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(2023) Ireland, JakeThesisPluripotent stem cell-derived cardiomyocytes (hPSC–CM) have great importance for predicting safety parameters for pharmaceutical compounds and models of healthy versus disease states of the human heart. In recent years, there has been an insistence that all new pharmaceutical products are tested on in vitro models for potential proarrhythmic effects and the increased demand for improved biomimetic hPSC-CM in pharmaceutical safety assays such as the Comprehensive in vitro Proarrhythmic Assay (CiPA). In addition, hPSC-CM are being utilised in cell therapies to treat and reverse the effects of ischaemic heart disease, offering potential cures for cardiovascular diseases instead of treatments for delaying progressive heart failure. In the first part of this thesis, I will examine how purified extracellular matrix proteins (ECMPs) can influence pluripotent stem cell (PSC) behaviour and how we may use this to precondition cardiac progenitor lineage specifications. I use array-based techniques to investigate how protein combinations affect proliferation, pluripotency, germ layer, and cardiac progenitors. This method allows us to visualise how individual proteins can affect cells' behaviour in a larger array whilst highlighting how specific combinations can precondition pluripotent cells towards a cardiomyocyte lineage. This combinatorial approach led to the identification of several unique matrices that promote differentiation, which will aid efforts at producing therapeutically useful cell types with greater efficiency. In the second part of this thesis, I demonstrate a novel bioreactor that attenuates a magnetic field to dynamically modulate the stiffness of magnetoactive hydrogel to look at how biomimetic dynamic stiffening of a substrate can influence cardiomyocyte lineage specification. We investigate how biomimetic in vivo mechanics may influence cell fate by following the expression profiles of cells in different dynamic environments. Non-invasive electromagnetic signals affect substrate stiffness when combined with magnetic particles and magnetic fibres and how this can help direct cell orientation and accompanying lineage specification Finally, I investigate how variability in cell phenotypes and expression patterns are influenced by biomimetic cues and how these variabilities could be utilised in future safety assessment protocols and cell therapy treatments for cardiovascular disease.
Design, Synthesis and Biological Evaluation of Phenylglyoxamide- and Benzothiazole-Based Small Molecular Peptidomimetics as Antibacterial Agents(2022) Yu, Tsz TinThesisThe rapid emergence and development of antibacterial resistance is a major global threat to public health. There is an urgent need for the development of antibacterial agents with novel therapeutic strategy to tackle the increasing incidence of antibacterial resistance. In recent years, antimicrobial peptides (AMPs) and their synthetic mimics have been under the spotlight of the development of a novel class of antibiotics to combat antibiotic resistance. This research project focused on the utilisation of phenylglyoxamide and benzothiazole scaffolds in the development of antimicrobial peptidomimetics. The synthesis of phenylglyoxamide-based peptidomimetics was achieved via the ring-opening reactions of N-sulfonylisatins with primary amines followed by salt formation. Minimum inhibitory concentrations (MIC) of the peptidomimetics against different bacterial strains were determined to assess their antibacterial activity. Structure-activity relationship (SAR) studies revealed the inverse relationship between the alkylsulfonyl chain length and the bulkiness of the phenyl ring system for high antibacterial activity. The most active peptidomimetics exhibited high antibacterial activity with the lowest MIC to be 4, 16 and 63 μM against S. aureus, E. coli and P. aeruginosa, respectively. These peptidomimetics also showed significant biofilm disruption (up to 50%) and inhibition (up to 70%) against S. aureus at 2–4× MIC. In addition, terphenylglyoxamide-based peptidomimetics synthesised by the ring-opening reaction of N-acylisatins with amines and amino acid esters were evaluated for their quorum sensing inhibition (QSI) activity against P. aeruginosa MH602. The most potent peptidomimetic possessed high QSI activity of 82%, 65% and 53% at 250, 125 and 62.5 μM, respectively, with no bacterial growth inhibition. On the other hand, benzothiazole-based peptidomimetics were synthesised via the Jacobson method of cyclisation of phenylthioamides, followed by the installation of cationic groups. 2-Naphthyl and guanidinium hydrochloride as the hydrophobic and cationic groups, respectively, were important for high antibacterial activity of the peptidomimetics against both Gram-positive and Gram-negative bacteria. The most potent peptidomimetics against S. aureus, E. coli and P. aeruginosa possessed MIC values of 2, 16 and 32 μM, respectively. These active peptidomimetics inhibited 39% of S. aureus biofilm formation and disrupted 42% of preformed S. aureus biofilms at sub-MIC.
(2022) Gadde, SatyanarayanaThesisHigh-risk neuroblastoma is one of the most aggressive and treatment-refractory childhood malignancies. MYCN (v-myc avian myelocytomatosis viral related oncogene, neuroblastoma derived) is a major oncogenic driver for neuroblastoma (NB) tumorigenesis. Developing direct inhibitors of MYCN has been challenging due to several limitations. Hence, targeting MYCN-binding proteins which regulate the stability of MYCN protein is a promising alternative approach. This study is aimed at developing novel inhibitors of ubiquitin specific protease 5 (USP5), a deubiquitinating enzyme, which is known to prevent MYCN protein degradation by deubiquitination. The first results chapter describes the synthesis of novel pyrido[1,2-a]benzimidazole compounds and their cytotoxicity against MYCN amplified NB cells with high expression of USP5 protein (SK-N-BE(2)-C and Kelly cells). However, none of the tested compounds displayed better cytotoxicity than the parental compound, SE486-11. The second results chapter describes a one-pot synthesis of novel γ-carbolinone, γ-carboline and spiro[pyrrolidinone-3,3′]indoles. One of the γ-carboline compounds (42d) displayed promising cytotoxicity against NB cells (SK-N-BE(2)-C (IC50 = 1.21 μM) and Kelly (IC50 = 3.09 μM)) but showed little therapeutic selectivity when compared to normal human fibroblasts, MRC-5 cells (IC50 = 3.75μM). The synthesis and cytotoxicity of novel pyrimido[1,2-a]benzimidazoles is described in the third results chapter. The active compound, 65a displayed promising cytotoxicity against SK-N-BE(2)-C (IC50 = 0.78 μM) and Kelly (IC50 = 2.00 μM) cells with a reasonable therapeutic window compared to MRC-5 cells (IC50 = 15.0 μM). 65a bound to USP5 protein by microscale thermophoresis assay (Kd = 0.47 µM). USP5 and MYCN protein levels were decreased in NB cells by treatment with 65a. Moreover, the cytotoxicity of 65a was dependant on the expression of USP5 and MYCN proteins. 65a showed synergy in combination with HDAC inhibitors, SAHA and panobinostat. In the fourth results chapter, the synthesis of more potent pyrimido[1,2-a]benzimidazoles with di- and tri- substitutions on the pendant phenyl ring (86b (SK-N-BE(2)-C IC50 = 0.31 μM; Kelly IC50 = 0.65 μM) and 91 (SK-N-BE(2)-C IC50 = 0.03 μM; Kelly IC50 = 0.07 μM)) are described. Importantly, 86b displayed significant in vivo efficacy in TH-MYCN homozygous NB mice when treated with 60 mg/kg for three weeks. The last results chapter describes the synthesis and cytotoxicity of novel benzo[4,5]imidazo[2,1-b]thiazole and pyrido[2,3-b]indole compounds. Collectively, this thesis identifies promising novel scaffolds with great potential for further development.