<p>This study reports on a proteomic analysis of sorghum proteomes in response to salt and hyperosmotic stresses. Two-dimensional gel electrophoresis (2DE) in combination with mass spectrometry (MS) was used to separate, visualise and identify sorghum proteins using both sorghum cell suspension cultures and whole plants. The sorghum cell suspension culture system was used as a source of culture filtrate (CF) proteins. Of the 25 visualised CBB stained CF spots, 15 abundant and well-resolved spots were selected for identification using a combination of MALDI-TOF and MALDI-TOFTOF MS, and database searching. Of these spots, 14 were positively identified as peroxidases, germin proteins, oxalate oxidases and alpha-galactosidases with known functions in signalling processes, defense mechanisms and cell wall metabolism.</p>

Many pathogenic bacteria use their iron acquisition mechanisms to live inside hosts. Streptococcus pyogenes is a pathogenic bacterium that uses streptococcal iron acquisition ABC transporter to obtain heme. SiaA (HtsA, spy1795), a lipoprotein located on the cell surface, serves as a heme binding protein. To understand the iron-uptake mechanism, histidine 229, one of the two proposed axial ligands in SiaA, was mutated to alanine. SiaA H229A was expressed in E. coli, lysed by French Press, and purified by fast protein liquid chromatography (FPLC). SDS-PAGE indicated that pure protein was isolated. Nickel affinity FPLC gave purer H229A when 0.5 M imidazole was added to the binding buffer. Overall, histidine 229 is likely to be an axial ligand in wild type SiaA, as shown by the fact the mutant readily lost heme as evidenced by UV-vis spectra.

<p>This study reports on a proteomic analysis of sorghum proteomes in response to salt and hyperosmotic stresses. Two-dimensional gel electrophoresis (2DE) in combination with mass spectrometry (MS) was used to separate, visualise and identify sorghum proteins using both sorghum cell suspension cultures and whole plants. The sorghum cell suspension culture system was used as a source of culture filtrate (CF) proteins. Of the 25 visualised CBB stained CF spots, 15 abundant and well-resolved spots were selected for identification using a combination of MALDI-TOF and MALDI-TOFTOF MS, and database searching. Of these spots, 14 were positively identified as peroxidases, germin proteins, oxalate oxidases and alpha-galactosidases with known functions in signalling processes, defense mechanisms and cell wall metabolism.</p>

<p>This study reports on a proteomic analysis of sorghum proteomes in response to salt and hyperosmotic stresses. Two-dimensional gel electrophoresis (2DE) in combination with mass spectrometry (MS) was used to separate, visualise and identify sorghum proteins using both sorghum cell suspension cultures and whole plants. The sorghum cell suspension culture system was used as a source of culture filtrate (CF) proteins. Of the 25 visualised CBB stained CF spots, 15 abundant and well-resolved spots were selected for identification using a combination of MALDI-TOF and MALDI-TOFTOF MS, and database searching. Of these spots, 14 were positively identified as peroxidases, germin proteins, oxalate oxidases and alpha-galactosidases with known functions in signalling processes, defense mechanisms and cell wall metabolism.</p>

Resumo: O objetivo do estudo foi avaliar a transferência de imunidade passiva (TIP) de vacas Canchim aos seus bezerros. Um grupo experimental foi constituído por 13 vacas primíparas e seus bezerros, e o outro por 13 vacas pluríparas e seus bezerros. Foram coletadas amostras de sangue venoso dos bezerros e secreções lácteas das vacas até 1 hora e 1, 2, 7, 15 e 30 dias após o nascimento/parto. No hemograma, notaram-se alterações características do período neonatal, como redução na contagem de hemácias, teor de hemoglobina e volume globular após a ingestão do colostro, além de aumento na contagem de linfócitos e redução da contagem de neutrófilos segmentados. Os exames bioquímicos séricos revelaram aumento nas atividades de GGT (até 3.746 U/L) e ALP (até 1.030 U/L) e nos teores de proteína total (até 7,77 g/dL), globulinas (até 6,01 g/dL), IgA (até 322 mg/dL) e IgG (até 2.918 mg/dL) após a ingestão do colostro, seguidos por redução gradual nestes parâmetros até os 30 dias de idade. As avaliações bioquímicas das secreções lácteas revelaram alta concentração de todos os componentes colostrais analisados, com redução gradual nos seus teores no decorrer do período experimental, à exceção dos minerais, cujas concentrações oscilaram pouco entre os momentos. A TIP foi eficiente em ambos os grupos de bezerros, e a qualidade das secreções lácteas, embora diferentes entre vacas primíparas e pluríparas, não interferiu na passagem de imunoglobulinas da vaca para o bezerro. / Abstract: The aim of this study was to evaluate the passive immunity transfer from Canchim cows to its calves. One experimental group comprised 13 primiparous cows and its calves and the other group, 13 multiparous cows and its calves. Samples of calves' venous blood and cow's lacteal secretions were collected until 1 hour and 1, 2, 7, 15 and 30 days after birth/parturition. In the hemogram, characteristic alterations of this period, such as reduction in the erytocyte count, hemoglobin concentration and corpuscular volume after colostrum intake and rise in the lymphocyte count and reduction in the neutrophil count were noticed. The serum biochemistry revealed augmentation in the GGT (up to 3,746 U/L) and ALP (up to 1,030 U/L) activities and in the total protein (up to 7.77 g/dL), globulin (up to 6.01 g/dL), IgA (up to 322 mg/dL) and IgG (up to 2,918 mg/dL) concentrations after colostrum intake, followed by a gradual reduction in these parameters until 30 days of age. The biochemical evaluation of the lacteal secretions revealed high concentrations of all the colostral components analysed, with gradual reduction in their concentrations along the experimental period, except for the minerals, which showed little oscillation between the moments. The passive immunity transfer was efficient in both groups of calves, and the quality of the lacteal secretions, although different between primiparous and multiparous cows, did not interfere in the transference of immunoglobulins from the cows to the calves. / Orientador: José Jurandir Fagliari / Coorientador: Alexandre Amstalden Moraes Sampaio / Banca: Eduardo Harry Birgel Júnior / Banca: Daniela Gomes da Silva / Mestre

Philosophiae Doctor - PhD / This study reports on a proteomic analysis of sorghum proteomes in response to salt and hyperosmotic stresses. Two-dimensional gel electrophoresis (2DE) in combination with mass spectrometry (MS) was used to separate, visualise and identify sorghum proteins using both sorghum cell suspension cultures and whole plants. The sorghum cell suspension culture system was used as a source of culture filtrate (CF) proteins. Of the 25 visualised CBB stained CF spots, 15 abundant and well-resolved spots were selected for identification using a combination of MALDI-TOF and MALDI-TOFTOF MS, and database searching. Of these spots, 14 were positively identified as peroxidases, germin proteins, oxalate oxidases and alpha-galactosidases with known functions in signalling processes, defense mechanisms and cell wall metabolism. / South Africa

Plasmodium falciparum is a protozoan parasite responsible for causing the most severe form of malaria in humans. This species is responsible for over 90% of malaria mortalities which occur predominantly in Africa. An increase in drug resistant parasites in recent years is threatening the progress made against malaria and thus new antimalarial drugs and vaccines are needed to combat this disease. During the intraerythrocytic phase, merozoites egress from mature schizonts to invade new uninfected erythrocytes. Glycophosphatidylinositol (GPI) -anchored proteins cover most of the exterior surface of the merozoite prior to invasion, while other GPI-anchored proteins are released onto the merozoite surface through apical organelle secretions. These proteins are involved in interactions with erythrocytes and are thought to be vital to erythrocyte invasion. GPI-anchored proteins have also been implicated as a cause of pathogenic symptoms and activation of immune components. These proteins are then released or cleaved to enable merozoite entry into the erythrocyte. Several enzymes are thought to be involved in their cleavage including the serine proteases subtilisin-like proteases (SUB) 1 and 2, and phosphatidylinositol-phospholipase C (PIPLC); GPI-anchored proteins are also generally sensitive to phospholipase A2 (PLA2). Cleaved proteins are released into the host blood system, while uncleaved proteins are carried into the erythrocyte during invasion. Merozoites have a limited period in which they retain invasive capacity. A previous lack of available techniques that are specifically adapted to merozoite analysis has resulted in an incomplete understanding of invasion and GPI-anchored protein involvement in invasion. This study aimed to determine how GPI-anchored proteins on the merozoite surface are altered in the invasive phase, and explore the possibility of using merozoite GPI-anchored proteins as potential drug targets to block erythrocyte invasion. Optimised methods of in vitro parasite culturing which produce highly synchronised merozoites was essential to this study. Parasite culturing techniques were optimised by utilising low haematocrit cultures with frequent culture splitting and optimised synchronisation. The “Malarwheel” is a tool that was developed for this research to provide a means for scheduling sorbitol treatments and MACs isolations. This tool and optimised culturing methods enabled large volumes of highly synchronised invasive merozoites to be harvested. Four compounds (vanadate, edelfosine, dioctyl sodium sulfosuccinate (DSS), and gentamicin) suspected to interfere with GPIanchored cleavage or processes were screened on intraerythrocytic stages and merozoites. Antimalarial and anti-invasive properties of these compounds were screened by modified malaria SYBR Green I-based fluorescence (MSF) assay and merozoite invasion assays (MIA) respectively. DSS and gentamicin showed limited potential as antimalarials or as anti-invasive agents. Vanadate and edelfosine both showed antimalarial and anti-invasive activity, while edelfosine was the most potent anti-invasive agent at physiological concentrations. The merozoite GPI-anchored proteome was analysed by sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE) followed by complete gel lane analyses conducted by liquid chromatography-tandem mass spectrometry (LC-MS/MS) on soluble and pelleted merozoite proteins in samples from either invasive or non-invasive merozoites. Thirteen known or predicted GPI-anchored proteins were identified in samples. Several changes were identified in merozoite GPI-anchored proteins between the invasive phase and after its completion, and minor differences were observed following treatment with edelfosine. Edelfosine showed partial inhibition of erythrocyte invasion, however, the primary cause of inhibition cannot be directly related to interferences with GPI-anchored proteins. These results suggest that GPIanchored proteins are controlled by various complex processes, and are cleaved or processed by diverse mechanisms during the invasive phase. These mechanisms may be controlled by multiple signals which effect proteins or groups of proteins in specific ways. These signals may be influenced by “checkpoints” during invasion processes including the time period after egress from schizonts, and possibly the recognition of erythrocyte targets. These methods and results provide a foundation for future research to enable culturing of P. falciparum parasites specifically for merozoite research, and to identify merozoite proteins active during the invasive phase. These results confirm and challenge previous ideas reported in literature on the GPI-anchored processes of merozoites and further characterise less studied GPIanchored proteins. The results suggest that the processes controlling GPI-anchored proteins may be more complex than previously thought. These results form a basis to further identify and characterise GPI-anchored proteins in the aim to develop antimalarial medications and vaccines that target merozoites and their GPI-anchored processes. / Dissertation (MSc)--University of Pretoria, 2017. / Pharmacology / MSc / Unrestricted

While a decrease of wild felid population has led to disruption of conservation programme, recent studies have shown the importance of immune regulation for determining health outcomes and co-infection. Immunoglobulin G is important for detecting and evaluating responses to infectious diseases and vaccination. But, there is limited information on felid immunoglobulins and their role for functional immunity. This study aimed at isolating and characterizing lion’s immunoglobulin G. Lions’ sera (n = 68) were processed using the MagReSyn® magnetic beads and the final protein concentration was determined using the Xpose™ Trinean Spectrophotometer. The cross-reactivity of goat anti-cat immunoglobulin with sera of lions and other species was analysed using ELISA. High cross-reactivity was observed in lions ranging from 87.7 to 100%, and low reactivity with rhino (22.4%) followed by chicken (0.01%). The protein concentration from purified sera yielded 39.09 mg/ml. Molecular weight of lion IgG 150-160 kDa was detected with both chains at 54-56 kDa and 24-26 kDa on SDS PAGE. These results indicate a potential aid in developing serological tools to monitor exposure to micro-organisms of lions. / Agriculture and  Animal Health / M. Sc. (Agriculture)

Philosophiae Doctor - PhD / Sorghum (Sorghum bicolorï, a drought tolerant cereal crop, is not only an important food source in the semi arid/arid regions but also a potential model for studying and gaining a better understanding of the molecular mechanisms of drought and salt stress tolerance in cereals. This study reports on a proteomic analysis of sorghum proteomes in response to salt and hyperosmotie stresses. Two-dimensional gel electrophoresis (2DE) in combination with mass spectrometry (MS) was used to separate, visualise and identify sorghum proteins using both sorghum cell suspension cultures and whole plants. The sorghum cell suspension culture system was used as a source of culture filtrate (CF) proteins. Of the 25 visualised CBB stained CF spots, 15 abundant and well-resolved spots were selected for identification using a combination of MALDI- TOF and MALDI- TOFTOF MS, and database searching. Of these spots, 14 were positively identified as peroxidases, germ in proteins, oxalate oxidases and alpha-galactosidases with known functions in signalling processes, defense mechanisms and cell wall metabolism. Following 200 mM NaCl and 400 mM sorbitol stress treatments, the expression/abundance of a protein spot similar to a rice wall-associated protein kinase was upregulated in the sorghum secretome in response to both stresses. Amino acid sequence alignment of the matching peptides between these two proteins showed that the sorghum CF spot possesses a protein kinase domain. Therefore, this protein could possibly participate in cell signalling functions, which link the external environment with the cell's cytoplasm. Using whole plant systems, a comparative study of leaf protein expression between two sorghum varieties, AS6 (salt sensitive) and MN1618 (salt tolerant) was conducted. Forty well resolved spots of varying abundances were picked for MS analysis. Of these, 28 were positively identified, representing proteins with functions in carbohydrate metabolism (60.7%), proton transport (17.9%), protein synthesis (7.1%), hydrolytic functions (7.1%), nucleotide metabolism (3.6%) and detoxification (3.6%). Using PDQuest™ Advanced 2D Analysis Software version 8.0.1 (BIO-RAD), a comparative analysis of leaf proteome expression patterns between the two sorghum varieties was conducted. The results indicated proteins with similar expression patterns as well as qualitative and quantitative differences between the two leaf proteomes. The effect of 100 mM NaCI on leaf proteome expression between the two sorghum varieties was also studied. Western blotting analysis of leaf, sheath and root tissues using Hsp70 antibodies showed that this treatment induced Hsp70 expression, a known stress protein, in both varieties. Thereafter, the partially annotated leaf proteome map was used to landmark other salt responsive proteins. Examples of differential expression patterns included glutathione S transferase and hydroxynitrile lyase proteins whose abundances were upregulated in both varieties, while the large subunit of RuBisCo was downregulated in AS6 but upregulated in MN1618. Qualitative spot expression differences in response to salt stress were also observed between the two sorghum varieties but these remained unidentified after both MALDI-TOF and MALDI-TOF-TOF MS, possibly indicating novel and previously uncharacterised sorghum proteins. The results of this study can be used as reference tools by proteomics researchers worldwide as well as a foundation for future studies.

This thesis aims to investigate if the endothelial protein C receptor is expressed in the murine kidney. This was done by performing flow cytometry and Western blot analysis on cultivated murine kidney endothelial cells (mKECs) as well as SDS-PAGE and Western blot analysis on murine kidney tissue. Flow cytometry was also performed on cultivated ARPE19 and 4T1 cells for comparison. It was discovered that ≥95,5% of the mKECs, ≥93,6% of the ARPE19 cells and ≥60,9% of the 4T1 cells express the receptor according to the flow cytometry data. A dot blot was performed to validate the primary antibody used for detection of EPCR in Western blot and SDS-PAGE. According to the dot blot, the primary antibody can be visualised in the dilution range from 1:2000 to 1:10. The dot blot also showed that the secondary antibody binds specifically to the primary antibody. Yet, Western blot analysis did not detect the receptor neither in mKECs nor tissue lysate. This was likely due to the fact that the primary antibody used did not bind specifically to the receptor, and may not be applicable for this method. SDS-PAGE did not show any indication that the receptor was present in the kidney tissue. In conclusion, it was discovered that the EPCR was expressed in the murine kidneys endothelial cells through flow cytometry, but the presented methods for Western blot and SDS-PAGE could not confirm the expression of the receptor.