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Methotrexate is a folic acid analogue widely used as a chemotherapeutic agent. It is known to be a potent inhibitor of the enzyme dihydrofolate reductase, therefore, perturbing intracellular pools of purine and pyrimidine bases for DNA synthesis, as well as pools of reduced folates used in a variety of metabolic reactions. It has been postulated, and subsequently widely accepted, that methotrexate kills cells by perturbing the intracellular ratio of dUTP:dTTP thereby leading to dUMP misincorporation into DNA. This would initiate an excision repair pathway designed to rid cellular DNA of this aberrant base. However, because of the imbalance of nucleotide pools, dUMP may well be re-incorporated during repair thus initiating a futile cycle of dUMP misincorporation and repair eventually leading to single-strand breaks in the DNA. From the results presented in this thesis, no evidence for dUMP misincorporation could be found in the two human cell lines studied (HeLa and CCRF-HSB2), despite the drug exhibiting dose-dependent cytotoxicity to both cell lines. This was true after a variety of methotrexate treatment times and at two different drug concentrations. Subsequent analysis of the drug treated cells, using the nucleoid sedimentation technique, for evidence of single-strand breaks in DNA yielded some anomalous results. Single-strand breaks, in the form of slower sedimenting nucleoids, were easily detectable after exposure of cells to low doses of methotrexate. However, treatment with higher doses resulted in the creation of faster sedimenting nucleoids. Subsequent analysis using other techniques showed that this faster sedimentation was occurring in the presence of DNA single-strand breaks. Collaborative work involving electron microscopy revealed methotrexate induced gross morphological changes in chromatin structure. Analogies with other unrelated anti-tumour agents interacting with topoisomerase enzymes are discussed.

The study of the development of anti-cancer drugs and preclinical efficacy tests has until today encountered a major problem identified as lack of reliable in vitro tumour models which are able to reflect in vivo tumour conditions. These models provide a clear basis for understanding tumour development processes, assisting in the selection of agents from various chemicals and testing the efficacy of drugs. There are two important characteristics for an in vitro tumour model, i.e. tumour-like structure of cell aggregates, and the in vivo-like culture microenvironment. To meet these two requirements, an in vitro perfusion based three-dimensional tumour model was developed for the three dimensional culture of cancer cells and related anti-cancer drugs tests. In order to assess this model, DLD1 and NCI/ADR cells were cultured in four different models and compared their proliferation rate, cell viability, micro tumour formation and drug responses. In addition, the comparison of static and perfusion culture were done on monolayer and in 3D also. The cells in perfusion culture showed higher proliferation rates and significantly, higher cell viabilities after a 6-day culture compared to statically cultured cells, especially for the cells in the 3D culture. Microtumours (MTs) were formed from this model, which showed significant tumour-like morphological characteristics, a denser and highly stable structure, a higher cell viability, and varied drug responses compared with spheroids. The inhibition effect of paclitaxel and cisplatin, two common type anti-cancer drugs, were tested and a comparative study was carried out using conventional two-dimensional (2D) static culture, spheroids, and the developed 3D MTs model, as well as real human tumour tissues. The results showed that the cells in 2D culture were most greatly inhibited while human tumours showed the lowest drug responses. The efficacy of anti-cancer drugs, tested in conventional 2D static culture, was greatly amplified. Besides, the response of MTs to agents was much closer to that of human tumours, when the values of spheroids are relatively closer to the cells in 2D culture. It is further supported that MTs have more tumour-like characteristics than spheroids. When compared, the inhibition to proliferation of cells in static and perfusion culture showed significantly different drug responses except for the cells on the monolayer. The shown difference between static and perfusion culture can be due to the different culture environment, and further related to the different action mechanisms of anti-cancer agents. The perfusion culture provides a more homogenous and more physiological microenvironment for the in vitro tumour growth, and in vitro perfused 3D cancer model, developed in this thesis, proved valuable for the study of in vitro cancer and related anti-cancer drug tests.

This thesis consists of two major chapters, each with its own introduction, experimental section and discussion. The TG4T/daunomycin and G4/daunomycin complexes described in Chapter One are two out of only five crystallographic quadruplex/ligand structures reported to date. In both structures daunomycin molecules stack onto a terminal G quartet preventing the G4 quadruplex from destacking and unwinding. The number of interacting ligand molecules depends on the quadruplex structure itself. The G4 quadruplex can accommodate four daunomycin molecules within one layer, while the TG4T tetraplex only accommodates three. In both structures daunosamine moieties form hydrogen bonds with the quadruplex but only daunosamine moieties from the TG4T/daunomycin structure make slight incursions into the quadruplex grooves. Both structures are stabilised by π-π interactions, hydrogen bonds, Van der Waals contacts and electrostatic interactions. The daunomycin/TG4T complex is the first ever reported and the only structure where a ligand interacts directly with the quadruplex groove. Chapter Two describes nine crystal structures of Hoechst 33258 analogues with d(CGCAAATTTGCG)2 and d(CGCGAATTCGCG)2 oligonucleotides, and is divided into two sections. Section A includes seven structures with Halogenated Hoechst 33258 analogues that are potential agents in radiotherapy, phototherapy, radioimmunotherapy or photoimmunotherapy, and the structure of the precursor. In all of the examined complexes the ligand binds to the minor groove but not all halogen substituents refine to 100% occupancy. The refined occupancies of the halogen atoms reveal that the degree of carbon-halogen cleavage is highest for ortho and lowest for para substitution. Among meta substituents pointing outside the minor groove, bromine atoms had a higher occupancy than the larger iodines. The position of the halogen atom in the minor groove is influenced by additional substituents on the phenyl ring. In most cases the bulky halogen atom is facing outside of the minor groove. Only in the 3-iodo-5-isopropylHoechst complex is iodine positioned towards the floor of the groove allowing the big isopropyl group to face outside. Section B describes the structure of a carborane-containing ligand (JW-B) bound to the minor groove of d(CGCAAATTTGCG)2. The analysis shows that is possible to position boron-rich moieties close to the cell nucleus, and JW-B may have potential in Boron Neutron Capture Therapy. / Data file restricted at the request of the author, but available by individual request, use the feedback form to request access.

One of the main problems of current cancer chemotherapy is the lack of selectivity of anti-cancer drugs to tumor cells which leads to systemic toxicity and adverse side effects. In order to overcome these limitations, researches on controlled drug delivery systems have gained much attention. Nanoscale based drug delivery systems provide tumor targeting. Among many types of nanocarriers, superparamagnetic nanoparticles with their biocompatible polymer coatings can be targeted to an intented site by an external magnetic field. Thus, the drug can be carried to the targeted site safely. The aim of this study is to prepare poly(dl-lactic-co-glycolic acid) (PLGA) coated magnetic nanoparticles and load anti-cancer drug, doxorubicin to them. For this purpose, magnetite (Fe3O4) iron oxide nanoparticles were synthesized as a magnetic core material (MNP) and then coated with oleic acid. Oleic acid coated MNP (OA-MNP) was encapsulated into PLGA. Effects of different OA-MNP/PLGA ratios on magnetite entrapment efficiency were investigated. Doxorubicin loaded magnetic polymeric nanoparticles (DOX-PLGA-MNP) were prepared. After the characterization of prepared nanoparticles, their cytotoxic effects on MCF-7 cell line were studied. PLGA coated magnetic nanoparticles (PLGA-MNP) had a proper size and superparamagnetic character. The highest magnetite entrapment efficiency of PLGA-MNP was estimated as 63 % at 1:8 ratio. Cytotoxicity studies of PLGA-MNP did not indicate any notable cell death between the concentration ranges of 2 and 250 &mu / g ml-1. It was observed that DOX-PLGA-MNP showed significant cytotoxicity on MCF-7 cells compared to PLGA-MNP. The results showed that prepared nanoparticles have desired size and superparamagnetic characteristics without serious toxic effects on cells. These nanoparticles may be suitable for targeted drug delivery applications. The findings obtained from drug studies may contribute to further work.

This thesis consists of two major chapters, each with its own introduction, experimental section and discussion. The TG4T/daunomycin and G4/daunomycin complexes described in Chapter One are two out of only five crystallographic quadruplex/ligand structures reported to date. In both structures daunomycin molecules stack onto a terminal G quartet preventing the G4 quadruplex from destacking and unwinding. The number of interacting ligand molecules depends on the quadruplex structure itself. The G4 quadruplex can accommodate four daunomycin molecules within one layer, while the TG4T tetraplex only accommodates three. In both structures daunosamine moieties form hydrogen bonds with the quadruplex but only daunosamine moieties from the TG4T/daunomycin structure make slight incursions into the quadruplex grooves. Both structures are stabilised by π-π interactions, hydrogen bonds, Van der Waals contacts and electrostatic interactions. The daunomycin/TG4T complex is the first ever reported and the only structure where a ligand interacts directly with the quadruplex groove. Chapter Two describes nine crystal structures of Hoechst 33258 analogues with d(CGCAAATTTGCG)2 and d(CGCGAATTCGCG)2 oligonucleotides, and is divided into two sections. Section A includes seven structures with Halogenated Hoechst 33258 analogues that are potential agents in radiotherapy, phototherapy, radioimmunotherapy or photoimmunotherapy, and the structure of the precursor. In all of the examined complexes the ligand binds to the minor groove but not all halogen substituents refine to 100% occupancy. The refined occupancies of the halogen atoms reveal that the degree of carbon-halogen cleavage is highest for ortho and lowest for para substitution. Among meta substituents pointing outside the minor groove, bromine atoms had a higher occupancy than the larger iodines. The position of the halogen atom in the minor groove is influenced by additional substituents on the phenyl ring. In most cases the bulky halogen atom is facing outside of the minor groove. Only in the 3-iodo-5-isopropylHoechst complex is iodine positioned towards the floor of the groove allowing the big isopropyl group to face outside. Section B describes the structure of a carborane-containing ligand (JW-B) bound to the minor groove of d(CGCAAATTTGCG)2. The analysis shows that is possible to position boron-rich moieties close to the cell nucleus, and JW-B may have potential in Boron Neutron Capture Therapy. / Data file restricted at the request of the author, but available by individual request, use the feedback form to request access.

This thesis consists of two major chapters, each with its own introduction, experimental section and discussion. The TG4T/daunomycin and G4/daunomycin complexes described in Chapter One are two out of only five crystallographic quadruplex/ligand structures reported to date. In both structures daunomycin molecules stack onto a terminal G quartet preventing the G4 quadruplex from destacking and unwinding. The number of interacting ligand molecules depends on the quadruplex structure itself. The G4 quadruplex can accommodate four daunomycin molecules within one layer, while the TG4T tetraplex only accommodates three. In both structures daunosamine moieties form hydrogen bonds with the quadruplex but only daunosamine moieties from the TG4T/daunomycin structure make slight incursions into the quadruplex grooves. Both structures are stabilised by π-π interactions, hydrogen bonds, Van der Waals contacts and electrostatic interactions. The daunomycin/TG4T complex is the first ever reported and the only structure where a ligand interacts directly with the quadruplex groove. Chapter Two describes nine crystal structures of Hoechst 33258 analogues with d(CGCAAATTTGCG)2 and d(CGCGAATTCGCG)2 oligonucleotides, and is divided into two sections. Section A includes seven structures with Halogenated Hoechst 33258 analogues that are potential agents in radiotherapy, phototherapy, radioimmunotherapy or photoimmunotherapy, and the structure of the precursor. In all of the examined complexes the ligand binds to the minor groove but not all halogen substituents refine to 100% occupancy. The refined occupancies of the halogen atoms reveal that the degree of carbon-halogen cleavage is highest for ortho and lowest for para substitution. Among meta substituents pointing outside the minor groove, bromine atoms had a higher occupancy than the larger iodines. The position of the halogen atom in the minor groove is influenced by additional substituents on the phenyl ring. In most cases the bulky halogen atom is facing outside of the minor groove. Only in the 3-iodo-5-isopropylHoechst complex is iodine positioned towards the floor of the groove allowing the big isopropyl group to face outside. Section B describes the structure of a carborane-containing ligand (JW-B) bound to the minor groove of d(CGCAAATTTGCG)2. The analysis shows that is possible to position boron-rich moieties close to the cell nucleus, and JW-B may have potential in Boron Neutron Capture Therapy. / Data file restricted at the request of the author, but available by individual request, use the feedback form to request access.

This thesis consists of two major chapters, each with its own introduction, experimental section and discussion. The TG4T/daunomycin and G4/daunomycin complexes described in Chapter One are two out of only five crystallographic quadruplex/ligand structures reported to date. In both structures daunomycin molecules stack onto a terminal G quartet preventing the G4 quadruplex from destacking and unwinding. The number of interacting ligand molecules depends on the quadruplex structure itself. The G4 quadruplex can accommodate four daunomycin molecules within one layer, while the TG4T tetraplex only accommodates three. In both structures daunosamine moieties form hydrogen bonds with the quadruplex but only daunosamine moieties from the TG4T/daunomycin structure make slight incursions into the quadruplex grooves. Both structures are stabilised by π-π interactions, hydrogen bonds, Van der Waals contacts and electrostatic interactions. The daunomycin/TG4T complex is the first ever reported and the only structure where a ligand interacts directly with the quadruplex groove. Chapter Two describes nine crystal structures of Hoechst 33258 analogues with d(CGCAAATTTGCG)2 and d(CGCGAATTCGCG)2 oligonucleotides, and is divided into two sections. Section A includes seven structures with Halogenated Hoechst 33258 analogues that are potential agents in radiotherapy, phototherapy, radioimmunotherapy or photoimmunotherapy, and the structure of the precursor. In all of the examined complexes the ligand binds to the minor groove but not all halogen substituents refine to 100% occupancy. The refined occupancies of the halogen atoms reveal that the degree of carbon-halogen cleavage is highest for ortho and lowest for para substitution. Among meta substituents pointing outside the minor groove, bromine atoms had a higher occupancy than the larger iodines. The position of the halogen atom in the minor groove is influenced by additional substituents on the phenyl ring. In most cases the bulky halogen atom is facing outside of the minor groove. Only in the 3-iodo-5-isopropylHoechst complex is iodine positioned towards the floor of the groove allowing the big isopropyl group to face outside. Section B describes the structure of a carborane-containing ligand (JW-B) bound to the minor groove of d(CGCAAATTTGCG)2. The analysis shows that is possible to position boron-rich moieties close to the cell nucleus, and JW-B may have potential in Boron Neutron Capture Therapy. / Data file restricted at the request of the author, but available by individual request, use the feedback form to request access.

In this thesis, I used a two-phase gas-liquid segmented microfluidic platform to synthesize drug-loaded block copolymer nanoparticles. In Chapter 2 and 3, the anti-cancer drug 7-ethyl-10-hydroxycamptothecin (SN-38) was physically encapsulated in poly(6-methyl-caprolactone-co-ε-caprolactone)-block-poly(ethylene oxide) (P(MCL-co-CL)-b-PEO) nanoparticles with various drug-to-polymer loading ratios, under different flow conditions. The effects of chemical and flow conditions on the size, morphology, drug loading efficiency, in vitro release and cytotoxicity of the nanoparticles were determined. For various loading ratios, the intermediate total flow rate (Q = 200 µL/min) produced the smallest nanoparticle sizes and pure spheres. The various nanoparticle preparation conditions showed flow-variable release rates and cytotoxicities against MCF-7 cancer cell line. Specifically, we found that release half times of SN-38 from the nanoparticles were from τ1/2 = 0.8 to 3.3 h as the total flow rate increased from Q = 50 to 200 µL/min. We also found that most conditions of SN-38 formulations generated stronger cytotoxicity than free SN-38. As well, at short and intermediate incubation time (48 and 72 h), the cytotoxic potency of microfluidic nanoparticles prepared at Q = 200 µL/min were slightly higher than nanoparticles prepared using a conventional bulk method, while potencies of microfluidic nanoparticles prepared at higher and lower flow rates were slightly lower than the bulk control. In Chapter 4, in order to pursue even higher shear rate and increased throughput, we switched the microfabrication material to silicon/glass from polydimethylsiloxane (PDMS) used in earlier chapters, maintaining the gas-liquid microfluidic reactor design. A comparison between the two microfluidic reactor materials at constant liquid flow rate showed that channel material affected both flow behaviour and the resulting nanoparticle morphologies. A new, single-phase microfluidic strategy was also proposed in order to generate high shear, in which variable high and low shear would arise from periodic changes in channel dimensions. However, issues regarding clogging of the more narrow microchannels require future work of improvements in either reactor design or the microfabrication process. / Graduate / 2019-01-12

An important role of Pin1 is to catalyze the cis-trans isomerization of pSer/Thr-Pro bonds; as such, it plays an important role in many cellular events through the effects of conformational change on the function of its biological substrates, including Cdc25, c-Jun, and p53. The expression of Pin1 correlates with cyclin D1 levels, which contributes to cancer cell transformation. Overexpression of Pin1 promotes tumor growth, while its inhibition causes tumor cell apoptosis. Because Pin1 is overexpressed in many human cancer tissues, including breast, prostate, and lung cancer tissues, it plays an important role in oncogenesis, making its study vital for the development of anti-cancer agents. Many inhibitors have been discovered for Pin1, including 1) several classes of designed inhibitors such as alkene isosteres, non-peptidic, small molecular Pin1 inhibitors, and indanyl ketones, and 2) several natural products such as juglone, pepticinnamin E analogues, PiB and its derivatives obtained from a library screen. These Pin1 inhibitors show promise in the development of novel diagnostic and therapeutic anticancer drugs due to their ability to block cell cycle progression. In order to develop potent Pin1 inhibitors, the concept of transition-state analogues was used for the design of three classes of compounds: ketoamide, ketone, and reduced amide analogues. Specifically, a convergent synthesis of α-ketoamide inhibitors of Pin1 was developed. An α-hydroxyorthothioester derivative of Ser was reacted directly with an aminyl synthon. The reaction was catalyzed by HgO and HgCl2 to form an α-hydroxyamide. Hydrolysis and coupling were combined in one step in 80% yield. Two diastereomers of a phospho-Ser-Pro α-ketoamide analogue were synthesized. The resulting IC50 values of 100 µM and 200 µM were surprisingly weak for the Pin1 peptidyl-prolyl isomerase. Diastereomeric ketones were synthesized by coupling cyclohexenyl lithium to the serine Weinreb amide, via the Michael addition of a carboxylate synthon. The IC50 values of the two ketone diastereomers were determined to be 260 μM and 61 μM, respectively. Five reduced amide inhibitors for Pin1 were synthesized through a selective reduction using borane. The most potent inhibitor was found to be Fmocâ pSerâ Ψ[CH2N]-Proâ tryptamine, which had an IC50 value of 6.3 µM. This represents a 4.5-fold better inhibition for Pin1 than a comparable cis-amide alkene isostere. The co-crystal structure of Acâ pSerâ Ψ[CH2N]-Proâ tryptamine bound to Pin1 was determined to 1.76 Ã resolution. Towards understanding the two proposed mechanisms of Pin1 catalysis, nucleophilic-additition mechanism and twisted-amide mechanism, three classes of Pin1 inhibitors (ketoamide, ketone, and reduced amide analogues) involving a total of nine compounds were synthesized and evaluated. The weak inhibitory activities of ketoamide and ketone analogues do not support the nucleophilic-addition mechanism, while the twisted-amide mechanism of Pin1 catalysis is promising based on the reduced amide inhibitors with good potencies. / Ph. D.