Identification of differentially expressed proteins involved in wheat (Triticum aestivum) resistance against Russian Wheat Aphid (Diuraphis noxia) SA2 using SWATH-MS analysis
- Authors: Ntlokwana, Sitha Emmanuel
- Date: 2020
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10353/18872 , vital:42888
- Description: Wheat (Triticum aestivum) is the second most-consumed cereal crop in the world, second only to maize. In South Africa it is mainly produced in three provinces; the Free State, Western Cape, and the Northen Cape. The average total land area used for wheat cultivation in these three Provinces, is 533 000 hectares, with a resultant annual production of between 1.3 to 2 million tons. The total wheat requirement in South Africa is currently 2.7 million tons according to the production of wheat guidelines published by the Department of Agriculture, Forestry and Fisheries, which is higher than the total annual production. One of the primary reasons that contribute to the lower production levels is insect and pest infestation, in particular, the Russian wheat aphid (Diurophis noxis), which can result in crop loss of up to sixty percent. Russian wheat aphids (RWA) are invasive insects that feed on the phloem of the plant through their specialised thin stylet-like mouthparts and release toxic agents contained in their saliva during feeding, which disrupt cellular functions inside the host plant. This damage leads to symptoms like; chlorosis, necrosis, wilting, stunting and curling of leaves, also known as leaf rolling. This study aimed to identify differentially expressed proteins in resistant and susceptible wheat (Triticum aestivum) cultivars during Russian wheat aphid Biotype South African 2 (RWA-SA2) infestation, in order to identify proteins involved in the wheat resistant mechanism against RWA-SA2. Two wheat cultivars SST398 (resistant to RWA-SA2) and SST356 (susceptible to RWA-SA2) were used, and a total number of 126 plants were planted and divided according to four different harvest times (Control day 0; day 5; day 7 and day 12). The wheat plants were infested with approximately 10 aphids per plant at the three-leaf stage, and placed within an insect cage inside a growth chamber (Conviron, Winnipeg, Canada) set at 24ºC, light intensity of 352 µmol.m-2.sec-1, ambient CO2 levels (410 ppm) and humidity of 60% with a 16h/8h day/night cycle. Wheat leaves were harvested at various time frames, and total protein extraction was performed. The protein samples were reduced with 10 mM dithiothreitol (DTT) and alkylated with 30 mM iodoacetamide (IAA). Sample clean-up and on-bead trypsin digest were performed on megReSyn Hilic columns, over four hours. The resultant peptides were vacuum dried and resuspended in 2% acetonitrile (ACN) before submitting them to LC-MS/MS (SWATH) for analysis. A total number of 611 proteins were differentially expressed, of which 19 were identified to be involved in the resistance response of wheat, and an additional 20 involved in biotic stress responses. This study showed that reactive oxidative species (ROS) such as hydrogen peroxide and hydroxyl radicals (OH-) play a significant role in the early stages of wheat resistance against RWA-SA2 infestation. They are involved in cell wall strengthening, activation of defence genes, involvement of phytohormone signals such as salicylic and jasmonic acid, which also mediates the systemic defences such as, systemic acquired resistance (SAR), leading to the expression of pathogen-related proteins such as (thaumatin-like proteins, oxalate oxidase, defensin, chitinase, and thionins). Although up-regulation of photosynthesis proteins such as (chlorophyll a/b binding protein, photosystem I and II, rubisco and divinyl chlorophyllide a, and 8 vinyl-reductase) were seen on both infested cultivars (susceptible and resistance), higher numbers of these classes of proteins were identified in the resistance cultivar, suggesting that they also play a critical role in resistance. In conclusion, the SWATH analysis used in this study was able to identify numerous proteins involved in the wheat resistance mechanism against RWA-SA2, most of which have not yet been reported to be involved in either biotic stress or RWA-SA2 resistance responses. Future studies are required to biochemically confirm the involvement of these enzymes and proteins the specific metabolic pathways where they are found, in the wheat resistance mechanism against Russian wheat aphid Biotype 2
- Full Text:
- Date Issued: 2020
- Authors: Ntlokwana, Sitha Emmanuel
- Date: 2020
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10353/18872 , vital:42888
- Description: Wheat (Triticum aestivum) is the second most-consumed cereal crop in the world, second only to maize. In South Africa it is mainly produced in three provinces; the Free State, Western Cape, and the Northen Cape. The average total land area used for wheat cultivation in these three Provinces, is 533 000 hectares, with a resultant annual production of between 1.3 to 2 million tons. The total wheat requirement in South Africa is currently 2.7 million tons according to the production of wheat guidelines published by the Department of Agriculture, Forestry and Fisheries, which is higher than the total annual production. One of the primary reasons that contribute to the lower production levels is insect and pest infestation, in particular, the Russian wheat aphid (Diurophis noxis), which can result in crop loss of up to sixty percent. Russian wheat aphids (RWA) are invasive insects that feed on the phloem of the plant through their specialised thin stylet-like mouthparts and release toxic agents contained in their saliva during feeding, which disrupt cellular functions inside the host plant. This damage leads to symptoms like; chlorosis, necrosis, wilting, stunting and curling of leaves, also known as leaf rolling. This study aimed to identify differentially expressed proteins in resistant and susceptible wheat (Triticum aestivum) cultivars during Russian wheat aphid Biotype South African 2 (RWA-SA2) infestation, in order to identify proteins involved in the wheat resistant mechanism against RWA-SA2. Two wheat cultivars SST398 (resistant to RWA-SA2) and SST356 (susceptible to RWA-SA2) were used, and a total number of 126 plants were planted and divided according to four different harvest times (Control day 0; day 5; day 7 and day 12). The wheat plants were infested with approximately 10 aphids per plant at the three-leaf stage, and placed within an insect cage inside a growth chamber (Conviron, Winnipeg, Canada) set at 24ºC, light intensity of 352 µmol.m-2.sec-1, ambient CO2 levels (410 ppm) and humidity of 60% with a 16h/8h day/night cycle. Wheat leaves were harvested at various time frames, and total protein extraction was performed. The protein samples were reduced with 10 mM dithiothreitol (DTT) and alkylated with 30 mM iodoacetamide (IAA). Sample clean-up and on-bead trypsin digest were performed on megReSyn Hilic columns, over four hours. The resultant peptides were vacuum dried and resuspended in 2% acetonitrile (ACN) before submitting them to LC-MS/MS (SWATH) for analysis. A total number of 611 proteins were differentially expressed, of which 19 were identified to be involved in the resistance response of wheat, and an additional 20 involved in biotic stress responses. This study showed that reactive oxidative species (ROS) such as hydrogen peroxide and hydroxyl radicals (OH-) play a significant role in the early stages of wheat resistance against RWA-SA2 infestation. They are involved in cell wall strengthening, activation of defence genes, involvement of phytohormone signals such as salicylic and jasmonic acid, which also mediates the systemic defences such as, systemic acquired resistance (SAR), leading to the expression of pathogen-related proteins such as (thaumatin-like proteins, oxalate oxidase, defensin, chitinase, and thionins). Although up-regulation of photosynthesis proteins such as (chlorophyll a/b binding protein, photosystem I and II, rubisco and divinyl chlorophyllide a, and 8 vinyl-reductase) were seen on both infested cultivars (susceptible and resistance), higher numbers of these classes of proteins were identified in the resistance cultivar, suggesting that they also play a critical role in resistance. In conclusion, the SWATH analysis used in this study was able to identify numerous proteins involved in the wheat resistance mechanism against RWA-SA2, most of which have not yet been reported to be involved in either biotic stress or RWA-SA2 resistance responses. Future studies are required to biochemically confirm the involvement of these enzymes and proteins the specific metabolic pathways where they are found, in the wheat resistance mechanism against Russian wheat aphid Biotype 2
- Full Text:
- Date Issued: 2020
An investigation of Grade 11 Oshindonga teachers' understanding and implementation of the learner-centered approach adopted in Namibia : a case study
- Mbangula, Christofina Nalweendo
- Authors: Mbangula, Christofina Nalweendo
- Date: 2011
- Subjects: Student-centered learning -- Namibia Ndonga language -- Study and teaching -- Namibia Teachers -- Training of -- Namibia Educational change -- Namibia Education -- Namibia
- Language: English
- Type: Thesis , Masters , MEd
- Identifier: vital:1628 , http://hdl.handle.net/10962/d1003510
- Description: Before the Republic of Namibia achieved independence in 1990, Bantu Education was the prevailing structure used to promote the social, economic, and political ethos of apartheid through a teacher-centered education system. After 1990, Namibia underwent a major restructuring of education. Learner-centered education was introduced as an inclusive and participatory approach to achieve the reform goals. One of the aims was to review the existing Language policy and to promote mother tongue teaching, since it is through language we internalize our experience and construct our own understanding. In other words, our cognitive, emotional and social development is dependent on language. In this case study, the understanding and implementation of a learner-centered approach in Namibia were investigated in order to gain insights about how the participating education officer, responsible for Oshindonga understands and assists teachers to implement this approach. At the same time, this study aimed at investigating Grade 11 teachers‟ understanding and implementation of LCE in their classrooms. The qualitative methodology in this case study used semi-structured interviews, classroom observations and document analysis for data collection. The data revealed that there are a number of misconceptions. In some cases, what teachers say is not what they do. The findings suggest that teachers, while attempting to implement a learner-centered approach, are not confident about its underlying theory, and therefore the degree of implementation depends on how the teachers used their understanding of that theory in their practice within these conceptual constraints. The study highlights particular challenges and problems that hinder the effective implementation of learner-centered education.
- Full Text:
- Date Issued: 2011
- Authors: Mbangula, Christofina Nalweendo
- Date: 2011
- Subjects: Student-centered learning -- Namibia Ndonga language -- Study and teaching -- Namibia Teachers -- Training of -- Namibia Educational change -- Namibia Education -- Namibia
- Language: English
- Type: Thesis , Masters , MEd
- Identifier: vital:1628 , http://hdl.handle.net/10962/d1003510
- Description: Before the Republic of Namibia achieved independence in 1990, Bantu Education was the prevailing structure used to promote the social, economic, and political ethos of apartheid through a teacher-centered education system. After 1990, Namibia underwent a major restructuring of education. Learner-centered education was introduced as an inclusive and participatory approach to achieve the reform goals. One of the aims was to review the existing Language policy and to promote mother tongue teaching, since it is through language we internalize our experience and construct our own understanding. In other words, our cognitive, emotional and social development is dependent on language. In this case study, the understanding and implementation of a learner-centered approach in Namibia were investigated in order to gain insights about how the participating education officer, responsible for Oshindonga understands and assists teachers to implement this approach. At the same time, this study aimed at investigating Grade 11 teachers‟ understanding and implementation of LCE in their classrooms. The qualitative methodology in this case study used semi-structured interviews, classroom observations and document analysis for data collection. The data revealed that there are a number of misconceptions. In some cases, what teachers say is not what they do. The findings suggest that teachers, while attempting to implement a learner-centered approach, are not confident about its underlying theory, and therefore the degree of implementation depends on how the teachers used their understanding of that theory in their practice within these conceptual constraints. The study highlights particular challenges and problems that hinder the effective implementation of learner-centered education.
- Full Text:
- Date Issued: 2011
A study of the biopharmaceutics and pharmacokinetics of the macrolide antibiotic, erythromycin
- Authors: Terespolsky, Susan Ann
- Date: 1992
- Subjects: Erythromycin -- Bioavailability , Erythromycin -- Pharmacokinetics
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3795 , http://hdl.handle.net/10962/d1003273 , Erythromycin -- Bioavailability , Erythromycin -- Pharmacokinetics
- Description: Erythromycin, a macrolide antibiotic isolated from Streptomyces erythreus, was first introduced into clinical medicine in 1952. It is active against most gram-positive bacteria, some gram-negative bacteria and is currently the agent of choice for Legionella pneumophila. Erythromycin is an acid-labile compound rapidly degrading in acidic solutions such as the acid environment of the stomach. As such, erythromycin absorption following oral administration of solid dosage forms is relatively poor. Accordingly there have been various approaches used to protect the drug against gastric inactivation. These precautions include enteric-coating of tablets, capsules or pellets of erythromycin base, the synthesis of acid stable 2' esters of erythromycin (ethylsuccinate and propionate) and salts of these esters (erythromycin estolate), and more recently, the synthesis of a range of new acid-stable, semi-synthetic macrolide antibiotics. The 2' esters are antimicrobially inactive or much less active than the parent compound and must be converted to the free erythromycin base in vivo in order to exhibit antibacterial activity. Intrinsic dissolution rates determined on raw material can provide extremely useful information relating to the gastrointestinal absorption of drugs from solid dosage forms. The large inter- and intrasubject variability associated with erythromycin base has, to date, mainly been attributed to gastric acid inactivation of the drug. However, changes in duodenal pH resulting in altered solubility and intrinsic dissolution rates may account for the observed variability. Thus, the intrinsic dissolution rates of erythromycin base at pH 6.0, 6.5, 7.0, 7.5 and 8.0 were compared in order to investigate the possible effects of pH changes which may occur in the duodenal contents, on the in vivo dissolution and subsequent absorption of this compound. The standard intrinsic dissolution rate test procedure employing a rotating disc of pure erythromycin base powder which only allows for dissolution from a constant surface area, was adapted and the drug quantitatively determined by reversed phase high performance liquid chromatography (HPLC) using ultraviolet detection. Results of intrinsic dissolution studies at both 22°C and 37°C indicate that the solubility, and therefore the rate of dissolution of erythromycin base is pH dependent, being more soluble at pH 6.0 than pH 8.0 (an approximate 800 times and 1000 times reduction in the amount dissolved after 30 minutes, at 22°C and 37°C respectively, when the pH of the medium was increased from 6 to 8). Although the stability of erythromycin and its ester derivatives in aqueous acidic solutions has been well documented, very little has been reported on the compound's stability in organic solvents. Methanol is recommended by official drug compendia (U.S.P. and B.P.) for use in erythromycin identification tests as well as in the sample preparation steps during assay procedures. Thus, the effect of methanol and acetonitrile, organic solvents of similar polarities and densities, on the stability of erythromycin base, erythromycin ethylsuccinate, propionyl erythromycin and erythromycin estolate at room temperature (22°C ± 0.5°C), using HPLC with electrochemical detection, was investigated. Erythromycin base is relatively stable in both methanol and acetonitrile, remaining intact for over 168 hours in acetonitrile and showing less than 5% degradation in methanol over the same period. Erythromycin ethylsuccinate in acetonitrile shows less than 5% degradation over 168 hours whereas in methanol, rapid hydrolysis occurs resulting in almost total conversion to base within 40 hours. Approximately 87% of erythromycin propionyl ester remained intact after 168 hours in acetonitrile whilst methanol caused rapid hydrolysis to erythromycin base (35% remaining after 28 hours). Erythromycin estolate appeared to be unstable in both acetonitrile and methanol. In acetonitrile, only 13% of the estolate remained intact after 168 hours, whereas in methanol, the reaction was much more rapid with 35% of the estolate remaining after 28 hours. The use of methanol as a solvent for erythromycin estolate reference standards is thus contraindicated. A number of conflicting reports on the half- life as well as the body compartment model that best describes erythromycin base serum concentration-time profiles (lBCM generally used to describe orally administered erythromycin, whilst a 2BCM has been used to describe erythromycin administered intravenously), appear in the literature. These differences may be largely attributed to the sampling period (between 6 and 12 hours) used in the repective studies. The objective of this study was to determine the body compartment model that best describes erythromycin base serum concentration-time curves by increasing the sampling time to 24 hours. In addition, the effect of chronic dosing of erythromycin on erythromycin pharmacokinetics, in the same group of subjects, was investigated. The single and multiple oral dose pharmacokinetics of erythromycin enteric coated base pellets within a gelatin capsule (250mg), were studied in 6 healthy, normal volunteers (19.5 ± 0.76 years, 71.5 ± 8.18 kg, 180.33 ± 5.99 cm). Furthermore, steady state concentrations were predicted using the pharmacokinetic parameters obtained from the single dose study, and compared with those obtained in the multiple dose study. Plasma concentrations were determined using a sensitive high-performance liquid chromatographic method with electrochemical detection. For the single dose study, after a tlag of 2.5 ± 0.71 hr, Cmax (1.12 ± 0.47 μ/ml) was reached at a tmax of 4.08 ± 0.93 hr post dose, with serum concentrations ranging from 0.31 - 1.62 μ/ml. The half-life was found to be 5.42 ± 1.31 hr. On multiple dosing (250mg six hourly), serum concentrations for the fifth, ninth and thirteenth dosing intervals ranged from 0.67 - 2.92 μ/ml, 1.69 - 3.65 μ/ml and 0.61 - 3.01 μ/ml, occurring at 3.75 ± 0.69 hr, 3.17 ± 1.03 hr and 3.17 ± 1.03 hr post dose with a Cmax of 1.89 ± 0.68 μ/ml, 2.35 ± 0.70 μ/ml and 1.94 ± 0.74 μ/ml, respectively. The area under the serum concentration- time curve for the single dose study (AUC₀₋∞) was 4.67 ± 0.88 hr.μ/ml, whilst the AUC₀₋τ. for the fifth, ninth and thirteenth dosing intervals of the multiple dose study were 5.77 ± 1.76 hr.μ/ml, 6.46 ± 1.33 hr.μ/ml and 5.97 ± 2.36 hr.μ/ml respectively, indicating an approximately 33% increase in AUC on chronic dosing of erythromycin. The observed increase in AUC may be a result of increased bioavailability or a decrease in clearance on chronic dosing.
- Full Text: false
- Date Issued: 1992
- Authors: Terespolsky, Susan Ann
- Date: 1992
- Subjects: Erythromycin -- Bioavailability , Erythromycin -- Pharmacokinetics
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3795 , http://hdl.handle.net/10962/d1003273 , Erythromycin -- Bioavailability , Erythromycin -- Pharmacokinetics
- Description: Erythromycin, a macrolide antibiotic isolated from Streptomyces erythreus, was first introduced into clinical medicine in 1952. It is active against most gram-positive bacteria, some gram-negative bacteria and is currently the agent of choice for Legionella pneumophila. Erythromycin is an acid-labile compound rapidly degrading in acidic solutions such as the acid environment of the stomach. As such, erythromycin absorption following oral administration of solid dosage forms is relatively poor. Accordingly there have been various approaches used to protect the drug against gastric inactivation. These precautions include enteric-coating of tablets, capsules or pellets of erythromycin base, the synthesis of acid stable 2' esters of erythromycin (ethylsuccinate and propionate) and salts of these esters (erythromycin estolate), and more recently, the synthesis of a range of new acid-stable, semi-synthetic macrolide antibiotics. The 2' esters are antimicrobially inactive or much less active than the parent compound and must be converted to the free erythromycin base in vivo in order to exhibit antibacterial activity. Intrinsic dissolution rates determined on raw material can provide extremely useful information relating to the gastrointestinal absorption of drugs from solid dosage forms. The large inter- and intrasubject variability associated with erythromycin base has, to date, mainly been attributed to gastric acid inactivation of the drug. However, changes in duodenal pH resulting in altered solubility and intrinsic dissolution rates may account for the observed variability. Thus, the intrinsic dissolution rates of erythromycin base at pH 6.0, 6.5, 7.0, 7.5 and 8.0 were compared in order to investigate the possible effects of pH changes which may occur in the duodenal contents, on the in vivo dissolution and subsequent absorption of this compound. The standard intrinsic dissolution rate test procedure employing a rotating disc of pure erythromycin base powder which only allows for dissolution from a constant surface area, was adapted and the drug quantitatively determined by reversed phase high performance liquid chromatography (HPLC) using ultraviolet detection. Results of intrinsic dissolution studies at both 22°C and 37°C indicate that the solubility, and therefore the rate of dissolution of erythromycin base is pH dependent, being more soluble at pH 6.0 than pH 8.0 (an approximate 800 times and 1000 times reduction in the amount dissolved after 30 minutes, at 22°C and 37°C respectively, when the pH of the medium was increased from 6 to 8). Although the stability of erythromycin and its ester derivatives in aqueous acidic solutions has been well documented, very little has been reported on the compound's stability in organic solvents. Methanol is recommended by official drug compendia (U.S.P. and B.P.) for use in erythromycin identification tests as well as in the sample preparation steps during assay procedures. Thus, the effect of methanol and acetonitrile, organic solvents of similar polarities and densities, on the stability of erythromycin base, erythromycin ethylsuccinate, propionyl erythromycin and erythromycin estolate at room temperature (22°C ± 0.5°C), using HPLC with electrochemical detection, was investigated. Erythromycin base is relatively stable in both methanol and acetonitrile, remaining intact for over 168 hours in acetonitrile and showing less than 5% degradation in methanol over the same period. Erythromycin ethylsuccinate in acetonitrile shows less than 5% degradation over 168 hours whereas in methanol, rapid hydrolysis occurs resulting in almost total conversion to base within 40 hours. Approximately 87% of erythromycin propionyl ester remained intact after 168 hours in acetonitrile whilst methanol caused rapid hydrolysis to erythromycin base (35% remaining after 28 hours). Erythromycin estolate appeared to be unstable in both acetonitrile and methanol. In acetonitrile, only 13% of the estolate remained intact after 168 hours, whereas in methanol, the reaction was much more rapid with 35% of the estolate remaining after 28 hours. The use of methanol as a solvent for erythromycin estolate reference standards is thus contraindicated. A number of conflicting reports on the half- life as well as the body compartment model that best describes erythromycin base serum concentration-time profiles (lBCM generally used to describe orally administered erythromycin, whilst a 2BCM has been used to describe erythromycin administered intravenously), appear in the literature. These differences may be largely attributed to the sampling period (between 6 and 12 hours) used in the repective studies. The objective of this study was to determine the body compartment model that best describes erythromycin base serum concentration-time curves by increasing the sampling time to 24 hours. In addition, the effect of chronic dosing of erythromycin on erythromycin pharmacokinetics, in the same group of subjects, was investigated. The single and multiple oral dose pharmacokinetics of erythromycin enteric coated base pellets within a gelatin capsule (250mg), were studied in 6 healthy, normal volunteers (19.5 ± 0.76 years, 71.5 ± 8.18 kg, 180.33 ± 5.99 cm). Furthermore, steady state concentrations were predicted using the pharmacokinetic parameters obtained from the single dose study, and compared with those obtained in the multiple dose study. Plasma concentrations were determined using a sensitive high-performance liquid chromatographic method with electrochemical detection. For the single dose study, after a tlag of 2.5 ± 0.71 hr, Cmax (1.12 ± 0.47 μ/ml) was reached at a tmax of 4.08 ± 0.93 hr post dose, with serum concentrations ranging from 0.31 - 1.62 μ/ml. The half-life was found to be 5.42 ± 1.31 hr. On multiple dosing (250mg six hourly), serum concentrations for the fifth, ninth and thirteenth dosing intervals ranged from 0.67 - 2.92 μ/ml, 1.69 - 3.65 μ/ml and 0.61 - 3.01 μ/ml, occurring at 3.75 ± 0.69 hr, 3.17 ± 1.03 hr and 3.17 ± 1.03 hr post dose with a Cmax of 1.89 ± 0.68 μ/ml, 2.35 ± 0.70 μ/ml and 1.94 ± 0.74 μ/ml, respectively. The area under the serum concentration- time curve for the single dose study (AUC₀₋∞) was 4.67 ± 0.88 hr.μ/ml, whilst the AUC₀₋τ. for the fifth, ninth and thirteenth dosing intervals of the multiple dose study were 5.77 ± 1.76 hr.μ/ml, 6.46 ± 1.33 hr.μ/ml and 5.97 ± 2.36 hr.μ/ml respectively, indicating an approximately 33% increase in AUC on chronic dosing of erythromycin. The observed increase in AUC may be a result of increased bioavailability or a decrease in clearance on chronic dosing.
- Full Text: false
- Date Issued: 1992
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