Philadelphia positive (Ph+) Acute Lymphoblastic Leukaemia (ALL) is characterised by the presence of the BCR-ABL fusion gene, which encodes a protein tyrosine kinase with aberrant activity. Imatinib, a chemical Bcr-Abl inhibitor, is rarely effective in Ph+ ALL patients as a single agent. In this study, insight into molecular and signalling changes occurring in Ph+ ALL during Imatinib therapy were investigated using cDNA microarrays. An optimal microarray assay was established to examine the gene expression changes in leukaemic cells from Ph+ ALL patients treated with Imatinib. Over 500 genes with ≥1.5-fold up- or down-regulation were identified. Based on gene ontology and novelty to Bcr-Abl signalling, six genes were selected and expression changes in five of these genes (PKCε, PINK1, SPRY2, ATF4 and PECAM1) confirmed by real time RT-PCR in Imatinib treated primary Ph+ ALL cells or the SUP-B15 cell line. The functional role of Protein Kinase C epsilon (PKCε) in response to Imatinib was further investigated using the Ph+ lymphoid and myeloid cell lines, SUP-B15 and K562. Detection of Imatinib-induced apoptosis by annexin V and PI staining demonstrated that SUP-B15 cells were less sensitive to Imatinib compared to K562 cells. PKCε mRNA was 50-fold higher in Ph+ ALL cells than Ph+ myeloid cells. In SUP-B15 cells, Imatinib upregulated PKCε mRNA but the protein was reduced by proteolytic cleavage. Inhibition of caspases showed that this cleaved product was not required for Imatinib induced-apoptosis. The treatment of SUP-B15 and primary Ph+ ALL cells with TAT-εV1-2 peptide, a specific inhibitor of PKCε, increased Imatinib-induced apoptosis. While the forced overexpression of PKCε in K562 cells reduced Imatinib-induced apoptosis. This increased expression of PKCε was associated with the increase of survival and anti-apoptotic proteins, Akt and Bcl-2. In summary, Gene expression profiling of Ph+ ALL cells during Imatinib therapy identified PKCε as an Imatinib responsive gene. A novel role of PKCε in Ph+ ALL response to imatinib is proposed. Experimental data presented in this thesis indicate that PKCε mediates pro-survival/anti-apoptosis signals in Ph+ ALL thereby reducing Imatinib-induced death. Thus, targeting PKCε during Imatinib therapy may be beneficial for the future treatment of Ph+ ALL.

The rapid determination of glycogen on indicator muscle immediately after slaughter is advantageous as it permits the prediction of a muscle’s ultimate pH (pHu) and allows the identification of high pHu meat carcasses by extrapolation. This thesis examines the development of two rapid glycogen determination methods. The first aim of this thesis was to find a new glucometer to replace the Bayer ESPRIT™ (Bayer) glucometer currently used in the Rapid pH (RpH) method. Roche’s Accuchek® Advantage II (Accuchek) and Abbott’s Medisense Optium™ (Medisense) glucometers were compared. Accuchek measurements exhibited a positive linear relationship in glucose standards made with water, RpH buffer and glucose spiked meat/buffer slurries ranging from 0 to 500 mg dL-1 (r2 = 0.999, 0.998 and 0.995 respectively). Medisense also exhibited a strong positive relationship for glucose standards made with water and RpH buffer; however, a non-linear trend in spiked meat slurries was observed. The second aim of this thesis was to explore the calibration of the KES K201 (KES Analysis Inc., NY, USA) near-infrared (NIR) diode array spectrometer to measure glycogen and pH at approximately 45 minutes after slaughter (pH45), and to predict pHu in pre-rigor M. longissimus dorsi (LD) from beef. This first required finding a reference method to calibrate against the NIR instrument. The RpH, Iodine and Bergmeyer methods were compared. Analysis of glycogen in replicate samples of three beef LD muscles at timepoints post-mortem (1, 4, 9 and 20 hours) was conducted. No significant difference in glycogen concentration was found between an enzymatic and an iodine based colorimetric method at each timepoint; however, the Iodine method was more consistent than the Bergmeyer method at all timepoints. Glucose measurements from the RpH method were consistent; however the pattern of glycogen decline at increasing timepoints post-mortem did not correspond with existing published studies. NIR spectra (538 to 1677 nm) of LD muscles from steers (n = 47), cows (n = 28) and bulls (n = 20) routinely slaughtered in a commercial abattoir were collected on-line approximately 45 minutes after slaughter. Poor results were obtained for Partial Least Squares (PLS) models generated from the mean reflectance spectra of each animal to measure glycogen and pH45, and predict pHu (r2 = 0.23, 0.37 and 0.20 respectively). A high mean square error of prediction (MSEP) for glycogen was also obtained (7.75). Validation of qualitative models generated with Generalised Partial Least Squares regression (GPLS) found that the optimum model was able to correctly categorise only 42% of high pHu samples with the remaining portion being wrongly classified as normal pHu meat. When the effect of gender was removed, only 21% of high pHu carcasses were correctly categorised. Exploratory analysis of the absorbance spectra of the LD muscles showed that a group composed predominantly of steers had a significantly lower pH45 than other existing groups. Further work is recommended for NIR to be successfully utilised on-line to measure glycogen or predict pHu in pre-rigor carcasses.

Thèse (M.Sc.)--Université Laval, 2003. / Bibliogr.: f. 83-96. Publié aussi en version électronique.

Th. doct.-ing.--Nancy, I.N.P.L., 1980.

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Submitted by Samique Kyene de Carvalho Araujo Camargo null (samiquekyene@hotmail.com) on 2017-04-16T19:19:16Z No. of bitstreams: 1 dissertacaoversaofinal2.pdf: 1378540 bytes, checksum: 81cd59c1f012a91616b94f76446b6c66 (MD5) / Approved for entry into archive by Luiz Galeffi (luizgaleffi@gmail.com) on 2017-04-18T17:41:00Z (GMT) No. of bitstreams: 1 camargo_skca_me_guara.pdf: 1378540 bytes, checksum: 81cd59c1f012a91616b94f76446b6c66 (MD5) / Made available in DSpace on 2017-04-18T17:41:00Z (GMT). No. of bitstreams: 1 camargo_skca_me_guara.pdf: 1378540 bytes, checksum: 81cd59c1f012a91616b94f76446b6c66 (MD5) Previous issue date: 2017-02-21 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O branqueamento objetiva a remoção de compostos que dão cor à polpa: lignina, extrativos, grupos carbonilas e carboxilas, complexo lignina carboidrato (LCC’s) e metais, para atingir a alvura mínima de 90± 0,5% ISO, exigida para comercialização da polpa e esta deve se manter estável em todo processo que compreende o transporte, manuseio e armazenamento, até o consumidor final. A utilização de diferentes reagentes de branqueamento efetiva a melhor eficiência na remoção destes compostos e, consequentemente, melhoram a estabilidade da alvura. O objetivo deste trabalho é avaliar o efeito das diferentes sequências de branqueamento na remoção dos grupos cromóforos, especialmente dos grupos carbonilas, minimizando assim a reversão de alvura. Para isso utilizou-se a espectroscopia na região do infravermelho. Também foi avaliado o efeito do pH nos estágios finais de branqueamento na estabilidade da alvura. Os experimentos foram realizados no Laboratório de Celulose da Unesp de Itapeva, onde foi efetuado o branqueamento das polpas de celulose deslignificada com oxigênio em sacos de polietileno, e para controle da temperatura utilizou-se o banho termostatizado. As sequências de branqueamento estudadas foram: D0(E+P)D1D2, AHTD0(E+P)D1D2, AHTD0(E+P)D1P, AHTD0(PO)D1, e AHTD0(PO), após o branqueamento foram efetuadas as análises de alvura, viscosidade e reversão de alvura. O efeito do pH na estabilidade da alvura pôde ser observado variando o pH do estágio final na faixa de 3 a 10 e efetuando a reversão induzida. A análise por espectroscopia na região do infravermelho comprova a eficiência dos reagentes na remoção dos grupos carbonilas, pois com a utilização do peróxido de hidrogênio nos estágios P final e PO houve diminuição da intensidade da banda de absorção na região do infravermelho destes grupos. Pôde-se observar com este estudo que a hidrólise ácida melhorou a alvura e sua estabilidade, e proporcionou uma economia na quantidade de reagente como cloro. A substituição do estágio final com dióxido de cloro por peróxido de hidrogênio proporcionou uma maior alvura, menor reversão de alvura e consumo de reagente como cloro, no entanto, ocasionou queda na viscosidade. A utilização de peróxido de hidrogênio pressurizado com oxigênio trouxe melhor resultado de reversão de alvura, mas menor viscosidade, tanto no estágio final, como intermediário. Neste último, houve economia na quantidade de reagente como cloro utilizado. / The bleaching process aims at the removal of compounds that give color to the pulp: lignin, extractives, carbonyl and carboxyl groups, lignin carbohydrate complex (LCC's) and metals, in order to reach the minimum whiteness of 90 ± 0.5% ISO, required for the commercialization of pulp And it must remain stable throughout the process, including transportation, handling and storage, to the final consumer. The use of different effective bleaching reagents gives better efficiency in the removal of these compounds and, consequently, enhances the brightness stability. The objective of this work is to evaluate the effect of the different bleaching sequences on the removal of the chromophore groups, especially the carbonyl groups, thus minimizing the reversal of whiteness. And, for that, spectroscopy was used in the infrared region. The effect of pH in the final stages of bleaching on brightness stability was also evaluated. The experiments were carried out at the Pulp Laboratory of Unesp de Itapeva, where the pulps of cellulose pulp were delignified with oxygen in polyethylene bags and the thermostated bath was used to control the temperature. The bleaching sequences studied were D0(E+P)D1D2, AHTD0(E+P)D1D2, AHTD0(E+P)D1P, AHTD0(PO)D1, and AHTD0(PO). After bleaching, of whiteness, viscosity and reversal of whiteness. The effect of pH on the brightness stability could be observed by varying the pH of the final stage in the range of 3 to 10 and effecting the induced reversal. The analysis by infrared spectroscopy proves the efficiency of the reagents in the removal of carbonyl groups, because with the use of hydrogen peroxide in the final stages P and PO there was a decrease in the intensity of the absorption band in the infrared region of these groups. It could be seen from this study that acid hydrolysis improved the brightness and stability, and provided savings in the amount of reactant as chlorine. The substitution of the final stage with chlorine dioxide by hydrogen peroxide provided a greater brightness, less reversion of brightness and consumption of reagent as chlorine, however, caused a drop in viscosity. The use of pressurized hydrogen peroxide with oxygen brought better blast reversion results, but lower viscosity, both in the final and intermediate stages. In the latter, there was savings in the amount of reagent such as chlorine used.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.