<p>Apoptosis is a conserved mode of cell death executed by a group of proteases named caspases, which collectively ensure tissue homeostasis in multicellular organisms by triggering a program of cellular "suicide" in response to developmental cues or cellular damage. </p><p>Accumulating evidence suggests that cellular metabolism impinges directly upon the decision to initiate cell death. Several links between apoptosis and metabolism have been biochemically characterized. Using <italic>Xenopus</italic> oocyte extracts, our laboratory previously discovered that caspase-2 is suppressed by NADPH metabolism through an inhibitory phosphorylation at S164. However, the physiological relevance of these findings has not been investigated at the whole organism level. Studies presented in this dissertation utilize both Schneider's <italic>Drosophila</italic> S2 (S2) cells and transgenic animals to untangle the influence of metabolic status on fly apoptosis.</p><p>We first demonstrate a novel link between <italic>Drosophila</italic> apoptosis and metabolism by showing that cellular NADPH levels modulate the fly initiator caspase Dronc through its phosphorylation at S130. Biochemically and genetically blocking NADPH production removed this inhibitory phosphorylation, resulting in the activation of Dronc and the subsequent apoptotic cascade in cultured S2 cells and specific neuronal cells in transgenic animals. Similarly, non-phosphorylatable Dronc was found to be more potent than wild-type in triggering neuronal apoptosis. Moreover, upregulation of NADPH prevented Dronc-mediated apoptosis upon abrogation of <italic>Drosophila</italic> Inhibitor of Apoptosis (IAP) protein 1 (DIAP1) by double-stranded RNA (dsRNA) or cycloheximide (CHX) treatment, revealing a novel mechanism of DIAP1-independent apoptotic regulation in <italic>Drosophila</italic>. Mechanistically, the CaMKII-mediated phosphorylation of Dronc hindered its activation, but not its catalytic activity. As NADPH levels have been implicated in the regulation of oocyte death, we demonstrate here that a conserved regulatory circuit also coordinates somatic apoptosis and NADPH levels in <italic>Drosophila</italic>.</p><p>Given the regulatory role of NADPH in the activation of Dronc in <italic>Drosophila</italic> and caspase-2 in vertebrates, we then attempted to further elucidate the underlying signaling pathways. By tracking the catabolic fate of NADPH, we revealed that fatty acid synthase (FASN) activity was required for the metabolic suppression of Dronc, as both the chemical inhibitor orlistat and FASN dsRNA abrogated NADPH-mediated protection against CHX-induced apoptosis in S2 cells. Interestingly, it has been previously demonstrated that blocking FASN induces cell death in numerous cancers, including ovarian cancer; however, the mechanism is still obscure. As our results predict that suppression of FASN activity may prevent the inhibitory phosphorylation of Dronc and caspase 2 (at S130 and S164 respectively), we examined the contribution of caspase-2 to cell death induced by orlistat using ovarian cancer cells. Indeed, caspase-2 S164 was dephosphorylated upon orlistat treatment, initiating the cleavage and activation of caspase-2 and its downstream target, Bid. Knockdown of caspase-2 significantly alleviated orlistat-induced cell death, further illustrating its involvement.</p><p>Lastly, we developed an assay based on bimolecular fluorescence complementation (BiFC) to monitor the oligomerization of Dronc in S2 cells, a crucial step in its activation. The sensitivity of this assay has been validated with several apoptotic stimuli. A future whole-genome screen employing this assay is planned to provide new insights into this complex apoptotic regulatory network by unbiasedly identifying novel apoptotic regulators.</p> / Dissertation

Made available in DSpace on 2015-03-26T13:36:32Z (GMT). No. of bitstreams: 1 texto completo.pdf: 233114 bytes, checksum: c512aebdda22870fd45ba41c414294f0 (MD5) Previous issue date: 2007-10-31 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Aluminum effects on malic acid exudation and root elongation were evaluated in two maize genotypes: UFVM 100 e UFVM 200, selected from a group of six genotypes developed by the Agronomy Department of the Federal University of Viçosa, Viçosa, MG, Brazil. Five days-old seedlings of the six genotypes were exposed to Al 50 µM in 0.4 mM CaCl2, pH 4.0, for 24 h and, based on growth inhibition of the main root, they were classified into two categories: a) Al sensitive (&#8805; 68% inhibition): UFVM 8 and UFVM 100; and b) Al tolerant (&#8804; 64% inhibition): UFVM 6, UFVM 7, UFVM 9 and UFVM 200. The genotypes: UFVM 100 and UFVM 200 were selected for the following experiments. Seedlings of both genotypes were treated with different Al concentrations and different time of exposition to the metal and then the Al effect on root elongation was evaluated. The elongation of the main root decreased with increasing Al concentration in both genotypes, especially in the UFVM 100 genotype. Differences between cultivar were higher at lower concentration but decreased as Al concentration was increased in the growth medium. Inhibition in the elongation of the main root was evident from the beginning of Al treatment and increased linearly with the time of exposition to Al. The UFVM 200 genotype always showed higher Al tolerance than the UFVM 100 genotype and the differences between them were bigger at Al concentration between 25 and 50 µM. Aluminum contents in leaves were always low in comparison with the roots, but the UFVM 200 genotype showed higher Al contents than the UFVM 100 genotype. In the roots Al contents increased with increasing Al concentration in the growth medium and they were always higher in the UFVM 100 genotype. The activity of the malate dehydrogenase (MDH) was higher in roots, but it increased with Al treatment only in leaves of the tolerant genotype. Malic acid exudation to the growth medium was observed in control plants of both genotypes. In Al-treated plants of both genotypes malic acid exudation increased with increasing Al concentration in the growth medium but UFVM 200 genotype always showed higher exudation at all Al concentrations. Maximum malic exudation occurred at estimated Al concentration of 133 and 197 µM for the genotypes UFVM 200 e UFVM 100, respectively. Although, both genotypes have exudated malic acid to the growth medium, in none of them the amount of malic acid was enough to detoxify the Al in the nutrient solution. / Os efeitos do alumínio sobre o alongamento radicular e a exsudação de ácido málico foram avaliados em dois genótipos de milho: UFVM 100 e UFVM 200, selecionados de um grupo de seis genótipos desenvolvidos pelo Departamento de Fitotecnia da Universidade Federal de Viçosa, Viçosa, MG, Brasil. Plântulas, com cinco dias de idade, dos seis genótipos foram expostas a Al 50 µM em solução de CaCl2 0,4 mM, por 24 horas e, baseado na inibição do alongamento radicular, foram classificados em duas categorias: a) sensíveis ao Al (&#8805; 68% de inibição): UFVM 8 e UFVM 100; e b) tolerantes ao Al (&#8804; 64% de inibição): UFVM 6, UFVM 7, UFVM 9 e UFVM 200. Os genótipos UFVM 100 e UFVM 200 foram selecionados para os demais experimentos. Plântulas dos dois genótipos foram expostas a diferentes concentrações de Al e diferentes tempos de exposição e, então, o efeito do Al sobre o alongamento da raiz principal foi avaliado. O alongamento da raiz principal decresceu com o incremento na concentração de Al nos dois genótipos, especialmente no genótipo UFVM 100. Diferenças entre os genótipos foram maiores em baixas concentrações de Al e decresceram à medida que a concentração de alumínio foi aumentando no meio de cultivo. Inibições no crescimento da raiz principal foram evidentes desde o início da aplicação do tratamento e aumentaram linearmente com o tempo de exposição ao Al. O genótipo UFVM 200 mostrou sempre maior tolerância ao Al que o genótipo UFVM 100, tendo as maiores diferenças entre eles ocorrido entre 25 e 50 µM de Al. Os teores de Al nas folhas foram sempre baixas em comparação aos teores nas raízes, mas o genótipo UFVM 200 apresentou teores mais elevados que o genótipo UFVM 100. Nas raízes, os teores de Al aumentaram com a elevação na concentração de Al na solução de cultivo e foram sempre mais elevados no genótipo UFVM 100. A atividade da enzima desidrogenase malato (MDH) foi maior nas raízes, mas só foi modificada pelo tratamento com Al nas folhas do genótipo tolerante. Exsudação de ácido málico para o meio de cultivo ocorreu nas plantas controle dos dois genótipos. A exposição das plantas ao Al resultou em aumento na exsudação de ácido málico para o meio de cultivo, tendo o genótipo UFVM 200 apresentado maior exsudação deste ácido em todas as concentrações de Al. A exsudação máxima de ácido málico ocorreu nas concentrações estimadas de Al de 133 e 197 µM para os genótipos UFVM 200 e UFVM 100, respectivamente. Embora, os dois genótipos tenham exsudado ácido málico para o meio de cultivo, em nenhum deles a quantidade de ácido málico foi suficiente para eliminar a toxidez do Al na solução nutritiva.

<p>C₄-dicarboxylic acids appear to be metabolized via the TCA cycle in N₂-fixing bacteria (bacteroids) within legume nodules. In <em>Sinorhizobium meliloti</em> bacteroids from alfalfa, NAD⁺-malic enzyme (DME) is required for symbiotic N₂-fixation and this activity is thought to be required for the anaplerotic synthesis of pyruvate. In contrast, in the pea symbiont <em>Rhizobium leguminosarum</em> pyruvate synthesis can occur via either the DME pathway or a pathway catalyzed by phosphoenolpyruvate carboxykinase (PCK), pyruvate kinase (PYK), and pyruvate dehydrogenase. Here we report that <em>dme</em> mutants of <em>Sin</em>or<em>hizobium sp</em>. NGR234 formed root nodules on a broad range of plants and that the level of N₂-fixation varied from 90% to 20% of wild type depending on the host plants inoculated. NGR234 bacteroids had significant PCK activity and while single <em>pckA</em> and single <em>dme</em> mutants fixed N₂ on <em>Macroptilium atropurpureum</em> and <em>Leucaena leucocephala</em> (albeit at a reduced rate), a <em>pckA</em> <em>dme</em> double mutant had no N₂-fixing activity (Fix^-). Thus, NGR234 bacteroids appear to synthesize pyruvate from TCA cycle intermediates via DME or PCK pathways. These NGR234 data, together with other reports, suggested that the completely Fix^- phenotype of <em>S. meliloti dme </em>mutants may be specific to the alfalfa-<em>S. meliloti </em>symbiosis. We therefore examined the ME-like genes <em>azc3656 </em>and <em>azc0119 </em>from <em>Azorhizobium caulinodans</em>, as <em>azc3656 </em>mutants were previously shown to form Fix^- nodules on the tropical legume <em>Sesbania rostrata</em>. We found that purified AZC3656 protein is an NAD (P)⁺-malic enzyme whose activity is inhibited by acetyl-coenzyme A (acetyl-CoA) and stimulated by succinate and fumarate. Thus, whereas DME is required for symbiotic N₂ fixation in <em>A. caulinodans </em>and <em>S. meliloti</em>, in other rhizobia this activity can be bypassed via another pathway(s).</p> <p>In <em>S. meliloti</em> both malic enzymes DME and TME share similar apparent <em>K_m</em>s for substrate and cofactors, but differ in their responses to TCA cycle intermediates, with DME activity inhibited by acetyl-CoA and induced by succinate and fumarate. Previous results in our laboratory indicated that DME is essential for symbiotic N₂ fixation, while TME fails to functionally replace DME. One possible reason for it is that a high ratio of NADPH/NADP⁺in<em> S. meliloti </em>bacteroids prevents TME from functioning in nodules. We sought to lower the<em> </em>NADPH/NADP⁺ratio by overexpressing a soluble pyridine nucleotide transhydrogenase (STH). However, metabolite measurements indicated that overproducing STH failed to lower the ratio of NADPH/NADP⁺ in<em> S. meliloti</em>.</p> <p>Previous studies assumed that DME and TME might play different roles in central carbon metabolism. To gain insight of their physiological functions, genome-wide microarray analysis was conducted in <em>S. meliloti</em> single<em> dme and</em> <em>tme</em> mutants grown on glucose or succinate. The most striking changes of gene expression were observed in <em>S. meliloti</em> <em>dme</em> mutants grown on succinate. The functions of upregulated genes suggested that DME might play an important role in regulating TCA cycle intermediates, important for the maintenance of metabolic flux through TCA cycle during C₄-dicarboxylate oxidation. However, changes of gene expression found in <em>tme </em>mutants were not significant enough to predict the physiological functions of TME protein in central carbon metabolism.</p> / Doctor of Philosophy (PhD)

No / This review summarises the recent developments in the synthesis and applications of polymers derived from malic acid. There has been an increased interest in the design of sustainable and biodegradable polymers as a result of the drive to use renewable feedstocks as an alternative to petrochemicals in addition to their significant potential in biomedical applications. Synthetic strategies to access polymers from malic acid based on both condensation and ring-opening polymerization, across a broad range of conditions, are reviewed along with their advantages and limits. The role that such materials are studied for in biomedical applications is discussed, and their environmental impact based on the biodegradability of the malic polymer backbone is outlined. / The Royal Society, EPSRC, BBSRC

Immune systems are fundamentally based on the differentiation of self and non-self. Unlike mammals, plants have an innate immune system responding to the pathogen only at the site of attack. One of these pathogens is Blumeria graminis f. sp. hordei which is an obligate biotrophic pathogen causing powdery mildew disease and resulting in up to 30% yield loss for both cultivated and wild barley. In this study, Pallas-01 (P-01) and Pallas-03 (P-03) barley lines were inoculated with powdery mildew race Bgh103 (64/01) resulting incompatible and compatible interactions, respectively. 6, 12, 24, 48 and 72 hour-post-inoculation (hpi) samples were used in order to define the differential gene expression of NAD malic enzyme, chloroplast lipocalin, phosphoglyceromutase (PGM), Mg chelatase and 26S protease regulatory subunit 6B homolog. In the proteomics study previously conducted in the laboratory, except for the NAD-dependent malic enzyme, the other four proteins were shown to be involved in the incompatible interaction of P-01 and Bgh103 at protein level, whereas NAD-dependent malic enzyme was changing in the compatible interaction. The expression level for both proteomics and transcriptomics were assumed to be similar. However, the level of transcript would not always reflect its protein level or correlate with the level of proteins, due to complex cellular regulation processes. Post-transcriptional modifications such as synthesis, processing, degradation and post-translational modifications are changing the level of proteins expressed, thus a parallel correlation between the protein and mRNA levels can be lost. Other possible reasons for this variation can be changes in mRNA and protein stability, efficiency of translation and protein&rsquo / s turnover rate. The transcription level changes of the genes investigated in this study are found to be differentially expressed, supporting the proteomics data indicating that these genes are possibly involved in resistance. For further investigations, genetic tools such as gene silencing with RNAi and knockout experiments are required in order to elucidate the mechanism of these candidate genes in the plant-pathogen interaction.

Delivery of biologically relevant ions such as drugs, neurotransmitters and hormones have been recognized as powerful a tool to control physiology of animals and plants for research purposes and practical applications. In the plant research community, ions are most commonly delivered as part of a solvent by soaking, spraying, pipetting or by adding to the soil. These methods have low control of the delivery dynamics and quantity of ion uptake. These issues motivated the development of the Organic Electronic Ion Pump (OEIP), which delivers only ions of interest by applying an external electric field through a polyelectrolyte membrane of high fixed charge concentration. A miniaturized, implantable version of the OEIP based on capillary fibres (c-OEIP), where the polyelectrolyte is enclosed in a capillary, enabled even higher precision of the delivery. In this master thesis, c-OEIP has been applied in the tropical plant Kalanchoe Blossfeldiana, chosen due to its characteristic skill to gradually learn to save water: while maturing it shifts to night time photosynthesis and transpiration, called Constitutive Crassulacean Acid Metabolism. A better understanding of this metabolism and water saving ability could guide engineering of enhanced drought tolerance in crop plants, which is motivated by the increasing global warming. One of the biologically relevant ions that is potentially involved in this water-saving learning process is the malate ions. The aim of this thesis is to test the hypothesis that c-OEIP is able to deliver malate ions to cause a reduction in stomatal conductance and transpiration of intact leaves of Kalanchoe Blossfeldiana. To test this hypothesis, firstly, the capillary-based OEIP were fabricated using polyimide coated glass capillaries filled with AETMAC polyelectrolyte. The ability of these devices to deliver malic acid (MA) was verified by using current-voltage characterisation during loading and delivery of MA. Secondly, the setup for MA delivery with c-OEIP to intact kalanchoe leaf was developed, optimising the insertion method to minimize the wounding of the plant and increase assay reproducibility. Finally, the MA was delivered to intact kalanchoe leaves via c-OEIP, where the plant transpiration response was evaluated using standard gas exchange porometer and also novel infrared camera, as plant temperature can be correlated with plant transpiration status. The results indicate that c-OEIP can deliver MA and trigger reduction of transpiration of young kalanchoe leaves, supporting the hypothesis that malate ions act to reduce stomatal conductance, potentially conveying a feedback message from the mesophyll to the guard cells. / <p>Examensarbetet är utfört vid Institutionen för teknik och naturvetenskap (ITN) vid Tekniska fakulteten, Linköpings universitet</p>

<i>Cryptosporidium parvum</i> is a protozoan parasite historically associated with waterborne and more recently foodborne outbreaks of diarrheal illness. Contamination of certain foods, such as unpasteurized apple cider, with infective oocysts may occur as oocysts are shed in the feces of common ruminants like cattle and deer that graze in and around orchards. Cryptosporidiosis can result in a severe illness for previously healthy individuals and a life-threatening illness in immunocompromised individuals. Disease occurs after the ingestion of small infective oocysts (4 to 5 mm in size). The relatively thick membrane of the oocysts allows them to be resistant to chlorine and many other environmental pressures, making oocysts difficult to inactivate. In this study, alternative treatments to pasteurization were evaluated for their ability to inhibit <i>C. parvum</i> oocyst viability in fruit juices. Oocyst viability was analyzed with a cell culture infectivity assay, using a human illeocecal cell line (HCT-8) that is most similar to human infection. The percent inhibition of infection by each treatment was determined along with the corresponding log reduction for the treatments found to be most effective. Infection by treated oocysts was compared to that of control untreated oocysts. Cell monolayers were infected with 10⁶ treated oocysts or a series of 10-fold dilutions. Parasitic life stages were visualized using an immunohistochemistry system and 100 microscope fields counted per monolayer. Organic acids and H₂O₂ were added on a wt/vol basis to apple cider, orange juice, and grape juices. Malic, citric, and tartaric acids at concentrations from 1%-5% inhibited <i>C. parvum</i> infectivity of HCT-8 cells by up to 88%. Concentrations ranging from 0.025%-3% H₂O₂ were evaluated where addition of 0.025% H₂O₂ to each juice resulted in a >5 log reduction of C. parvum infectivity as determined with an MPN-based cell culture infectivity assay. Treating apple cider, orange juice, and grape juice with ozone for a time period of 30 seconds up to 15 minutes at 6° and 22°C (0.9 g/L flow rate) inhibited C. parvum viability to > 90% as monitored in the cell culture assay. It is hypothesized that oocyst wall proteins that are necessary for infection are oxidized by the reactive oxygen species generated from the decomposition of the ozone and hydrogen peroxide treatments. These treatments or combinations thereof may offer potential alternatives to traditional pasteurization for fruit juices to successfully inhibit <i>C. parvum</i> viability. / Ph. D.

Le stress oxydant est un acteur clé dans les réponses des plantes à des conditions contraignantes. En raison de la complexité de la régulation de l’état redox cellulaire, il reste beaucoup à élucider concernant les interactions entre différentes composantes dans ces conditions. Grâce à une approche de génétique inverse basée sur un mutant d’Arabidopsis déficient en catalase (cat2) qui présente des modifications d’état redox prévisibles et bien définies, cette étude a exploré les interactions entre le stress oxydant et (1) un gène spécifique impliqué dans la déphosphorylation des protéines, (2) des enzymes spécifiques impliquées dans les systèmes antioxydants réducteurs. Les résultats obtenus révèlent que la sous-unité B'γ de la protéine phosphatase de type 2A (PP2A-B'γ) est importante dans la détermination des phénotypes et des réponses de défense photopériode-dépendantes chez cat2. En conditions de jours courts (SD), un double cat2 pp2a-b'γ mutant montrait une gamme de réponses qui n’étaient pas observées chez cat2. Ces effets comprenaient l’apparition de lésions ainsi que l’accumulation de l’acide salicylique et d’autres composés de défense. Des analyses métabolomiques et protéomiques ont permis de démontrer que ces effets étaient accompagnés de modifications de l’abondance de métabolites et protéines spécifiques, ainsi que des changements dans le statut de phosphorylation de certains polypeptides. Dans un deuxième volet du travail, l’importance d’une enzyme productrice du NADPH a été évaluée en produisant des doubles cat2 nadp-me2 mutants chez lesquels l’isoforme majeure de l’enzyme malique cytosolique n’est plus exprimée. Malgré une induction de cette enzyme par le stress oxydant aux niveaux de transcrits et d’activité, et une diminution importante de l’activité foliaire associée aux mutations nadp-me2, peu de différence a été observée entre les lignées cat2 et cat2 nadp-me2. De même, la mutation nadp-me2 n’a pas affecté la réponse phénotypique de plantes exposées à l’ozone. Dans la troisième partie du travail, le couplage entre les pools ascorbate et glutathion lors du stress oxydant a été exploré par l’introduction de mutations pour la déshydroascorbate réductase (DHAR) dans le fond génétique cat2. L’activité extractible de cette enzyme a été diminuée à des niveaux très faibles chez des lignées portant à la fois les mutations dhar1 et dhar3. Cependant, peu de différence a été observée dans les phénotypes et les statuts d’ascorbate et de glutathion chez un triple mutant cat2 dhar1 dhar3 par rapport à cat2. Des analyses préliminaires d’un quadruple cat2 dhar1 dhar2 dhar3 mutant semblent pourtant indiquer que les trois DHARs jouent des rôles fonctionnellement redondants dans le stress oxydant. Dans son ensemble, ces travaux apportent des données nouvelles sur les enzymes qui régulent les réponses aux stress oxydants et ont généré des outils intéressants pour des études ultérieures. / Oxidative stress is a key player in plant responses to challenging environmental conditions. The intricate nature of the regulation of cellular redox state means that much remains to be elucidated on interactions between different components in these conditions. By using a genetic approach based on a catalase-deficient Arabidopsis mutant (cat2) that presents well-defined, predictable changes in redox state, this study explored interactions between oxidative stress and (1) a specific gene involved in protein dephosphorylation, and (2) specific enzymes involved in the antioxidative/reducing system. The results showed that protein phosphatase 2 subunit B'γ (PP2A-B'γ) is involved in determining day length-dependent phenotypes and related defense responses in cat2. A cat2 pp2A-B'γ double mutant showed a range of responses that were not observed in cat2 grown in short days, including lesion formation and accumulation of salicylic acid (SA) and related metabolites. Metabolomics and proteomics analyses showed that these effects were associated with altered abundance of specific metabolites and proteins, as well as changes in protein phosphorylation status. A second part of the study investigated the importance of NADP-generating enzymes in oxidative stress by production of cat2 nadp-me2 double mutants, in which the cytosolic isoform of NADP-malic enzyme is knocked out. Although NADP-ME2 was shown to be induced by oxidative stress, and mutants for this gene had much decreased leaf NADP-malic enzyme activity, no effects on cat2 phenotypes or redox profiles were apparent. Similarly, phenotypic responses to ozone were not affected in an nadp-me2 single mutant. In the third part, coupling between ascorbate and glutathione pools during oxidative stress was investigated by introduction of loss of function mutations for dehydroascorbate reductase (DHAR) into the cat2 background. In lines carrying a combination of dhar1 and dhar3 mutations, extractable leaf activity was decreased to very low levels. Despite this, cat2 dhar1 dhar3 and cat2 phenotypes and ascorbate and glutathione pools were similar. However, preliminary functional analysis of a cat2 dhar1 dhar2 dhar3 quadruple mutant suggested that the three DHARs play functionally redundant roles in oxidative stress. Overall, the work provides new data on enzymes that regulate responses to oxidative stress and has produced interesting genetic tools for further study.

Made available in DSpace on 2017-07-21T18:53:09Z (GMT). No. of bitstreams: 1 Mary Dias Queji.pdf: 909281 bytes, checksum: 0364cd27c13b09019850a0944168a898 (MD5) Previous issue date: 2008-04-05 / The agro industry of the apple generates, during the processing, the pomace which is considered the main by-product. Studies show that of the total amount of fruit that is processed to obtain the apple juice, 20 to 40 correspond to this by-product, which, is usually destined as a complement in animal feed or delivered onto the soil as organic fertilizer. The chemical composition, in moist base, is constituted by moisture (80), fibers (5) and soluble solids (14) represented, mainly, by fructose, glucose and sucrose, as well as, by organic acids, represented, in the most part by malic acid. The apple pomace also presents phenolic compounds that are target nutrients of great sensory and nutritional importance. Therefore, the quantification of its constituents represents an important source of data in the characterization of apple pomace for biotechnological purposes, seeking to attribute an appropriate use for this by-product. The conventional methodologies used for the quantification of sugars, organic acids and total phenols, although being part of the routine analyses in the quality control laboratories, are onerous, time consuming and generate residues. The aim of this study was to develop a fast, versatile analytical technique, of low cost and no pollutant, using the diffuse reflectance infrared spectroscopy (DRIFTS) allied to methods of multivariate calibration (PLSR). For the construction of the multivariate models, the averages of the concentrations of the simple sugars, malic acid and total phenols were used, obtained by the conventional methodologies, as well as the data in the medium infrared spectroscopy (MID) and near (NIR), obtained in duplicate, and of the 52 spectra obtained for the samples of apple pomace, 47 made part of the set calibration and 5 of the set validation. The regression models for the prediction of the concentration of fructose, sucrose, total phenols and malic acid obtained better results in MID, with averages of relative standard errors of 3.9 (with 5 Latents Variable), 6.6 (with 5 Latents Variable), 6.4 (with 4 Latents Variable) and 5.9 (with 5 Latents Variable), respectively. Already the best capacity prediction for glucose concentration was obtained by NIR, in which the average of relative standard error was 7.4 (with 6 Latents Variable). The obtained results demonstrate the good capacity of prediction of the multivariate models based in infrared spectroscopy and characteristic advantages of the association DRIFTPLSR. / A agroindústria da maçã gera, durante o processamento, o bagaço que é considerado o principal subproduto deste setor. Levantamentos mostram que da quantidade total de fruta que é processada para a obtenção do suco de maçã, 20 a 40 correspondem a este subproduto, que, normalmente, é destinado como complemento na alimentação animal ou dispensado no solo como adubo orgânico. Sua composição química, em base úmida, é constituída por umidade (80), fibras (5) e sólidos solúveis (14), estes últimos representados, principalmente, por frutose, glucose e sacarose, bem como, ácidos orgânicos, representados, majoritariamente pelo ácido málico. O bagaço da maçã também apresenta compostos fenólicos, que são fito-nutrientes de grande importância sensorial e nutricional. Portanto, a quantificação desses constituintes representa uma importante fonte de dados na caracterização do bagaço de maçã para finalidades biotecnológicas, visando atribuir um fim mais nobre a este subproduto. As metodologias convencionais empregadas para a quantificação de açúcares, ácidos orgânicos e fenóis totais, embora façam parte das análises de rotina nos laboratórios de controle de qualidade, são onerosas, demoradas e geram resíduos. O objetivo deste trabalho foi desenvolver uma técnica analítica rápida, versátil, de baixo custo e não poluente. Para tal, utilizou-se a espectroscopia no infravermelho por refletância difusa (DRIFTS) aliada a métodos de calibração multivariada (Regressão de Mínimos Quadrados Parciais - PLSR). Para a construção dos modelos multivariados, foram utilizadas as médias das concentrações dos açúcares simples, ácido málico e fenóis totais, obtidas pelas metodologias convencionais, bem como, os dados de espectroscopia no infravermelho médio (MID) e próximo (NIR). Os espectros foram obtidos em duplicata, sendo que dos 52 espectros das amostras de bagaço de maçã, 47 fizeram parte do conjunto de calibração e 5 do conjunto de validação externa. Os modelos de regressão para a previsão da concentração de frutose, sacarose, fenóis totais e ácido málico obtiveram melhores resultados no MID, com médias de erro padrão relativo de 3.9 (com 5 Variáveis Latentes), 6.6 (com 5 Variáveis Latentes), 6.4 (com 4 Variáveis Latentes) e 5.9 (com 5 Variáveis Latentes), respectivamente. Já a melhor capacidade de previsão para a concentração de glucose foi obtida pelo NIR, na qual a média de erro padrão relativo foi de 7.4 (com 6 Variáveis Latentes). Os resultados obtidos demonstram a boa capacidade de previsão dos modelos multivariados fundamentados em espectroscopia no infravermelho e vantagens características da associação DRIFT-PLSR.

The influence of rootstock and canopy density on grape berry composition was investigated over the summer of 2003-2004 on a commercial vineyard at Waipara, North Canterbury. This experiment was designed to investigate the influence of rootstock and canopy density on the acid composition, potassium (K) content and final pH of harvested fruit (Pinot Noir AM 10/5 Lincoln Selection). The trial block consisted of eight rootstocks laid out to an 8 x 8 latin square, each plot consisting of five vines of the same rootstock. Two canopy treatments were overlaid the block (down whole rows, assigned randomly, four rows to each treatment); one treatment allowed to grow naturally, in the other treatment the canopy was thinned removing double burst shoots and laterals. The bunch numbers were adjusted in the Unthinned canopy treatment (UCT) to match the Thinned canopy treatment (TCT). Information was gathered to assess: the canopy size and density (Pinot Quadrat Leaf Layer and Percent Gaps and canopy porosity), the plant yield (and berry size, berries per cluster, cluster weight, clusters per plant), plant K levels at flowering and veraison (from petioles and leaf blades) and berry composition at harvest (soluble solids (as brix), K, titratable acidity (TA), tartaric acid concentration, malic acid concentration and pH). The trial area was non-irrigated on clay loam soils and viticultural management was to best commercial practice. It was found that although rootstock influenced the levels of K in the plant and in the juice at harvest, the level of K in the juice did not influence pH in this experiment (range of rootstock juice K: 808 ppm to 928 ppm, l.s.d. = 75 ppm). The level of tartaric acid concentration in the juice was found to be the dominant influence on the level of pH in this experiment (rootstock pH range: 3.21 to 3.39, l.s.d. = 0.05). The juice concentration of tartaric acid was influenced by both rootstock (rootstock range 4.0 to 4.7 g/L, l.s.d = 0.4) and canopy density (UCT = 4.1, TCT = 4.7, l.s.d. = 0.4), decreased shading positively increasing the level of tartaric acid. The malic acid concentration in the juice was positively influenced by increasing canopy density (UCT = 4.7 g/L, TCT = 4.1 g/L, l.s.d = 0.4) and this played a minor role in the determination of pH in this experiment; an influence of rootstock on the level of malic acid concentration was found. The malic acid concentration strongly influenced the determination of TA (UCT = 11.0 g/L, TCT = 10.2 g/L, l.s.d = 0.5); tartaric acid concentration had a minor influence on the recorded TA. Attempts to characterise the influence of rootstock on malic acid, tartaric acid and pH were inconclusive. Rootstock was found to influence the canopy variables measured in this experiment and the recorded average plant yield. Crosses of Vitis rupestris were found to exhibit the most canopy vigour and those derived from Vitis berlandieri and Vitis riparia the least. The Canopy treatment did not show an influence over yield but the rootstock was found to influence plant yield, through the numbers of berries set in a cluster and the final harvest cluster weight. The influence of rootstock on pH may be described by the influence it exerts on canopy growth and yield but this was thought unlikely. Further research is required to describe the nature of the rootstock influence on K, malic acid, tartaric acid and pH.