We developed an approach to the β-thioenamide linkage contained in the S-(2-aminovinyl)cysteine (avCys) residue of thioviridamide.1,2 Kinetic and thermodynamic control of radical additions of thiols to ynamides were studied for the formation of β-thioenamide linkage. Thiyl radicals are electrophilic and ynamides are electron-rich alkynes. This complementary polarity of the radical and acceptor increases the likelihood of a successful radical addition reaction. Because little is known about these types of compounds (β-thioenamides), we were unsure what kinds of yields and stereoselectivities (cis vs. trans) to expect. The adduct stability is another issue to consider. Fortunately, under typical radical addition conditions, the two separable isomers (cis and trans) are formed in good yield. Selective formation of kinetic (cis) and thermodynamic (trans) isomers are controlled by reaction time and equivalents of thiol. We converted the kinetic isomer to the thermodynamic isomer to confirm that isomerization can occur under the reaction conditions. Alkyl and aryl thiols including cysteine-derived thiols with different ynamides were used in this process.

The analysis of Gene Regulatory Networks (GRNs) is essential to understanding the complete process of embryo development. Elucidating every gene regulatory circuit from maternal regulatory inputs all the way to the activation of differentiation gene batteries is an important step in increasing our understanding of developmental biology. In this work I study the cis-regulatory architecture of a pigment cell differentiation gene, polyketide synthase (SpPks) in the sea urchin Strongylocentrotus purpuratus. SpPks encodes an enzyme that is responsible for the biosynthesis of the sea urchin pigment echinochrome in larval pigment cells. The analysis of the promoter of a differentiation gene will lead to identifying the direct upstream regulators and ultimately to elucidating the structure of the upstream gene regulatory network, which is mostly uncharacterized. From previous studies the transcription factors SpGcm and SpGatae are predicted to be positive regulators of SpPks. Here, I identify a minimal 1kb promoter region containing putative DNA-binding sites for both GCM and GATAE that is able to recapitulate the expression of SpPks. I further show by mutagenesis that a putative DNA-binding site for GCM located 1,179 base pairs upstream of the start of transcription is a direct target for the positive cis-regulation of SpPks. Quantitative analysis of the transcriptional regulatory function of the GCM-mutagenized construct suggests that GCM is not necessary for the start of SpPks transcription but is required for its maintenance. Several GATA E binding sites have been identified within the minimal promoter for SpPks by means of consensus sequence. My analysis suggests that GATA E may be a direct positive regulator and could potentially be required for the onset of transcription of SpPks, though further experimentation will be necessary to characterize the exact regulatory function of GATA E.

Mosquitoes are notorious vectors for multiple diseases like malaria, yellow fever and dengue fever. To manipulate gene expression in mosquito and spread desired genes among natural population for vector control, a thorough understanding of mosquito development and gene regulation is critical. Early embryogenesis is a rapid, complex yet crucial process in the very beginning of development. Previous research in other species indicated genes transcribed that early evolved fast and played essential roles. The study of mosquito early zygotic genes (EZGs) would offer unique insights into mosquito gene evolution as well as potential targets for mosquito control. In this study, I identified 61 pure EZGs (pEZGs) in mosquito Aedes aegypti. These pEZGs were enriched in architectures adapting to the rapid embryonic cell cycles and were over represented by domains or functions related to maternal zygotic transition. Phylogenetic analysis showed that pEZGs originated mainly from duplication, retrotransposition and de novo emergence. The comparison of pEZGs in Ae. aegypti with those in Drosophila revealed an interesting evolutionary paradox where the early zygotic genes turned over fast but the regulatory motif was conserved in two species. Curiously, the motif binding protein in Drosophila (zelda) seemed unable to initiate the earliest zygotic transcription in Ae. aegypti due to late temporal expression. The regulatory motif (VBRGGTA) found in Ae. aegypti pEZGs was shown necessary and sufficient for driving early zygotic gene expression by transient reporter assays and one motif-bearing promoter was tested with success in driving gene expression as early as 2-4h after egg laying in transgenic Ae. aegypti. This was the first characterized promoter with early zygotic but no maternal expression in Ae. aegypti that can be used for future genetic studies and mosquito control strategies. As important gene regulators, miRNAs also play essential roles in early embryogenesis. The genome-wide predictions and systematic analysis of miRNAs in Ae. aegypti and Anopheles stephensi were conducted in this study. The first miRNA profiling in mosquito across all developmental stages was also performed to provide basis for future functional study. Several lineage-specific miRNAs were found highly expressed in embryos, indicating their special roles in the embryogenesis of mosquitoes. / Ph. D.

Under the Dobzhansky-Muller model, postzygotic isolation results from incompatibility between interacting genes. Evidence points to regulatory networks as a rich source of incompatibilities that impact hybrid fitness. Pleiotropy is a natural feature of regulatory networks because regulatory elements generally have multiple targets. Both pleiotropy and hybrid incompatibility arise due to genetic interactions; therefore we can expect an intimate association between them. In the following chapters, I investigate the relationship between pleiotropy and hybrid incompatibility in the context of regulatory networks. In chapter one, I extend a general network-based study of hybrid incompatibility by incorporating a sequence-based thermodynamic model of transcriptional regulation. In the absence of pleiotropy, hybrid misregulation of a positively selected trait evolves quickly as a consequence of non-recognition or spurious binding in regulatory interactions across species boundaries. In a conserved trait, hybrid incompatibility evolves much slower as a product of compensatory drift. In chapter two, I show that pleiotropy can promote or constrain the evolution of hybrid incompatibility in a regulatory network depending on its fitness landscape, which emerges from the thermodynamic properties of molecular binding. Pleiotropy may promote hybrid incompatibility in accordance with the "selection, pleiotropy, and compensation model" of evolution, in which compensation for the pleiotropic side-effects of adaptation accelerates incompatibility in conserved traits. Pleiotropy can limit the evolution of hybrid incompatibility by constraining change in trans-acting regulatory elements in favor of adaptation at less pleiotropic downstream cis-regulatory targets. Without change in both interactors, incompatibility does not occur under the Dobzhansky-Muller model. In chapter three, I evaluate the hypothesis that pleiotropy facilitates the onset of hybrid incompatibility under antagonistic coevolution, an ubiquitous and persistent source of natural selection. When infectivity and resistance in a host-parasite system are determined epistatically by network interactions, reciprocal selective pressure results in a genotypic chase. This causes pleiotropic mutations to accumulate and be compensated over time, producing intrinsic hybrid incompatibility in both species independent of local adaptation. Thus, cyclical antagonistic coevolution eventually overcomes constraint on pleiotropic loci, facilitating the evolution of regulatory incompatibilities commonly observed in hybrids.

Thesis advisor: Shih-Yuan Liu / Thesis advisor: Amir H. Hoveyda / Chapter 1. Senphos–Palladium-Catalyzed cis-Carboboration of Internal 1,3-Enynes with Carbon–Bound Boron Enolates: Reaction Development and Mechanistic Analysis. A new family of carbon-bound boron enolates (C–boron enolates) that are created through a kinetically controlled halogen exchange process between B–chlorocatecholborane and silylketene acetals is presented. These C–boron enolates are demonstrated to activate 1,3-enynes substrates in the presence of a Senphos-Pd complex to achieve carboboration reaction of an alkyne unit. This carboboration reaction produced highly substituted dienyl boron building blocks in high site-, regio-, and diastereoselectivity. A combined experimental and computational study of this carboboration reaction by Density-Functional Theory (DFT) calculations, 31P NMR study, kinetic study, Hammett analysis and Arrhenius/Eyring analysis will also be described. Mechanistic study supports a syn outer-sphere oxidative addition mechanism featuring a Pd-π-allyl intermediate followed by coordination-assisted rearrangement instead of the conventional inner-sphere β-migratory insertion mechanism. Chapter 2. trans-Hydroalkynylation of Internal 1,3-Enynes Enabled by Cooperative Catalysis. A trans-hydroalkynylation reaction of internal 1,3-enynes enabled by a cooperative catalysis system that comprises of Senphos–Pd complex, tris(pentafluorophenyl)borane, copper(I) bromide, and 2,2,6,6-tetramethylpiperidine, is described. The tris(pentafluorophenyl)borane as Lewis acid catalyst is shown to promote the reaction involving the emerging outer-sphere oxidative reaction step. This hydroalkynylation reaction affords the cross-conjugated dieneynes that serve as versatile synthons for organic synthesis. The photophysical properties of these cross-conjugated dieneynes depend on the position of electron donor/acceptor substituents along the conjugation path, as characterized by UV–vis absorption and emissions spectroscopy. Chapter 3. Senphos–Palladium/B(C6F5)3-Catalyzed trans-Hydrothiolation of 1,3-Enynes: Reaction Development and Mechanistic Analysis. A trans-hydrothiolation reaction of 1,3-enynes enabled by a cooperative catalysis system that comprises of Senphos–Pd complex and tris(pentafluorophenyl)borane is detailed. The tris(pentafluorophenyl)borane is shown to alter the reaction pathway, leading to a trans-addition product over cis-addition one. Experimental mechanistic study that includes 31P NMR, kinetic study, kinetic isotope effect (KIE) study, Hammett analysis, is consistent with a cooperative activation mechanism that features an outer-sphere protonation step. / Thesis (PhD) — Boston College, 2023. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

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High-risk neuroblastoma (NB) is an aggressive childhood tumor that originates from progenitor neural crest cells. Even if the therapeutic protocol for NB is articulate and aggressive, the outcome remains dismal, with the 5-year disease-free overall survival below rating 50%. A novel drug combination strategy can possibly provide a new solution to this unmet therapeutic need. 13-cis retinoic acid (13-cis-RA, isotretinoin) is an anti-proliferative and pro-differentiative agent currently used in the post-consolidation phase of NB therapy. To identify molecules able to potentiate the anti-proliferative activity of 13-cis-RA, NB cells were treated with a library of 169 naturally occurring polyphenols in combination with the retinoid. This in vitro screen led to the identification of isorhamnetin as a synergistic partner of 13-cis-RA, producing an 80% reduction in cell viability. At the molecular level, this synergistic effect is followed by a marked increase in the expression of a member of the catecholamine receptor superfamily: the adrenergic receptor alpha-1B (ADRA1B) suggesting that this receptor might represent a key mediator of the synergistic effect of 13-cis-RA and isorhamnetin observed in vitro. This finding redirected our attention to the class of adrenergic receptors (ARs) as novel targets in NB. To investigate the role of ADRA1B in the synergism, we generated CHP134 NB cell lines knocked-out (KO) for the receptor and observed that exposure of CHP134 KO cell to 13-cis-RA leads to a reduction of cell viability and neural differentiation. We, therefore, substituted the genetic KO strategy with the alpha-1B adrenergic antagonist, L765,314, obtaining the same results. Subsequently, we extended the analysis on the role of adrenergic receptors (AR) performing a biased screen using two libraries of AR-ligands. The screen results confirm that the molecules working as alpha-1-ARs antagonists are those that greatly increase cell sensitivity to 13-cis-RA with reduction of cell viability and increase in differentiation. We confirmed our observation in NB xenograft mice models in vivo, treating mice with a combination of 13-cis-RA and the FDA approved alpha-1 AR antagonist doxazosin. The proposed pharmacological treatment was effective in slowing tumor growth, leading to tumors of smaller size. From our results, we can conclude that the deletion or inhibition of alpha-1-AR sensitizes NB cells to 13-Cis-RA, both in terms of induction of apoptosis and neural differentiation. Since NB is a catecholamine-rich tumor, we propose that antagonization of alpha-1-AR disrupts the established autocrine pro-survival circuit generated by catecholamines in NB and restores the ability of the cells to follow the pro-differentiative and pro-apoptotic programs endorsed by 13-cis-RA. Considering the druggable nature of the alpha-1-AR receptors, we indicate this class of receptors as a novel pharmacological target for the treatment of neuroblastoma.

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