<p>Sol-gel derived silica provides a bio-compatible material for the solid-phase entrapment of viable cells. A selection of <em>E. coli</em> cells containing unique promoter-linked GFP expression vectors were applied to fluorescence microwell plate assays, plate counting and various microscopy methods to assess changes in the entrapped bacteria and compatibility towards compound screening. Materials screening showed that a fastgelation sol-gel composition from sodium silicate precursor and PBS buffer provided a consistently greater fluorescence signal than non-entrapped cells. It is shown for the first time that entrapped cells are capable of dividing within pockets of the silica gel, and can di vide at a comparable rate to free cells. The entrapment of cells within a silica matrix does not induce the basal expression level of promoters tested here. Silica entrapment provides improved storage capabilities over non-entrapped cells in solution. A set of 12 related GFP-linked promoters were induced in solution and within silica when screened by two DNA gyrase inhibitors, providing similar expression profiles but greater signal-tonoise ratios in silica. The sol-gel derived material is amenable in an array format, and is a prospective material for the fabrication of sol-gel cell microarrays.</p> / Master of Science (MSc)

<p>Using the model organism <em>Escherichia coli</em>, we discuss herein two novel antimicrobial targets: namely, the protein YjeE and the process of ribosome assembly.</p> <p>YjeE is essential for viability and widely conserved amongst bacterial pathogens and has no human homologue. We searched for a small molecule probe of the function of YjeE to help circumvent the inadequate genetic tools that are available for studying this protein. Sensitive methods for detecting ligand binding were optimized; however, this effort yielded no inhibitors. A second approach to studying the function of YjeE was the development of a reporter using a promoter that is directly upstream of <em>yjeE </em>in <em>E. coli</em>. The activity of this promoter was tested in the presence of small molecules of known function and in diverse gene deletion backgrounds. YjeE found to be linked to the inhibition of DNA and protein translation as well as central metabolism and respiration. These interactions prompted experiments that revealed YjeE to be dispensable under anaerobic conditions.</p> <p>Many antibiotics target ribosomal protein synthesis; however, no current antibiotics target the process of ribosome biogenesis. In order to identify new biogenesis factors, the non-essential fraction of the <em>E. coli </em>genome was screened for deletions that gave rise to cold-sensitive growth. We found that genes associated with ribosome function were the most represented cold sensitive factors amongst the genes of known function. We identified and present here two new putative ribosome biogenesis factors, <em>prfC</em> and <em>ychF</em>, which had phenotypes associated with ribosome assembly defects.</p> / Doctor of Philosophy (PhD)

Technetium-99m (99mTc) radiopharmaceuticals are widely used for diagnostic imaging of heart, kidney, and liver disease, and cancer. Evolution from perfusion type tracers to targeted agents however has proven difficult. 99mTc labeled antibodies for imaging specific disease biomarkers would be of great interest, however the disparity between the isotopes half-life (6 hours) and the long circulation time of most antibodies (multiple days) has been a significant barrier. Furthermore, the conjugation of bifunctional 99mTc-chelate complexes to small molecules often has a detrimental impact on targeting. The use of bioorthogonal chemistry derived from tetrazines and trans-cyclooctene derivatives, along with pretargeting has the potential to overcome these issues and create a new generation of targeted 99mTc radiopharmaceuticals. Initially, the synthesis of three generations of imidazole based tridentate chelates linked to a tetrazine was completed. These new ligands were labeled with 99mTc under mild conditions (60 °C, 20 min, pH 3.5) with modest to good radiochemical yields ranging from 31 to 83%. Biodistribution studies revealed that compound 14, which contains a polyethylene glycol 5 (PEG5) linker had the best clearance from non-target tissues. Compound 14 was also used successfully in a pretargeting strategy along with a transcyclooctene (TCO) derivative of the bone targeting bisphosphonate, alendronate (ALN). One hour following the administration of TCO-ALN to BALB/c mice, compound 14 was injected intravenously where uptake at sites of high calcium turn over (i.e. the joints) was observed. At 6 hours post injection, for example, uptake reached as high as 20.1 ± 4.91 and 16.1 ± 4.84 %ID/g in the knee and shoulder, respectively. Pretargeted imaging studies were performed subsequently with a TCO-functionalized huA33 antibody in mice bearing SW122 xenografts. The TCO-huA33 antibody was injected 24 hours before the administration of two radiolabeled tetrazines at high and low specific activities. At 6 hours post injection tumour uptake was minimal, with tumour: blood ratios <1 in all cases. Blood clearance studies determined that the tetrazines were being cleared rapidly, with a blood residence half-life of 1.3-2.1 minutes. The hypothesis is that the low concentration of the antibody (owing to its high molecular weight), combined with the rapid clearance of the tetrazine and significant off-target uptake resulted in unfavorable kinetics and low tumor binding. Studies of the clearance pathway of 14 were investigated with clinically approved hepatobiliary transport inhibitors to help understand the mechanism of clearance, which could in turn be used to optimize the pharmacokinetics of the tetrazine ligands. A range of different inhibitors of key clearance pathways were evaluated with limited success. However, co-administration of 14 with ALN resulted in a 75% decrease in gall bladder uptake of 14 (216 ± 75.9 to 33.6 ± 3.93 %ID/g). Pretargeting studies of 14 with TCO-ALN in the presence of excess ALN revealed that ALN did not hinder the uptake of TCO-ALN in the bone, with all organs and tissues having the same uptake with TCO-ALN or TCO-ALN + ALN (knee: 20.1 ± 4.91 and 14.9 ± 2.43 %ID/g, respectively). There was also a concomitant decrease in gall bladder uptake (91.5 ± 17.1 to 28.8 ± 2.63 %ID/g). Further work on improving the distribution of the tetrazine ligands involved investigating the effect of the chelate. The core chelate found in 14 without the tetrazine moiety (compound 11a) was labeled with 99mTc to produce 11b in a 31% radiochemical yield. Biodistribution studies of 11b and 14 at 6 hours post injection demonstrated that the imidazole-based 99mTc-chelate was a major factor in the rapid and significant uptake and retention in the liver and gallbladder. A new triazole based chelate with optimal clearance from Kluba and coworkers was synthesized in 45% yield and successfully labeled with 99mTc (compound 23a). Biodistribution studies were performed where at 6 hours post injection, 23a had five times lower uptake in all non-target organs compared to 11b. The synthesis of a tetrazine derivative of 23a (compound 32) unfortunately demonstrated high hepatobiliary uptake compared to the original triazole chelate (gall bladder: 228 ± 251 and 8.77 ± 0.73 %ID/g, large intestine: 85.5 ± 83.5 and 6.88 ± 0.30 %ID/g, respectively). This particular derivative had a lipophilic linker as a result of the synthetic challenges faced during the preparation of a more hydrophilic triazole-tetrazine derivative. In addition to pretargeting applications, the 99mTc-tetrazine was used as a reagent to create multimodal imaging agents. Nanoscale gas vesicle (GV) ultrasound contrast agents were functionalized with TCO via an amide coupling to lysine residues. TCO-GVs were then radiolabeled by adding compound 6 where the desired product, a new multimodal probe, was obtained in 59% radiochemical yield. SPECT imaging and biodistribution studies in mice were completed where the labeled GV’s showed uptake in the gall bladder (120 ± 29.1 %ID/g), liver (16.8 ± 7.50 %ID/g), lungs (3.26 ± 1.53 %ID/g), small intestines (14.5 ± 5.30 %ID/g), and spleen (5.47 ± 2.71 %ID/g) at 120 min post injection. In addition to radiolabelling, the TCO-GVs were also functionalized with a near IR-tetrazine dye to produce a multimodal ultrasound/photoacoustic (US/PA) imaging agent in a 68% yield. / Thesis / Doctor of Philosophy (PhD)

<p dir="ltr">Post-translational modification (PTM) is an important biological process by which cells regulate their signaling pathways. De-regulation of these signaling pathways often leads to many diseases. Protein AMPylation is a recently discovered PTM that caught a great amount of attention for its involvement in neurodevelopment and neurodegeneration. However, the mechanisms by which protein AMPylation modulates these biological processes remain mostly unknown. FIC domain protein adenylyltransferase (FICD)<b> </b>is one of the only two known AMPylators in eukaryotes, and its physiological role remains largely unexplored. By using a chemical approach, we identified two direct substrates of FICD: Peroxiredoxin 1 (PRX1) and Peroxiredoxin 2 (PRX2). These are antioxidant enzymes responsible for protecting cells from oxidative stress, which has been implicated in many neurodegenerative diseases. In addition, we found that FICD-mediated AMPylation increased PRX1 and PRX2 enzymatic activity <i>in vitro</i> and their protein levels in cells. These findings established a link between FICD-mediated AMPylation and oxidative stress, suggesting a potential neuroprotective role of FICD in neurodegenerative diseases.</p><p dir="ltr">Protein phosphorylation is another PTM that has been under extensive study due to its widespread role in cell signaling in many biological processes such as growth, division, metabolism, membrane transport, etc... Deregulation of protein kinases, which catalyze phosphorylation reaction, is often implicated in many diseases, including cancer. To elucidate disease mechanisms and explore alternative therapeutic targets, identifying direct protein substrates of a given disease-relevant kinase is crucial but remains a major challenge. Conventional methods to study phosphorylation involved the use of radiolabeled ATP, which poses health hazards and lacks reliability due to rapid decay of radioactive isotope. In this research, we developed an alternative method with a series of novel γ-modified ATP analog probes bearing a phospho-alkyne reporter handle, and their effectiveness and efficiency for<i> </i><i>in vitro</i> phosphorylation of recombinant proteins and proteomic substrate labeling in cell lysate were examined.</p>

No / Since the development of the first cytotoxic agents, synthetic organic chemistry has advanced enormously. The synthetic and medicinal chemists of today are at the centre of drug development and are involved in most, if not all, processes of drug discovery. Recent decreases in government funding and reformed educational policies could, however, seriously impact on drug discovery initiatives worldwide. Not only could these changes result in fewer scientific breakthroughs, but they could also negatively affect the training of our next generation of medicinal chemists.

Yes / The extensive use of mollusc shell as a versatile raw material is testament to its importance in prehistoric times. The consistent choice of certain species for different purposes, including the making of ornaments, is a direct representation of how humans viewed and exploited their environment. The necessary taxonomic information, however, is often impossible to obtain from objects that are small, heavily worked or degraded. Here we propose a novel biogeochemical approach to track the biological origin of prehistoric mollusc shell. We conducted an in-depth study of archaeological ornaments using microstructural, geochemical and biomolecular analyses, including ‘palaeoshellomics’, the first application of palaeoproteomics to mollusc shells (and indeed to any invertebrate calcified tissue). We reveal the consistent use of locally-sourced freshwater mother-of-pearl for the standardized manufacture of ‘double-buttons’. This craft is found throughout Europe between 4200–3800 BCE, highlighting the ornament-makers’ profound knowledge of the biogeosphere and the existence of cross-cultural traditions. / Ministry of Education, Universities and Research Young Researcher: European Commission PERG-GA-2010-26842: Leverhulme Trust: Centre National de la Recherche Scientifique: Campus France, Universita` Italo-Francese PHC Galile´ programme

<p dir="ltr">Post-translational modifications (PTMs) play a crucial role in regulating protein function and location. Protein AMPylation, the addition of adenosine monophosphate (AMP), significantly influences protein trafficking, stability, and pathogenic virulence. The Fic Domain family of proteins targets hydroxyl-containing amino acid residues (Ser, Thr, or Tyr), catalyzing the addition of various phosphate-containing moieties such as nucleoside monophosphates (NMPs), phosphocholine, and phosphate. Using gene mining techniques, Dr. Seema Mattoo’s group has identified a clade of Fic domain containing proteins typified by the enzyme originating from <i>Bordetella bronchiseptica</i> (BbFic) which prefers utilizing guanosine triphosphate (GTP) as a substrate over other nucleotides. To understand the physiological role of GMPylation, identifying the proteins modified by BbFic is a first critical step and can be accomplished via mass spectrometry-based proteomics. For a low stoichiometry PTM like GMPylation, however, there is a need to develop chemical tools that enable the targeted enrichment of modified protein. Identifying key interactions between substrate proteins and the BbFic nucleotide binding site will enable development of highly specific molecular tags for Fic substrates.</p><p dir="ltr">The goal of this research project, therefore, is to design chemical probes to tag Fic enzyme substrates, thereby facilitating the identification of GMPylated proteins in chemical proteomics workflows. A set of ATP and GTP analogues carrying either alkyne or azide handles were proposed as possible probes. While 8-azido guanosine showed a high docking score in our in-silico study, literature reports highlight its chemical instability upon exposure to air and light. An alternative probe, the 8-ethynyl guanosine, also showed a high docking score and docks in the same position and orientation as guanosine (the natural ligand) but necessitates synthetically challenging via cross-coupling reactions.</p><p dir="ltr">We considered multiple GMP analogues as potential molecular tags with the assistance of molecular docking with the BbFic enzyme. With predicted binding affinities in hand, we prioritized candidate GTP analogs for synthesis to probe the BbFic-mediated protein GMPylation process. While N6 propargyl guanosine serves as a lead probe for AMPylation, computational analysis reveals challenges with O6 due to its altered hydrogen bond donor/acceptor presentation. The distinctive chemical properties of guanosine, compared to adenosine, require a thorough evaluation of protective group strategies, as not all synthetic methodologies used for ATP analogue synthesis are applicable to GTP analogues. Isolating the triphosphate analogue proved challenging, although purification of the monophosphorylated counterpart is feasible. The Protide analogue benefits from phosphate charge masking, which facilitates purification. While much work remains until the physiological role of GMPylation can be determined, important progress has been made in the design and synthesis of chemical tools for studying this newly discovered PTM.</p>

The overall goal of my work was to develop and apply new chemical methods for the study of epigenetic DNA and protein modifications. In Chapter 3 the development of Suzuki-Miyaura cross coupling (SMcc) for the post-synthetic modification of DNA is described. DNA modification by SMcc is efficient (4-6h) and proceeds under mild conditions (37°C, pH 8.5). The incorporation of various groups useful for biological investigations is demonstrated using this methodology. Using a photocrosslinker, introduced into the DNA by SMcc capture experiments are performed to identify potential binding partners of modified DNA. In Chapter 4 a dehydroalanine (Dha) based chemical protein modification method is described that enables the introduction of posttranslational modification (PTM) mimics into histones. The PTM mimics introduced by this method are tested using western- and dot-blot and binding and enzymatic assays, confirming they function as mimics of the natural modifications. Chapter 5 describes the use of a generated PTM mimics to elucidate the function of O-linked β-Nacetylglucosamine (GlcNAc) of histones in transcriptional regulation. It is shown that GlcNAcylation of Thr-101 on histone H2A can destabilize nucleosome by modulating the H2A/B dimer – H3/H4 tetramer interface. N- and C-terminal histone tails play an important role in transcriptional regulation. In Chapter 6, nuclear magnetic resonance is used to investigate the structure of the histone H3 N-terminal tail in a nucleosome. The H3 tail, while intrinsically disordered, gains some α-helical character and adopts a compact conformation in a nucleosome context. This H3 tail structure is shown to be modulated by Ser-10 phosphorylation. The effect of a new covalent DNA modification, 5- hydroxymethylcytosine (5hmC), on transcription factor binding is investigated in Chapter 7. 5hmC influences HIF1α/β, USF and MAX binding to their native recognition sequence, implying involvement of this modification in epigenetic regulation.

Well-regulated Ca2+ signalling is essential for every living organism, and disruption of this signalling can lead to diseases including heart failure, neurological disorders and diabetes. Intracellular Ca2+ levels are regulated by influx of extracellular Ca2+ through channels located in the cell membrane. In addition, release of Ca2+ from intracellular stores also plays an important role in controlling intracellular Ca2+ concentration. Of the three types of intracellular Ca2+ stores that have been characterised those with D-myo-Inositol 1,4,5 trisphosphate receptors (InsP3Rs) showed a close relationship with cell proliferation. Hence, selective blockage of InsP3Rs will allow better understanding of Ca2+ signalling and might also unveil novel treatment for cancers, in the long term. There were no selective InsP3Rs antagonists known at the start of these studies. Based on the crystal structure of InsP3Rs bound to InsP3 and SAR studies of InsP3, we designed and tested several InsP3 analogues.1 Compound 15, 16 and 23 acted as InsP3R antagonists, though their selectivity for InsP3Rs was not completely determined. Furthermore, we also attempted to improve the potency of 16 via substitution at the 1-postion phosphate. By considering the interaction formed between adenophosphostins and InsP3Rs compounds (53-55) were designed and synthesised. In addition, analogues of compound 92, selected from an in silico screen, have led to the discovery of another novel scaffold that acts as an InsP3R antagonist.

Site-selective protein modification has become an important tool to study protein functions in chemical biology. In the preliminary work, allyl sulfides were found to be reactive substrates in aqueous cross-metathesis (CM) enabling the first examples of protein modification via this approach. In order to access the enhanced CM reactivity of allyl sulfide on proteins, facile chemical methods to install S-allyl cysteine on protein surface were developed. In particular, a cysteine-specific allylating reagent – allyl selenocyanate was used on protein substrate for the first time. The substrate scope of allyl sulfide-tagged proteins and factors that affect the outcome of CM was also investigated. A range of metathesis substrates containing different olefin tether of various lengths were screened; allyl ethers were found to be most suitable as CM partners. By reducing the steric hindrance around the allyl sulfide on protein surface through a chemical spacer, the rate and conversion of metathesis reaction on proteins was greatly enhanced. Moreover, allyl selenides were found to be more reactive than allyl sulfides in CM and enabled reactions with substrates that were previously impossible for the corresponding sulfur-analogue. Through this work, substrate selection guidelines for successful metathesis reaction on proteins were established. Rapid Se-relayed CM was further investigated through biomimetic chemical access to Se-allyl selenocysteine (Seac) via dehydroalanine. On-protein reaction kinetics revealed rate constants of Seac-mediated CM to be comparable or superior to off-protein rates of many current bioconjugations. This CM strategy was applied to histone proteins to install a mimic of acetylated lysine (K9Ac, an epigenetic marker). The resulting synthetic H3 was successfully recognized by antibody that binds natural H3-K9Ac. A Cope-type selenoxide elimination subsequently allowed the removal of such modification to regenerate dehydroalanine. Finally, preliminary research efforts towards metabolic incorporation of allyl sulfide-containing amino acid into proteins, and CM on cell surfaces were discussed.