The main function of the cardiovascular system is to deliver a sufficient quantity of oxygenated blood to the tissues, cells, and organs of the body in order to provide the cells with essential nutrients for metabolism and for the removal of waste products. All cells require and utilize oxygen. Oxygen is transported to various cells and tissues via red blood cells flowing through the microcirculation of an organism. Measurement of oxygen transport in the microcirculation has shown that about ten times more oxygen appears to leave the blood of arterioles than can be accounted for by diffusion. One possibility to explain the high oxygen loss is an increased release of oxygen due to exposure of blood to light. In the present in vitro study the release of oxygen from red blood cells was measured during exposure of the sample to light by monitoring the change in PO2 of the suspension during light exposure. A PO2 electrode was calibrated using PBS solution and utilized to monitor the change in current in the present study. Red blood cell suspensions were made using blood withdrawn from male Sprague-Dawley rats. The red blood cell suspension was placed in a closed sample chamber and exposed to light for 5 minutes. A method to correct for the drift of the PO2 electrode and temperature change during the experiment was implemented. The calculated change in PO2 of the RBC suspension due to light exposure was small. The change of PO2 in the sample chamber during light exposure was an average of 1.60 ± 0.9 mmHg (SEM). The contribution of photo-dissociation of oxygen from oxygenated hemoglobin molecules to the observed oxygen loss per RBC can account for only about 0.01% of the observed in vivo results. Therefore, light-associated oxygen release is negligible. These findings disprove the hypothesis of the present study, in which light exposure does not have a significant effect on oxygen release and thus rules out this possible explanation for the discrepancy between experiment and theory.

Thesis (MScAgric (Horticulture))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Peel colour is an important quality factor in the production of bi-coloured apple fruit. Most markets set minimum requirements for red colour coverage. Fruit that do not meet these requirements are downgraded and has a major impact on the profitability of apple production in South Africa. South African apple production areas are amongst the warmest in the world. Since anthocyanin accumulation requires induction at low temperature and synthesis require mild temperatures, experiments were conducted to investigate optimum day and night temperatures for red colour development throughout fruit development for red and bi-coloured apple cultivars grown in South Africa. We found that redder strains of bi-coloured apple cultivars did not appear to owe their enhanced pigmentation to higher temperature optima for anthocyanin synthesis. The optimum day temperatures for red colour development in the different cultivars seemed to differ between seasons, but not between production areas. In general, red colour in the cultivars evaluated developed maximally between 17 ºC and 25 ºC. The optimum day temperature for red colour development remained constant throughout fruit development for most cultivars, but increased roughly from 14 ºC to 22 ºC in ‘Cripps’ Pink’ between January and April. The extent of red colour development increased during fruit development in all the cultivars assessed. We were unable to determine optimum induction temperatures for red colour development. ‘Royal Gala’ from Ceres seemed to benefit from induction at 4 ºC while red colour in ‘Fuji’ decreased with decreasing temperature. To explain the presence of anthocyanins in immature apple fruit, we tested the hypothesis that anthocyanins protect the peel from photoinhibition and photooxidative damage during conditions of increased light stress. First we established that the rate of colour change in response to a passing cold front appears to be sufficient to provide photoprotection during a cold snap. Also in agreement with the hypothesis, ‘Cripps Pink’ peel incurred significantly more photoinhibition at low temperature (16 ºC) compared to mild (24 and 32 ºC) and high (40 ºC) temperature under high irradiance with visible light. Recovery rate was temperaturedependent, being the slowest at low temperature and increasing with temperature. The photoapparatus in ‘Cripps Pink’ peel appears to be particularly sensitive to light stress at low temperature throughout the season, with significant photoinhibition occurring even at moderate temperature (24 ºC). The sensitivity of the apple peel to photoinhibition increased throughout the season at lower irradiance levels, but remained the same at higher irradiance. In our final experiment, fruit were exposed to high irradiance at low and mild temperature before exposure to high temperature in combination with high irradiance. This was done to test the hypothesis that photoinhibition incurred during cold snaps predisposes peel to photothermal damage when temperature increases again after the cold snap. Unfortunately, due to the severity of the stress incurred in response to high temperature treatment, the results were inconclusive. / AFRIKAANSE OPSOMMING: Vrugkleur is ‘n belangrike kwaliteitsfaktor in die produksie van tweekleurappels. Die meeste markte stel minimum vereistes vir rooi kleurbedekking. Vrugte wat nie aan hierdie vereistes voldoen nie, word afgegradeer. Suid-Afrika se appel produksie areas word beskou as van die warmste ter wêreld. Antosianien akkumulasie benodig induksie by lae temperature gevolg deur sintese in lig by matige temperature. Gevolglik het swak rooi kleurontwikkeling onder plaaslike toestande ‘n groot impak op die winsgewendheid van appelproduksie in Suid-Afrika. Eksperimente is uitgevoer om die optimum dag- en nagtemperature vir rooi kleurontwikkeling tydens vrugontwikkeling vir die rooi en tweekleur appel kultivars wat in Suid-Afrika geproduseer word te bepaal. Ons het gevind dat die verhoogde pigmentasie van rooier seleksies van tweekleurappel kultivars nie aan ‘n hoër temperatuur optimum vir antosianiensintese toegeskryf kan word nie. Die optimum dag temperature vir rooi kleurontwikkeling vir die onderskeie kultivars verskil klaarblyklik tussen seisoene, maar nie tussen produksie areas nie. Oor die algemeen het kleurontwikkeling maksimaal plaasgevind tussen 17 ºC en 25 ºC. Die optimum dagtemperatuur vir rooi kleurontwikkeling het konstant gebly tydens vrugontwikkeling, buiten vir ‘Cripps’ Pink’ waar dit toegeneem het van ongeveer 14 ºC tot 22 ºC vanaf Januarie tot April. Die mate van rooi kleurontwikkeling het in al die kultivars toegeneem deur die loop van vrugontwikkeling . Ons kon nie daarin slaag om optimum induksie temperature vir rooi kleurontwikkeling vas te stel nie. Rooi kleurontwikkeling van ‘Royal Gala’ uit Ceres is moontlik bevorder deur induksie by 4 ºC, terwyl ‘Fuji’ se rooi kleur afgeneem het met ‘n verlaging in induksie temperatuur. Ten einde die teenwoordigheid van antosianien in onvolwasse appelvruggies te verduidelik, het ons die hipotese getoets dat antosianien die vrugskil beskerm teen fotoinhibisie en fotooksidatiewe beskadiging gedurende tydperke van verhoogde ligstres. Eerstens het ons bevestig dat die tempo van kleurontwikkeling in reaksie op ‘n koue front waarskynlik vinnig genoeg is om fotobeskerming te verleen. Vervolgens is gevind dat ‘Cripps’ Pink’ vrugskil aansienlik meer fotoinhibisie ervaar het by lae temperatuur (16 ºC) in vergelyking met matige (24 ºC en 32 ºC) en hoë (40 ºC) temperatuur onder hoë irradiasie met sigbare lig. Die hersteltempo was temperatuur-afhanklik; dit was die stadigste by lae temperatuur en het toegeneem met ‘n toename in temperatuur. Die foto-apparaat in ‘Cripps’ Pink’ vrugskil blyk besonder sensitief te wees vir ligstres by lae temperatuur regdeur die groeiseisoen met aansienlike fotoinhibisie by selfs matige temperatuur (24 ºC). Die sensitiwiteit van die vrugskil vir fotoinhibisie het toegeneem deur die groeiseisoen by laer ligvlakke, maar het dieselfde gebly by hoër vlakke van irradiasie. Laastens is vrugte blootgestel aan hoë irradiasie by lae en matige temperatuur voordat dit vervolgens blootgestel is aan hoë temperatuur in kombinasie met hoë irradiasie. Dit was om die hipotese te toets dat fotoinhibisie wat opgedoen word gedurende ‘n onverwagte koue periode, die skil meer vatbaar maak vir fototermiese skade sodra die temperatuur weer styg na die koue periode verby is. Ongelukkig het die hoë temperatuur stres al die behandelings tot so ‘n mate geaffekteer dat dit onmoontlik was om enige gevolgtrekkings vanuit ons resultate te maak.

Cellulose is a biopolymer of great relevance in the plant cell walls, where it constitutes the most important skeletal component. Cellulose is also an important raw material in the pulp- and paper, forest, and textile industries, among others. Cellulose biosynthesis in particular, and xylogenesis in general are processes which are currently poorly understood. Yet, research in cellulose synthesis is progressing and different applications of cellulose, mainly cellulose derivatives for e.g. pharmaceuticals and coatings, are constantly emerging. This thesis depicts how cellulose synthase (CesA) genes in Populus were identified and characterized by gene expression- and bioinformatics analyses. Within an EST database of more than 100,000 clones from wood forming tissues of three different Populus taxa, ten CesA genes were identified in Populus tremula x tremuloides. Subsequent gene expression analyses by using microarrays and real-time PCR experiments in woody tissues, revealed distinct regulation patterns among the genes of interest. This enabled proper classification and characterization of the secondary cell wall related CesA genes, in particular. Bioinformatic analyses of the genome sequence of Populus trichocarpa further provided a complete picture of the number of putative CesA genes retained after several duplication events during tree evolution. In contrast to the previously reported set of ten 'true' CesA genes in many other plant species, the genome of P. trichocarpa encodes 18 putative proteins, which could be assembled into nine groups according to their sequence similarities. Interestingly, studies in the EST database suggested that paralogs within at least two groups have corresponding orthologs in P. tremula x tremuloides, which are furthermore transcribed. This implies that at least some of the duplicated genes have remained functional, or may have acquired a modified function. By focusing on the CesA genes associated with secondary cell wall formation, cellulose synthesis was also studied in poplar cell suspension cultures. Selection of CesA enriched material was performed by determining expression intensities of the CesA genes using RT-PCR, whereupon membrane protein extraction was initiated. CesA proteins are part of large cellulose synthesizing complexes in the plasma membrane. Subsequent proteomic approaches comprised partial purification of these cellulose synthesizing complexes from protein enriched culture material and in vitro cellulose synthesis experiments. De novo synthesized material was successfully characterized and the acquired yields were as high as 50% cellulose (compared to previously reported yields of 30% in other plant systems) of the total in vitro synthesized product. Elevated CesA gene expression levels can thus be correlated to increased protein activity in poplar cell suspension cultures. In addition, antibodies raised against CesA antigens were used in Western blot analyses comprising samples along the protein extraction- and purification procedure. Proteins with corresponding molecular weight to the theoretical 120kDa of CesA proteins were recognized by a range of different specific antibodies. The study demonstrates that poplar cell suspension cultures can provide a valuable model system for studies of cellulose synthesis and different aspects of xylogenesis. / QC 20101005

The objective of the proposed research is to design an experimental setup to assess the ability of impedance measurements to characterize mouse embryonic stem cell (ESC) and embryoid body (EB) growth and differentiation. Existing quality assurance measurements used to stage the growth and differentiation of embryoid bodies are labor intensive and most often destructive to the cells, thus present methods are typically valid for a single time point. Bioimpedance measurements are non-invasive and non-destructive, presenting an alternative approach to this challenge. These measurements can be done continuously for real-time measurements on the changes in embryoid body growth and differentiation. A system capable of making bioimpedance measurements of ESC and EB suspensions was designed along with a biocompatible test device to hold the cells and Ag-AgCl electrodes. The system uses a lock-in amplifier to record the magnitude and phase changes of the ESC and EB suspensions when a 1 Vpp signal sweeping frequencies from 100 Hz to 100 kHz is applied. The system performance was validated with a test case of 1 mL of 0.1 M KCl. Then experiments with cell culture media, ESCs, and EBs were performed, with varying concentrations of cells and EBs. Experimental results for single ESC suspensions showed promise in detecting a difference in cell concentration between 2 million and 4 million cells in 0.5 mL of media. Results for four day old EBs were ambiguous, and we conclude that a different experimental set up is required due to EB settling during experimentation.

Plant cell and tissue cultivations are of growing interest for the production of structurally complex and expensive plant-derived products, especially in pharmaceutical production. Problems with up-scaling, low yields and high-priced process conditions result in an increased demand for models to provide comprehension, simulation, and optimization of production processes. In the last 25 years, many models have evolved in plant biotechnology; the majority of them are specialized models for a few selected products or nutritional conditions. In this article we review, delineate, and discuss the concepts and characteristics of the most commonly used models. Therefore, the authors focus on models for plant suspension and submerged hairy root cultures. The article includes a short overview of modeling and mathematics and integrated parameters, as well as the application scope for each model. The review is meant to help researchers better understand and utilize the numerous models published for plant cultures, and to select the most suitable model for their purposes.

Les anthocyanes sont une famille de polyphénols très répandus chez les végétaux. Chez la vigne, elles sont responsables de la coloration des baies des cépages rouges, et sont impliquées dans les propriétés organoleptiques des vins. Une nutrition azotée faible induit la production des anthocyanes dans les cellules de la pellicule de raisin des cépages rouges via des mécanismes de régulation qui ne sont pas encore totalement élucidés. Dans ce contexte, nous avons étudié les mécanismes moléculaires impliqués dans la réponse de l’accumulation des anthocyanes pour différents niveaux d’apports azotés. Deux matériels biologiques complémentaires ont été utilisés : des suspensions cellulaires de vigne (lignée GT3) et des plants de Cabernet-Sauvignon, cultivés au vignoble.L’augmentation de la synthèse d’anthocyanes en réponse à la diminution de la nutrition azotée a été confirmée dans les baies et les cellules de vigne en culture. Les analyses transcriptomiques globales (génome complet) et ciblées (qPCR) ont mis en lumière des modifications de l’expression génique, notamment de gènes liés au métabolisme des flavonoïdes, en réponse à la nutrition azotée. L’expression de nombreux gènes structuraux impliqués dans la voie de biosynthèse des anthocyanes est induite par une faible nutrition azotée. La variation de l’apport azoté influence également de façon coordonnée l’expression des gènes régulateurs positifs (facteurs de transcription de type MYB) et négatifs (protéine de type Lateral organ Boundary Domain (LBD)) des gènes de la biosynthèse des flavonoïdes chez la Vigne. L’expression de gènes liés à la production d’énergie (NADH, NADPH), est également affectée.En parallèle, une approche intégrative a été développée sur les suspensions cellulaires, en combinant des mesures d’activités enzymatiques, des dosages de métabolites primaires et secondaires, avec un modèle de balance de flux (Flux Balance Analysis, FBA). Les cartes de flux obtenues prédisent que la diminution de l’apport azoté entraîne une augmentation des flux métaboliques dans la voie du shikimate et des phénylpropanoïdes ; ainsi qu’une répression de la majorité des flux dans les différentes voies du métabolisme primaire, à l’exception de la voie des pentoses phosphates, dont le flux est maintenu, et de la voie de synthèse de l’amidon qui est accrue. Les résultats obtenus plaident en faveur d’un lien fort entre synthèse des anthocyanes et statut énergétique (ATP, NADPH) des cellules vigne. / Anthocyanins are polyphenol compounds very abundant in most of the plants. In grapevine, they give color to red berries and they improve red wine quality and increase the organoleptic properties of the wine. Low nitrogen supply stimulates anthocyanin production in berry skin cells of red grape varieties through regulation mechanisms that are far from being fully understood. In this context, we worked on the molecular mechanisms involved in anthocyanin biosynthesis response to nitrogen supply. Two complementary biological materials were used: grapevine cell suspensions (GT3 line) that originate from a teinturier cultivar and produce anthocyanins under normal conditions; and red grape berries of cv. Cabernet-Sauvignon cultivated in a commercial vineyard. Increases of anthocyanins synthesis in response to low nitrogen levels were confirmed in the field-grown berries and the cells suspensions. Both comparative global (microarrays) and targeted (qPCR) transcriptomic analysis showed different regulations on the expression of the genes involved in the secondary (especially the anthocyanin) and nitrogen metabolisms. The expression of most structural genes of the anthocyanin biosynthesis pathway was induced by a low nitrogen supply. Nitrogen controls also the expression of the positive (MYB transcription factors) and negative (Lateral organ Boundary Domain family protein LBD39) regulatory genes of the flavonoid pathway in grapevine. Furthermore, some genes improved in energy production (ATP, NADPH) were affected. In parallel, an integrative approach combining enzymatic activities and primary and secondary metabolites measurements with developing a Flux Balance Analysis (FBA) modeling approach was used on cells suspensions GT3. The flux maps deciphered that low nitrogen increases metabolic fluxes in shikimate and phenylpropanoid pathways and represses the majority metabolic fluxes in different pathways of primary metabolism. The two exceptions included the pentose phosphate pathway, which the flux metabolism was maintained, and the starch synthesis pathway, which was enhanced. The results obtained showed a strong link between anthocyanin synthesis and energy status (ATP, NADPH) in the berry cell suspensions.

Foi investigada a resposta antioxidante de culturas em suspensão celular de café (Coffea arabica L.) ao cádmio (Cd) e níquel (Ni). As células de café foram tratadas por doze dias com zero (controle), 0,05 e 0,5 mM de NiCl2 ou CdCl2. O Cd e o Ni acumularam rapidamente nas células e este acúmulo foi diretamente correlacionado com o aumento na concentração de metal no meio externo. Em 0,05 mM CdCl2 e 0,05 mM NiCl2, o crescimento foi estimulado, mas em 0,5 mM CdCl2 e 0,5 mM NiCl2 a taxa de crescimento foi reduzida. As alterações no metabolismo do oxigênio ativo foram detectadas pela análise visual, assim como pelo aumento na peroxidação lipídica, nos tratamentos com 0,5 mM CdCl2 e 0,5 mM NiCl2. As atividades das enzimas catalase (CAT; EC 1.11.1.6), glutationa redutase (GR; EC 1.6.4.2) e superóxido dismutase (SOD; EC 1.15.1.1) aumentaram após exposição ao Cd, particularmente na maior concentração de CdCl2. A atividade da ascorbato peroxidase (APX; EC 1.11.1.11) foi elevada por 0,05 mM CdCl2, mas não pode ser detectada em células cultivadas na maior concentração de CdCl2 após 24 h de cultivo, enquanto que a guaiacol peroxidase (GOPX; EC 1.11.1.7) não demonstrou um padrão claro de resposta ao tratamento com Cd. As atividades das enzimas CAT, GR, APX, GOPX e SOD foram aumentadas, particularmente nos períodos iniciais de exposição ao NiCl2 e as atividades foram maiores na dosagem 0,5 mM NiCl2, na maioria dos tempos de exposição testados. A análise em PAGE não desnaturante seguido pela revelação da atividade enzimática, apresentou uma isoenzima da CAT, nove isoenzimas da SOD e quatro isoenzimas da GR. As isoenzimas da SOD foram diferencialmente afetadas pelo tratamento com CdCl2 e NiCl2 e uma isoenzima da GR mostrou responder especificamente ao CdCl2 e ao NiCl2. Os resultados sugerem que 0,5 mM CdCl2 e 0,5 mM NiCl2 podem levar ao estresse oxidativo. O CdCl2 e o NiCl2 a 0,05 mM não induziram peroxidação lipídica e a principal resposta aparenta ser via indução das atividades das enzimas SOD, CAT e APX nas células tratadas com Cd e das enzimas SOD, CAT, GOPX e APX nas células tratadas com Ni, para a remoção das espécies ativas de oxigênio (EAOs), e pela indução da GR para garantir a disponibilidade de glutationa reduzida. A síntese de peptídeos de ligação ao Cd, deve também estar relacionada com a inibição da atividade da APX, provavelmente devido ao esgotamento da glutationa e ascorbato na maior concentração de CdCl2. / The antioxidant responses of coffee (Coffea arabica L.) cell suspension cultures to cadmium (Cd) and nickel (Ni) were investigated. Coffee cells were treated for twelve days with 0 (control), 0.05 and 0.5 mM NiCl2 or CdCl2. Cd and Ni accumulated very rapidly in the cells and this accumulation was directly correlated with an increase in applied metal concentration in the external medium. At 0.05 mM CdCl2 and 0.05 mM NiCl2, growth was stimulated, but at 0.5 mM CdCl2 and 0.5 mM NiCl2 the growth rate was reduced. An alteration in activated oxygen metabolism was detected by visual analysis as well as by an increase in lipid peroxidation at 0.5 mM CdCl2 and 0.5 mM NiCl2. Catalase (CAT; EC 1.11.1.6), glutathione reductase (GR; EC 1.6.4.2) and superoxide dismutase (SOD; EC 1.15.1.1) activity increased after Cd exposure, particularly at the higher concentration of CdCl2. Ascorbate peroxidase (APX; EC 1.11.1.11) activity was higher at the lower CdCl2 concentration used, but could not be detected in cells growing in the higher CdCl2 concentration after 24 h of growth, whilst guaiacol peroxidase (GOPX; EC 1.11.1.7) did not show a clear response to Cd treatment. CAT, GR, APX, GOPX and SOD activities were increased due to Ni treatment, particularly at earlier NiCl2 exposure times and the activities were higher at 0.5 mM NiCl2 for most of exposure times tested. An analysis by non-denaturing PAGE followed by staining for enzyme activity, revealed one CAT isoenzyme, nine SOD isoenzymes and four GR isoenzymes. The SOD isoenzymes were differently affected by CdCl2 and NiCl2 treatment and one GR isoenzyme was shown to specifically respond to CdCl2 and NiCl2. The results suggest that the higher concentrations of CdCl2 and NiCl2 may lead to oxidative stress. CdCl2 and NiCl2 at 0.05 mM did not induce lipid peroxidation and the main response appeared to be via the induction of SOD, CAT and APX activities in Cd treated cells and SOD, CAT, GOPX and APX activities in Ni treated cells, for the removal of reactive oxygen species (ROS), and by the induction of GR to ensure the availability of reduced glutathione. The synthesis of Cd-binding proteins may also be related to the inhibition of APX activity probably due to glutathione and ascorbate depletion at higher CdCl2 concentration.

<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>

<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>