Browsing by Author "Belisle, John, committee member"

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Open Access
Establishing base-catalyzed halogen transfer as a general platform for C–H functionalization
(Colorado State University. Libraries, 2024) Bone, Kendelyn I., author; Bandar, Jeffrey, advisor; Crans, Debbie, committee member; Henry, Chuck, committee member; Belisle, John, committee member
In contrast to traditional multi-step routes, C–H functionalization offers a resource and time efficient route to desired products. Current methods for oxidative C–H functionalization are developed on three predominate reactivity platforms (1) hydrogen atom transfer, (2) single- electron transfer, and (3) C–H insertion. Despite their synthetic power, methods built on these platforms are restricted to similar bond conversions, substrates, and selectivities. Thus, there remains a strong demand for new mechanistic approaches to oxidative C–H functionalization that offer a departure from traditional reactivity. In efforts to address this need, new methods for oxidative coupling based on a base-catalyzed halogen transfer (X-transfer) reactivity platform are described herein. Chapter one provides an overview on the development of a X-transfer enabled direct C–H hydroxylation of mildly acidic N-heteroarenes and benzenes. Hydroxylated (hetero)arenes are valued in many industries as both key constituents of end products and diversifiable synthetic building blocks. Accordingly, the development of reactions that complement and address the limitations of existing methods for the introduction of aromatic hydroxyl groups is an important goal. To this end, this chapter discusses the development of a protocol that employs an alkoxide base to catalyze X-transfer from sacrificial 2-halothiophene oxidants to aryl substrates, forming SNAr-active intermediates that undergo nucleophilic hydroxylation. Key to this process is the use of 2-phenylethanol as an inexpensive hydroxide surrogate that, after aromatic substitution and ii rapid elimination, provides the hydroxylated arene and styrene byproduct. Use of simple 2-halothiophenes allows for C–H hydroxylation of 6-membered N-heteroarenes and 1,3-azole derivatives, while a rationally designed 2-halobenzothiophene oxidant extends the scope to electron-deficient benzene substrates. Mechanistic studies indicate that aromatic X-transfer is reversible, suggesting that the deprotonation, halogenation, and substitution steps operate in synergy, manifesting in unique selectivity trends that are not necessarily dependent on the most acidic aryl position. The utility of this method is further demonstrated through streamlined target molecule syntheses, examples of regioselectivity that contrast alternative C–H hydroxylation methods, and the scalable recycling of the thiophene oxidants. Chapter two describes the elaboration the X-transfer enabled C–H functionalization platform to encompass benzylic C(sp3)–H bonds. Thus, a benzylic C–H oxidative coupling reaction with alcohols that proceeds through a synergistic deprotonation, halogenation and substitution sequence is discussed. In contrast to existing radical-based pathways for C–H functionalization, this process is guided by C–H acidity trends. This gives rise to new synthetic capabilities, including the ability to functionalize diverse methyl(hetero)arenes, tolerance of oxidizable and nucleophilic functional groups, precision regioselectivity for polyalkylarenes and use of a double C–H etherification process to controllably oxidize methylarenes to benzaldehydes.
Open Access
Exosomes: a potential novel source of biomarkers for tuberculosis
(Colorado State University. Libraries, 2017) Diaz, Gustavo, author; Dobos, Karen M., advisor; Kruh-Garcia, Nicole, advisor; Belisle, John, committee member; Gonzalez-Juarrero, Mercedes, committee member; Di Pietro, Santiago, committee member
To view the abstract, please see the full text of the document.
Open Access
Investigating the biochemistry and genetics of chrysolaminarin metabolism in a model marine diatom
(Colorado State University. Libraries, 2017) Caballero, Michael Adan, author; Peers, Graham, advisor; Belisle, John, committee member; Bush, Daniel, committee member; Prasad, Ashok, committee member
To view the abstract, please see the full text of the document.
Open Access
Molecular characterization of novel transcription of antisense toxin-antitoxin RNA in regulating Mycobacterium tuberculosis
(Colorado State University. Libraries, 2020) Dawson, Clinton C., author; Slayden, Richard A., advisor; Basaraba, Randall, committee member; Belisle, John, committee member; Karkhoff-Schweizer, RoxAnn, committee member; Tjalkens, Ronald, committee member
Despite more than seventy years of available anti-tuberculosis (TB) treatments, Mycobacterium tuberculosis (Mtb) remains the deadliest human pathogen. Novel short-course therapies are needed that effectively treat latent TB infection (LTBI), which is like a major source for new infections. However, the molecular determinants of LTBI, including a large repertoire of regulators encoded by Mtb that mediate survival, are largely uncharacterized. Gene expression studies have implicated numerous regulators and particularly toxin-antitoxin (TA) systems in Mtb pathogenesis. Whole genome sequencing (i.e. WGS) studies have linked the massive genomic expansion of TA systems along with other pathogen-specific gene families to the emergence of TB-causing mycobacteria. In addition, a multitude of TA systems show genotypic differences that distinguish between ancient and modern lineages of Mtb. These predominantly include lineage-specific changes in amino acids, altering antitoxin DNA-binding, and nucleotides, generating new promoters. These mutations have led to an overrepresentation of differentially expressed Mtb TA genes responsible for mediating epigenetic changes that are associated with gains in virulence of modern lineages. Thus, the work presented in this dissertation begins to define the novel co-regulation of TA systems that underlie Mtb pathogenesis. Unraveling of more complex regulation of Mtb TA systems will provide keen insights into the phenotypic changes responsible for Mtb survival and persistence in vivo. This will ultimately help to streamline research and development of novel antibiotics as well as host directed immunotherapies against hard-to-treat tubercle bacilli, effectively shortening the duration of TB treatment. TA systems are ubiquitous among bacteria, especially pathogens, and increasingly found to be essential for adaptation to host immune defenses and in vivo drug pressures, resulting in the development of persistent or chronic infections. Phylogenomics comparisons have revealed that Mtb encodes a significantly expanded repertoire of TA systems that are solely conserved by tubercle bacilli, including homologous ParDE/RelBE systems like RelBE2 (i.e. Rv2865-Rv2866). Herein, we report a novel antisense (as)RNA, we call asRelE2, which is uniquely encoded by Mtb and involved in differentially post-transcriptionally regulating relE2 mRNA levels as part of the response to host-associated stress such as low pH in a cAMP-dependent manner. This dynamic regulation of the tripartite relBE2/asrelE2 TA locus appear to be essential for long-term survival under acidic stress in vitro. In addition, the overexpression of relE2 is found to mediate phenotypic development of a persistent state in Mtb associated with increasing tolerance towards frontline anti-TB drugs isoniazid (Inh) and rifampicin (Rif). In mice, asRelE2 acts in differentially regulating bi-cistronic relB2 and relE2 mRNA levels in a host tissue-specific manner dependent upon the downstream effector functions of interferon gamma (i.e. IFN-γ) in murine TB. Specifically, relE2 and relB2 mRNA levels are found to steadily increase in lungs and in spleens, respectively, in the development of the chronic phase of Mtb infection. To our knowledge, this is the first time a Mtb TA system has been shown to be co-regulated by an asRNA antitoxin. Furthermore, this is linked with the development of the adaptive host immune response to Mtb, demonstrating that the post-transcriptional regulation of TA systems is an important mechanism, coordinating the epigenetic changes that are a hallmark of Mtb persistence and pathogenesis.
Open Access
Reframing viral infections as acute metabolic disorders: dengue viruses and their dependency on host metabolic pathways
(Colorado State University. Libraries, 2022) St. Clair, Laura A., author; Perera, Rushika, advisor; Belisle, John, committee member; Nachappa, Punya, committee member; Wilusz, Jeff, committee member; Zabel, Mark, committee member
Dengue viruses (DENVs) are the etiological agent of the world's most aggressive arthropod-borne disease. At present, there are no available antivirals against DENVs. This fact underscores a dire need to examine host-virus interactions to identify and develop novel therapeutic approaches. As obligate intracellular parasites, DENVs are reliant upon and hijack several host metabolic pathways both to fulfill their replicative needs, and to evade the host immune response. We and others have previously established that infection with DENVs causes significant perturbation to host lipid metabolism, including elevations in sphingolipids in both the human and mosquito host. In addition, we and others previously discovered that the DENV NS1 protein increases sialidase activity in both in vitro and in vivo models leading to increased endothelial hyperpermeability and vascular leakage which are hallmarks of severe dengue. To further clarify and characterize these previous works, we have performed siRNA-mediated loss of function studies using human hepatoma cells (Huh7 cells) on several metabolic pathways altered during DENV2 infection. First, we examined the role of acyl-CoA thioesterases, enzymes responsible for controlling the intracellular balance of activated fatty acids and free fatty acids, on the DENV2 lifecycle. In these analyses, we determined that the cytosolic ACOT1 enzyme had an inhibitory effect on DENV2 replication and release, while mitochondrial ACOT (ACOTs 2 and 7) functionality was critical for viral replication and release. Moreover, we identified several enzymes within the ACOT family whose expression was dependent on ACOT2 and ACOT7 expression. These results highlighted complex relationships between ACOTs and DENVs, as well as identified yet unknown functional interdependence between ACOT enzymes. Next, we expanded our previous understanding of the relationship between DENVs and the human sialidase enzymes (NEU1-4). While previously studies linked upregulation of these enzymes with DENV2 pathology, we provide the first evidence showing that NEU1-4 functionality is vital for DENV2 genome replication and viral egress. Moreover, our analyses also revealed previously unknown functionality of NEU4 or its downstream products as transcriptional regulators for NEU1-3. Finally, we provide the first profile of the effect of loss of function of enzymes within the entire sphingolipid metabolic pathway (as identified through KEGG pathway database) on the DENV2 life cycle. In this study, we identified that enzymes involved the sphingomyelinase and salvage pathways of ceramide synthesis as opposed to de novo ceramide synthesis were critical to DENV2 release from Huh7 cells. In addition, we determined that enzymes involved in the synthesis and degradation of glycosphingolipids were vital for DENV2 release. An especially intriguing result within this arm of sphingolipid metabolism was that the two enzymes which hydrolyze GluCer had differential effects on DENV2 replication and release. GBA1 (lysosomal) had an antiviral effect on DENV2, while GBA2 (non-lysosomal) was required for DENV2 replication and release. This prompted us to profile the changes that occur to glycosphingolipids (GSLs) during infection, and we uncovered several species of GSLs that are elevated during infection. Moreover, we identified that Ambroxol HCl, a pharmaceutical GBA1 chaperone/GBA2 inhibitor, was able to abrogate these elevations in GSLs. Combined, our results allowed us to propose a novel function for GBA2 as a GluCer recycling enzyme during DENV2 infection. In conclusion, together, the work in this dissertation highlights critical metabolic nodes that impact virus replication and provides new directions for investigating viral infections as acute metabolic diseases.
Open Access
Targeted computational analysis of the C3HEB/FEJ mouse model for drug efficacy testing
(Colorado State University. Libraries, 2020) Asay, Bryce Clifford, author; Lenaerts, Anne J., advisor; Belisle, John, committee member; Munsky, Brian, committee member; Lyons, Michael, committee member
Efforts to develop effective and safe drugs for the treatment of tuberculosis (TB) require preclinical evaluation in animal models. Alongside efficacy testing of novel therapies, effects on pulmonary pathology and disease progression are monitored by using histopathology images from these infected animals. To compare the severity of disease across treatment cohorts, pathologists have historically assigned a semi-quantitative histopathology score that may be subjective in terms of their training, experience, and personal bias. Manual histopathology, therefore, has limitations regarding reproducibility between studies and pathologists, potentially masking successful treatments. This report describes a pathologist-assistive software tool that reduces these user limitations while providing a rapid, quantitative scoring system for digital histopathology image analysis. The software, called 'Lesion Image Recognition and Analysis' (LIRA), employs convolutional neural networks to classify seven different pathology features, including three different lesion types from pulmonary tissues of the C3HeB/FeJ tuberculosis mouse model. LIRA was developed to improve the efficiency of histopathology analysis for mouse tuberculosis infection models. The model approach also has broader applications to other diseases and tissues. This also includes animals that are undergoing anti-mycobacterial treatment and host immune system modulation. A complimentary software package called 'Mycobacterial Image Analysis' (MIA) had also been developed that characterizes the varying bacilli characteristics such as density, aggregate/planktonic bacilli size, fluorescent intensity, and total counts. This further groups the bacilli characteristic data depending on the seven different classifications that are selected by the user. Using this approach allows for an even more targeted analysis approach that can determine how therapy and microenvironments influence the Mtb response.