Dissertations / Theses on the topic 'Multiple resistance mechanisms'

The aim of this thesis was to investigate expression of putative drug resistance markers in myeloma both by examining clinical material and through the development of a xenograft model. Pgp expression was examined in 57 samples from 37 patients with myeloma. Of 23 samples at presentation and 37 at relapse, 7 and 26 respectively were Pgp positive. A myeloma xenograft model was established to examine the acute effects of cytotoxic drugs on the expression of "classical" drug resistance markers and genes involved in regulation of apoptosis. The untreated xenografts were Pgp negative, expressed low levels of glutathione S-transferase-P (GSTP) and had readily detectable topo I and II. Little p62 myc or p53 were detected, whereas bcl-2 was strongly expressed. Treated xenografts contained only scattered apoptotic cells, but the majority demonstrated cell cycle arrest at the G2/M transition, and GSTP and topo IIα expression were increased. Pgp expression was also increased in animals treated on 3 consecutive days. C-myc was detected in dead or dying cells, but there was no mutational inactivation of p53, and bcl-2 expression was unaltered. The increased Pgp and GSTP expression following therapy, rather than inducing a resistant phenotype, may reflect activation of expression by drug administration. Cellular resistance occurred despite evidence of DNA damage suggesting that resistance arose from failure to engage apoptosis, possibly due to the strong bcl-2 expression. Bcl-2 expression was therefore evaluated in 40 samples from 31 individuals, with strong expression observed in over 80% of cases. This was not associated with rearrangement of the bcl-2 locus. The presence of abundant bcl-2 protein in the majority of cases has potentially important implications for drug resistance in this disease and suggests that future assessment of drug resistance in myeloma may be better directed downstream of immediate drug-target interactions to regulation of engagement of apoptosis.

Tumor cells in cancer patients acquire drug resistance as a result of chemotherapy. One type of acquired drug resistance is multidrug resistance (MDR) caused by the overexpression of P-glycoprotein, a transmembrane efflux protein. Inhibitors of P-glycoprotein or chemosensitizers such as verapamil are used to reverse MDR in cancer patients. Clinical studies have shown that some patients with P-glycoprotein positive cancer cells respond to the chemosensitizing effect of verapamil. However, this response is short lived and tumor cells become resistant to chemosensitizers. In order to study the mechanism of resistance to chemosensitizers, a human myeloma cell line, 8226/MDR₁₀V, was selected from a P-glycoprotein positive cell line, 8226/Dox₄₀, in the continuous presence of doxorubicin and verapamil. MDR₁₀V cells are consistently more resistant to MDR drugs than the parent cells, Dox₄₀. Chemosensitizers were less effective in reversing resistance in the MDR₁₀V compared to D0X₄₀ cells. Despite higher resistance to cytotoxic agents, MDR₁₀V expresses less P-glycoprotein in the plasma membrane compared to Dox₄₀. However, total cellular P-glycoprotein was the same in both cell lines suggesting a relocation of P-glycoprotein from plasma membrane into cytoplasm. Confocal immunofluorescence microscopy showed 2.5X more P-glycoprotein in the cytoplasm of MDR₁₀V cells as compared to D0X₄₀ cells. The relocation of P-glycoprotein was associated with a redistribution of doxorubicin. In D0X40 cells, doxorubicin was concentrated in the nucleus, whereas in MDR₁₀V cells, 90% of doxorubicin was found in the cytoplasm. We hypothesized that P-glycoprotein trafficking from the endoplasmic reticulum to the plasma membrane may be interrupted resulting in a higher concentration in the cytoplasm. To test this hypothesis, endoglycosidase H sensitivity of newly sensitized P-glycoprotein was examined. Medial Golgi processing of P-glycoprotein was identical between the two cell lines and the N-glycosylation of P-glycoprotein was complete by 3 hours. No mutations were found in MDR1 cDNA from MDR₁₀V cells compared to Dox₄₀ cells. These results suggest that increased resistance to cytotoxic drugs and chemosensitizers is associated with an altered intracellular location of P-glycoprotein which in turn causes a redistribution of doxorubicin.

Anthelmintic treatment of livestock is an important aspect of the control of gastrointestinal parasites. Resistance to anthelmintics is common, and an understanding of resistance requires knowledge of an anthelmintic's mode(s) of action and mechanism(s) of resistance. The parasitic nematode, Haemonchus contortus, has developed resistance to benzimidazoles and avermectins/milbemycins. Proposed mechanisms of resistance are here supported by genetic changes observed in genes whose protein products are believed to interact with these anthelmintics. Statistically significant differences in allele frequencies were observed between untreated and ivermectin- and moxidectin-treated strains in a gene encoding a putative glutamate-gated chloride channel alpha subunit, a proposed target of avermectins/milbemycins. One allele appeared to be associated with resistance. Similar changes in allele frequencies in the same strains occurred in a gene encoding a subunit of a gamma-aminobutyric acid receptor. Significant differences in allele frequencies of a gene encoding a P-glycoprotein were found in strains of H. contortus treated with ivermectin and moxidectin compared to derived, untreated strains. In all treated strains, one allele appeared to be associated with resistance. Similarly, allele frequencies of this gene were significantly different between a cambendazole-treated strain and its derived, untreated strain. These results implicate glutamate-gated chloride channels and gamma-aminobutyric acid receptors in mechanisms of resistance to avermectins/milbemycins and implicate P-glycoprotein in a mechanism of resistance to avermectins/milbemycins and benzimidazoles in H. contortus.

In this study, mechanisms of resistance to Imatinib-induced apoptosis in human K562 and Meg-1 chronic myeloid leukemia (CML) cells were examined. Continuous exposure of cells to step-wise increasing concentrations of Imatinib resulted in the selection of 0.2 and 1 &
#956
M imatinib resistant cells. Measurement of endogenous ceramide levels showed that treatment with Imatinib increased the generation of C18-ceramide significantly, which is mainly synthesized by the human longevity assurance gene 1 (hLASS1), in sensitive, but not in resistant cells. Mechanistically, analysis of mRNA and enzyme activity levels of hLASS1 in the absence or presence of Imatinib did not show any significant differences in the resistant cells when compared to its sensitive counterparts, suggesting that accumulation and/or metabolism, but not the synthesis of ceramide, might be altered in resistant cells. iv Indeed, further studies demonstrated that expression levels, and enzyme activity of sphingosine kinase-1 (SK-1), increased significantly in resistant K562 or Meg-1 cells. The expression levels of glucosyl ceramide synthase (GCS) also increased in resistant cells, comparing to the sensitive counterparts, which indicates conversion of pro-apoptotic ceramide to glucosyl ceramide. Expression analyses of BCR-ABL gene demonstrated that expression levels of BCR-ABL gene increased gradually as the cells acquired the resistance. However, Nucleotide sequence analyses of ABL kinase gene revealed that there was no mutation in Imatinib binding region of the gene in resistant cells. There was also an increase in expression levels of MDR1 gene in resistant cells, which transport the toxic substances outside of cells. In conclusion, these data show, for the first time, a role for endogenous ceramide synthesis via hLASS1 in Imatinib-induced apoptosis, and those alterations of the balance between the levels of ceramide and S1P. Mainly the overexpression of SK-1 seems to result in resistance to Imatinib in K562 cells. The cellular resistance may also result from conversion of ceramide to glucosyl ceramide, from overexpression of BCR-ABL and MDR1 genes but not due to mutations in Imatinib binding site of ABL kinase.

Resistance to broad spectrum of chemotherapeutic agents in cancer cell lines and tumors has been called multiple drug resistance (MDR). In this study, the molecular mechanisms of resistance to two anticancer agents (paclitaxel and vincristine) in mammary carcinoma cell line MCF-7 were investigated. MCF-7 cells were selected in the presence of paclitaxel and vincristine by stepwise dose increments. The cell viability and growth profiles of resistant sublines were examined. As the resistance indices increased, the growth rates of sublines were found to decrease. Gene and protein expression levels of the basic drug resistance proteins P-gp and MRP1 were studied in sensitive and drug resistant MCF-7 cells. It was shown that P-gp overexpression is significantly contributing to the developed drug resistance phenotype. Mutation analysis of beta tubulin gene which encodes the target of paclitaxel and vincristine was performed. Single histidine to proline mutation was identified near GTP binding site of beta tubulin in vincristine resistant subline which was not reported before. Apoptosis related BCL-2 and BAX were examined at both gene and protein expression levels and they were not found to be significantly related to the developed resistance in the sublines. The reversal of drug resistance by various inhibitory agents of P-gp and MRP1 was investigated by using flow cytometry. Synthetic silicon compounds were found to be the most effective MDR reversal agents. The effects of various combinations of anticancer drugs and reversal agents on cell proliferation were examined by checkerboard microplate method. ALIS409-paclitaxel and paclitaxel-doxorubicin pairs seem to have highest antiproliferative effects on resistant sublines. The microarray expression profiling of sensitive and resistant MCF-7 cells was performed for a much detailed and comprehensive analysis of drug resistance. The results indicated that the upregulation of MDR1 gene is the dominating mechanism of paclitaxel and vincristine drug resistance. Additionally up regulation of the genes encoding the detoxifying enzymes (i.e. GSTP1) was observed. Significant down regulation of apoptotic genes (i.e. PDCD2/4/6/8) and alterations in expression levels of genes related to invasion and metastasis (MMPs, ADAMs, COL4A2, LAMA etc.) were detected. Upregulation of some oncogenes (i.e. ETS, RAS) and cell cycle regulatory genes (CDKN2A, CCNA2 etc.) was seen which may be in close relation to MDR in breast cancer. Further studies will demonstrate the relationship between the components contributing to drug resistance phenotype in breast cancer cells.

Mechanisms underlying the interactions between the proteasome inhibitor carfilzomib and HDAC inhibitors were examined in both germinal center (GC) and activated B-cell (ABC) subtypes of human diffuse large B-cell lymphoma (DLBCL). Simultaneous exposure to minimally toxic concentrations of carfilzomib and HDAC inhibitor vorinostat resulted in the release of mitochondrial pro-apoptotic proteins SMAC and cytochrome c, pro-apoptotic caspase activation, and synergistic induction in apoptosis in both ABC and GC DLBCL subtypes. These events were associated with a marked increase in the stress kinase JNK, ROS generation, G2-M cell cycle arrest, as well as induction of DNA damage. Genetic knockdown of JNK resulted in a significant decrease in carfilzomib/vorinostat induced cell death. Co-administration of the antioxidant MnTBAP significantly reduced carfilzomib/vorinostat induced cell death, and resulted in a marked decrease in caspase-3 as well as a striking decrease in JNK phosphorylation. Tumor growth reduction was also observed in animal models that were treated with a combined regimen of carfilzomib and vorinostat. Finally, the combined treatment of carfilzomib/vorinostat was able to overcome any cross-resistance to carfIlzomib in bortezomib resistant cells. Collectively, these finding indicate that the combined regimen of carfilzomib and HDAC inhibitors promote lethality in ABC and GC human DLBCL cells by a variety of mechanisms both in vitro and in vivo. Further studies are necessary for clinical development of this drug regimen.

Kyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第17830号
農博第2015号
新制||農||1016(附属図書館)
学位論文||H25||N4787(農学部図書室)
30645
京都大学大学院農学研究科農学専攻
(主査)教授 稲村 達也, 教授 冨永 達, 教授 奥本 裕
学位規則第4条第1項該当

Doctor of Philosophy
Department of Agronomy
Mithila Jugulam
Palmer amaranth (Amaranthus palmeri) is one of the most aggressive, troublesome and damaging broadleaf weeds in many cropping systems including corn, soybean, cotton, and grain sorghum causing huge yield losses across the USA. As a result of extensive and intensive selection of pre- and -post emergence herbicides, Palmer amaranth has evolved resistance to multiple herbicide modes of action, microtubule-, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS)-, acetolactate synthase (ALS)-, photosystem II (PS II)-, hydroxyphenylpyruvate dioxygenase (HPPD)- and more recently to protoporphyrinogen oxidase (PPO)-inhibitors. A Palmer amaranth population from Kansas was found resistant to HPPD-, PS II-, and ALS-inhibitors. The overall objective of this research was to investigate the target-site and/or non-target-site resistance mechanisms in Palmer amaranth from KS (KSR) to mesotrione (HPPD-inhibitor), atrazine (PS II-inhibitor), and chlorsulfuron (ALS-inhibitor) relative to known susceptible Palmer amaranth from Mississippi (MSS) and KS (KSS). Whole plant dose-response assays showed high level of resistance in KSR to mesotrione, atrazine and chlorsulfuron. KSR was 10-18, 178-237 and >275 fold more resistant to mesotrione, atrazine, and chlorsulfuron, respectively, compared to MSS and KSS. Metabolism studies using [¹⁴C] labeled mesotrione and atrazine demonstrated non-target-site resistance to both herbicides, particularly, enhanced metabolism of [¹⁴C] mesotrione likely mediated by cytochrome P450 monooxygenases and rapid degradation of [¹⁴C] atrazine by glutathione S-transferases (GSTs). In addition, molecular and biochemical basis of mesotrione resistance was characterized by quantitative PCR (qPCR) and immunoblotting. These results showed 4-12 fold increased levels of the HPPD transcript and positively correlated with the increased HPPD protein. Sequencing of atrazine and chlorsulfuron target genes, psbA and ALS, respectively, showed interesting results. The most common mutation (serine264glycine) associated with atrazine resistance in weeds was not found in KSR. On the other hand, a well-known mutation (proline197serine) associated with chlorsulfuron resistance was found in 30% of KSR, suggesting ~70% of plants might have a non-target-site, possibly P450 mediated metabolism based resistance. Over all, KSR evolved both non-target-site and target-site based mechanisms to mesotrione and chlorsulfuron with only non-target-site based mechanism of resistance to atrazine leaving fewer options for weed control, especially in no-till crop production systems. Such multiple herbicide resistant Palmer amaranth populations are a serious threat to sustainable weed management because metabolism-based resistance may confer resistance to other herbicides and even those that are yet to be discovered. The findings of this research are novel and valuable to recommend appropriate weed management strategies in the region and should include diversified tactics to prevent evolution and spread of multiple herbicide resistance in Palmer amaranth.

The feasibility of using superelastic shape memory alloys in the retrofit of multiple frame bridges is investigated. First, three shape memory alloy constitutive models with various levels of complexity are compared in order to determine the significance of including subloops and cyclic loading effects on the structural response. The results show that the structural response is more sensitive to the shape memory alloys strength degradation and residual deformation than the sublooping behavior. Next, two parametric studies are conducted to explore the sensitivity of hinge opening to the mechanical behavior of the superelastic shape memory alloys. The first study is focused on the hysteretic properties of the alloy that could vary depending on the chemical composition or the manufacturing process of the alloy, while the second study targets the changes in the mechanical behavior of shape memory alloys resulting from the variability in the ambient temperature. The results show that the hysteretic behavior of shape memory alloys has only a slight effect on the bridge hinge opening as long as the recentering property is maintained. A detailed study on the effect of temperature shows that a reduction in the ambient temperature tends to negatively affect the hinge opening while an increase in temperature results in a slight improvement. Next, a parametric study is conducted to examine the effectiveness of shape memory alloy retrofit devices in limiting hinge openings in bridges with various properties. In addition, a comparison is made with other devices such as conventional steel restrainers, metallic dampers, and viscoelastic solid dampers. The results illustrate that superelastic shape memory alloys are superior in their effectiveness compared to other devices in the case of bridges with moderate period ratios and high level of ductility, especially when subjected to strong earthquakes.

La chimiothérapie a une faible efficacité sur les tumeurs cérébrales humaines (taux de réponse objective allant de 13 a 33%). Nous avons étudié les mécanismes moléculaires de chimiorésistance intrinsèque sur une série de 66 tumeurs cérébrales humaines comportant 31 gliomes (1 gangliogliome grade 1, 9 astrocytomes grade 2 et 10 grade 3, 11 gliomes grade 4), 18 méningiomes, 12 métastases cérébrales (de 2 cancers bronchiques épidermoïdes; de 8 adénocarcinomes du sein (3 cas), du poumon (2 cas), d'origine indéterminée (2 cas) et du colon (1 cas); d'1 sarcome d'Ewing; d'1 cancer bronchique peu différencié), 1 médulloblastome, 1 tératome malin, 3 épendymomes. L'analyse en southern blot de l'ADN de 33 tumeurs cérébrales humaines, hybride avec la sonde de la résistance pléiotropique (MDR1) ne montre jamais d'amplification de ce gène. Cette résistance pléiotropique et les 3 autres mécanismes principaux de chimiorésistance ont été étudiés par la technique du northern blot avec les sondes de la résistance pléiotropique (MDR1), de la glutathion-s-transférase (GSTPIL), de la dihydrofolate réductase (DHFR) et de la topoisomérase 2 (topo 2). Le principal résultat de ce travail est la non-expression du gène de la topoisomérase 2 dans le tissu cérébral humain normal (100%) et dans 75% (44/59) des échantillons tumoraux. Le deuxième gène, GSTPI, est surexprimé dans 23% (12/53) des tumeurs cérébrales (6/32 des tumeurs cérébrales primitives, 3/10 métastases, 3/11 des méningiomes). Les deux autres gènes lies a la chimiorésistance sont trouvés exceptionnellement surexprimés 2% pour MDR1 (1 gliome grade 4 sur 61 tumeurs), 8% pour DHFR (4/49: 2 gliomes grade 2,3; 1 métastase; 1 méningiome). Nos résultats suggèrent qu'il existe une chimiorésistance intrinsèque dans les tumeurs cérébrales humaines; elle est associée à 2 mécanismes majeurs de résistance (topo 2, GSTPI) en cause avec les drogues utilisées dans le traitement de ces tumeurs.

Glyphosate (N-(phosphonomethyl)glycine) is a post-emergent, systemic and non-selective herbicide for the control of annual and perennial weeds. This herbicide has very low toxicity to the mammals. The target enzyme for glyphosate in plants is 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Glyphosate inhibits the biosynthesis of the aromatic amino acids phenylalanine, tyrosine and tryptophan in the plant. The first case of glyphosate resistance was reported in Lolium rigidum in Australia after 15 years of persistence use of this herbicide and the number weeds reported resistant to glyphosate has increased around the world. So far, two mechanisms known to be involved in resistance to glyphosate are target-site mutation and reduced herbicide translocation. Recently, two populations of L. rigidum from Australia have been discovered with very high levels of resistance to glyphosate. This project aims to determine the levels of glyphosate resistance in these populations, investigate glyphosate resistance mechanisms in the populations and finally assess the mode of inheritance of resistance. In this project, four resistant (NLR70, SLR77, SLR80 and SLR88) and one susceptible (VLR1) L. rigidum populations were evaluated for their response to glyphosate. From the dose response experiments, the susceptible population of VLR1 was completely controlled with the recommended rate of glyphosate (450 g a.e ha⁻ ¹). In contrast, the resistant populations were not fully controlled by this herbicide rate. There was considerable variation between the populations in their resistance to glyphosate. In comparison to the susceptible population VLR1, SLR77 was 2.2 to 3.5 fold resistant to glyphosate, NLR70 was 3.7 to 8.4 fold resistant to glyphosate, SLR88 was 5.6 to 11.4 fold resistant to glyphosate and SLR80 was 8.2 to 76.7 fold resistant to glyphosate. The mechanism of glyphosate resistance in the populations was investigated. ¹⁴ C-glyphosate was used to determine the absorption and translocation of glyphosate among the populations. There was no significant difference on the absorption of ¹⁴ C-glyphosate 48 hours after treatment in the population. However, the accumulation of ¹⁴ C-glyphosate in the stem region was higher in the susceptible VLR1 population (25.9%) and in resistant SLR77 (25%) than the other three populations. The resistant populations NLR70, SLR88 and SLR80 had about half the amount of glyphosate accumulating in the stem region. These three resistant populations appear to be resistant to glyphosate as a result of reduced translocation of glyphosate to the shoot meristem. Part of the EPSP synthase gene of the susceptible and four resistant populations was amplified and sequenced to identify any changes in the nucleotide sequence. The predicted amino acid sequence from the susceptible population VLR1 was the same as the consensus sequence from other plant species in the conserved region sequenced. However, the resistant populations of NLR70, SLR77, SLR80 and SLR88 showed polymorphisms within the nucleotide sequence in this region. Single nucleotide substitutions of A for C at codon 106 were observed in the resistant populations SLR77 and SLR80. This nucleotide change is predicted to substitute threonine for proline at position 106. In the resistant population SLR88, a nucleotide substitution of T for C was observed at the same codon. This nucleotide substitution is predicted to change the amino acid from proline 106 to serine. Therefore, these three populations appear to be resistant to glyphosate as a result of a target-site mutation. An inheritance study was conducted by cross pollinating the susceptible VLR1 and resistant SLR88 population. From the dose response, the parent susceptible was completely killed with the recommended rate of glyphosate and higher rates of glyphosate were required to control parental resistant and both F₁ progenies (maternal susceptible and resistant). Both F₁ progenies showed an intermediate response to glyphosate compared with the parental populations. This indicated that the resistance to glyphosate in population SLR88 is inherited by nuclear gene(s) through the transfer of pollen during the cross pollination. It is suggested that SLR88 and SLR80 population contain both glyphosate resistant mechanisms due to the cross pollination between individuals with different resistant mechanisms. Having two resistant mechanisms results in populations being highly resistant to glyphosate compared to those with one resistance mechanism. The higher level of glyphosate resistance in these multiple glyphosate resistance populations will likely make them harder to manage.
Thesis (M.Ag.Sc.) -- University of Adelaide, School of Agriculture, Food and Wine, 2010

AMP-activated protein kinase (AMPK) is an energy-sensing enzyme that is activated when cellular energy is low and causes muscle and other cells to increase glucose uptake and fat oxidation, diminish lipid synthesis, and alter expression of various genes. AMPK activity is diminished in animals with the metabolic syndrome, though the mechanisms causing this reduction are unknown. To examine nutrient-induced changes in AMPK activity over time and factors that may regulate it, we compared rat muscle incubated with high glucose (HG) (30min-2h) and muscle of glucose-infused rats (3-8h) with appropriate controls. In addition to diminished AMPK activity (measured by the SAMS peptide assay) and phosphorylation of its activation loop at Thr172, we observed increased muscle glycogen, phosphorylation of AMPK’s α1/α2 subunit at Ser485/491, and PP2A activity, and decreased SIRT1 expression, all of which have been shown to diminish AMPK activity. Dysregulation of one or more of these factors could contribute to pathophysiological changes leading to metabolic syndrome associated disorders. Since recent studies suggest phosphorylation at Ser485/491 may play an important role in AMPK inhibition, we sought to determine how phosphorylation of this site is regulated. We investigated whether insulin or diacylglycerol (DAG) signaling pathways may be involved, since both are increased in at least one of the HG models. Akt and Protein Kinase (PK)D1 phosphorylated AMPK at Ser485/491 and diminished its activity in C2C12 myotubes, downstream of insulin and the DAG-mimetic PMA, respectively. Additionally, p-AMPK Ser485/491 was increased in muscle and liver of fed versus fasted mice and liver of diabetic mice. Our results suggest that Akt- and PKD1-mediated inhibition of AMPK via Ser485/491 phosphorylation may inhibit energy-metabolizing processes, while favoring energy-storing processes. Our results highlight the fact that phosphorylation of Ser485/491 can inhibit AMPK activity independent of changes in p-AMPK Thr172, a measure which is often used as a readout of AMPK activity. We hypothesize that Akt-mediated inhibition of AMPK is an acute, physiological response to insulin, whereas PKD1-mediated inhibition may be associated with more chronic pathophysiological changes. Thus, PKD1 inhibition or prevention of Ser485/491 phosphorylation may represent new strategies for therapeutic AMPK activation as treatment for the metabolic syndrome.

Resistance to therapeutic drugs is detrimental to treatment efficacy in chronic myeloid leukaemia (CML). CML is driven by the constitutive activity of the tyrosine kinase, Bcr-Abl, and has been treated effectively, in the majority of patients, using tyrosine kinase inhibitor (TKI) therapy. However, therapeutic TKI resistance often results in suboptimal treatment response. The molecular mechanisms of TKI resistance include: overexpression of Bcr-Abl; perturbed activity of cell membrane influx/efflux transporters; amino acid substitutions in the Bcr-Abl kinase domain precluding TKI binding (known as kinase domain mutations); and loss of dependence on Bcr-Abl kinase activity, via the gain of alternate driver function (known as Bcr- Abl independence). There is a temporal order to the acquisition of resistance mechanisms, with shifts in clonal architecture over time and treatment altering therapy sensitivity. This thesis explores the emergence of resistance to the TKIs dasatinib and ponatinib with regard to CML, investigating the molecular mechanisms of TKI resistance, to identify novel therapeutic strategies for circumventing resistance and improving treatment outcomes. The initial experiments described development of a leukaemic cell model of TKI resistance. Using gradually increasing concentrations of the TKI dasatinib, TKI resistance was induced. A dasatinib resistant K562 cell line was established by culture in increasing concentrations of dasatinib over several months. Intermediate cell line samples were harvested over the course of TKI dose escalation and were interrogated for TKI sensitivity by TKI-induced cell death assay and phospho-CrkL IC50. Results indicated the loss of dasatinib and ponatinib sensitivity and efficacy with prolonged dasatinib exposure. The expression of BCR-ABL1 gene transcript and protein levels were analysed, demonstrating BCR-ABL1 overexpression as an early occurring resistance mechanism. The Bcr-Abl kinase domain mutation, T315I, was observed emerging late in dasatinib dose escalation, conferring complete dasatinib resistance. Dose escalation intermediates were further examined by transcriptome sequencing. By interrogating global gene expression perturbation and genetic structural rearrangements, putative resistance mechanisms were identified: a) overexpression of the drug transporter ABCG2, and b) expression of the rare BCR-ABL1 transcript isoform, e6a2. The identification of ABCG2 expression in mRNAseq experiments was validated by assays to demonstrate ABCG2 function and involvement in TKI resistance, confirming ABCG2 overexpression as a critical early resistance mechanism. Cell death and IC50 assays were performed in the presence of the ABCG2 inhibitor, Ko143, which sensitised ABCG2 overexpressing cells to TKI-based inhibition. It was demonstrated, for the first time, that ABCG2 is able to confer decreased ponatinib sensitivity. Interestingly, the loss of ABCG2 expression coincided with the gain of T315I, demonstrating the competitive advantage of T315I-positive cells. The role of the e6a2 BCR-ABL1 fusion transcript in TKI resistance was investigated. BCR-ABL1 e6a2 gene expression was quantified in dasatinib resistant cells using RT-qPCR and DNA qPCR, demonstrating a transient peak in expression, followed by a distinct reduction. Intriguingly, this reduction in BCR-ABL1 expression coincided with the gain of ABCG2. To determine whether e6a2 was less sensitive to TKI than the typical e14a2 (p210) or e1a2 (p190) Bcr-Abl fusions, these were cloned into a Ba/F3 pro-B cell line. All isoforms were able to transform cells to IL-3 independence, demonstrating the leukaemia driving activity of Bcr-Abl. TKIinduced cell death experiments determined that e6a2 expressing cells were less sensitive to dasatinib than Ba/F3 cells harbouring the e14a2 isoform. Overall, several resistance mechanisms were identified and explored, however more may remain undetected. Results suggest a model of selective pressure in drug resistance, whereby leukaemic cells expressing the most efficient drug resistance mechanisms are selected for, eventually becoming the predominant cell population. These data and conclusions have bearing on the treatment of Bcr-Abl driven leukaemia, guiding the development of better therapeutic targets and strategies.
Thesis (Ph.D.) -- University of Adelaide, Adelaide Medical School, 2019

Conyza bonariensis (L.) Cronquist is a global weed and considered one of the most problematic species in modern agriculture. As a species it has developed resistance to herbicides of nine different active ingredients globally including 5-enolpyruvylskikimate 3-phosphate synthase (EPSPS) inhibitors, photosystem I (PSI) electron diverters, photosystem II (PSII) inhibitors and acetolactate synthase (ALS) inhibitors. Examination of 88 C. bonariensis populations collected across northeast Victoria identified that 40% of populations (or plants in specific populations) were resistant to 1080 g a.e. ha⁻¹ glyphosate. Multiple resistance was found to glyphosate and the ALS inhibitors chlorsulfuron, metsulfuron-methyl and sulfometuron-methyl in five of the nine populations fully characterised. This is the first reported case of multiple resistance to EPSPS- and ALS-inhibiting herbicides in C. bonariensis. Nine populations collected as part of a resistance survey conducted across northeast Victoria showed varying levels of glyphosate resistance; glyphosate susceptible (GS) biotypes DL4, IR7 and IR11; low level glyphosate-resistant (Gr) biotypes DL3, DL13 and IR14 with Resistance Indices (RI’s) between 2.3 and 2.8; and high level glyphosate resistance (GR) biotypes DL19, IR5 and IR10 with RI’s over 6. Results of laboratory evaluation for herbicide translocation demonstrated that this was not involved in the resistance present in these populations. GR, Gr and GS populations showed differential accumulation of shikimate suggesting insensitive EPSPS may be involved in the resistance found in these C. bonariensis populations. Sequencing both genomic DNA and plasmid DNA identified Pro106-Thr and Pro106-Ser mutations, these mutations have previously been found to confer glyphosate resistance. As these mutations occurred in all three population groups, therefore (an) other mechanism(s) must be contributing to the resistance. Future investigation focused on expression of EPSPS and ABC transporter genes may provide greater insight into the mechanisms conferring resistance in these C. bonariensis populations. C. bonariensis is a successful ruderal invader common on irrigation channel banks in Victoria and New South Wales, Australia. Options approved for herbicide control on channel banks are limited and field experimentation conducted in New South Wales over two years demonstrated that there are no effective herbicide control options for managing the weed in these sites. The lack of effective herbicide options highlights the need for further research into both new herbicides and non-chemical control options. Little is known about the use of defoliation as a strategic management tool of C. bonariensis and information available shows variable results. Field experiments were therefore conducted in Dookie, Victoria and Goolgowi, New South Wales to investigate using defoliation in conjunction with herbicide applications. Greatest control over the two experiments was provided by the sequential applications of paraquat + diquat applied 5-10 days after defoliation; and MCPA + dicamba applied 8-9 days prior to defoliation. These experiments demonstrated effective control could be achieved by the use of defoliation with herbicide application in a double-knock system. In a region where widespread resistance to EPSPS and ALS inhibitors has been demonstrated, additional strategies for management of C. bonariensis are critical.
Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2018.

by Cheung Yuen-Nei.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2002.
Includes bibliographical references (leaves 194-208).
Abstracts in English and Chinese.
Acknowledgement --- p.i
Abstract --- p.ii
摘要 --- p.iv
Contents --- p.vi
List of Figures --- p.xii
List of Tables --- p.xv
Abbreviations --- p.xvi
Chapter Chapter 1 --- Introduction --- p.1
Chapter 1 --- Introduction --- p.2
Chapter 1.1 --- Characteristic of Saponins --- p.3
Chapter 1.1.1 --- Occurrence of Saponins --- p.3
Chapter 1.1.2 --- General Properties of Saponins --- p.3
Chapter 1.1.2.1 --- Emulsifying Agents --- p.3
Chapter 1.2.2.2 --- Forming Complex with Cholesterol --- p.4
Chapter 1.1.2.3 --- Hemolytic Property --- p.4
Chapter 1.1.3 --- Structure of Saponins --- p.5
Chapter 1.1.3.1 --- Categories of Saponins --- p.5
Chapter 1.1.3.1.1 --- Triterpene Saponins --- p.5
Chapter 1.1.3.1.2 --- Steroid Saponins --- p.5
Chapter 1.1.3.2 --- Monodesmosidic and Bidesmosidic Saponins --- p.7
Chapter 1.1.4 --- Biological and Pharmacological Properties of Saponins --- p.9
Chapter 1.1.4.1 --- Anti-microbial Activity --- p.9
Chapter 1.1.4.1.1 --- Anti-fungal Activities --- p.9
Chapter 1.1.4.1.2 --- Anti-bacterial Activities --- p.10
Chapter 1.1.4.1.3 --- Anti-viral Activities --- p.10
Chapter 1.1.4.2 --- Insecticidal Activity --- p.10
Chapter 1.1.4.3 --- Molluscicidal Activity --- p.10
Chapter 1.1.4.4 --- Hypocholesterolemic Activity --- p.11
Chapter 1.1.4.5 --- Anti-ulcer Activity --- p.11
Chapter 1.1.4.6 --- Contraceptive Activity --- p.12
Chapter 1.1.4.7 --- Immunomodulatory Activities --- p.12
Chapter 1.1.4.7.1 --- Direct Immunostimulation --- p.12
Chapter 1.1.4.7.2 --- Acting as Immuno-adjuvants --- p.13
Chapter 1.1.4.8 --- Anti-tumor Activity --- p.14
Chapter 1.1.4.8.1 --- Anti-carcinogenesis --- p.15
Chapter 1.1.4.8.2 --- Suppression of Tumor Growth --- p.16
Chapter 1.1.5 --- Anti-tumor Activity of Steroid Saponins --- p.18
Chapter 1.1.5.1 --- Diosgenin Steroid Saponin --- p.18
Chapter 1.1.5.2 --- Hong Kong Compounds --- p.18
Chapter 1.1.5.3 --- Hong Kong18 --- p.21
Chapter 1.2 --- Human Hepatocellular Carcinoma (HCC) --- p.24
Chapter 1.2.1 --- The Incidence of Liver Cancer --- p.24
Chapter 1.2.2 --- Classification of Liver Cancer --- p.24
Chapter 1.2.3 --- Human Hepatocellular Carcinoma Cell Lines --- p.25
Chapter 1.2.3.1 --- Human Hepatocellular Carcinoma Cell Line HepG2 --- p.25
Chapter 1.2.3.2 --- Multidrug Resistant Human Hepatocellular Carcinoma Cell Line R-HepG2 --- p.27
Chapter 1.2.3.2.1 --- Mechanisms of Multidrug Resistance --- p.28
Chapter 1.2.3.2.2 --- Structure and Characteristics of P-glycoprotein --- p.29
Chapter 1.2.3.2.3 --- Methods in Dealing with P-glycoprotein Over-expressed MDR Cells --- p.31
Chapter 1.3 --- Objectives of the Project --- p.32
Chapter 1.3.1 --- Study of the Anti-tumor Activities of Hong Kong 18 on Human Hepatocellular Carcinoma Cell Line HepG2 and Unravel the Underlying Mechanisms --- p.32
Chapter 1.3.2 --- Study of the Anti-tumor Activities of Hong Kong 18on Multidrug Resistant Human Hepatocellular Carcinoma Cell Line R-HepG2 and Unravel the Underlying Mechanisms --- p.32
Chapter Chapter 2 --- Materials and Methods --- p.33
Chapter 2.1 --- Materials --- p.34
Chapter 2.1.1 --- Cell Culture --- p.34
Chapter 2.1.1.1 --- Cell Lines --- p.34
Chapter 2.1.1.2 --- Culture Media --- p.35
Chapter 2.1.2 --- Reagents and Buffers --- p.36
Chapter 2.1.2.1 --- Phosphate Buffered Saline (PBS) --- p.36
Chapter 2.1.2.2 --- Reagents and Buffers for DNA Fragmentation --- p.36
Chapter 2.1.2.3 --- Reagents and Buffers for Western Analysis --- p.37
Chapter 2.1.2.4 --- Reagents and Buffer for Caspases Activities --- p.39
Chapter 2.1.2.5 --- Fluorescent Dyes used for Flow Cytometry --- p.39
Chapter 2.1.3 --- Chemicals --- p.39
Chapter 2.2 --- Methods --- p.46
Chapter 2.2.1 --- MTT Assay --- p.46
Chapter 2.2.2 --- Determination of Cell Viability --- p.46
Chapter 2.2.3 --- Purification of Macrophages from balb/c Mice --- p.47
Chapter 2.2.4 --- Hemolysis Assay --- p.47
Chapter 2.2.5 --- In vivo Studies of the Toxicity of HK18 --- p.48
Chapter 2.2.6 --- DNA Fragmentation Assay --- p.50
Chapter 2.2.7 --- Detection of Apoptotic and Necrotic / Late Apoptotic Cells Death by Flow Cytometry with Annexin V-FITC / PI --- p.51
Chapter 2.2.8 --- Detection of Mitochondrial Membrane Potential by JC-1 Fluorescent Dye --- p.52
Chapter 2.2.9 --- Detection of Intracellular Ca Level by Flow Cytometry with Fluo-3 Fluorescent Dye --- p.52
Chapter 2.2.10 --- Detection of Intracellular Hydrogen Peroxide Level by Flow Cytometry with DCF Fluorescence Dye --- p.53
Chapter 2.2.11 --- Simultaneous Detection of Mitochondrial Membrane Potential and Intracellular Ca2+ or Mitochondrial Membrane Potential and Intracellular Hydrogen Peroxide --- p.54
Chapter 2.2.12 --- Western Analysis --- p.55
Chapter 2.2.12.1 --- Total Protein Extraction --- p.55
Chapter 2.2.12.2 --- Extraction of Cytosolic Proteins --- p.59
Chapter 2.2.13 --- Determination of Caspases Enzymatic Activity --- p.63
Chapter 2.2.14 --- Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) --- p.67
Chapter 2.2.14.1 --- RNA Extraction by TRIzol Reagent --- p.67
Chapter 2.2.14.2 --- Reverse Transcription --- p.68
Chapter 2.2.14.3 --- Polymerase Chain Reaction --- p.68
Chapter 2.3 --- Statistic Analysis --- p.71
Chapter Chapter 3 --- Cytotoxicity of HK18 --- p.72
Chapter 3.1 --- Cytotoxicity of HK18 on HepG2 Cells --- p.73
Chapter 3.1.1 --- Study of the Cytotoxic Activity of HK18 on HepG2 Cells by MTT Assay --- p.73
Chapter 3.1.2 --- Study of the Cytotoxic Activity of HK18 on HepG2 Cells by Tryphan Blue Exclusion Assay --- p.76
Chapter 3.2 --- Cytotoxicity of HK18 on R-HepG2 Cells --- p.78
Chapter 3.2.1 --- Study of the Cytotoxic Activity of HK18 on R-HepG2 Cells by MTT Assay --- p.78
Chapter 3.2.2 --- Study of the Cytotoxic Activity of HK18 on R-HepG2 Cells by Tryphan Blue Exclusion Assay --- p.81
Chapter 3.3 --- Cytotoxicity of HK18 on Macrophages --- p.83
Chapter 3.4 --- Hemolytic Activity of HK18 --- p.85
Chapter 3.5 --- In vivo Study of the Toxicity of HK18 --- p.87
Chapter Chapter 4 --- Mechanistic Study of HK18 on HepG2 Cells --- p.90
Chapter 4.1 --- Hallmarks of Apoptosis Induced by HK18 on HepG2 Cells --- p.91
Chapter 4.1.1 --- Induction of Phosphatidylserine Externalization by HK18 on HepG2 Cells --- p.91
Chapter 4.1.2 --- Induction of DNA Fragmentation by HK18 of HepG2 Cells --- p.97
Chapter 4.2 --- Study of the Underlying Mechanisms of HK18 Induced Apoptosis in HepG2 Cells --- p.99
Chapter 4.2.1 --- The Role of Mitochondria in HK18 Induced Apoptosis of HepG2 Cells --- p.99
Chapter 4.2.1.1 --- HK18 Induced Mitochondrial Membrane Depolarization in HepG2 Cells --- p.101
Chapter 4.2.1.2 --- Addition of Bongkrekic Acid Reduced HK18 Cytotoxicity on HepG2 Cells --- p.105
Chapter 4.2.1.3 --- Elevation of Intracellular Hydrogen Peroxide Level in HK18 Treated HepG2 Cells --- p.108
Chapter 4.2.1.4 --- Elevation of Intracellular Ca2+ Level in HK18 Treated HepG2 Cells --- p.114
Chapter 4.2.1.5 --- HK18 Induced Cytochrome c and AIF Released from Mitochondria of HepG2 Cells --- p.120
Chapter 4.3 --- Downstream Biochemical Changes Induced by HK18 on HepG2 Cells --- p.123
Chapter 4.3.1 --- Activation of Caspase 3 of HepG2 Cells by HK18 as Demonstrated by Western Blot --- p.123
Chapter 4.3.2 --- Induction of Caspases Activities of HepG2 Cells by HK18 as Demonstrated by Enzymatic Activity Assays --- p.125
Chapter 4.4 --- Down-regulation of Anti-apoptotic Bcl-2 Family Members of HepG2 Cells by HK18 --- p.129
Chapter Chapter 5 --- Mechanistic Study of HK18 on R-HepG2 Cells --- p.133
Chapter 5.1 --- Hallmarks of Apoptosis Induced by HK18 on R-HepG2 Cells --- p.134
Chapter 5.1.1 --- Induction of Phosphatidylserine Externalization by HK18 on R-HepG2 Cells --- p.134
Chapter 5.1.2 --- Induction of DNA Fragmentation by HK18 of R-HepG2 Cells --- p.137
Chapter 5.2 --- Study of the Underlying Mechanisms of HK18 Induced Apoptosis in R-HepG2 Cells --- p.139
Chapter 5.2.1 --- The Role of Mitochondria in HK18 Induced Apoptosis of R-HepG2 Cells --- p.139
Chapter 5.2.1.1 --- HK18 Induced Mitochondrial Membrane Depolarization in R-HepG2 Cells --- p.139
Chapter 5.2.1.2 --- Addition of Bongkrekic Acid Reduced HK18 Cytotoxicity on R-HepG2 Cells --- p.142
Chapter 5.2.1.3 --- Elevation of Intracellular Hydrogen Peroxide Level in HK18 Treated R-HepG2 Cells --- p.144
Chapter 5.2.1.4 --- Elevation of Intracellular Ca2+ Level in HK18 Treated R-HepG2 Cells --- p.146
Chapter 5.3 --- Downstream Biochemical Changes Induced by HK18 on R-HepG2 Cells --- p.148
Chapter 5.3.1 --- Activation of Caspase 3 of R-HepG2 Cells by HK18 as Demonstrated by Western Blot --- p.148
Chapter 5.3.2 --- Induction of Caspases Activation of R-HepG2 Cells by HK18 as Demonstrated by Enzymatic Activity Assays --- p.150
Chapter 5.4 --- Down-regulation of the Anti-apoptotic Bcl-2 Protein of R-HepG2 Cells by HK18 --- p.154
Chapter 5.5 --- HK18 was Not a Substrate of P-glycoprotein Contents --- p.156
Chapter Chapter 6 --- Discussion --- p.158
Chapter 6.1 --- Cytotoxicity of HK18 --- p.159
Chapter 6.1.1 --- HK18 was Cytotoxic to the Human Hepatocellular Carcinoma Cell Line HepG2 and Multidrug Resistant Human Hepatocellular Carcinoma Cell Line R-HepG2 --- p.159
Chapter 6.1.2 --- Study of the Toxicity of HK18 --- p.160
Chapter 6.2 --- Mechanistic Studies of the Cytotoxic Effects of HK18 on HepG2 Cells --- p.161
Chapter 6.2.1 --- Apoptotic Cell Death Induction of HK18 on HepG2 Cells --- p.161
Chapter 6.2.2 --- Studies of the Underlying Mechanisms of HK18 Induced Apoptosis of HepG2 Cells --- p.162
Chapter 6.3 --- Mechanistic Studies of the Cytotoxic Effects of HK18 on R-HepG2 Cells --- p.181
Chapter 6.3.1 --- Apoptotic Cell Death Induction of HK18 on R-HepG2 Cells --- p.181
Chapter 6.3.2 --- Studies of the Underlying Mechanisms of HK18 Induced Apoptosis of HepG2 Cells --- p.181
Chapter Chapter 7 --- Future Perspectives --- p.190
Chapter Chapter 8 --- References --- p.193

博士
國立中興大學
生命科學系所
100
Acinetobacter baumannii is one of the most common pathogens causing nosocomial infection in recent decades. Increasing trends in multiple drug resistance and its rapid spread in hospitals have caused a serious impact on the treatment for A. baumannii nosocomial infections. Therefore, it is very important to study the molecular epidemiology of A.baumannii, including the outbreak of multiple drug resistant A.baumannii, the transfer and spread of drug resistant genes, and the control of multiple drug resistant A.baumannii. Nowadays pulsed-field gel electrophoresis (PFGE) is the best tool for studying molecular epidemiology. In order to obtain clear and consistent PFGE band patterns for inter-laboratory comparison and discussion, establishing a standard experiment model is necessary, and the restriction enzyme used to cut genomic DNA is critical, it should be cost-effective and discriminative. This study aimed to select a suitable rare-cutting restriction enzyme for analyzing A. baumannii genome with PFGE. Among the enzymes tested, ApaI, AscI and AsiSI exhibited similar discriminative indices. ApaI which has been used by some laboratory was the most cost-effective restriction enzyme for PFGE analysis for its low price, but it generated more than 40 fragments that were a little crowded and not easy to resolve, different laboratories would have less consistent results. AscI exhibited a very close discriminative ability to that of AsiSI, both enzymes generated 10 to 20 fragments that were easy to resolve, but AscI was at half of the cost of AsiSI for PFGE analysis, therefore AscI is a more suitable restriction enzyme for standardizing the PFGE analysis of A. baumannii and for inter-laboratory comparison. Recent researches have indicated that multiple drug resistant A. baumannii produce class D (OXA) carbapenemases to degrade antibiotic carbapenem. This study collected total 90 carbapenem resistant A. baumannii (CRAB) isolates from the clinical microbiological laboratory of three medical centers in northern, central, and southern Taiwan, their MIC of carbapenem were determined, and their genomes were screened the existence of OXA genes by multiplex PCR. It was found that all the 90 isolates had OXA-51-like gene, twenty-six out of 30 isolates (86.67%) from northern Taiwan had OXA-51-like gene only, 25 out of 30 isolates (83.33%) from central Taiwan had both OXA-51-like and OXA-23-like genes, and 23 out of 30 isolates (76.67%) from southern Taiwan had both OXA-51-like and OXA-24-like genes. It looked like different part of Taiwan had different distribution of CRAB producing specific OXA type carbapenemase. We also found the appearance of OXA gene types related to the resistant strength of CRAB isolates to carbapenem (MIC value). CRAB isolates harboring OXA-51-like gene has imipenem MIC50 value of 8 ug/mL, CRAB isolates harboring OXA-51-like and OXA-23-like genes has imipenem MIC50 value of 16 ug/mL, whereas CRAB isolates harboring OXA-51-like and OXA-24-like genes has imipenem MIC50 value of 64 ug/mL. All the 90 CRAB isolates were susceptible to colistin and tigecycline. As A.baumannii is difficult to be eradicated from hospital environment, we tried using bacteriophage for environment control. Ten phages infecting A.baumannii were isolated. We chose two hospitals with respiratory care ward (RCW) to sample from patients, beds and environments. The isolated A.baumannii were carried out MIC determination and PFGE analysis. Then the mixture of the ten phages was used for environment eradication. After spraying the phages mixture to the beds and environments previously sampled, no A.baumannii was isolated any more. This is the first study to use bacteriophage for controling A.baumannii in environments and the result suggested the eradication is effective.

碩士
長庚大學
醫學生物技術研究所
96
Multiple drug resistance (MDR) is a major obstacle to attenuate the effectiveness of chemotherapy to many human malignancies. Despite extensively investigation, MDR inhibitors have been discovered unwanted severe side effects. Recently, scientists have reported that proteasome inhibition induced apoptosis in a variety of cancer cells and recognized as a novel anticancer therapy approach. Despite its success, some patients are resistant or become refractory to ongoing proteasome inhibitor-bortezomib treatment. These finding suggested that chemoresistant multiple myeloma may develop a novel mechanism to against proteasome inhibitor. To test hypotheses, we studied: (1) to evaluate proteasome inhibitor effect in MDR cell line model. (2) to investigate the potential mechanisms of MDR cell line attenuates apoptosis induced by proteasome inhibitor. Our results shown: compared to parental human uterus sarcoma cell line MES-SA cells, the Dx5-C5 cells with highly expressed P-glycoprotein was more resistant to MG132 and didn’t amend the proteasome inhibitor induced apoptosis pathway. Interestingly, when co-treatment of MDR inhibitor and MG132 together, the survival rates of Dx5-C5 cells shown significant declined compared with MES-SA cells. These data indicated that P-glycoprotein may play a role in efflux out MG132 from Dx5-C5 cells and reduced MG132 induced apoptosis. Furthermore, we also found that the Canonical Wnt pathway was only activated in Dx5-C5 cells through active β-catenin and its related transactivation activity. Western blot analysis demonstrated that Wnt targeting genes, including c-Myc and Cyclin D1, were up-regulated and relevant to inhibit the expression of p21, whereas MES-SA cells expressed the high level of p21 and down-regulated the Cyclin D1 and caused cell cycle arrest. In conclusion, our study demonstrated that existence of P-glycoprotein and Wnt pathway in MDR cell line would attenuate proteasome inhibitor induced apoptosis and may help us to develop a new strategy for treatment of multiple drug resistance cancer cells.

博士
國防醫學院
醫學科學研究所
105
Part 1 Acinetobacter baumannii is an increasing threat of nosocomial infections in recent years, especially the emergence of multi-drug resistant strains (multi-drug resistant Acinetobacter baumannii; MDRAB). Infections caused by multi-drug resistant A. baumannii could cause longer hospital stay and higher treatment costs. Tigecycline, a board-spectrum tetracycline derivative, is considered as the last antibiotic choice for the MDRAB. However, resistance to Tigecycline was reported following the drug usage worldwide. These resistances are mainly associated with overexpression of efflux pump, especially AdeABC, which is regulated by a corresponding two-component system AdeRS. In the research, we found that the regulatory factor AdeR could recognize the direct repeat on intercistronic region between adeR and adeA. This interaction inhibits the downstream efflux pump expression. In addition, mutations on AdeR DNA binding domain show lower affinity to the direct repeat sequences, and elevate the expression level of efflux pump, leading to high resistance to tigecycline (MIC = 16 μg/mL). This result is very useful for understanding the mechanism of tigecycline resistance of A. baumannii. Part 2 We generate a silver nanoparticles using green synthesis. In this process, silver nitrate is used as a precursor of silver ions, and then glucose and trimethyl nitrate chitosan (TMCN) are used as a reducing agent and stabilizer, respectively. The whole reaction of silver nanoparticle synthesis could be done at room temperature after adding alkaline solution and mixing thoroughly. There is no need to consume energy or to use expensive equipment. Adjusting the concentration of sodium hydroxide, glucose and TMCN will affect the particle size, zeta potential and formation yield of silver nanoparticles. The average size of this silver nanoparticles (TMCN-AgNPs) is around 60 nm with positive surface charge. The physical and chemical properties of this nanoparticles were characterized by UV-Vis spectroscopy, X-ray diffraction and X-ray photoelectron spectroscopy. The catalytic activity of TMCN-AgNPs was determined by reduction of 4-nitrophenol using NaBH4 as a reducing agent. The antibacterial activity of TMCN-AgNPs was evaluated by broth microdilution method, and was proved to have antibacterial activity against Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus. The minimum inhibitory concentration (MIC) was < 6.13 μg/mL. Moreover, TMCN-AgNPs also showed antibacterial activity against multidrug-resistant Acinetobacter baumannii from clinical isolated, and the MIC value was < 12.25 μg/mL.

BACKGROUND AND OBJECTIVES: Streptococcus agalactiae (Group B Streptococcus, GBS) is the leading cause of neonatal infections and deaths in human. It can also cause infections in pregnant women and non-pregnant adults. Penicillin and ampicillin are antibiotics of choice for the treatment of GBS infections. Erythromycin and clindamycin are used as alternative therapy in penicillin allergic patients, however resistance to these agents has been increasingly observed. This present study was undertaken to determine the colonization rate of GBS, susceptibility profile and the mechanism of antibiotic resistance in pregnant women and their babies at Dr. George Mukhari Academic Hospital in Pretoria. METHODS: Rectal and vaginal swabs were collected from pregnant women; ear and umbilical swabs from newborns over an 11 month period. Samples were cultured on selective media (CNA agar and Todd-Hewitt broth) and GBS positively identified using morphological and biochemical tests including Gram staining, hemolytic activity, catalase test, bile esculin, CAMP test and Latex agglutination test. The susceptibility testing was done using the Kirby-Bauer and E-test methods. The D-test method was used to determine the inducible clindamycin resistance. Multiplex PCR with were used to detect different genes coding for resistance. RESULTS: Out of the 413 patients evaluated, 128 (30.9%) were positive with GBS. All isolates were sensitive to penicillin and ampicillin. Erythromycin and clindamycin resistance was 21.1% and 17.2% respectively; of which 69% harbouring constitutive MLBB, 17.4% inducible MLSB. The alteration of ribosomal target encoded by ermB genes was the commonest mechanism of resistance observed in 55% of isolates, 38% of isolates had both ermB and linB genes and efflux pump mediated by mefA genes was detected in one of isolates. Conclusion: This study reaffirms the appropriateness of penicillin as the antibiotic of choice for treating GBS infection. However it raises the challenges of resistance to the macrolides and lincosamides. More GBS treatment options for penicillin allergic patients need to be researched.
Health Studies
M.Sc. (Life Sciences (Microbiology))

碩士
台南應用科技大學
生活應用科學研究所
100
(I) Colorectal cancer is the leading cause of cancer mortality, and metastasis is responsible for approximately 40% of death in colon cancer patients. Matrix metalloproteinases (MMPs) are key enzymes in the degradation of extracellular matrix, and MMP-2 and -9 are critical for cell migration leading to invasion and metastasis of cancer. The inhibition of MMP-2 and -9 is therefore considered that might depress the occurrence of tumor invasion and metastasis. Rosmarinic acid (RA) is a bioactive polyphenol that widely presented in Rosmarinus officinalis L. However, the literature regarding the effect of RA on invasion of cancer cells is still limited. In this study, we investigated the anti-invasion activity of RA against human colorectal adenocarcinoma Colo205 and HT-29 cells. The cells were treated with 0, 10, 50, 100, or 200 M of RA at 37C for 24 h, and the MMP-2, -9, and uPA activities as well as cell-matrix adhesion, motility, and invasion activities were determined. The MMP-2, -9, TIMP-1 and -2 mRNA levels were assayed by RT-PCR. The molecular signaling was determined by Weastern blot. The results showed that the MMP-2 and uPA activities of Colo205 and HT-29 cells were significantly inhibited by RA at a concentration of > 50 μM. The migration and invasion activities of Colo205 and HT-29 cells were also suppressed after treating with RA at a concentration higher than 50 M. The mRNA level of MMP-2 in HT-29 and Colo205 were significantly inhibited by RA at a concentration of > 50 M. The mRNA levels of TIMP-1 and -2 in HT-29 and TIMP-1 in Colo205 were significantly increased by RA. The signaling of p-ERK and p-p38 in Colo205 and HT-29 were signigicantly inhibited by RA. Furthermore, the activations of NF-κB and AP-1 in colorectal cancer cells were also suppressed by RA. Our results suggest that RA might be a bioactive with potential anti-invasive activity against colorectal cancer cells by inhibiting uPA and MMP-2 activity and reducing motility capabilities through inhibiting the activations of MAPKs signaling pathways and NF-κB and AP-1 transcription factors. (II) Tuberculosis (TB) is a contagious disease which causes a serious public health risk. WHO estimates that there were approximately 9,400,000 new TB cases globally in 2008, and South-East Asia Region accounted for 34% of incident cases. Multidrug-resistant TB (MDR-TB) is a particularly dangerous form of TB, which is responsible for the majority of failures in TB therapy. Mycobacterium tuberculosis is the major pathogeny of TB, and uridine diphosphate-glucose pyrophosphorylase (UGPase; EC 2.7.7.9) is the major enzyme that involved to the synthesis of cell wall. The inhibition of UGPase might depress the proliferation of the bacilli. Rosmarinic acid (RA), a polyphenol which is widely presented in natural plants of Lamiaceae and Boraginaceae family, has been reported that possesses several biological activities such as anti-virus, anti-inflammation, and anti-bacteria. The aim of this study was to investigate the inhibitory effects of RA on the viability of multidrug-resistant M. tuberculosis, and their impact on UGPase was also evaluated. Because the biological activity of a bioactive sometimes having an association with their antioxidant power, we therefore first determined the antioxidant activity of RA by trolox equivalent antioxidant capacity (TEAC) method. The viability of the bacilli was measured by counting the colony growing on 7H11 agar plates, and the expression of UGPase was performed by RT-PCR. The results showed RA having a strong antioxidant activity, and the viability of M. tuberculosis was reduced by treating with RA at a concentration of > 250 g/ml for 24 h. Furthermore, the expression of UGPase was also decreased by treating with 250 g/ml RA for 24 h. Based on the data mentioned above, it is suggested that RA can be used to inhibit the proliferation of multidrug-resistant M. tuberculosis, and the anti-proliferous activity of RA might be through inhibiting the expression of UGPase.

碩士
台南應用科技大學
生活應用科學研究所
100
(I) Colorectal cancer is the leading cause of cancer mortality, and metastasis is responsible for approximately 40% of death in colon cancer patients. Matrix metalloproteinases (MMPs) are key enzymes in the degradation of extracellular matrix, and MMP-2 and -9 are critical for cell migration leading to invasion and metastasis of cancer. The inhibition of MMP-2 and -9 is therefore considered that might depress the occurrence of tumor invasion and metastasis. Rosmarinic acid (RA) is a bioactive polyphenol that widely presented in Rosmarinus officinalis L. However, the literature regarding the effect of RA on invasion of cancer cells is still limited. In this study, we investigated the anti-invasion activity of RA against human colorectal adenocarcinoma Colo205 and HT-29 cells. The cells were treated with 0, 10, 50, 100, or 200 M of RA at 37C for 24 h, and the MMP-2, -9, and uPA activities as well as cell-matrix adhesion, motility, and invasion activities were determined. The MMP-2, -9, TIMP-1 and -2 mRNA levels were assayed by RT-PCR. The molecular signaling was determined by Weastern blot. The results showed that the MMP-2 and uPA activities of Colo205 and HT-29 cells were significantly inhibited by RA at a concentration of > 50 μM. The migration and invasion activities of Colo205 and HT-29 cells were also suppressed after treating with RA at a concentration higher than 50 M. The mRNA level of MMP-2 in HT-29 and Colo205 were significantly inhibited by RA at a concentration of > 50 M. The mRNA levels of TIMP-1 and -2 in HT-29 and TIMP-1 in Colo205 were significantly increased by RA. The signaling of p-ERK and p-p38 in Colo205 and HT-29 were signigicantly inhibited by RA. Furthermore, the activations of NF-κB and AP-1 in colorectal cancer cells were also suppressed by RA. Our results suggest that RA might be a bioactive with potential anti-invasive activity against colorectal cancer cells by inhibiting uPA and MMP-2 activity and reducing motility capabilities through inhibiting the activations of MAPKs signaling pathways and NF-κB and AP-1 transcription factors. (II) Tuberculosis (TB) is a contagious disease which causes a serious public health risk. WHO estimates that there were approximately 9,400,000 new TB cases globally in 2008, and South-East Asia Region accounted for 34% of incident cases. Multidrug-resistant TB (MDR-TB) is a particularly dangerous form of TB, which is responsible for the majority of failures in TB therapy. Mycobacterium tuberculosis is the major pathogeny of TB, and uridine diphosphate-glucose pyrophosphorylase (UGPase; EC 2.7.7.9) is the major enzyme that involved to the synthesis of cell wall. The inhibition of UGPase might depress the proliferation of the bacilli. Rosmarinic acid (RA), a polyphenol which is widely presented in natural plants of Lamiaceae and Boraginaceae family, has been reported that possesses several biological activities such as anti-virus, anti-inflammation, and anti-bacteria. The aim of this study was to investigate the inhibitory effects of RA on the viability of multidrug-resistant M. tuberculosis, and their impact on UGPase was also evaluated. Because the biological activity of a bioactive sometimes having an association with their antioxidant power, we therefore first determined the antioxidant activity of RA by trolox equivalent antioxidant capacity (TEAC) method. The viability of the bacilli was measured by counting the colony growing on 7H11 agar plates, and the expression of UGPase was performed by RT-PCR. The results showed RA having a strong antioxidant activity, and the viability of M. tuberculosis was reduced by treating with RA at a concentration of > 250 g/ml for 24 h. Furthermore, the expression of UGPase was also decreased by treating with 250 g/ml RA for 24 h. Based on the data mentioned above, it is suggested that RA can be used to inhibit the proliferation of multidrug-resistant M. tuberculosis, and the anti-proliferous activity of RA might be through inhibiting the expression of UGPase.