1 / Xiaoye Su

Toxoplasma gondii is an obligate intracellular parasite that can cause lifethreatening disease in immunocompromised individuals. Host cell invasion is therefore central to the pathology of the disease and parasite survival. Unlike many intracellular pathogens, T. gondii does not enter cells by manipulating the host’s phagocytic machinery; instead, the parasite enters the cell by a process of active penetration. Gliding motility and active penetration are driven by a complex of proteins termed the glideosome. The glideosome consists of four major proteins: TgMyoA, an unconventional myosin XIV, myosin light chain (TgMLC1) and glideosome-associated proteins 45 and 50 (TgGAP45, TgGAP50). TgMyoA has been shown to be essential for parasite motility, but the role of TgMLC1 in regulating myosin function remains unknown. Our lab has identified an inhibitor of T. gondii motility and invasion that results in a post-translational modification (PTM) to TgMLC1. Using molecular genetic and mass spectrometry methods we have shown cysteine 53 and cysteine 58 of TgMLC1 are essential for the modification to occur. To determine if the TgMLC1 PTM alters TgMyoA activity, glideosomes were isolated from DMSO- and 115556-treated parasites. Using an in vitro motility assay we have shown that the TgMyoA actin filament displacement velocities are decreased after 115556 treatment. This is the first evidence that TgMLC1 plays a role in regulating TgMyoA activity. The TgMLC1 PTM is responsible, at least in part, for the invasion and motility defects seen in the parasite after compound treatment. During the course of our investigations we have shown that TgMLC1 is dimethylated on lysine 95. This is an unusual modification for cytosolic proteins and has not been previously described for MLCs. Experiments using parasites expressing a non-methylatable form of TgMLC1 (TgMLC1-K95A) show that dimethylation is not necessary for TgMLC1 peripheral localization, TgMLC1 protein-protein interactions and is not required for TgMyoA activity in vitro. However, TgMLC1-K95A does not appear to be phosphoryalted indicating that TgMLC1 dimethylation is necessary for efficient phosphorylation of TgMLC1. These experiments will provide new insight into the ways in which TgMLC1 regulates this unconventional myosin motor complex.

Display technology has become a vital and ubiquitous part of our daily life. Undoubtedly, today’s technologically minded society is living in the era of the digital image. After high resolution and efficiency could successfully be realized, the major trends in display technology now aim towards achieving high color purity for natural looking display colors. Organic light-emitting diodes (OLEDs), as one strong contender for high performance displays and lighting, have been undergoing tremendous industrial and commercial development. Despite the great progress, though, there is still space for improvement, especially in the case of blue light emitting devices. Blue OLEDs are always challenging, since they traditionally suffer from low efficiencies and lifetimes. Both, novel materials and device architectures, are driving ongoing developments while still always aiming to lower the overall costs. In a continual effort to search for robust materials for blue devices, small molecules (SMs) and polymers, are shown to be promising candidates. In this thesis is presented the results of the detailed study of photophysical and electroluminescence (EL) properties in the case of thin films based on blends of the conjugated polymer Poly(9,9-di-n-octylfluorenyl-2,7-diyl) (PFO) and the of novel SMs; 4,4'-(anthracene-9,10-diyl)bis(N,N-bis(4-methoxyphenyl)aniline) (TPAA) and 4,4'-(pyrene-1,6-diyl)bis(N,N-bis(4-methoxyphenyl)aniline) (TPAP). Finally, devices based on these systems are optimized step by step as a solution processable emissive layer (EML), for applications in sky blue OLEDs.

Small molecules are powerful reagents for biological investigation. They provide an alternative to genetic perturbation and may offer more control over a target’s activity. C. elegans has recently gained prominence as a platform to discover new chemical tools. Through large-scale screens for compounds that induce phenotypes consistent with the disruption of conserved pathways, we identified two previously uncharacterized molecules of interest that we named nemadipine and migrazole. Here, I describe my efforts to understand their mechanism of action. Nemadipine is structurally analogous to 1,4-dihydropyridines (DHPs), which target the Cav1 calcium channel and are used clinically to lower blood pressure. Phenotypic and genetic evidence suggest that nemadipine targets the worm Cav1 channel, EGL-19. To identify the target of nemadipine in an unbiased manner, I performed a forward genetic screen for mutants resistant to its effects. The majority of the mutants from my screen had polymorphisms in EGL-19, providing additional evidence that it is the target of nemadipine. I also found that nemadipine is the only DHP that robustly elicits phenotypes in the worm. Therefore, I used this unique chemical to investigate the in vivo interactions between DHPs and the Cav1 channel. I identified residues in EGL-19 important for DHP-sensitivity in worms and showed that some of these residues are also important for mammalian DHP-interaction. Other labs have since exploited nemadipine’s in vivo properties to demonstrate new biological insights for EGL-19. Chemical genetic analyses indicated that migrazole disrupts multiple signal transduction pathways. This, together with experiments that I performed in yeast, suggests that migrazole may affect multiple pathways by perturbation of protein transport. To identify migrazole’s target, I performed a forward genetic screen for mutants resistant to migrazole’s effects. However, I was unable to identify the target of migrazole through analysis of the mutants I isolated. This result illustrates that while forward genetic screens can be very successful for target identification, their effectiveness is likely dependent on the nature of the compound-target interaction. My work shows that all aspects of developing a small molecule into a tool for biological analysis, from its discovery to its characterization, can be accomplished using C. elegans.

Small molecules are powerful reagents for biological investigation. They provide an alternative to genetic perturbation and may offer more control over a target’s activity. C. elegans has recently gained prominence as a platform to discover new chemical tools. Through large-scale screens for compounds that induce phenotypes consistent with the disruption of conserved pathways, we identified two previously uncharacterized molecules of interest that we named nemadipine and migrazole. Here, I describe my efforts to understand their mechanism of action. Nemadipine is structurally analogous to 1,4-dihydropyridines (DHPs), which target the Cav1 calcium channel and are used clinically to lower blood pressure. Phenotypic and genetic evidence suggest that nemadipine targets the worm Cav1 channel, EGL-19. To identify the target of nemadipine in an unbiased manner, I performed a forward genetic screen for mutants resistant to its effects. The majority of the mutants from my screen had polymorphisms in EGL-19, providing additional evidence that it is the target of nemadipine. I also found that nemadipine is the only DHP that robustly elicits phenotypes in the worm. Therefore, I used this unique chemical to investigate the in vivo interactions between DHPs and the Cav1 channel. I identified residues in EGL-19 important for DHP-sensitivity in worms and showed that some of these residues are also important for mammalian DHP-interaction. Other labs have since exploited nemadipine’s in vivo properties to demonstrate new biological insights for EGL-19. Chemical genetic analyses indicated that migrazole disrupts multiple signal transduction pathways. This, together with experiments that I performed in yeast, suggests that migrazole may affect multiple pathways by perturbation of protein transport. To identify migrazole’s target, I performed a forward genetic screen for mutants resistant to migrazole’s effects. However, I was unable to identify the target of migrazole through analysis of the mutants I isolated. This result illustrates that while forward genetic screens can be very successful for target identification, their effectiveness is likely dependent on the nature of the compound-target interaction. My work shows that all aspects of developing a small molecule into a tool for biological analysis, from its discovery to its characterization, can be accomplished using C. elegans.

Doctor of Philosophy / Department of Chemistry / Duy H. Hua / Small molecules are of great importance in drug discovery currently. The first three chapters discussed the design, synthesis and bio-evaluation of three different classes of small molecules and exploration of their biological targets. Triacsin C analogs were designed as long chain fatty acyl-CoA synthetase (ACSL) inhibitors for attenuating ischemia and reperfusion (I/R) injury. Oxadiazole derivatives were designed as T-type calcium channel inhibitors, which have potential application in the treatment of seizure and epilepsy. Tricyclic pyrone derivatives were reported as anti-Alzheimer lead compounds in previous research done by the Hua group. TP70 and CP2 were synthesized to explore their pharmacokinetics properties. Chapter 4 described chiral-substituted poly-N-vinylpyrrolidones (CSPVP) supported Cu/Au nanoclusters mediation of cyclization reaction of 5-substituted nona-1,8-dien-5-ols. A five-member cyclized lactone possessing a stereogenic tetrasubstituted carbon center was formed in a one-step Cu/Au nanoclusters-hydrogen peroxide oxidation reaction. This developed a novel and simple method to synthesize tetrasubstituted carbon stereogenic center. Drawbacks of the method in my initial study were low reaction yield and moderate enantioselectivity. The chemical yield and enantioselectivity have been significantly improved by introducing bulkier substitution in C3 and C4 positions of CSPVP according to the updates of ongoing research.

Bestrophin-1 is a homopentameric Ca2+-gated anion channel which localises to the basolateral plasma membrane of retinal pigment epithelium (RPE) cells. Homozygous and compound heterozygous mutations in the BEST1 gene are associated with autosomal recessive bestrophinopathy (ARB), a retinopathy characterised by altered electrooculogram (EOG), deposits in the retina, and is often associated with the risk of developing angle-closure glaucoma. The mechanism by which mutations in bestrophin-1 cause disease remains unknown. Expression of four ARB-causing bestrophin-1 proteins in polarised MDCKII cells, a cell model for RPE, results in mutant proteins mislocalisation and degradation. Furthermore, when the ability of the mutant proteins to conduct Cl- ions was investigated in HEK293 cells by whole-cell patch-clamp, a reduction in the Cl- current was observed in all mutants compared to the WT.The use of a combination of the small molecules bortezomib and 4-phenylbutyrate (4PBA) successfully restored the expression and trafficking of all four ARB-causing bestrophin-1 proteins. Importantly, 4PBA was also able to restore the ability of the mutant channel to conduct Cl- ions. Biotinylation of cell surface proteins shows that the number of active channels at the plasma membrane of HEK293 cells increases following 4PBA treatment. The functional rescue achieved with 4PBA supports the hypothesis that ARB-associated missense mutations reduce the number of functional channels that reach the cell membrane rather than altering other aspects of channel function. The results presented in this thesis suggest that 4PBA may be a promising therapy for the treatment of ARB and the other bestrophinopathies resulting from missense mutations in BEST1, particularly as 4PBA is already approved for long-term use in infants and adults. These finding also pave the way for the use of small molecule therapies to treat conformational diseases caused by mutation in other protein expressed in the RPE.

No description available.

No description available.

The numerous benefits associated with natural products isolated from the environmental sources, including soil bacteria, plants and fungi, are long known and well appreciated. Interestingly, the immense number of microorganisms that reside within our bodies and whose cell counts greatly outnumber our own represents a potentially new and practically untapped reservoir of bioactive compounds. With the advent of next generation sequencing we are only now starting to realize the complexity and biological diversity of the human microbiome. With this ever-increasing flow of genomic information, more bioactive potential in these microbes can be identified. For instance, biosynthetic assembly lines responsible for the production of two largest classes of bioactive compounds, polyketides and nonribosomal peptides, can be readily identified within the microbial genomes, providing us with a view of their bioactive profiles. In addition to the identification of biosynthetic assembly lines, the building blocks of polyketide and nonribosomal peptide products can also be accurately predicted, given the well-understood logic of assembly line operations. Nonetheless, the identification of actual products is still lagging behind. The discovery of these bioactive molecules can be achieved, however, by establishing a unique connection between genomes and molecules. Using several concrete examples, this thesis demonstrates how both metabolomic and biochemoinformatic platforms can assist in discovery of bioactive small molecules. More specifically, investigations involving three members of the human microbiome, Streptococcus mutans, Lactobacillus plantarum and Pseudomonas aeruginosa, provide distinct examples of identification of bioactive agents and assessment of their immunomodulatory potential. Interrogating the human microbiome form the angle of small molecules is critical for evaluation of microbial effects on our cells, and ultimately our health. Studying these agents will hopefully reveal interesting principles on how microorganisms speak to human cells and how this communication could lead to therapeutic strategies or downstream mechanistic revelations. / Thesis / Master of Science (MSc) / The numerous benefits associated with natural products isolated from the environmental sources, including soil bacteria, plants and fungi, are long known and well appreciated. Interestingly, the immense number of microorganisms that reside within our bodies and whose cell counts greatly outnumber our own represents a potentially new and practically untapped reservoir of bioactive compounds. With the advent of next generation sequencing we are only now starting to realize the complexity and biological diversity of the human microbiome. With this ever-increasing flow of genomic information, more bioactive potential in these microbes can be identified. For instance, biosynthetic assembly lines responsible for the production of two largest classes of bioactive compounds, polyketides and nonribosomal peptides, can be readily identified within the microbial genomes, providing us with a view of their bioactive profiles. In addition to the identification of biosynthetic assembly lines, the building blocks of polyketide and nonribosomal peptide products can also be accurately predicted, given the well-understood logic of assembly line operations. Nonetheless, the identification of actual products is still lagging behind. The discovery of these bioactive molecules can be achieved, however, by establishing a unique connection between genomes and molecules. Using several concrete examples, this thesis demonstrates how both metabolomic and biochemoinformatic platforms can assist in discovery of bioactive small molecules. More specifically, investigations involving three members of the human microbiome, Streptococcus mutans, Lactobacillus plantarum and Pseudomonas aeruginosa, provide distinct examples of identification of bioactive agents and assessment of their immunomodulatory potential. Interrogating the human microbiome form the angle of small molecules is critical for evaluation of microbial effects on our cells, and ultimately our health. Studying these agents will hopefully reveal interesting principles on how microorganisms speak to human cells and how this communication could lead to therapeutic strategies or downstream mechanistic revelations.