Background Globally, the HIV/AIDS epidemic has cost over 35 million lives and approximately a further 37 million people are currently infected with HIV. In South Africa alone, more than 7 million people are HIV positive. Since the initiation of combination antiretroviral therapy (cART), viral replication can be supressed below the limit of detection by conventional testing. There is, however, no approved therapy for the cure of HIV. This is because HIV establishes viral reservoirs in memory CD4+ T-cells, where replication is low or arrested, allowing prolonged survival. Since there is little or no replication, a therapeutic strategy which targets the viral production and replication becomes ineffective and upon cessation of antiretroviral therapy a dramatic viral relapse occurs. The eradication of HIV, therefore, requires the targeted killing of the reservoir cells, or latency reversal followed by the prevention of further infection using cART. Targeting of cell-surface antigens for therapeutic purposes is the basis of immunotherapy. FDA-approved monoclonal antibodies such as Trastuzumab have been used to treat breast cancer via the human epidermal growth factor 2 (HER2) receptor. Immunotoxins (ITs) composed of an antibody fragment fused to apoptosis-inducing protein toxins targeting cellsurface antigens have been used for therapy of refractory leukaemia. The anti-CD22 recombinant IT Moxetumomab pasudotox based on Pseudomonas aeruginosa exotoxin A (ETA) has been FDA approved to treat hairy cell leukaemia. Moxetumomab pasudotox targets the antigen CD22 found on the surface of tumour cells. The HIV neutralizing VHH-nanobody J3, isolated from an immunised Llama has demonstrated anti-HIV properties against more than 95 % of HIV strains in vitro. As part of an ongoing project to develop a J3-ETA IT, this work sought to produce a J3-SNAP fusion protein by osmotic stress expression in the presence of compatible solutes in the periplasmic space of E. coli. SNAP-tag is a self-labelling protein that covalently binds benzylguanine (BG)-modified substrates in a 1:1 stoichiometric ratio. When recombinantly fused to any protein of interest, SNAP-tag allows the stable labelling of the protein of interest of in vitro and in vivo imaging. The periplasmic space of bacteria has been reported as a dedicated compartment to express functional proteins of interest. Furthermore, osmotic stress expression in the presence of compatible solutes has been reported to result in up to a thousand-fold increase in protein yield for difficult to express proteins. This study ultimately aimed to understand whether a functional J3-SNAP or J3-ETA can be expressed under osmotic stress in the presence of compatible solutes, in the periplasmic space of E. coli. 11 Experimental work In this study, a SNAP-tag-based fusion protein and an ETA-based IT were designed using J3, an anti-HIV-1 Env VHH-nanobody isolated from an immunised llama. Using the SnapGene® software (v.5.0.8, GSL Biotech LLC, USA), in silico design and cloning of an ETA-based IT J3-ETA and SNAP-tag-based fusion protein J3-SNAP was performed. Molecular cloning of designed open reading frames (ORFs) was performed into appropriate bacterial expression plasmid vectors. Plasmid vectors confirmed to contain the required ORFs by Sanger sequencing were transformed into E. coli BL21-DE3. Histidine-tagged J3-SNAP was expressed by osmotic stress in the presence of compatible solutes. J3-SNAP was purified by IMAC and assessed by SDS-PAGE and Western blot analysis. To ascertain the binding of J3- SNAP to cells expressing HIV-1 Env in vitro, recombinant Env protein was transiently transfected into HEK293T-cells to generate an Env expressing cell line. Cell-surface binding of SNAP-Surface® Alexa Fluor® 488 -conjugated J3-SNAP on Env expressing HEK293Tcells was assessed by confocal microscopy analysis. Results Successful expression of J3-SNAP in E. coli BL21-DE3 was confirmed by SDS-PAGE and Western blot analysis. The J3-SNAP fusion protein was subsequently purified by IMAC. Purified J3-SNAP was conjugated to the benzyl guanine-modified fluorophore SNAPSurface® Alexa Fluor® 488 and full-length conjugated protein was confirmed by combinations of SDS-PAGE and Western blot analysis. Cell-surface binding of J3-SNAP to HIV-1 Env-expressing HEK293T-cells was demonstrated in vitro by confocal microscopy analysis. These results prompted the generation of the IT, J3-ETA, by replacing SNAP-tag with ETA. Conclusion Successful binding studies suggest using J3 to target HIV-1 Env. Accessing patient probes would allow for the confirmation of these results for future human applications. Future in vitro studies would need to confirm the selective elimination of Env expressing T-cells by J3-ETA and thereafter confirmed on Env-positive patient probes.

Background: South Africa has a large HIV disease burden, with the highest rate of infection occurring in young women of the childbearing age. This gave impetus to the Prevention-of-Mother-To-Child-Transmission (PMTCT) program, that has been successfully implemented. Due to the success of the PMTCT program, HIVexposed-uninfected (HEU) infants represent a growing population in South Africa. However, these infants have been found to have increased morbidity and mortality rates compared to their HIV Unexposed Uninfected (HUU) peers, as well as altered immune and vaccine responses. The reasons for this remain unclear, but one hypothesis is that altered gut microbiomes in HEU adversely affect the developing infant immune system. The microbiome (a collection of an array of microorganisms, their genes, genomes, proteomes, and metabolites) is an area of emerging research interest; dysbiosis of the gut microbiome has recently been associated with disease outcomes and progression in several disease areas. The microbial colonisation of the infant begins in utero and continues after birth. It is affected by several factors: birth mode, age of gestation, feeding mode, maternal health status as well as environmental factors. Aim: To elucidate the microbiomes of HEU infants in Africa, compared to HUU controls Design: Ultra-high-performance liquid chromatography mass spectrometry was used to analyse and characterize a subset of existing stool samples stored from the InFANT cohort study. The infant gut metaproteome of 34 HEU versus 29 HUU infants, from the South African arm of the study was analysed. Cross-sectional samples were collected and analysed at two-time points, namely at birth and within the first week of life (between 4 to 7 days after birth). Results: Comparative analysis of the HEU and HUU reveal differences in the microbial composition between the two groups at birth and day 4-7, with the most apparent difference occurring at birth. In our comparison we found that the relative abundances of Bacteroidetes and Firmicutes were different between the HEU and HUU at both birth and day 4-7. There was a dramatic shift in the microbial composition within the first week of life. Conclusion: It is evident from our analysis that the HEU infant has a different gut microbiome to that of the HUU infant at birth. The HEU microbiome is characterised by a high microbial diversity at birth. This could be associated with more severe outcomes from childhood ailments. The human breast milk (HBM) microbiome greatly influences and mitigates the differences upon subsequent breastfeeding, but differences in the measured microbiomes of HEU and HUU nonetheless remain. Recommendations: A longitudinal study should be carried out to better monitor the long-term effects of the microbiome on infant immune priming. A study of the HBM microbiome should also be investigated to better understand the role of HBM in mediating and priming the infant's immune system. Further, a study of the metabolome of the infant gut and the matching HBM of the mother may identify potential metabolites that could be used as biomarkers for vaccine responses.

Chromatin organization is at the heart of deciphering gene regulation as it is instructive to transcription. Current technological advances in next-generation sequencing approaches have offered unprecedented opportunities to interrogate the genomic landscape in multiple pathological and clinical presentations. Historically, mutations and alterations at the genomic loci of protein-coding genes were thought to be exclusively causal to many human diseases. However, the non-coding genome has emerged as the master regulator of chromatin dynamics and transcriptional activity. With cancer increasingly becoming the greatest health epidemic of our time, the comprehensive genomic characterization of tumor genotypes has become central to current therapeutic approaches. Functioning as the basic unit of chromatin organisation, chromatin loops and topologically associating domains (TADs) compartmentalize genomic loci and their corresponding molecular transcriptional elements in three-dimensional space. Transcription of the human genome is proximity-dependent requiring the cooperative engagement of non-coding elements and epigenetic modifiers to create permissive topological chromatin contacts and structures. The repertoire of chromatin contacts at any given time is regulated by the threedimensional structure and organization of the chromatin. TAD structures are formed and maintained by chromatin insulating proteins such as CTCF (CCCTC-binding factor) and multiprotein complex, cohesin. The dysfunction of which, through mutational and epigenetic aberrations, directly impacts a plethora of chromatin contacts and the resultant transcriptional profiles within each cell. Loops and TADs are formed by the binding of CTCF on the conserved 19 bp CTCF binding motif as the chromatin is protruded through the "ring-like" multi-protein complex, cohesin. When two convergently oriented and CTCF enriched CTCF-binding sites (CBSs) come into contact within the ring, cohesin is thought to "hand-cuff" the chromatin resulting in the formation a chromatin loop. These loop structures then serve to compartmentalize and restrict the chromatin contacts and their frequency within each loop. Promoter-resident CBSs can also function as "docking sites” for tissue- and context-specific enhancers. The dysregulation of CTCF binding has been repeatedly demonstrated to directly alter chromatin contacts in a vast array of cellular contexts including cancer. Fundamentally, CTCF functions as a potent regulator of chromatin contacts, which directly instruct transcriptional status. Thus, CTCF binding has become an attractive regulatory target for manipulating the topological and transcriptional activity of chromatin. In this study, we sought to identify CBS swith differential, specifically abrogated CTCF enrichment that may be hijacked by oncogenes in an attempt to modify transcriptional programmes to favour cancer progression. To this end, we developed an integrated bioinformatic pipeline to identify promoter-associated lower-CTCF enrichment sites (PA-LCes) in colorectal cancer (CRC) cell lines as compared to primary colonic tissue from CTCF ChIP-Seq data. With ever-growing catalogues of nextgeneration sequencing datasets, including ChIP-Seq, in the public domain, the use of ENCODE datasets proved to be an economical option and added layer of standardization in our analysis. Briefly the pipeline developed in this study takes ENCODE ChIP-Seq FASTQ files from the NCBI SRA using fastqdump as input files. The FASTQ files undergo a quality control and dataset filtration with FASTQC. The filtered datasets are then aligned to the hg38 human genome and fed back into FASTQC to ensure aligned reads pass quality control metrics. The mapped reads are then processed using samtools and duplicate reads are marked with the picard markduplicates argument. Narrow peaks are then called from processed reads using MACS2 and processed using bedtools. Called peaks then undergo a final quality control step using ChIPQCr and are visualized using IGV before undergoing differential enrichment analysis. Differential CTCF enrichment analysis between the peaks in primary sigmoidal colon cells and CRC cell lines is then conducted using DeSeq2 within DiffBind. Lower CTCF enrichment peaks are then used for the discovery of the canonical CTCF MA00139.1 motif using homer and compared to similar annotations in the primary consensus peakset. The resultant lower CTCF enrichment peaks are then annotated using homer and ChiPpeakAnno to determine their genomic locations and extract LCes located proximal (<1kb) to annotated TSS or promoter regions i.e. PA-LCes. The PA-LCe discovery pipeline developed in this study is highly robust, resulting in some previously validated CBSs implicated in oncogenesis. Intriguingly, the PA-LCe sites identified in this study emanate from bidirectional promoters at oncogenes with differential methylation and transcriptional patterns in cancer. Additionally these PA-LCes transcribe antisense lncRNAs such as the tumor-suppressive aslncRNA ZNF582-AS1. This data adds to the recent body of evidence that suggests that disruption of promoter-associated CBSs leads to fluctuations in promoter activity. Recent studies have implicated the requirement of CTCFlncRNA complexes at promoter regions in facilitating and regulating CTCF docking on chromatin which subsequently influences transcriptional activity. In accordance with this, our data suggests that the lncRNAs at PA-LCe loci may be molecular targets for the regulation ofCTCF binding and transcriptional activity in CRC. Perturbation of CTCF enrichment at PALCes in CRC result in differential chromatin contacts, epigenetic context and, the transcriptional activity of the promoters in which they reside. As CTCF binding at CBSs sites is highly modular, the use of targeted CRISPR-mediated gene-editing and DNA methylation at PA-LCe CBSs may represent viable and druggable oncogenic targets.

Angiotensin converting enzyme (ACE) is a zinc metalloprotease comprised of two highly homologous, catalytically active domains (90% active site identity and 60% sequence similarity). The C-domain is responsible for blood pressure regulation via angiotensin I cleavage while the N-domain inactivates an antifibrotic peptide Acetyl-Ser-Asp-Lys-Pro (AcSDKP). Since selective N-domain inhibition will result in AcSDKP accumulation, it shows promise for the treatment of fibrosis without affecting blood pressure. Low bioavailability, however, precludes the use of currently available N-selective ACE inhibitors in a clinical setting. Inhibition of ACE by a phosphinic, peptidomimetic compound, 33RE, was characterized using a continuous assay with quenched fluorogenic substrate. The N-domain displayed nanomolar (Ki = 11.21±0.74nM) and the C-domain micromolar (Ki = 11 278±410nM) inhibition, thus 1000-fold selectivity. Residues predicted to contribute to selectivity based on the N-domain-33RE co-crystal structure were subsequently mutated to their C-domain counterparts. S2 subsite mutation with resulting loss of a hydrogen bond drastically decreased 33RE affinity (Ki = 2794±156nM), yet did not entirely account for the selectivity. Additional substitution of all unique S2’ residues, however, completely abolished N-selectivity (Ki = 10 009±157nM). Interestingly, these residues do not directly bind 33RE. All mutants were therefore subjected to molecular dynamics (MD) simulations in the presence and absence of 33RE in addition to co-crystallization of 33RE with the N-domain mutant having all S2 and S2’ residues mutated. Trajectory analyses highlighted the S2’ residues’ importance in formation of a favourable interface between the ACE subdomains and thus a closed, ligand-bound complex. This was supported by X-ray crystallography and provides a molecular basis for the inter-subsite synergism governing 33RE’s 1000-fold N-domain selectivity. Enzyme kinetics were also used to study the concentration-dependent competitive inhibition and time-dependent irreversible oxidative inactivation of ACE catalysed by the Cu-Gly-GlyHis-lisinopril (CuGGHLis) metallodrug. Although both domains displayed nanomolar affinity for metallodrug binding (N-domain Ki = 44.94±1.84nM and C-domain Ki = 15.57±1.30nM), rapid and complete CuGGHLis-mediated inactivation occurred exclusively in the N-domain upon incubation with ascorbate and H2O2 redox co-reactants (k2 = 59 710 M-1 min-1 ). Michaelis-Menten characterization of the residual activity after partial N-domain inactivation revealed a decreased rate for hydrolysis of a non-domain selective substrate. This suggests that although CuGGHLis binds with similar affinity to both domains, the metal-chelate is optimally orientated in the N- but not the C-domain to catalyze oxidation of residues involved in substrate hydrolysis. The C-domain, in contrast, showed increased susceptibility to oxidative inactivation by diffuse radicals. This is of physiological significance as C-domain inactivation in normotensive individuals could result in accumulation of pro-inflammatory peptides. Since the N-domain is more heavily glycosylated, the potential role of unique glycans in diffuse radical shielding was studied using glycoprotein MD simulations. Unique C-domain solvent tunnels were identified that could increase diffuse radical access and, additionally, the mechanism whereby glycosylation contributes to ACE thermal stability was described for each site. This has implications for future ACE crystallography studies and the design of ACE-modulating agents with potential anti-inflammatory activity. This study demonstrated the utility of combining in vitro and in silico approaches to reveal how subtle amino acid or glycosylation site differences between the highly homologous domains control dynamic behaviour. It furthermore elucidated how two inhibitors with different mechanisms of action selectively target the N-domain active site by exploiting these differences and provided valuable insight for future anti-fibrotic ACE inhibitor design.

Tuberculosis is a curable disease with an average treatment success rate of 86 %. Despite this, there were an estimated 1.5 million deaths due to tuberculosis in 2013, most of which occurred in low and middle income countries. In order to overcome tuberculosis in developing countries innovation in diagnostics is key to administering treatment. While detection of whole mycobacteria has been favoured in the past to diagnose tuberculosis, culturing mycobacteria is costly and microscopy is often not sensitive enough due to low bacterial loads. Detection of Mycobacterium tuberculosis biomarkers in urine, a safe and easy specimen to test, could offer a cost effective and simple solution to identify patients with tuberculosis. Enzyme linked immunosorbent assays (ELISAs) were performed on concentrated tuberculosis patient urine to detect two M. tuberculosis biomarkers: lipoarabinomannan (LAM) and early secreted antigen-6 kDa (ESAT-6). Concentrating urine improved the detection of LAM in human immunodeficiency virus (HIV) negative patients and patients with a CD4 count > 200 cells/µl. ESAT-6 was not detected by ELISA due to a high background signal caused by the available antibodies cross reacting with a human protein present in urine which was identified by western blot and mass spectrometry. Targeted mass spectrometry did not detect ESAT-6 or its dimer partner, culture filtrate protein-10 kDa (CFP-10) in tuberculosis positive patient urine. Since concentrating urine samples is impractical in a clinical setting a more sensitive diagnostic is needed to detect LAM in urine and ESAT-6 or CFP-10 in other samples. Aptamers can be packed more densely on biosensor surfaces increasing the dynamic range of detection while matching the affinity that an antibody has for a biomarker. Chemically modified DNA aptamers were isolated for LAM and the ESAT-6.CFP-10 dimer. The aptamers were characterised by enzyme linked oligonucleotide assays (ELONAs) and biolayer interferometry. One aptamer bound with high affinity to ESAT-6 while one aptamer bound with low affinity to LAM. The use of aptamers as capture agents for detecting biomarkers in biological specimens thus appears to be a viable option for diagnosing tuberculosis, although availability and concentration of individual biomarkers seems likely to remain key to the choice of specimen in which to make diagnostic measurements.

Cutaneous melanoma is the deadliest form of skin cancer, which arises from epidermal pigment-producing cells called melanocytes. In melanoma, surgical excision of primary nonmetastatic tumor remains the gold standard of therapy worldwide. Upon metastases, melanoma becomes highly resistant to conventional radio-and chemotherapy. Chemotherapy, using dacarbazine (DTIC), remains the standard treatment option. In melanoma, chemotherapy failure has partly been attributed to a resistant population which is endowed with higher clonogenic potential, and aberrant expression of membrane proteins known as ABC transporters (ABCB5 and ABCG2), which mediate cellular resistance by extruding cytotoxic molecules from cells. To palliate these adverse effects, this study primarily aimed to investigate the efficacy of the synergistic actions of chemotherapy (Dacarbazine:DTIC) and hypericin-activated photodynamic therapy (HYP-PDT) in reducing chemoresistance in DTIC resistant (UCT Mel1DTICR2) and non-resistant melanoma cells (UCT Mel-1). To achieve these goals, the therapeutic efficacy of conventional therapies (DTIC, HYP-PDT and DTIC+HYP-PDT) was evaluated based on their ability to reduce cell viability; therapeutic resistant subpopulations; clonogenicity and ABC transporters (ABCB5 and ABCG2) in both melanoma cells. Additionally, the ability of the therapeutic treatments to efficiently halt cell division and activating cell death mechanisms, was assessed using cell cycle analysis and an Annexin-V assay. The results obtained showed that combination therapy was the most efficient therapy which was associated with a reduction in main populations (therapeutic resistant sub-population not expressing ABC transporters: MP), and clonogenic capacity in both melanoma cell types. Similarly, DTIC displayed a therapeutic efficacy which significantly reduced side populations (therapeutic resistant sub-population which were expressing ABC transporters: SP), and clonogenicity in UCT Mel-1 only. Interestingly, both ABCG2 and ABCB5 expressions were significantly increased in both melanoma cells, post combination therapy. Lastly, combination therapy and PDT were equally shown to induce a G1 cell cycle arrest, as opposed to DTIC which induced an S phase arrest. These cell cycle arrests were associated with efficient activation of apoptosis and necrosis, depending on the melanoma cell type, post HYP-PDT and combination therapy. Nevertheless, the efficacy of DTIC and HYP-PDT might respectively be limited by off target effects harming normal cells and low dosage in tumour cells which limiting their clinical utility. To address these challenges, this study aimed to develop a targeted tumor therapy, using the self-labelling activity of SNAP-tag fusion protein to conjugate synthetic small molecule lead substance toxin such as monomethyl Auristatin F (MMAF or AURIF). This cytotoxic payload was delivered to targeted cells, through genetic fusion of SNAP-tag to three different single chain fragments of an antibody (scFv1711, LsFv49 and scFv#34), which specifically treated tumor cells expressing epidermal growth factor (EGFR), melanotransferrin (p97) orfibroblast activation protein alpha (FAP-α) receptors, respectively. Achievement of this targeted therapy was performed through construction of three scFv-SNAP fusion proteins, which were expressed in HEK 293T cells. Thereafter, the purified scFv-SNAP fusion proteins were conjugated to BG-substrates to investigate their efficacy in specifically killing tumor cells. Binding and cytotoxic activities of the scFv-SNAP fusion proteins were performed using flow cytometry, and the XTT cell viability assay. All scFv-SNAP-fusion proteins were specifically bound to their target cells, indicating that AURIF conjugation did not compromise the binding activity of scFv-SNAP fusions. Finally, the cytotoxic assay confirmed that all scFv-SNAPAURIF conjugates induced a 50 percent reduction in cell viability at nanomolar concentrations in targeted cells which expressed their cognate antigens. To conclude, combination therapy was shown to be more efficient than monotherapies in killing chemoresistant melanoma cells, while SNAP-tag technology provided a superior, targeted therapeutic efficacy by sparing normal cells from unwanted toxic effects, and reducing the therapeutic requirement for high concentration of cytotoxic payloads.

Basal cell carcinoma (BCC) is the most common skin cancer worldwide, with South Africa having the highest incidence rate only after Australia. The most effective treatment modality for BCC is tumor excision via Mohs surgery (pioneered by Dr. Frederic Mohs of the University of Wisconsin in 1930), a microscopically controlled surgery that removes a tumor piecemeal in layers until each layer is free of any neoplastic tissue. The major drawback of Mohs excision is that the surgeon might miss any neoplastic tissue as the tumor margin is not always well defined, and the tumor often could extend beyond the superficial layers of skin. Moreover, it's a time-consuming, expensive procedure that takes generally 3-4 h, at times even more, if several rounds of excisions are warranted. In South Africa, at the time of writing, therapy using the surgery cost around R45,000. The status quo thus necessitates identifying BCC cells both in the superficial layers and beyond the layers of the skin in individual patients. Our aim was to identify BCC-specific cell surface proteins and design, engineer, and test a range of SNAPtag–based antibody fusion proteins that would specifically bind to and detect such BCC cell surface receptors. The SNAP-tag antibody technology is based on the genetic fusion of a disease-specific ligand to a protein tag derived from O6-alkylguanine-DNA alkyltransferase, which would allow for covalent auto-labeling of the corresponding antibody based fusion proteins with benzylguanine-modified (BG) substrates (e.g., fluorophores) under physiological conditions with high efficiency at 1:1 stoichiometry. This would allow to develop a unique immunological screening modality which should allow to visually label BCC lesions for a more precise surgical excision. The best-performing SNAP-tag–based diagnostic antibodies resulting from these studies would be further evaluated in the future in suitable mouse models, thus aiming to reduce the time needed for surgical removal of BCC lesions and complete removal of the tumor from both superficial and deep layers of the skin by a single-excision procedure. We used an integrated computational tool to re-analyze publicly available cDNA microarray data in combination with theoretical search to identify BCC-associated antigens. Accordingly, six different antigens were selected and single-chain variable fragments (scFv) targeting these antigens were cloned in fusion with SNAP-tag encoding gene into a custom expression vector for production in a secretory mammalian system. scFv-SNAP-tag protein was isolated from the cell culture supernatant by immobilized metal affinity chromatography and eluted protein samples were analyzed by gel electrophoresis and immunoblotting. The absolute amount of the full-length protein was quantified by densitometry. Purified scFv-SNAP-tag proteins were validated for specific binding to corresponding antigen-positive cells by flow cytometry and confocal microscopy. Of the six different scFv-SNAP-tag fusion proteins cloned, four were successfully expressed in HEK293T cells. The specific binding to EpCAM, EMA, CSPG4, and CD138 antigenexpressing cell lines was observed on incubation with scFvUBS54-SNAP-tag, scFvID405- SNAP-tag, 9.2.27scFv-SNAP-tag, and scFvh-STL002-SNAP-tag, respectively. In addition, we showed the selective cell killing effect of scFvUBS54-SNAP after conjugating it with the cytotoxic drug BG-modified auristatin-F (BG-AF). In conclusion, we identified various cell surface antigens along with one possibly novel antigen for BCC detection and therapy. Further, we successfully designed and synthesized SNAP tag based antibody fusion proteins and showed their functional activity by selective binding to the corresponding antigens on the surface of tumor cells. Based on these findings, we presume that these antibodies can effectively bind to BCC and can confirm EpCAM as one of the target antigens, which has already been reported to be a standard immunophenotypic marker for differential BCC diagnosis.

A range of fluorescein derivatives were synthesised via Pd0-mediated cross-coupling chemistry of the mono triflate of fluorescein with a variety of boronic acids and the optical properties of each dye was studied. Among these derivatives, a new multicolour pH–dependant anthofluorescein which was highly sensitive to pH changes and viscosity changes was identified. Work was carried out to explore intracellular catalysis based on the immobilisation of Pd0 nanoparticles on microspheres. The entrapped Pd0 nanoparticles were rapidly taken up by cells, stay harmlessly within the cytoplasm for days and were shown to carry out novel cell-based chemistry. This included an allylcarbamate cleavage and a Suzuki-Miyaura cross-coupling reaction for the in situ generation of a mitochondria-localized “switch-on” fluorophore.

The sequencing of individual DNA strands with nanopores is being developed as a rapid, low-cost platform in which bases are identified in order as a DNA strand is transported through a pore under an electrical potential. Several challenges remain and this thesis focuses on one major area, the base identification properties of the a-hemolysin (aHL) nanopore. Under the potentials required for threading, DNA translocates too fast for single bases to be identified. However, immobilization of the DNA within the pore increases the residence time and therefore improves the precision of the electrical current reading and allows for the small differences in current flow, associated with different sequences, to be observed. DNA molecules with a 3'-terminal biotin-tag were complexed with streptavidin. Streptavidin is too large to be transported through the aHL pore and therefore the DNA-btnestreptavidin complex is not fully translocated; thus, the DNA strand is immobilized within the pore. Using this approach the nucleobase recognition properties of the aHL pore were mapped. The data suggest that the transmembrane 13 barrel domain of the pore contains at least three nucleobase recognition sites, termed R1, R2 and R3. Additional sequence information can be gained when multiple recognition sites are employed within a single aHL pore, as compared to the simple case of a single recognition site. Recognition site R1, which is located near the central constriction, can be modified by site-directed mutagenesis of Met-113. It was observed that amino acids with related side chains produce similar patterns of nucleobase recognition. Amino acids that provide an energy barrier to ion flow (e.g. bulky or hydrophobic residues) strengthen base identification, while amino acids that lower the barrier, weaken identification. Deletion and site-directed mutagenesis were used to remove one recognition site and generate an αHL pore. With truncated β barrel domain that contains only two recognition sites.

Thesis advisor: Eranthie Weerapana / Functional amino acids that play critical roles in catalysis and regulation are known to display elevated nucleophilicity and can be selectively targeted for covalent modification by reactive electrophiles. Chemical-proteomic platforms, such as activity-based protein profiling (ABPP), exploit this reactivity by utilizing chemical probes to covalently modify active-site residues to inform on the functional state of enzymes within complex proteomes. These and other applications rely on the availability of a diverse array of electrophiles and detailed knowledge of the reactivity and amino-acid specificity of these groups. The sulfonyl fluoride activity-based probe (ABP) DAS1 was discovered to label and inhibit both serine proteases and glutathione S-transferases (GSTs). In the case of GSTs, DAS1 covalently bound to a tyrosine residue, despite predicted serine reactivity. Investigation of potential aryl halide electrophiles for ABPP found that chloronitrobenzene RB2 and dichlorotriazine RB7 covalently modify cysteine and lysine residues in target proteins. Applying an existing ABP, iodoacetamide alkyne (IA-alkyne), demonstrated the ability of ABPP to discover novel reactive residues in short open reading frame (sORF)-encoded peptides, as well as previously unannotated cysteine residues on glycolysis enzymes. These studies illustrate the development and characterization of novel electrophiles and demonstrate the application of ABPs to interrogate biological systems. Looking further ahead, the novel electrophiles also provide new tools for the development of covalent inhibitors for treatment of disease. / Thesis (PhD) — Boston College, 2015. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.