Colorectal cancer (CRC) is the second leading cause of cancer-related deaths in Canada each year. Currently, most therapeutic approaches target rapidly dividing cancer cells by inhibition of normal cellular processes, however these therapies are not selective for cancer cells and unwanted side effects occur. Accordingly, novel cancer-targeted therapeutic strategies and drug targets are urgently needed to diminish the morbidity and mortality rates associated with CRC. Synthetic lethality is a new therapeutic approach that is designed to better target and kill cancer cells by exploiting a cancer-associated mutation (i.e. RAD54B-deficiency) thereby minimizing adverse side effects. We hypothesize that RAD54B-deficient CRC cells will be selectively killed via a synthetic lethal (SL) interaction with PARP1. We have identified and validated a novel drug target, PARP1, within CRC cells harboring RAD54B-deficiencies through a SL paradigm. This study represents the first steps necessary to identify and develop precision medicine based therapeutic strategies to combat CRC. / October 2016

Das Projekt „Balanced Lethal System in Salmonella typhi Ty21a“ der AG Bakterielle Tumortherapie des MSZ zielt auf die Entwicklung eines Vakzinstammes, der in der humanen Krebstherapie zum Einsatz kommen soll. Ziel dieser Arbeit war das zuvor etablierte Balanced Lethal System in Salmonella typhi Ty21a zu optimieren. / The aim of this project is to develop a bacterial vaccine that will be used in cancer immunotherapy. The aim of this dissertation was to improve the established balanced lethal system in Salmonella typhi Ty21a.

Mangroves and seagrass beds serve as essential fish habitat for many economically- and ecologically-valuable species. Depending on their location, these shallow-water habitats are often characterized by substantial fluctuation in salinity levels, which can represent a source of osmoregulatory stress for associated organisms. In South Florida, one of the most important fish species that utilizes these habitats is the gray snapper (Lutjanus griseus). Although this species constitutes a significant portion of the region?s total recreational fishery harvest, the effects of salinity on its distribution, physiology and behavior remain poorly understood. The main goal of this thesis was then to investigate the ecophysiological basis of habitat selection by the gray snapper. Specific objectives include to: (1) examine patterns of distribution and abundance across gradients in environmental salinity; (2) measure physiological status and responses to controlled salinity challenges and; (3) conduct behavioral trials to examine for salinity preferenda (if any). To begin investigating if salinity could be a primary factor structuring the gray snapper assemblages, I examined empirical data collected from Biscayne Bay to test the null hypothesis that gray snapper abundances were evenly distributed along the full salinity range at which samples have been collected. Using the delta approach, three abundance metrics (frequency of occurrence, concentration and delta density) were used as an index for the distribution and abundance of this species. Results indicated that abundance patterns for the smaller gray snapper were consistent with a strategy of reducing osmoregulatory costs by selecting intermediate salinities. However, corresponding abundance patterns for subadult gray snapper were inconsistent with this strategy of minimizing energetic costs, suggesting that this life stage may be indifferent to the range of salinities at which they were observed. These patterns helped developed further hypotheses regarding the ecophysiology of juvenile and subadult gray snapper, the latter of which was then tested via laboratory experiments. Subsequently, I challenged fish in the laboratory with six different salinity treatments (0, 5, 30, 50, 60 and 70ppt, including control) for 192 consecutive hours and collected blood samples at different time points. Results indicated that physiological stress to salinity changes is unlikely to occur at a salinity range of 5 to 50 ppt. At salinities of 0 and 60 ppt transient significant changes in plasma osmolality and/or blood haematocrit were observed, but were corrected after an initial adjustment period of approximately 96 hours. At the highest salinity treatment (70 ppt), a constant osmolality could not be maintained, resulting in death for all fish within 48 hours of exposure. Overall, these findings demonstrate the strong euryhalinity and extraordinary tolerance of this species to both extreme hypo- and hypersaline environments. Finally, I investigated the salinity preference and effects on swimming behavior of the gray snapper in an automated salinity choice shuttlebox via 48-hr trials. In general, gray snapper tested displayed either one of two distinctively different salinity preferences. Half of gray snappers displayed a salinity preference in the range of 9-15 ppt, whereas the other half displayed a salinity preference in the range of 19-23 ppt. Recorded swimming speeds in all fish tested reflected a significant, but weak negative linear relationship with salinity during both time periods of the day (light and dark); however, gray snapper were usually most active during the dark period across all salinities. Overall, these findings reveal that gray snapper prefer slightly hyperosmotic salinities that may minimize the physiological costs of osmoregulation compared to extreme salinities.

Dosage compensation is the key regulatory process employed in <i>Drosophila melanogaster</i> to equalize the level of gene transcripts between the single X chromosome in males (XY) and the two X chromosomes in females (XX). Dimorphic sex chromosomes evolved by the severe degeneration of the Y chromosome, giving rise to an imbalance between the heterogametic sex and the homogametic sex. Vital to the viability of male Drosophila is the dosage compensation complex (DCC), a ribonucleoprotein complex that mediates the precise two-fold transcription of the single male X chromosome. The DCC is comprised of five proteins: male-specific-lethal proteins (MSL) 1, 2, and 3, male absent-on-the-first (MOF), maleless (MLE), and two non-coding RNAs. The complex specifically co-localizes along the male X chromosome in a reproducible manner, resulting in acetylation of lysine 16 of the N-terminal tail of histone H4. The exact mechanism of recruitment and spreading of the DCC along the male X chromosome remains unclear; recent studies propose a multi-step mechanism involving DNA sequence elements, epigenetic marks, and transcription. Understanding how dosage compensation functions provides insight into the interplay between gene regulation and chromatin remodelling. The goal of this project was to better understand how <i>Drosophila</i> MSL1, MSL3, and MOF interact and how their interaction modulates MOFs acetyltransferase activity. Recombinant protein constructs were cloned and over-expressed in a bacterial expression system permitting future structure determination by X-ray crystallography. The dMSL1820-1039 construct consisted of the C-terminal domain, reported to be able to interact with both dMSL3 and dMOF. dMSL3186-512 contained the domain required for the interaction with dMSL1 and dMOF. dMOF371-827 was comprised of the catalytic domain, the CCHC zinc finger, and the chromodomain, as the N-terminal region does not encode any known domains. All three recombinant proteins were successfully cloned, over-expressed, and purified to homogeneity. Recombinant dMOF371-827 was determined to acetylate histones. Interaction studies using GST pull-down assays and size exclusion chromatography determined that dMSL1820-1039 and dMOF371-827 did not interact above background levels. Moreover, size exclusion chromatography revealed dMSL3186-512 and dMOF371-827 did not interact nor did the three recombinant proteins form a stable complex.

Control of mRNA stability is an important aspect in the regulation of gene expression. A well studied signal for rapid transcript decay in mammalian cells is the AU-rich element (ARE), which is found in the 3’ untranslated region (UTR) of many labile transcripts. These sequence elements confer destabilization of transcripts by binding to AU-binding proteins (AUBPs) that can recruit cellular decay enzymes. The stability of ARE-containing mRNAs can be regulated by extracellular stimuli, which allows for cells to adapt to the changing environment. AREs are found in many transcripts that encode for inflammatory genes, including TNF, GM-CSF, and IL-8. Pathogens evolve and devise mechanisms to subvert the immune response of the host to aid in its infection. Bacillus anthracis is one such infectious agent that can disable numerous arms of the host immune response. Its secreted toxin, anthrax lethal toxin (LeTx), causes the accelerated decay of the IL-8 mRNA. IL-8 is a dual function cytokine and chemokine that can recruit and activate neutrophils at the site of infection. Through the inactivation of MAPK pathways, LeTx activity causes the destabilization of IL-8 transcripts through its ARE. In this thesis, I show that an AUBP, TTP, is dephosphorylated by LeTx and MAPK inhibitors, and knock-down of its expression stabilized IL-8 transcripts. LeTx activity also increased the colocalization of TTP to P-bodies, cytoplasmic sites concentrated with RNA decay enzymes. This suggests that the post-translational modification of TTP induced by LeTx led to its enhanced destabilization function. Identified TTP-associated proteins, non-muscle myosin heavy chain 9 (myosin-9) and HSC-70, were examined for their role in IL-8 transcript decay. Knock-down of each protein led to a slower rate of IL-8 mRNA destabilization. However, treatment of LeTx continued to mediate accelerated destabilization of IL-8 in these siRNA-transfected cells. This suggests that LeTx, myosin-9, and HSC-70 modulate the destabilization function of TTP independently.

Dosage compensation is the key regulatory process employed in <i>Drosophila melanogaster</i> to equalize the level of gene transcripts between the single X chromosome in males (XY) and the two X chromosomes in females (XX). Dimorphic sex chromosomes evolved by the severe degeneration of the Y chromosome, giving rise to an imbalance between the heterogametic sex and the homogametic sex. Vital to the viability of male Drosophila is the dosage compensation complex (DCC), a ribonucleoprotein complex that mediates the precise two-fold transcription of the single male X chromosome. The DCC is comprised of five proteins: male-specific-lethal proteins (MSL) 1, 2, and 3, male absent-on-the-first (MOF), maleless (MLE), and two non-coding RNAs. The complex specifically co-localizes along the male X chromosome in a reproducible manner, resulting in acetylation of lysine 16 of the N-terminal tail of histone H4. The exact mechanism of recruitment and spreading of the DCC along the male X chromosome remains unclear; recent studies propose a multi-step mechanism involving DNA sequence elements, epigenetic marks, and transcription. Understanding how dosage compensation functions provides insight into the interplay between gene regulation and chromatin remodelling. The goal of this project was to better understand how <i>Drosophila</i> MSL1, MSL3, and MOF interact and how their interaction modulates MOFs acetyltransferase activity. Recombinant protein constructs were cloned and over-expressed in a bacterial expression system permitting future structure determination by X-ray crystallography. The dMSL1820-1039 construct consisted of the C-terminal domain, reported to be able to interact with both dMSL3 and dMOF. dMSL3186-512 contained the domain required for the interaction with dMSL1 and dMOF. dMOF371-827 was comprised of the catalytic domain, the CCHC zinc finger, and the chromodomain, as the N-terminal region does not encode any known domains. All three recombinant proteins were successfully cloned, over-expressed, and purified to homogeneity. Recombinant dMOF371-827 was determined to acetylate histones. Interaction studies using GST pull-down assays and size exclusion chromatography determined that dMSL1820-1039 and dMOF371-827 did not interact above background levels. Moreover, size exclusion chromatography revealed dMSL3186-512 and dMOF371-827 did not interact nor did the three recombinant proteins form a stable complex.

Control of mRNA stability is an important aspect in the regulation of gene expression. A well studied signal for rapid transcript decay in mammalian cells is the AU-rich element (ARE), which is found in the 3’ untranslated region (UTR) of many labile transcripts. These sequence elements confer destabilization of transcripts by binding to AU-binding proteins (AUBPs) that can recruit cellular decay enzymes. The stability of ARE-containing mRNAs can be regulated by extracellular stimuli, which allows for cells to adapt to the changing environment. AREs are found in many transcripts that encode for inflammatory genes, including TNF, GM-CSF, and IL-8. Pathogens evolve and devise mechanisms to subvert the immune response of the host to aid in its infection. Bacillus anthracis is one such infectious agent that can disable numerous arms of the host immune response. Its secreted toxin, anthrax lethal toxin (LeTx), causes the accelerated decay of the IL-8 mRNA. IL-8 is a dual function cytokine and chemokine that can recruit and activate neutrophils at the site of infection. Through the inactivation of MAPK pathways, LeTx activity causes the destabilization of IL-8 transcripts through its ARE. In this thesis, I show that an AUBP, TTP, is dephosphorylated by LeTx and MAPK inhibitors, and knock-down of its expression stabilized IL-8 transcripts. LeTx activity also increased the colocalization of TTP to P-bodies, cytoplasmic sites concentrated with RNA decay enzymes. This suggests that the post-translational modification of TTP induced by LeTx led to its enhanced destabilization function. Identified TTP-associated proteins, non-muscle myosin heavy chain 9 (myosin-9) and HSC-70, were examined for their role in IL-8 transcript decay. Knock-down of each protein led to a slower rate of IL-8 mRNA destabilization. However, treatment of LeTx continued to mediate accelerated destabilization of IL-8 in these siRNA-transfected cells. This suggests that LeTx, myosin-9, and HSC-70 modulate the destabilization function of TTP independently.

The distribution of apple snail Pomacea canaliculata are island-wide but Pomacea scalaris is only found in southern Taiwan. In order to gain more information on the not well-known alien invasive species, comparative studies on the reproduction and thermal tolerance of P. canaliculata and P. scalaris were conducted. Sexual dimorphism in shell morphology has been found in both species, with wider operculum in males. Positive correlation between shell length and penis sheath length or penis length has been observed in male P. canaliculata and P. scalaris, based on the samples collected during the period of December 2004 and March 2006, The width of penis sheath in P. canaliculata was greater than in P. scalaris. Positive correlation between shell length and the height and width of vestigial penis in female P. canaliculata and P. scalaris was also found. The width of vestigial penis in P. scalaris was greater than in P. canaliculata. Gonadosomatic index (GSI) in male P. canaliculata and P. scalaris was 68 and 60% and 31 and 33% in females. Their reproductive cycle was annual without seasonal peaks. Significant difference in thermopreferrenda was found between P. canaliculata and P. scalaris. The 24, 48 and 72-h lethal thermal minima temperatures in P. canaliculata and P. scalaris were similar, i.e. 9.8¡V11.8¢J. And, the 24, 48 and 72-h lethal thermal maxima temperatures were 33.1¡V35.9¢J. Based on the results, it is known that P. canaliculata and P. scalaris reproduce year-round and temperature is not a major factor in shaping the species distribution pattern in Taiwan.

Thesis (Ph. D.)--University of Missouri--Columbia, 2001. / "May 2001." Typescript. Vita. Includes bibliographical references (leaves 97-110). Also available on the Internet.

Thesis (Ph. D.)--York University, 2000. Graduate Programme in Biology. / Typescript. Includes bibliographical references. Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://wwwlib.umi.com/cr/yorku/fullcit?pNQ56253.