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La surexpression rétrovirale du facteur de transcription HOXB4 résulte en une expansion sélective des cellules souches hématopoïétiques (CSH) in vitro et in vivo et ce, sans induire de leucémie. Par contre, la demi-vie intracellulaire de la protéine est de seulement une heure et le fait que la protéine disparaît du milieu de culture après environ 4 heures représente un obstacle majeur à l’utilisation clinique de la protéine HOXB4. Trois mutants HOXB4 ayant une substitution d`un seul acides aminés (AA) parmi les 31 premiers AA ont démontré une augmentation de la stabilité de la protéine. Nous avons donc évalué l’effet de HOXB4 et de ses trois mutants sur la production de cellules progénitrices myéloïdes. L’expression ectopique de HOXB4 sauvage (s-HOXB4) et HOXB4 mutant (m-HOXB4) a un effet comparable sur la fréquence des cellules progénitrices myéloïdes en essai clonogénique. Par contre, la capacité de prolifération des cellules progénitrices myéloïdes qui surexpriment s-HOXB4 et 1423 m-HOXB4 a été supérieure à celle des cellules contrôles (GFP seul) et des deux autres mutants. De plus, malgré le fait que toutes les variantes de HOXB4 confèrent une capacité d’autorenouvellement similaire aux cellules progénitrices multipotents (GEMM), la production des progéniteurs granulocytaires (CFU-G) est compromise lorsque les cellules surexpriment 1426 et 1427 m-HOXB4. D’autre part, la densité cellulaire des colonies myéloïdes qui surexpriment ces deux mutants est diminuée, ce qui suggère que ces mutations ont non seulement augmenté sa stabilité, mais potentiellement affecté certaines fonctions biologiques de s-HOXB4. Enfin, 1423 m-HOXB4 semble n’avoir perdu aucune fonction de s-HOXB4 dans nos évaluations clonogéniques in vitro, ce qui fait de ce mutant une molécule intéressante pour des applications cliniques d’expansion des cellules progénitrices hématopoïétiques. / Over-expression of the homeobox transcription factor HOXB4 results in a selective expansion of HSC in vitro and in vivo. However, the intracellular half-life of the protein is only one hour, which represents an important obstacle for its clinical use. Three HOXB4 mutants with single amino acid (AA) substitution in the first 31 AA have demonstrated increased intracellular stability of the protein. We evaluated the effect of wild type (wt) and mutant (m) HOXB4 on expansion and differentiation of myeloid progenitors. Ectopic expressions of wt- and m- HOXB4 had a comparable effect on the frequency of myeloid progenitors in semi-solid assay. However, the proliferative capacity of myeloid progenitors expressing wt-HOXB4 and 1423 m-HOXB4 was considerably better than that of control (GFP only) and other two mutants. Additionally, while all HOXB4 variants conferred similar self-renewal capacity to multipotent (GEMM) progenitors, the production of single-lineage granulocytic progenitors (CFU-G) was severely compromised when cells were expressing 1426 and 1427 m-HOXB4. Conversely, the cellularity of myeloid colonies was also diminished with these mutants suggesting, that both mutations affect certain biologic function(s) of wt-HOX4 in addition to increasing its stability. On the other hand, 1423 m-HOXB4 did not seem to lose any wt-HOXB4 functions tested in in vitro assays making this mutant an interesting target for further investigation.

La surexpression rétrovirale du facteur de transcription HOXB4 résulte en une expansion sélective des cellules souches hématopoïétiques (CSH) in vitro et in vivo et ce, sans induire de leucémie. Par contre, la demi-vie intracellulaire de la protéine est de seulement une heure et le fait que la protéine disparaît du milieu de culture après environ 4 heures représente un obstacle majeur à l’utilisation clinique de la protéine HOXB4. Trois mutants HOXB4 ayant une substitution d`un seul acides aminés (AA) parmi les 31 premiers AA ont démontré une augmentation de la stabilité de la protéine. Nous avons donc évalué l’effet de HOXB4 et de ses trois mutants sur la production de cellules progénitrices myéloïdes. L’expression ectopique de HOXB4 sauvage (s-HOXB4) et HOXB4 mutant (m-HOXB4) a un effet comparable sur la fréquence des cellules progénitrices myéloïdes en essai clonogénique. Par contre, la capacité de prolifération des cellules progénitrices myéloïdes qui surexpriment s-HOXB4 et 1423 m-HOXB4 a été supérieure à celle des cellules contrôles (GFP seul) et des deux autres mutants. De plus, malgré le fait que toutes les variantes de HOXB4 confèrent une capacité d’autorenouvellement similaire aux cellules progénitrices multipotents (GEMM), la production des progéniteurs granulocytaires (CFU-G) est compromise lorsque les cellules surexpriment 1426 et 1427 m-HOXB4. D’autre part, la densité cellulaire des colonies myéloïdes qui surexpriment ces deux mutants est diminuée, ce qui suggère que ces mutations ont non seulement augmenté sa stabilité, mais potentiellement affecté certaines fonctions biologiques de s-HOXB4. Enfin, 1423 m-HOXB4 semble n’avoir perdu aucune fonction de s-HOXB4 dans nos évaluations clonogéniques in vitro, ce qui fait de ce mutant une molécule intéressante pour des applications cliniques d’expansion des cellules progénitrices hématopoïétiques. / Over-expression of the homeobox transcription factor HOXB4 results in a selective expansion of HSC in vitro and in vivo. However, the intracellular half-life of the protein is only one hour, which represents an important obstacle for its clinical use. Three HOXB4 mutants with single amino acid (AA) substitution in the first 31 AA have demonstrated increased intracellular stability of the protein. We evaluated the effect of wild type (wt) and mutant (m) HOXB4 on expansion and differentiation of myeloid progenitors. Ectopic expressions of wt- and m- HOXB4 had a comparable effect on the frequency of myeloid progenitors in semi-solid assay. However, the proliferative capacity of myeloid progenitors expressing wt-HOXB4 and 1423 m-HOXB4 was considerably better than that of control (GFP only) and other two mutants. Additionally, while all HOXB4 variants conferred similar self-renewal capacity to multipotent (GEMM) progenitors, the production of single-lineage granulocytic progenitors (CFU-G) was severely compromised when cells were expressing 1426 and 1427 m-HOXB4. Conversely, the cellularity of myeloid colonies was also diminished with these mutants suggesting, that both mutations affect certain biologic function(s) of wt-HOX4 in addition to increasing its stability. On the other hand, 1423 m-HOXB4 did not seem to lose any wt-HOXB4 functions tested in in vitro assays making this mutant an interesting target for further investigation.

Upland cotton (Gossypium hirsutum L.) is the world's most prominent fiber crop. Cotton transformation is labor intensive and time consuming, taking 12 to 18 months for rooted T0 plants. One rate limiting step is the necessary production of somatic embryos. In other recalcitrant species, ectopic expression of three genes were shown to promote somatic embryogenesis: WUSCHEL (WUS), SHOOT MERISTEMLESS (STM), and BABY BOOM (BBM). WUS is responsible for maintaining stem-cell fate in shoot and floral meristems. STM is needed to establish and maintain shoot meristems. STM and WUS have similar functions but work in different pathways; overexpression of both together converts somatic cells to meristematic and embryogenic fate. BBM encodes an AP2/ERF transcription factor that is expressed during embryogenesis and ectopic expression of BBM reprograms vegetative tissues to embryonic growth. In prior studies, these genes were constitutively expressed, and cultures did not progress beyond embryogenesis because the embryogenic signal was not turned off. In our study, we set out to use these genes to increase the efficiency of cotton transformation and decrease the time it takes to regenerate a plant. A disarmed cotton leaf crumple virus (dCLCrV) vector delivers WUS, STM, or BBM into cotton tissue cultures through Agrobacterium tumefaciens infection. We propose that virus delivery of embryo-inducing genes is a better approach for transformation because A) inserts more than 800 nucleotides are unstable, and will spontaneously inactivate, B) virus DNA can migrate through plasmodesmata to cells around the infected cell, creating a gradient of embryonic potential, C) the virus DNA does not pass through the germ line and the seed will not contain virus. We propose this method of inducing embryogenesis will facilitate the stable transformation of cotton and will be beneficial to the cotton industry. Ectopic expression of AtBBM, AtSTM, and AtWUS GrWUS:meGFP from a constitutive CaMV 35S promoter produced plants with phenotypes similar to those described in previous studies overexpressing AtBBM, indicating that the AtBBM gene was functional. The cotton cotyledon infiltration of the pART27 constructs showed transformed cells in Coker 312 by GFP localization in the nucleus. Although GFP was detected, no visible embryos appeared from the cotyledon. Cotyledons infiltrated with Agrobacterium harboring overexpression vectors withered and aborted after ~2 weeks. The virus-based vector in tissue culture failed to increase transformation efficiency, resulting in no embryos. The combination of hormone concentration showed no contribution to increasing the transformation efficiency.

Mature grains of wild-type (WT) and low phytic acid 1-1 (lpa 1-1) mutant from corn (Zea mays L.) were studied for total phosphorus (total P), phytic acid-phosphorus (PA-P), and mineral cations. Whole grain PA-P in lpa 1-1 was reduced 61.6% compared to WT whereas whole grain total P remained constant. Scutellum and root-shoot axis PA-P was 91.6% and 3.6% of WT whole-grain amounts respectively, compared to 89.3% and 4.0% in lpa 1-1. Relative partitioning of PA-P between the scutellum and root-shoot axis was not altered in lpa 1-1 mutant embryos as compared to WT. In lpa 1-1 the total P was slightly decreased in the scutella and increased in both root-shoot axes and rest-of-grain fractions. Whole grain Mg, Fe, and Mn amounts were higher in lpa 1-1 grains than in WT grains; K and Zn were similar, and Ca was lower. The lpa 1-1 whole grains and embryos contained 1/3 higher Fe than WT. For both grain types all measured metallic elements, except Ca, were more concentrated in embryos than the rest-of-grain fractions. Studies showed that WT grains contained larger globoids than lpa 1-1 grains in both scutellum and aleurone layer cells. This globoid size reduction reflected the PA-P decrease. Most lpa 1-1 aleurone globoids were non-spherical and lpa 1-1 scutellum globoids were clusters of spheres while both scutellum and aleurone globoids of WT were discrete spheres. The lpa 1-1 mutation had an impact on the globoid formation. X-ray analyses of scutellum and aleurone layer globoids from both grain types revealed major amounts of P, K, and Mg and traces of Ca, Fe, and Zn. Analysis demonstrated lower P, K and Mg and higher Ca, Fe and Zn in aleurone globoids than scutellum globoids. Both grain types contained almost no mineral nutrient stores in the starchy endosperm, whereas the scutellum was the major site of PA-P and mineral nutrient deposition. / Thesis / Master of Science (MSc)

De nouveaux enjeux émergent dans le domaine de la Santé en raison du vieillissement de la population et du développement inquiétant de la Maladie d’Alzheimer (MA). Chez le sujet sain ou pathologique, peu d’études ont porté sur le vieillissement du contrôle nerveux de la respiration, en dépit de son rôle crucial pour l’oxygénation du cerveau. Cette thèse présente des recherches translationnelles, réalisées chez la souris, pour étudier le vieillissement physiologique et pathologique du contrôle nerveux de la respiration. Chez des souris transgéniques, modèles reconnus de la MA et du syndrome de Rett, nous décrivons le développement de neuropathologies respiratoires graves, conduisant à un décès prématuré. Nous montrons pour la première fois qu’une tauopathie du tronc cérébral altère le fonctionnement des voies aériennes supérieures, la vocalisation et la respiration. De plus, nos travaux suggèrent un rôle délétère de l’anesthésie pour la MA et identifient des pistes thérapeutiques nouvelles. En conclusion, nos travaux chez la souris peuvent avoir des retombées particulièrement intéressantes notamment pour la MA. / New issues are emerging in the field of Health care due to ageing of the population and the alarming development of Alzheimer’s Disease (AD). In healthy or pathological living being, very few studies are dealing with the ageing of the respiratory nervous control, in spite of the crucial role of respiration for brain oxygenation. This thesis presents translational research performed in mice to examine the physiological and pathological ageing of the respiratory nervous control. In mice from two transgenic strains, recognized models for AD and Rett syndrome, we describe the development of drastic respiratory neuropathologies leading to premature death. In the AD mouse model, we show for the first time that brainstem tauopathy triggers dysfunctions of the upper airways, impairs vocalization and alters respiration and respiratory control. In addition, our work suggests a deleterious effect of anaesthesia for AD and identifies new therapeutic strategies. This mouse research could well contribute to significant improvements in AD care.

As Hsp60 acham-se conservadas em todos os organismos, participando da estruturação de proteínas e em processos crônico-degenerativos. Foi avaliada a ação das Hsp65r WT e sua mutante K409A de M. leprae no envelhecimento. Análises do Tempo Médio de Sobrevida (TMS), titulação dos isótipos, ensaios de avidez e análises histopatológicas foram realizadas em camundongos das linhagens HIII e LIII inoculados intraperitonealmente aos 120 ou 270 dias de vida com 2.5µg/mL das Hsp65r. Verificou-se redução no TMS de fêmeas HIII, envelhecidas e adultas, inoculadas com WT. A inoculação da Hsp65r WT em fêmeas LIII envelhecidas resultou no aumento do TMS. A dosagem dos anticorpos não revelou alterações marcantes na produção dos isótipos, e a avidez de IgG foi menor nas fêmeas HIII envelhecidas inoculadas com WT. A análise histopatológica mostrou inflamação nos rins de fêmeas HIII velhas tratadas com Hsp65r WT. Os resultados indicaram a interferência da Hsp65r WT na imunidade durante o processo de senescência de fêmeas envelhecidas constitutivamente boas produtoras de anticorpos. / The Hsp60 are conserved in all organisms, involved in proteins folding and chronic-degenerative processes. We evaluated the action of M. leprae rHsp65 WT and its mutant K409A in aging. Analyses of mean survival time (MST), antibody titration, avidity assays and histopathological examinations were performed in mice from HIII and LIII lines intraperitoneally inoculated at 120 or 270 days of life with 2.5µg/mL of rHsp65. There was a decrease in MST in adult and aged HIII females mice inoculated with WT protein. WT rHsp65 treatment in aged LIII females resulted in the increase of MST. The antibodies titration showed no marked changes in the production of isotypes and IgG avidity was lower in aged HIII females inoculated with WT. Histopathology showed inflammation in the kidneys of old HIII females treated with WT rHsp65. The results indicated the interference of WT rHsp65 in immunity during the senescence of aged females from HIII line constitutively producing good antibodies.

O conhecimento dos eventos biológicos que ocorrem no periápice dos dentes com necrose pulpar se torna importante para compreender o desenvolvimento das lesões periapicais. Muitas são as moléculas e mediadores que participam na instalação da lesão periapical, a partir da infecção bacteriana que ocorre no interior dos canais radiculares. Assim, o objetivo do presente trabalho foi avaliar moléculas do sistema imune inato, da osteoclastogênese e metaloproteinases em lesões periapicais (LP) induzidas experimentalmente em camundongos knockout e wild type. Para esse objetivo, o presente estudo foi dividido em dois trabalhos distintos. O primeiro teve como objetivo avaliar a expressão de metaloproteinase 2 (MMP2) e metaloproteinase 9 (MMP9) durante a progressão da LP em camundongos knockout para TLR2 (TLR2 KO) e MyD88 (MyD88 KO), em comparação com camundongos wild type (WT). O segundo estudo avaliou a correlação da expressão gênica e imunomarcação de RANK, RANKL, OPG, TLR2 e MyD88 durante a progressão da LP em camundongos WT. No primeiro estudo lesões periapicais foram induzidas em molares inferiores de 54 camundongos TLR2 KO, MyD88 KO e WT (n=18/grupo). Após 7, 21 e 42 dias, os animais foram eutanaziados e as mandíbulas foram dissecadas e submetidas a processamento histotécnico. Os cortes histológicos foram submetidos a imunohistoquímica e posteriormente foi avaliada presença ou ausência de MMP2 e MMP9 nos diferentes grupos. No segundo estudo, 35 camundongos WT foram utilizados. As lesões periapicais foram induzidas nos primeiros molares inferiores de ambos os lados. Após 0 (G0), 7 (G7), 21 (G21) e 42 (G42) dias, os animais foram anestesiados e eutanasiados para que as mandíbulas fossem dissecadas e divididas ao meio.O lado direito das mandíbulas foi para o processamento histotécnico, para posterior marcação de RANK, RANKL, OPG, TLR2 e MyD88, por meio da imuno-histoquímica do lado esquerdo da mandíbula foi utilizado para a extração de RNA, para a determinação da expressão gênica de RANK (Tnfrsf11a), RANKL (Tnfrsf11), OPG (Tnfrsf11b), TLR2 (Tlr2) e MyD88 (Myd88) utilizando quantificação em Tempo Real da Reação da Polimerase em Cadeia (qRT-PCR). Para ambos os estudos, testes paramétricos e não paramétricos foram realizados com nível de significância de 5%. Foi possível observar, no primeiro estudo, que nos períodos iniciais da progressão da lesão periapical, houve um aumento na imunomarcação de MMP9 nos camundongos TLR2 KO e MyD88 KO, quando comparados aos WT, diferente da MMP2 que não se observou nenhum aumento na imunomarcação. No entanto, aos 42 dias observou-se uma redução da imunomarcação de MMP2 e um aumento da MMP9 nos camundongos TLR2 KO. Adicionalmente, no segundo estudo, foi possível observar um aumento da imunomarcação para RANK, RANKL, OPG, TLR2 e MyD88 durante a progressão da lesão periapical (p<0,05). O aumento da expressão de Tnfrsf11 foi diferente entre os grupos G0 e G42, e G21 e G42 (p=0,006). No entanto, a expressão de Tnfrsf11b foi diferente entre os grupos G0 e G7, G7, G21 e G42, sendo possível observar uma diminuição dessa expressão ao longo do tempo (p<0,001). Tlr2 foi mais expresso entre os grupos G0 e G42 (p=0,03). E a expressão da molécula Myd88 foi estatisticamente significante entre os grupos G0 e G7, G21 e G42 (p=0,01). A razão Tnfrsf11/Tnfrsf11b aumentou durante a progressão da lesão periapical (p=0,002). Também foi possível observar uma correlação moderada entre Myd88 e Rankl (r=0,42; p=0,03) e entre Myd88 e Tlr2 (r=0,48; p<0,0001). Após as metodologias empregadas e os dados analisados, concluímos que a produção de MMP2 e MMP9 foi modulada por TLR2 e Myd88 durante a progressão da lesão periapical. Alem disso, podemos sugerir que existe uma correlação positiva entre o sistema RANK/RANKL/OPG e as proteínas do sistema imune inato, TLR2 e MyD88, durante a perda óssea decorrente da infecção bacteriana dos canais radiculares e posterior progressão da lesão periapical. / Knowledge of the biological events occurring inteeth apex with pulp necrosis becomes important to understand the development of periapical lesions. There are manymolecules and mediators that participate in the installation of the periapical lesion, from the bacterial infection that occurs inside the root canals. Thus, the aim of the present study was to evaluate molecules of the innate immune system, osteoclastogenesis and metalloproteinases in experimentally apical periodontitis (AP) induced in knockout and wild type mice. For this purpose, the present study was divided into two distinct studies. The first one aimed to evaluate the expression of metalloproteinases 2 (MMP2) and metalloproteinases 9 (MMP9) during the progression of AP in TLR2 knockout mice (TLR2 KO) and MyD88 knockout mice (MyD88 KO), compared to wild type mice (WT). The second study evaluated the correlation of gene expression and immunostaining of RANK, RANKL, OPG, TLR2 and MyD88 during LP progression in WT mice. In the first study AP were induced in lower molars of 54 TLR2 KO, MyD88 KO and WT mice (n = 18 / group). After 7, 21 and 42 days, the animals were euthanized and the jaws were dissected and submitted to histotechnical processing. The histological sections were submitted to immunohistochemistry and subsequently the presence or absence of MMP2 and MMP9 in the different groups was evaluated. In the second study, 35 WT mice were used. Periapical lesions were induced in the lower first molars on both sides. After 0 (G0) to 7 (G7), 21 (G21) and 42 (G42) days, the animals were anesthetized and euthanized so that the jaws were dissected and divided in half. The right side of the jaws was for the histotechnic processing, for subsequent imunostaining of RANK, RANKL, OPG, TLR2 and MyD88, through immunohistochemistry and the left side of the jaws was used for the extraction of RNA, for the determination of expression of RANK (Tnfrsf11a), RANKL (Tnfrsf11), OPG (Tnfrsf11b), TLR2 (Tlr2) and MyD88 (Myd88) using Quantification Real Time of Polymerase Chain Reaction (qRT-PCR). For both studies, parametric and non-parametric tests were performed with significance level of 5%. It was possible to observe in the first study that in the initial periods of AP progression there was an increase in MMP9 immunostaining in TLR2 KO and MyD88 KO mice when compared to WT, different from MMP2 that no increase in immunostaining was observed. However, at 42 days there was a reduction in MMP2 immunostaining and an increase of MMP9 in TLR2 KO mice was observed. Additionally, in the second study, it was possible to observe an increase in the immunostaining for RANK, RANKL, OPG, TLR2 and MyD88 during periapical lesion progression (p <0.05). The increase in Tnfrsf11 expression was different between groups G0 and G42, and G21 and G42 (p = 0.006). However, the expression of Tnfrsf11b was different between the G0 and G7, G7, G21 and G42 groups, and a decrease in expression over time (p <0.001) was observed. Tlr2 was more expressed between the G0 and G42 groups (p = 0.03). And the expression of the Myd88 molecule was statistically significant between the G0 and G7, G21 and G42 groups (p = 0.01). The Tnfrsf11 / Tnfrsf11b ratio increased during the AP progression (p = 0.002). It was also possible to observe a moderate correlation between Myd88 and Rankl (r = 0.42, p = 0.03) and between Myd88 and Tlr2 (r = 0.48, p <0.0001). After the methodologies used and the data analyzed, we conclude that the production of MMP2 and MMP9 was modulated by TLR2 and Myd88 during the AP progression. In addition, we can suggest that there is a positive correlation between the RANK / RANKL / OPG system and the proteins of the innate immune system, TLR2 and MyD88, during bone loss due to bacterial infection of the root canals and subsequent progression of the apical periodontitis.

As Hsp60 acham-se conservadas em todos os organismos, participando da estruturação de proteínas e em processos crônico-degenerativos. Foi avaliada a ação das Hsp65r WT e sua mutante K409A de M. leprae no envelhecimento. Análises do Tempo Médio de Sobrevida (TMS), titulação dos isótipos, ensaios de avidez e análises histopatológicas foram realizadas em camundongos das linhagens HIII e LIII inoculados intraperitonealmente aos 120 ou 270 dias de vida com 2.5µg/mL das Hsp65r. Verificou-se redução no TMS de fêmeas HIII, envelhecidas e adultas, inoculadas com WT. A inoculação da Hsp65r WT em fêmeas LIII envelhecidas resultou no aumento do TMS. A dosagem dos anticorpos não revelou alterações marcantes na produção dos isótipos, e a avidez de IgG foi menor nas fêmeas HIII envelhecidas inoculadas com WT. A análise histopatológica mostrou inflamação nos rins de fêmeas HIII velhas tratadas com Hsp65r WT. Os resultados indicaram a interferência da Hsp65r WT na imunidade durante o processo de senescência de fêmeas envelhecidas constitutivamente boas produtoras de anticorpos. / The Hsp60 are conserved in all organisms, involved in proteins folding and chronic-degenerative processes. We evaluated the action of M. leprae rHsp65 WT and its mutant K409A in aging. Analyses of mean survival time (MST), antibody titration, avidity assays and histopathological examinations were performed in mice from HIII and LIII lines intraperitoneally inoculated at 120 or 270 days of life with 2.5µg/mL of rHsp65. There was a decrease in MST in adult and aged HIII females mice inoculated with WT protein. WT rHsp65 treatment in aged LIII females resulted in the increase of MST. The antibodies titration showed no marked changes in the production of isotypes and IgG avidity was lower in aged HIII females inoculated with WT. Histopathology showed inflammation in the kidneys of old HIII females treated with WT rHsp65. The results indicated the interference of WT rHsp65 in immunity during the senescence of aged females from HIII line constitutively producing good antibodies.

Paclitaxel is one of the most successful drugs for the treatment of cancer because of its ability to target tubulin, block cell cycle progression at mitosis, and induce apoptosis. Despite the success of Paclitaxel, the development of drug resistance hampers its clinical applicability. Paclitaxel is used in malignant tumors in the present research, including non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC), head-neck scale epitheliomatous, urinary bladder cancer, tumor of the reproduction organ and so on. Clinical treatment of paclitaxel be injected 250mg/m2 to previously non-treated patients of small lung cancer its effect about 34% and previously non-treated patients of non-small lung cancer its effect about 21% to 24% . On the other hand we had established a taxol-resistant human lung carcinoma subline A549R by paclitaxel to compare the different proteins with A549 wild type and treat the different concention of paclitaxel with MTT assay so as to observe the tolerance dosage of subline growth.We obtained the patient¡¦s specimens of lung cancer to treat with paclitaxel some of resistant and some of non-resistant to compare differentially expressed proteins between normal and tumor. When we cultured taxol-resistant human lung carcinoma subline in which paclitaxel and calcium regulate growth, owing to the proteins of changes result to resistance. The extraction cell subline and specimens were analyzed by 2-D electrophoresis patterns and we found that interact paclitaxel with calcium it is the important factor of drug resistant. Verified from clinical treatment might have hypercalcemia in malignant tumors and calcium ion may increase paclitaxel drug resistance and hypercalcemia patient will be more insensitive to paclitaxel treatment.