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.

Thesis (Ph. D.)--University of Missouri-Columbia, 2000. / Typescript. Vita. Includes bibliographical references (leaves 185-226). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 2000. / Typescript. Vita. Includes bibliographical references (leaves 185-226). Also available on the Internet.

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.

Hereditary non-polyposis colon cancer syndrome (HNPCC) is associated with an increased risk of developing several types of cancer and is the most common cause of hereditary ovarian cancer after BRCA1 and BRCA2 mutations. HNPCC results from a germline mutation in one of the DNA mismatch repair (MMR) genes: MLH1, MSH2, PMS1, PMS2, MSH6, MSH3 and MLH3. While there has been extensive investigation of MMR deficiency in colorectal cancer, MMR in ovarian cancer is relatively under-investigated. The goal of this project was to study MMR deficiency in ovarian cancer at both the clinical and molecular level. The first aim was to examine the frequency of MMR loss in a large patient cohort and investigate the clinical consequences of MMR deficiency. The second aim was to describe the molecular characteristics of MMR deficiency in ovarian cancer cell lines and establish an in vitro cell line model of MMR deficiency in ovarian cancer. The third aim was to identify synthetic lethal strategies for the treatment of ovarian cancer to maximise cytotoxicity in a MMR-deficient background. In order to characterise the clinical consequences of MMR deficiency, a large patient cohort was studied with regard to MMR status. Three tissue microarrays consisting of 581 ovarian tumours were constructed, and expression of the four most frequently lost MMR proteins: MLH1, MSH2, PMS2 and MSH6 were detected by immunohistochemistry. Afterwards, MMR status and histology subtypes were analysed in combination with the associated clinical data. The overall incidence of MMR deficiency (loss of any MMR protein) was 15.7%, with PMS2 being the most frequently lost protein (9.7%). In addition, MMR deficiency tended to appear in a grouped fashion: MLH1 with PMS2; MSH2 with MSH6. Patients with non-serous subtypes of ovarian cancer, clear cell or mucinous especially, had higher incidence of MMR deficiency compared to patients with serous ovarian cancer. Overall MMR deficient patients were more likely to be diagnosed at early stages compared with MMR proficient patients, and this is probably due to the association between MMR deficiency and non-serous histology. However, platinum-based treatment for patients with MMR deficiency gives no advantage over those without MMR deficiency. Therefore better treatments for this subgroup of patients may be needed. The features of MMR deficiency in ovarian cancer were also characterized at the molecular level. After quantifying mRNA and protein expression of MMR genes in 19 ovarian cell lines, three cell lines (SKOV3, TOV21G and IGROV1) were found to have a defect in MLH1 expression at both the mRNA and protein level. Interestingly, the three cell lines also carried a defect in PMS2 expression at the protein level but not at the mRNA level, which is consistent with our clinical data demonstrating that MLH1 protein and PMS2 protein are paired in loss. In addition, across the 19 cell lines, MLH1 and PMS2 showed positive correlation at both the mRNA level (R=0.53, p=0.02) and protein level (R=0.72, p=0.0006). In order to study co-expression of MLH1 and PMS2, a plasmid encoding the cDNA for MLH1 was transfected into the three MLH1 deficient cell lines; and conversely siRNA targeting MLH1 was transfected into the MMR proficient cell line A2780 and expression of MLH1 protein and PMS2 protein was quantified. The results showed that re-introduction of MLH1 into MLH1 deficient cells resulted in increased expression of PMS2 protein, while knocking down MLH1 in MMR proficient cells leads to decreased PMS2 protein expression. This indicates that MLH1 may play a crucial role in regulating PMS2 protein expression. As the three MLH1 and PMS2 protein deficient cell lines all express PMS2 mRNA, the regulation of PMS2 expression by MLH1 is likely to be at the translational or post-translational level. However, the expression of PMS2 protein was not increased in the absence of MLH1, even when the proteasomal and lysosomal protein degradation pathways were blocked (as seen with SKOV3 cells), suggesting decreased PMS2 protein expression is not due to rapid degradation in the absence of MLH1. Therefore MLH1 may play a role in regulating the synthesis of PMS2 protein at the translational level, rather than preventing the degradation of PMS2. Thus, to investigate the mechanism by which PMS2 protein levels are regulated by MLH1, future work should focus on translational regulation of PMS2. In order to identify synthetic lethal strategies to target MMR deficiency in ovarian cancer, an isogenic cell line model of MMR deficiency was established by stable transfection of a plasmid for MLH1 and its corresponding empty vector into SKOV3 cells. The MLH1+ cell line SAC-1 and MLH1- cell line SN-5 were selected for drug screening based on their phenotype and growth rate. The AlamarBlue assay, with z’ above 0.5, was chosen for drug screening and a kinase inhibitor library containing 362 drugs of known target was screened. Two drugs with similar structures that targeted PLK1 showed greater growth inhibition of SN-5 compared with SAC-1. When the two cell lines were treated with another PLK1 inhibitor, BI2536, with different structure, a 2-fold difference in growth inhibition between SAC-1 and SN-5 was also observed, suggesting PLK1 is a potential synthetic lethal target for MLH1 deficiency in ovarian cancer. Together these data demonstrate that clinically, MMR deficiency is associated with non-serous subtypes of ovarian cancer and specific MMR proteins are paired in loss. While current standard therapy offers no selective benefit to ovarian cancer patients with MMR deficiency, inhibiting PLK1 activity may confer selective benefit.

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

<div>G-quadruplexes (G4s) are non-canonical secondary structures formed in single-stranded guanine-rich nucleic acid sequences, such as those found in oncogene promoters and telomeres. <i>MYC</i>, one of the most critical oncogenes, has a DNA G4 (MycG4) in its proximal promoter region that functions as a transcriptional silencer. MycG4 is very stable and the pathological activation of <i>MYC</i> requires its active unfolding. However, it remains unclear what drives MycG4 unfolding in cancer cells. We have studied the interactions of DDX5 with the MycG4 at both molecular and cellular levels and discovered that DDX5 actively unfolds the MycG4 and is involved in the <i>MYC</i> gene transcriptional regulation, which is described in the first part of this dissertation. DDX5 is extremely proficient at unfolding the MycG4 and ATP hydrolysis is not directly coupled to the G4-unfolding of DDX5. In cancer cells, DDX5 is enriched at the <i>MYC</i> promoter and activates <i>MYC</i> transcription. G4-interactive small molecules inhibit the DDX5 interaction with the <i>MYC</i> promoter and DDX5-mediated <i>MYC</i> activation. The second part of this dissertation describes the study of interactions of indenoisoquinoline anticancer drugs with MycG4. The MycG4 transcriptional silencer is a very attractive therapeutic target. Compounds that bind and stabilize the MycG4 have been shown to repress <i>MYC</i> gene transcription and are antitumorigenic. Indenoisoquinolines are human topoisomerase I inhibitors in clinical testing. However, some indenoisoquinolines with potent anticancer activity do not exhibit strong topoisomerase I inhibition, suggesting a separate mechanism of action. Our studies show that indenoisoquinolines strongly bind and stabilize MycG4 and lower <i>MYC</i> levels in cancer cells. Moreover, the analysis of indenoisoquinoline analogues for their <i>MYC</i> inhibitory activity, topoisomerase I inhibitory activity, and anticancer activity reveals a synergistic effect of <i>MYC</i> inhibition and topoisomerase I inhibition on anticancer activity. Besides the MycG4, human telomeric G4s are also attractive targets for anticancer drugs due to their ability to inhibit telomere extension in cancer cells. The last part of this dissertation reviews two recent solution structural studies on small molecule complexes with the hybrid-2 telomeric G4 and the hybrid-1 telomeric G4. Structural information of those complexes can advance the design of telomeric G4-interactive small molecules in the cancer therapeutic areas.</div>