Diverse Applications of Flow Technology in Discovery Chemistry

The research presented herein describes the development of methods and applications of flow technology in discovery chemistry. The first two chapters highlight the use of several beneficial features offered by flow technology to increase the throughput, safety, and convenience of organic synthesis. The last two chapters describe the use of microfluidic technology as a platform for rapid reaction discovery. Working with researchers at Abbott Laboratories, a droplet-based library method was developed. This approach allowed for the preparation of a theoretically unlimited number of compounds in a single run using minimal amounts of material. The universal nature of this approach was subsequently demonstrated in the preparation of two 20-membered libraries based around thiazole and pyrazole cores. A second methodology study, also completed with researchers at Abbott Laboratories, took advantage of the intrinsic closed environment of flow systems which enabled the creation, reaction, and removal of noxious chemicals in situ. Using these features an in situ synthesis of isocyanides, reagents notorious for their unpleasant smell, was developed. Coupling this method to the Ugi four-component reaction, a series of medicinally relevant amides was synthesized. Reactions performed in the flow system experienced an overall reduction in transformation time from two-days to two-hours and gave yields that were generally higher than those for the same reactions performed on the benchtop or in the microwave. Expanding the application of this technology toward the discovery and development of new synthetic methodologies we partnered with the laboratory of Dr. John A. Porco, Jr. at Boston University to explore transformations of multifunctional substrates. Given the variant nature of these reactions, both a simple iminium ether and a densely functionalized iminium ether derived from a bicyclo[3.2.1]octanoid scaffold were explored. Multidimensional reaction screening on an automated microfluidic platform was employed to facilitate the simultaneous investigation of multiple reaction variables. While the majority of products obtained from the study resulted from expected modes of O- and N- alkylation, several interesting transformations were uncovered. These included the pseudo-dimerization of homophthalic anhydride, an unusual integration of the van Leusen sulfone, and an unexpected carbon-carbon bond forming event of ethyl diazoacetate and acetonitrile. Finally, in a follow-up study, collaboration with Boston University was continued to explore additional reactivity of the bicyclo[3.2.1]octanoid scaffold. Preliminary reaction screens uncovered the synthesis of a series of densely functionalized donor-acceptor cyclopropanes which resulted from the photochemical rearrangement of the bicyclic scaffolds. Expansion of the photochemical screening to a polycyclic iminium ether led to the first example of an aza-di-pi reaction of a charged iminium species. Subjection of the new cyclopropane scaffolds to a variety of reaction conditions led to the discovery of additional rearrangement reactions affording several structurally diverse chemotypes including a fused dihydropyran, a fused pyrrole, a bicyclic imide, and a complex cyclic imine

Beyond Recovery: Colonization, Health and Healing for Indigenous People in Canada

How do we limit our focus to mental health when Indigenous teaching demands a much wider lens? How do we respond to mental health recovery when Indigenous experience speaks to a very different approach to healing, and how can we take up the health of Indigenous people in Canada without a discussion of identity and colonization? We cannot, for the mental health and recovery of Indigenous people in Canada have always been tied to history, identity, politics, language and dislocation. Thus, in this paper, our aim is to make clear that history, highlight the impacts of colonization and expound on Indigenous healing practices taking place in Toronto. Based on findings from a local research project, we argue these healing practices go beyond limited notions of recovery and practice, offering profound and practical ways to address the physical, emotional, spiritual and mental health of Indigenous peoples

Patterns of Gene Expression and a Transactivation Function Exhibited by the vGCR (ORF74) Chemokine Receptor Protein of Kaposi's Sarcoma-Associated Herpesvirus

The ORF74 or vGCR gene encoded by Kaposi's sarcoma-associated herpesvirus (KSHV; also called human herpesvirus 8) has properties of a ligand-independent membrane receptor signaling protein with angiogenic properties that is predicted to play a key role in the biology of the virus. We have examined the expression of vGCR mRNA and protein in primary effusion lymphoma (PEL) cell lines, PEL and multicentric Castleman's disease (MCD) tumors, Kaposi's sarcoma lesions and infected endothelial cell cultures. The vGCR gene proved to be expressed in PEL cell lines as a large spliced bicistronic mRNA of 3.2 kb that also encompasses the upstream vOX2 (K14) gene. This mRNA species was induced strongly by phorbol ester (TPA) and sodium butyrate treatment in the BCBL-1 cell line, but only weakly in the HBL6 cell line, and was classified as a relatively late and low-abundance delayed early class lytic cycle gene product. A complex bipartite upstream lytic cycle promoter for this mRNA was nestled within the intron of the 5′-overlapping but oppositely oriented latent-state transcription unit for LANA1/vCYC-D/vFLIP and responded strongly to both TPA induction and cotransfection with the KSHV RNA transactivator protein (RTA or ORF50) in transient reporter gene assays. A vGCR protein product of 45 kDa that readily dimerized was detected by Western blotting and in vitro translation and was localized in a cytoplasmic and membrane pattern in DNA-transfected Vero and 293T cells or adenovirus vGCR-transduced dermal microvascular endothelial cells (DMVEC) as detected by indirect immunofluorescence assay (IFA) and immunohistochemistry with a specific rabbit anti-vGCR antibody. Similarly, a subfraction of KSHV-positive cultured PEL cells and of KSHV (JSC-1) persistently infected DMVEC cells displayed cytoplasmic vGCR protein expression, but only after TPA or spontaneous lytic cycle induction, respectively. The vGCR protein was also detectable by immunohistochemical staining in a small fraction (0.5 to 3%) of the cells in PEL and MCD tumor and nodular Kaposi's sarcoma lesion specimens that were apparently undergoing lytic cycle expression. These properties are difficult to reconcile with the vGCR protein's playing a direct role in spindle cell proliferation, transformation, or latency, but could be compatible with proposed contributions to angiogenesis via downstream paracrine effects. The ability of vGCR to transactivate expression of both several KSHV promoter-driven luciferase (LUC) reporter genes and an NFκB motif containing the chloramphenicol acetyltransferase (CAT) reporter gene may also suggest an unexpected regulatory role in viral gene expression

Whole-genome sequencing of patients with rare diseases in a national health system

Most patients with rare diseases do not receive a molecular diagnosis and the aetiological variants and causative genes for more than half such disorders remain to be discovered1. Here we used whole-genome sequencing (WGS) in a national health system to streamline diagnosis and to discover unknown aetiological variants in the coding and non-coding regions of the genome. We generated WGS data for 13,037 participants, of whom 9,802 had a rare disease, and provided a genetic diagnosis to 1,138 of the 7,065 extensively phenotyped participants. We identified 95 Mendelian associations between genes and rare diseases, of which 11 have been discovered since 2015 and at least 79 are confirmed to be aetiological. By generating WGS data of UK Biobank participants2, we found that rare alleles can explain the presence of some individuals in the tails of a quantitative trait for red blood cells. Finally, we identified four novel non-coding variants that cause disease through the disruption of transcription of ARPC1B, GATA1, LRBA and MPL. Our study demonstrates a synergy by using WGS for diagnosis and aetiological discovery in routine healthcare