimo meses de gestação, justamente quando o feto tem seu maior crescimento, sinalizando um provável envolvimento desta citocina neste fenômeno. O TNF-α surge em maior quantidade no final da gestação, tanto nas carúnculas quanto nos cotilédones, demonstrando o início de uma reação inflamatória, necessária para a adequada expulsão do concepto e da placenta durante o parto. / Doutor em Imunologia e Parasitologia Aplicadas

Philosophiae Doctor - PhD / Starch is a major carbohydrate reserve in many plants, providing energy during heterotrophic growth and it is contained in large amounts in staple foods such as potatoes, wheat, maize, rice, sorghum and cassava. Apart from being a major product for use in the food industry, starch is also attracting interest from the biofuels industry as a source of bioethanol. This study reports on the development of genetic tools aimed at increasing starch production in sorghum (Sorghum bicolor L Moench), a crop of key agronomic importance worldwide by exploiting a new discovery of a transcription factor gene that regulates starch accumulation in Arabidopsis thaliana namely LEAFY COTYLEDON I (LECl). Ectopic over expression of this gene in arabidopsis has previously been shown to induce a massive hyper accumulation of starch in vegetative tissues. Therefore, we set out to investigate the function of its orthologous gene counterpart in sorghum with the aim of manipulating starch yield directly. Deduced protein sequence analyses showed that the putative sorghum LEAFY COTYLEDON I gene (SbLEC1) cloned in this study shares an overall high amino acid sequence identity (70 %) with the arabidopsis LEC 1, while the functional central B domain shows an even higher percentage sequence identity (91 %) with the same region of arabidopsis LEC 1. The putative SbLEC1 protein shares 14 out of the 16, signature ammo acids characteristic of the Central B Domain with arabidopsis. Furthermore, the putative SbLEC1 protein was also shown to share a significantly high sequence identity (> 80 %) with other well-characterized LEC1 protein sequences from organisms such as maize, rice, rapeseed as well as other organisms documented in the NCB I database. Similarly, much of the sequence similarity lies within the functional central B domain compared to any other region. Gene expression profiling using semi-quantitative PCR showed that SbLEC1 transcripts accumulated in developing seeds as well as in embryogenic calli tissue and no SbLEC 1 transcripts were detectable in leaf, root or sheath tissue. In order to confirm that the identified transcription factor is a functional ortholog, the full cDNA encoding putative SbLEC 1 transcription factor was identified, isolated and cloned from the sweet sorghum MN 1812 genotype. Plant transformation gene constructs based on the pCAMBIA1305.2 binary vector harbouring the transcription factor gene under the control of different promoter sequences were then assembled and immobilized into Agrobacterium tumefaciens strain LBA4404 in preparation for sorghum and arabidopsis transformation. Transient GUS expression studies showed that the five SbLEC1 gene constructs developed in this study were successfully transformed into arabidopsis (Ws ecotype) and sorghum (variety MN1812) callus and cell suspension cultures. The transformed tissues thus represent essential tools that are useful to evaluate the effect of over expressing the putative SbLEC1 protein. Transient GUS expression assays also further revealed differences in efficiency among promoters in driving transgene expression. Transient GUS activity was highest for the maize ubiquitin promoter (MUbi1), followed by the sorghum LEC1 promoter (SLECP), the arabidopsis LEC1 promoter (ALECP) and lastly the maize alcohol dehydrogenase promoter (MAdh1). The ability of the putative SbLEC 1 gene to complement the arabidopsis lecI mutation was also investigated and our findings were not conclusive as they only revealed partial complementation. A detailed comparison of SbLECI full cDNA sequences isolated and cloned from twenty-eight different F2 population plants from different sorghum varieties revealed the existence of sequence variation within the SbLEC 1 gene, which appeared to be allelic. The allelic variation was further shown to affect the amino acid composition of the putative SbLEC 1 protein. Heterologous protein expression studies of the SbLECI gene using an E. coli system showed that the predicted 29.16 kDa putative SbLEC 1 protein could be expressed in vitro both as an development of an efficient tissue culture protocol is a prerequisite for plant genetic engineering, this study also reports on the evaluation of thirteen sorghum genotypes from different genetic backgrounds for their in vitro culture response. A tissue culture protocol for three previously unexplored sorghum genotypes namely Agricol white, AS4 and MNI812 was established. The effect of plant genotype, explant and medium composition on in vitro culture response was highly significant (95 % Cl) in this study. Taken together, the findings in our study demonstrate efforts to draw a baseline foundation for the development of molecular technologies that can be used to increase starch production in sweet sorghum as a water efficient and sustainable feedstock for biofuel production.

Made available in DSpace on 2014-07-29T15:22:59Z (GMT). No. of bitstreams: 1 Lilian C M Lopes - Dissertacao.pdf: 480404 bytes, checksum: edbfe2459d6604d32a4980d5fd7dcdca (MD5) Previous issue date: 2010-04-30 / The common bean (Phaseolus vulgaris L.) assumes great importance in human nutrition. It holds essential components to the diet such as proteins, carbohydrates and minerals. The bean cotyledon is a byproduct of the bean industry; however, its nutritional value is kept. An alternative to the exploitation of bean cotyledon is the use of extrusion in the development of food products. In this context, the objective of this research was to produce extruded flours from the common bean cotyledon (Phaseolus vulgaris L.), the Carioca type, in different conditions of temperature and humidity in order to achieve technological and biochemical parameters for their use as instant food ingredient. The grains were crushed to a granulometry between 0.75 and 0.85 mm. The extrusion was carried out in full line of extrusion using extruder model MI - 130 plus, screw rotation speed 414 rpm, single screw and long with 02 compression rates and helical shirt. A model 22 was applied to evaluate the conditions of extrusion. The variables studied were temperature (150, 154, 164, 174 and 178° C) and humidity (12.3, 14, 18, 22 and 23.7%). After extrusion, the products milled to obtain flour were evaluated for chemical composition, rate of water absorption, water solubility, ability to gel formation, viscoamylographic properties, trypsin inhibitor activity, in vitro digestibility of protein and starch and microbiological quality. The results showed no significant difference between treatments related to the composition of the flours, except for the humidity. Regarding technological properties analyzed, there was a decrease of water solubility of 33% in raw flour to 15 to 23% in the extrudates. There was an increase in water absorption (9.1 to 23.9%) and improvement in ability to form gels (9% versus 11% of raw flour). Regarding the parameters of viscosity of extruded flours, the initial viscosity increased with the decrease in humidity and temperature and was observed no peak viscosity. The high values of breakdown viscosity were found in median temperatures and high humidity values. On the other hand, the final viscosity and retrogradation of extruded flours were higher when median temperatures and low humidity were used. The process resulted in complete elimination of the activity of trypsin inhibitors and increase in protein (between 11.7 and 26.6%) and starch (between 14.4 and 38.07%) digestibility. The microbiological analysis showed that all samples of extruded bean flours and instant bean soup prepared were presented in accordance with standards established by the Legislature. These results indicate that it is possible to produce instant bean flour, as an appropriate ingredient of soups preparations, baby food and other foods, with better nutritional value and good technological and biochemical properties. / O feijão comum (Phaseolus vulgaris L.) assume enorme importância na alimentação humana. Ele possui componentes essenciais à dieta, como proteínas, carboidratos e minerais. A bandinha de feijão consiste em um subproduto da indústria de feijão, porém, os seus aspectos nutricionais são mantidos. Uma alternativa para o aproveitamento de bandinha de feijão é a utilização da extrusão no desenvolvimento de produtos alimentícios. Diante deste quadro, o objetivo desta pesquisa foi produzir farinhas extrusadas da bandinha de feijão comum (Phaseolus vulgaris L.) da variedade Carioca, em diferentes condições de temperatura e umidade, a fim de atingir parâmetros tecnológicos e bioquímicos para sua utilização como ingrediente para alimentos instantâneos. Os grãos foram triturados a uma granulometria, entre 0,75 e 0,85 mm. A extrusão foi realizada em linha completa de extrusão utilizando extrusora modelo MI 130 plus, velocidade de rotação da rosca 414 rpm, rosca simples e longa com 02 taxas de compressão e camisa helicoidal. Um modelo 22 foi aplicado para avaliar as condições de extrusão. As variáveis estudadas foram temperatura (150; 154; 164; 174 e 178ºC) e umidade (12,3; 14; 18; 22 e 23,7%). Após a extrusão, os produtos moídos para obtenção de farinhas, foram avaliados quanto à composição centesimal, ao índice de absorção de água, solubilidade em água, capacidade de formação de gel, propriedades viscoamilográficas, inibidor de tripsina, digestibilidade in vitro de proteína e amido e qualidade microbiológica. Os resultados indicaram que não houve diferença significativa entre os tratamentos, quanto à composição centesimal das farinhas, à exceção da umidade. Em relação às propriedades tecnológicas analisadas, houve diminuição da solubilidade em água de 33% na farinha crua para 15 a 23% nos extrusados. Houve aumento na absorção de água (9,1 a 23,9%) e melhora na capacidade de formação de gel (9% contra 11% da farinha crua). Quanto aos parâmetros de viscosidade das farinhas extrusadas, a viscosidade inicial aumentou com a diminuição da umidade e temperatura e foi observado ausência de pico de viscosidade. Os altos valores de quebra de viscosidade foram encontrados em temperaturas medianas e altos valores de umidade. Já a viscosidade final e tendência à retrogradação das farinhas extrusadas foi elevada quando foram usadas temperaturas medianas e baixas umidades. O processo provocou eliminação completa da atividade de inibidores de tripsina e aumento nas digestibilidades protéica (entre 11,7 a 26,6%) e de amido (entre 14,4 e 38,07%). Os resultados das análises microbiológicas indicaram que todas as amostras de farinhas de feijão extrusadas e o caldo de feijão instantâneo elaborado apresentaram-se de acordo com os padrões estabelecidos pela legislação. Estes resultados indicam que é possível produzir farinha de feijão instantânea, com características de ingrediente no preparo de sopas, caldos, papinhas, entre outros alimentos, com melhor valor nutricional e boas propriedades tecnológicas e bioquímicas.