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This thesis explores the synthesis of several different polymeric nanostructures using reversible addition-fragmentation chain transfer (RAFT) polymerisation in both aqueous and ethanolic solutions. A preface entailing an abstract, contents, common abbreviations and details of where this work has been presented is included. Chapter 1 provides an introduction to living polymer techniques with an emphasis on RAFT polymerisation as well as the biomedical applications of polymer nanoparticles synthesised via RAFT. Chapter 2 explores the synthesis of well-defined cross-linked star polymer model networks (CSPMNs) equipped with both acid-labile and non-degradable cores to study potential drug release in acidic conditions. The synthesis of the neutral star polymer precursors to the CSPMNs are also documented. This chapter includes the encapsulation and release of rhodamine B dye from an acid-labile CSPMN. Chapter 3 further investigates the synthesis of CSPMNs but incorporates a zwitterionic, phospholipid-like monomer alongside a cationic monomer to examine the effect of monomer composition on cell-internalisation properties. Chapter 4 highlights the formation of amphiphilic benzaldehyde-functionalised polymer nanoparticles using polymerisation induced self-assembly (PISA) alongside RAFT in aqueous conditions. Cross-linking of benzaldehyde-functionalised polymer micelles is explored through addition of an aminooxy cross-linker and analysed via SEM, TEM and DLS. Chapter 5 shows the formation of benzaldehyde-functionalised polymer latex particles through the use of the single-emulsion solvent evaporation method. The subsequent latex particles are then further functionalised by attachment of an alkene containing small molecule for biomedical applications. Chapter 6 includes the conclusions and potential future work the thesis results may encourage. An appendix showing crude proton NMR spectra and crude IR spectra is also included.

Chemistry plays an essential role in biological processes within all living organisms and better understanding of how specific chemical bonds can be either formed or broken gives chemical biologists opportunities to control and regulate these processes with high precision. Therefore, with growing importance of the field of chemical biology, expansion of the toolbox of available strategies for the formation or cleavage of specific chemical bonds under biocompatible conditions is needed for future advances in diverse topics in life sciences. The presented work aims to contribute to this growing field with a versatile method for the preparation of site-specific and plasma stable DNA-protein conjugates which are suitable for cellular applications. Using the method developed, various types of ONs can be easily conjugated to a range of proteins via solvent accessible cysteine residues, including therapeutic full-length IgG antibodies. Moreover, in order to optimise and simplify the characterisation of prepared DNA-protein conjugates, an improved and straightforward LC-MS method for the analysis of bioconjugation reactions between proteins and ONs was developed. Prepared and characterised DNA-antibody conjugates were then employed as probes in cellular imaging using a super- resolution DNA-PAINT method or fluorescence microscopy. Furthermore, this thesis reports new types of metal-mediated bond-cleavage reactions triggered by an active catalytic palladium species which can be either prepared as a complex or directly generated in living cells under biocompatible conditions. The cleavage reactions described are then used for on-demand extra- or intracellular release of highly cytotoxic small molecule drugs bearing phenolic hydroxyl or amine functional groups. Essentially, the strategy presented not only allows the control of the biological activity of small molecules, but also the reactivity of palladium species involved in palladium-assisted bioorthogonal reactions. In summary, the methods and findings presented in this thesis contribute to the portfolio of chemical transformation strategies available in the interdisciplinary field of chemical biology and provide new opportunities for future therapeutic applications.

Monoclonal antibodies (mAbs) have been clinically utilised as a monotherapy for cancer treatment, demonstrating mixed evidence of effectiveness as patients developed acquired resistance to their activity. Research has shown that mAbs biological effector function can be enhanced, by using antibodies as a targeting mechanism to deliver cytotoxic drugs as seen in antibody drug conjugates (ADCs). It has been shown, Sbi (Staphylococcus binder of IgG) enables Staphylococcus aureus to evade the host immune response by futile fluid-phase consumption of the complement protein C3 through Alternative pathway (AP) activation. The aim of this study was to conjugate Sbi to Trastuzumab (Tmab) and target it on the surface of HER2 positive cancer cells to induce AP complement cascade activation. This could cause complement-dependent cytotoxicity (CDC), cell recognition-opsonisation and cell lysis by the formation of the MAC (membrane attack complex) leading to cancer reduction or clearance. Sbi was recombinantly made for subsequent conjugation reactions with previously produced Fabs (antigen-binding fragment) or Trastuzumab for the production of the novel Fab-Sbi and Tmab-Sbi drug immunoconjugates. By site-directed mutagenesis (SDM) different mutants were developed and evaluated with variable immunostimulatory capabilities and stability. Terminal cysteines were introduced for subsequent conjugation reactions for the production of the final Sbi novel immunoconjugates. Novel Sbi-Tmab immunoconjugates were produced using previously characterised novel disulfide-rebridging agents. The factors affecting the construction of novel immunoconjugates with selected Sbi mutants were evaluated by two strategies (antibody functionalisation vs Sbi functionalisation). Strategy two (Sbi functionalisation) proved successful for the production of an anti-HER2 antibody Tmab-Sbi immunoconjugate at a good yield. The same strategy was then applied for the production of novel anti-HER2 and anti-MUC1 Fab-Sbi products. A novel eGFP-Sbi protein was explored as a diagnostic for the detection of C3 fragment deposition. The protein was used for C3 fragment deposition detection on HER2+ cancer cell lines surfaces using a combination of cell culturing techniques, confocal microscopy and by utilising the AP activation capabilities of the produced anti-HER2 Tmab-Sbi immunoconjugate. C3 deposition was detected under various conditions using two HER2+ human expressing cancer cell lines but also in a aHUS animal model. Preliminary findings imply the applicability of the tested conjugation method to construct multiple mAbs and Fabs immunoconjugates with variable Sbi mutants, allowing the production of specific targeted treatments as novel interventions in the field of oncology. Furthermore it was demonstrated the applicability of the eGFP-Sbi fusion protein to detect C3 fragment deposition that could be further explored in animal studies and human trials in multiple complement mediated diseases.

The environmental dimensions of the global health problem of antibiotic resistance have remained largely uncharted, particularly in regard to biotic interactions of waterborne bacteria. This thesis studied the effect of phytoplankton and zooplankton on horizontal gene transfer (HGT) in Enterococcus faecalis strains in experimental microcosms. HGT was investigated between an E. faecalis donor strain with the plasmid borne vancomycin resistance gene vanA and rifampicin-resistant recipient strains. Gene transfer frequency (GTF) of vanA in centrifugation-induced liquid mating assays at 37 oC (10-7 ) significantly exceeded GTF at 20 oC or 30 oC (10-8). Enhanced nutrient availability increased GTF from 10-7 to 10-5 in liquid mating assays, thus documenting the importance of energy resources to conjugation in E. faecalis. Active filter feeding increased vanA GTF in Daphnia-bacteria microcosms to a range of 1.5 × 10-8 – 3.3 ×10-7, and ingested enterococci remained viable inside Daphnia for at least four days. Filter feeding zooplankton may thus facilitate the emergence of multi-resistance and disseminate transconjugants in aquatic environments. Microalgae zooplankton feeds on may further enhance HGT. Microcosm experiments documented that Palmellopsis sp. enhanced vanA GTF by an order of magnitude, indicating that the algal cell mucilage was a suitable attachment substrate and energy source for conjugation. The ciliate Tetrahymena pyriformis also facilitated HGT in E. faecalis. In the presence of live ciliates vanA GTF was 10-6 – 10-5 , an order of magnitude higher than in ciliate-free controls. GTF was higher within vesicles than in the ambient medium; it increased with time and peaked after 24 h. Overall, this thesis highlights the potential effects of interactions between eukaryotic freshwater organisms and Enterococcus faecalis as a representative of Gram positive bacteria on the emergence and spread of antibiotic resistance. It also offers a projection of gene transfer frequencies under environmentally relevant conditions in lakes and wastewater treatment plants.

In this thesis will be described research towards the development of bioorthogonal bond-cleavage reactions, and their applications in targeted drug delivery (Figure 1). The first project relates to the development of a palladium mediated bond-cleavage or "decaging" reaction which can cause a propargyl carbamate to decompose and release an amine. This was further developed by the incorporation of a protein modification handle which allowed an amine-bearing drug to be covalently ligated to a protein by a palladium-cleavable linker. This chemistry was demonstrated by the conjugation of the anticancer drug doxorubicin to a tumour targeted anti-HER2 nanobody. The drug could then be delivered to cancer cells upon addition of a palladium complex. The second project relates to the development of a platinum mediated bond-cleavage reaction. This was developed with the aim of using platinum-containing anticancer drugs - such as cisplatin - as a catalyst to cause drug release reactions in tumours. In this reaction an alkyne-containing amide can decompose to release an amine upon addition of platinum complexes, and was applied to the release of prodrugs of the cytotoxins monomethylauristatin E and 5-fluorouracil in cancer cells. A cisplatin-cleavable antibody-drug conjugate was designed and synthesised, and progress towards its biological evaluation will be discussed.

As filterfeeders, freshwater sponges encounter bacteria in streams, rivers and lakes including those from faecal sources like enterococci and coliforms, which can exhibit antibiotic resistance with potential clinical impacts through e.g. infection of humans from recreational use of these environments. Filterfeeding trials verified the potential of Irish freshwater sponges Ephydatia fluviatilis and Spongilla lacustris, which occupy wide ranges in the northern hemisphere, to reduce the abundance of Escherichia coli in ambient water. Plate counts of bacterial abundance were more reliable than monitoring methods involving turbidity or fluorescence measurements. Laboratory and field studies tested the application of the sponges for biomonitoring of microbial water quality. In the laboratory both sponge species retained Enterococcus faecalis in lower abundances than E. coli. Although gradual changes of abundance of enterococci and coliforms in sponge samples were also observed along a longitudinal river reach transect, the between river differences in retention of enterococci and coliforms were greater than within a single river. The sponges' potential for facilitating conjugal antibiotic resistance transfer was explored in microcosms with E. faecalis strains resistant to either vancomycin or rifampicin. Lack of a significant difference between transconjugant numbers on double selection plates from microcosms with live or dead sponges suggested that filtration activity had no decisive role in conjugal transfer of monitored resistance traits. Sponge gemmule surfaces were found to be associated with bacteria resistant to ampicillin, erythromycin, rifampicin, tetracycline, trimethoprim and vancomycin. Methanol extracts from freshwater sponges inhibited the growth of some nosocomial bacteria, with adult sponge extracts having a higher inhibitory effect than extracts from gemmule-grown sponges, indicating the contribution of the sponge microbiome. The antimicrobial properties of sponge samples varied with collection site, and the combination of sponge extracts from several sites caused the better inhibitor to become diluted and less effective as an antimicrobial agent.

Biodegradable plastics are being adopted to reduce the environmental impacts associated with petroleum-based plastics, including long residence times, and reliance on fossil fuels as a feedstock. Microbial degradation of bioplastics is still poorly understood, which leads to inconsistent degradation outcomes and challenges for implementation. Bacillus pumilus B12 degrades the biodegradable bioplastic polylactic acid (PLA) via the protease AprE, but the regulatory mechanisms controlling aprE expression in B. pumilus B12 are largely unknown. To explore this question, we established developed a conjugation- based method to introduce DNA into B. pumilus B12 and diverse Bacillus species that is applicable to environmental and industrially relevant species. We are leveraging this conjugation technique to begin exploring aprE regulation in B. pumilus B12. Despite belonging to the same genus there is a considerable difference in the aprE upstream regulatory region sequence of B. pumilus B12 and B. subtilis. Thus, we wanted to begin defining subregions of the upstream regulatory region that influence expression, especially transcription factor binding sites. We generated a series of transcriptional reporters to GFP with varying lengths of the upstream regulatory region. These reporters provide indications of repressor and activator binding sites, including for Spo0A. Additionally, we used site directed mutagenesis (SDM) to mutate the bases corresponding to putative transcription factor DNA binding motifs. We observe that SinR acts as a repressor of aprE but ScoC has no effect.t We then show that expression of aprE is repressed by the addition of inosine, adenine and adenosine, which supports our previous work. In other Bacillus species, AprE is a highly regulated secreted protein expressed during stationary phase, but the mechanism for this nucleotide- mediated repression remains unknown, as no one transcription factor abrogated the repressive effect of inosine addition. We hypothesize that purine addition disrupts the activity of a pleiotropic regulator, which is reflected in the repression of additional genes. Overall, we show that aprE expression is controlled over 500 bases upstream of the predicted open reading frame and that there may be more regulatory elements than described in B. subtilis. Further, we describe aprE repression by inosine for the first time.

In disease, especially cancer, biological barriers impede the delivery of therapeutic agents to effectively reach their intended targets after administration. Developing delivery strategies that understand and take into account the biological environment is critical to improve the efficiency of drug delivery. Nature provides a biological toolkit from which effective drug delivery systems can be developed. In particular, albumin is an attractive delivery carrier with favorable intrinsic properties to overcome intracellular and extracellular delivery barriers in cancer. In a subset of cancers, the RAS family of oncogenes (KRAS, HRAS, NRAS) are the most frequent mutations in cancers and regulate key signaling pathways that drive tumor progression, including hyperactive cell proliferation and metabolism. As a result, drug delivery targeting RAS-driven tumors has been a long-standing challenge in cancer therapy. Looking for solutions inspired by nature, recent observations indicate that extracellular nutrients, including glucose, lipids, and albumin, were found to be actively scavenged by mutant RAS activated cancer cells via macropinocytosis to fulfill the energetic requirements of cancer cells to survive and proliferate. Here, we exploit this mechanism to deliver albumin nanoparticles in cancer cells harboring activating KRAS mutations. We synthesized stable albumin nanoparticles that demonstrate significantly greater uptake in cancer cells with activating mutations of KRAS than monomeric albumin (i.e. dissociated form of clinically used nab-paclitaxel). From pharmacological inhibition and semi-quantitative fluorescent microscopy studies, these nanoparticles exhibit significantly increased uptake in mutant KRAS cancer cells than wild-type KRAS cells by macropinocytosis. Importantly, we demonstrate that their uptake is driven by KRAS. This nanoparticle-based strategy targeting KRAS-driven macropinocytosis is a facile approach towards improved delivery into KRAS-driven cancers. Through our studies with albumin nanoparticles, it was realized that albumin itself potentially possesses excellent pharmacokinetics that could be leveraged to improve drug delivery. To further harness the intrinsic transport properties of albumin yet improve the therapeutic index of current in situ albumin-binding prodrugs, we developed albumin-drug conjugates with a controlled loading that achieved better antitumor efficacy. Model drug monomethyl auristatin E (MMAE) was conjugated ex vivo to Cys34 of albumin via a cathepsin B-sensitive dipeptide linker to ensure that all drugs would be bound specifically to albumin. The resulting albumin-drug conjugate with a drug to albumin ratio (DAR) of 1 (ALDC1) retained the native structure of albumin compared to conjugate with a higher DAR of 3 (ALDC3). ALDC1 exhibited improved drug release and cytotoxicity compared to ALDC3 in vitro. Slower plasma clearance and increased drug exposure over time of ALDC1 were observed compared to ALDC3 and MMAE prodrug. In single dose studies with MIA PaCa2 xenografts, cohorts treated with ALDC1 had the highest amount of MMAE drug in tumor tissues compared to other treatment arms. After multiple dosing, ALDC1 significantly delayed the tumor growth compared to control treatment arms MMAE, MMAE-linker conjugate, and ALDC3. When dosed with the maximum tolerated dose of ALDC1, there was complete eradication of 83.33% of the tumors in the treatment group. Ex vivo conjugated ALDC1 also significantly inhibited tumor growth in an immunocompetent syngeneic mouse model that recapitulates the phenotype and clinical features of human pancreatic cancers. In summary, site-specific loading of drug to albumin at 1:1 ratio allowed the conjugate to maintain the native structure of albumin and its intrinsic properties. By conjugating the drug to albumin prior to administration minimized premature cleavage and instability of the drug in plasma and enabled higher drug accumulation in tumors compared to in situ albumin-binding prodrugs. This strategy to control drug loading ex vivo ensures complete drug binding to the albumin carrier and achieves excellent antitumor efficacy, and it has the potential to greatly improve the outcomes of anticancer therapy. Collectively, these findings suggest that albumin can serve as an effective drug delivery carrier for drug delivery in solid tumors when the intrinsic properties of albumin were carefully utilized.

Protein-polymer conjugates have been studied for many decades for applications in drug delivery, tissue engineering, sensing and detection of specific biomarkers, antifouling surfaces, and detergents. The armoring of proteins with synthetic polymers has been known to enhance the native properties of the protein while also taking on the unique properties of the polymer. Conjugation has been shown to increase protein stability, improve solubility, reduce immunogenicity, improve biocompatibility, and increase circulation time in vivo. Additionally, covalent attachment of synthetic block copolymers, such as poly(ethylene oxide) (PEO)- poly(propylene oxide) (PPO) block copolymers, offers opportunity to improve targeting of conjugates to hydrophobic surfaces and interfaces as well as potential for catalysis in organic solvents. The goal of this project was to conjugate proteins with Pluronics, specifically F127, L64, and F68. Pluronics have an A-B-A triblock structure with differing hydrophobic and hydrophilic chain lengths. In this study, conjugation reaction conditions were screened for a range of polymers by studying their attachment to the protein, β-lactoglobulin (βLG). The synthesized conjugates were characterized using gel electrophoresis and amine quantification. The difference in molecular weights and weight percent of the hydrophobic to lipophilic chain ratios showed a direct impact on the degree of conjugation for each Pluronic.

Acinetobacter baumannii is Gram-negative bacterial human pathogen that can be foundin myriad environmental niches and, more notoriously, thrives in the hospital setting. Thisopportunistic pathogen is the causative agent in a range of disease outcomes includingwound infections, urinary tract infections, meningitis, and pneumonia. The ability of A.baumannii to persist on medical equipment in the hospital environment, colonize thehuman host, and express a number of antibiotic resistance mechanisms makes it aformidable nosocomial threat and justifies the investigation of the molecular mechanismsunderlying its virulence and overall physiology. A. baumannii uses various sensing andregulatory systems to coordinate a response to cues from its local environment. Ofparticular importance in the context of this study are the cues of light and temperature.The goal of the work presented in this manuscript was to elucidate the molecularmechanisms underlying light sensing and regulation in A. baumannii at two differenttemperatures, 24°C and 37°C. The dependence of the bacterial oxidative stress responseon the blue light using flavin protein BlsA at 24°C was tested using catalase activity,peroxide resistance, and superoxide dismutase (SOD) assays. BlsA’s regulation of thecatalase enzyme KatE was dependent on the BlsA residue lysine 144, and its regulationof SOD activity was dependent on the last five BlsA residues. Blue light-dependent, BlsAindependent regulation at 24°C was examined by RNA sequencing analysis andinvestigation of the role of the putative photolyase PhrB in light-dependent functions.Global blue light-dependent transcriptional regulation was apparent even in the absenceof BlsA at 24°C. Furthermore, PhrB did not play a detectable role in canonical photolyaseactivity yet did impact bacterial surface-associated motility, a light-regulated phenotype inA. baumannii. For bacteria cultured at 37°C, a temperature at which BlsA is not only lowin expression but also effectively inoperative based on conformational changes that inhibitits photocycle, RNA sequencing showed that there is nevertheless blue light-dependentregulation of a set of genes whose products are involved in bacterial conjugation. Theblue light-dependent transcriptional regulation of these genes has implications forconjugation itself: the transfer of the large, self-transmissible plasmid pAB3 by A.baumannii bacteria to sister cells is regulated in a blue light-dependent manner at 37°C.Taken together, this work provides new information concerning the molecularmechanisms underlying light sensing and regulation in the human pathogen A. baumanniiat two different temperatures, both of which it encounters in the hospital setting.

The rapid evolution of bacteria can be credited to the almost ubiquitous association to Mobile Genetic Elements, the means of Horizontal Gene Transfer enabling rapid exchange of genetic information between bacterial populations. Integrative and Conjugative Elements (ICEs) are Mobile Genetic Elements (MGEs) that are transferred largely by conjugation between two bacterial cells and able to minimise the impact of their acquisition by integration into the host chromosome. The scarce number of studies concerning the frequency of transfer have been predominantly conducted on highly similar strains or non-related bacterial species, impeding the comprehension of its ecological significance. In this study, the conjugative transfer of Horizontally Acquired Island 2 (HAI2), an ICE that enhances virulence of the potato pathogen Pectobacterium atrosepticum SCRI1043, was investigated by assessing frequency of acquisition into strains of SCRI1043 in which HAI2 had been removed. HAI2 was transferred into engineered recipients confirming it as indeed a functional ICE. Further functional analysis of the island using mutants with transposon insertions in selected core genes of HAI2 demonstrated that the process of conjugation is decoupled from the initial excision of the island from the host genome. HAI2 requires a group of genes related to cell partitioning systems and a DNA helicase for effective transfer by conjugation. To understand whether HAI2 was likely to be transferred to related bacteria within the ecological niche of Pectobacterium atrosepticum SCRI1043, conjugation assays were conducted in vitro using plant pathogenic Pectobacteriaceae strains belonging to the genera Pectobacterium and Dickeya from New Zealand and overseas. Thirty-three PCR-positive isolates for at least one HAI2-compatible bacterial attachment (attB) site were selected. Surprisingly, HAI2 was not acquired by any of the selected isolates. A screening of the genome sequences of these potential recipients and other publicly available Pectobacteriaceae genome sequences revealed that the necessary attachment sites were almost always occupied. Several different mobile elements are seemingly integrated at these spots in their chromosomes, suggesting that horizontal transfer of HAI2 may be restricted by previous acquisition of mobile elements. The influence of HAI2 excision as a strategy to manipulate the cell regulatory network has also been investigated. Transcriptome analysis comparing the wild-type and mutant strainslacking the expression of HAI2 core genes involved in DNA topology and processing prior conjugation was performed in vitro and in planta. A topoisomerase IIIβ (ECA0525) and a relaxase (ECA0613) able to induce hyper-excision of the island, and an integrase (ECA0614) and an engineered islandless mutant (ΔHAI2) with no detectable excision were assessed. Genome-wide differential expression was observed, with HAI2 as the most affected portion of the chromosome. The transcriptome profiles were strongly correlated to the excision status of HAI2 rather than to a particular mutation. Hyper-excision was associated with upregulation of HAI2 core genes and genes related to plasmid stability. Absence of HAI2 excision induced downregulation of transcription pathways energy use. Changes in HAI2 excision impacted the expression of HAI5, HAI6 and HAI10, MGEs also integrated in Pba SCRI1043 chromosome. In summary, we provided evidence to confirm that HAI2 is a fully functional and active ICE, able to control its expression and influence the expression of its host. The important association of HAI2 to P. atrosepticum does not seem to be extended to other Pectobacteriaceae by conjugative transfer to environmental strains because of intense competition for attachment sites in the past.

Abstract: Chapter 2 describes an optimized 7-step successful synthesis of a first of its kind permanently open-form ortho-hydroxyalkyl arylboronic acid, otherwise known as a “frustrated benzoxaborole”, with a columnless purification and complete structure elucidation. This novel compound is completely water soluble due to its pKa of 7.5, which is unexpectedly low for a non-deactivated boronic acid. The rationale behind this low pKa is due to the ortho hydroxy involvement in stabilizing the trihydroxyboronate conjugate base by H-bonding to one of the boronate oxygens. Conjugation of this new compound with biological amino and thiol groups was comparable with current methods involving ortho formyl and acetyl phenylboronic acid. Chapter 3 summarizes collaborative work with the National Research Council in Halifax for the development of a boronic acid polymer gel column “catch-and-release” system of diol containing marine neurotoxins. Such a system would provide pre-purification of toxins to eliminate background noise for easier detection in analytical methods. A survey of 22 boronic acids was conducted for efficient boronate ester formation and acid hydrolysis utilizing tetrodotoxin as a model. 2-(Trifluoromethyl)phenylboronic acid proved to be the ideal compound and was redesigned and synthesized with a conjugatable carboxylic acid for installation on a polymer gel. Chapter 4 highlights work done in collaboration with Prof. Brudno at the University of North Carolina for the in vivo imaging of a boronate/thiosemicarbazone systems developed by the Hall Group. The previously published synthesis of the precursors was improved in terms of yield, purity and reproducibility, as well as derivatized for detection by fluorescence microscopy. The imaging in live mice was shown to be promising and future work will be planned to optimize the system.

The advent of CRISPR based gene editing has transformed both basic scientific research and the development of new medicines and treatments. These complex proteins promise freedom to modify any gene at will but must be carefully delivered to prevent degradation or off-target editing. This dissertation describes work to encapsulate Cas9 within the bacteriophage MS2 protein scaffold, and later to modify the Cas9 protein itself to allow for more efficient delivery and editing. Cas9 was successfully encapsulated within the MS2 VLP, but showed high sensitivity to RNase degradation.Towards the modification of the Cas9 ribonuclear protein, a novel protein modification reaction using the enzyme tyrosinase was developed, enabling modification and delivery of Cas9 using cationic delivery peptides. The tyrosinase reaction was further used to fuse a variety of proteins to immunoglobulin derivatives including scFvs, nanobodies, and IgG1 Fc domains. Cas9-immunoglobulin fusions were shown to induce tolerance in in vivo mouse model studies, highlighting the potential of tyrosinase coupling to mediate Cas9 protein fusions. Prokaryotic tyrosinases were explored for their potential as universal protein activators, of which the tyrosinase from Bacillus megaterium showed exciting promise as a universal activator. Using substrate charge preference data from the tyrosinase screens we were able to design charged tyrosine tags that enabled sequential activation and coupling of protein trimers.

Antibiotic resistance genes (ARGs) may enter surface water with the discharge of treated wastewater. Conjugation is a main mechanism for the horizontal gene transfer of ARGs from wastewater microbiome to the microbiome indigenous to surface water. However, little is known about how environmental factors affect the conjugation process of ARGs. In this thesis, an extensive research related to spreading antibiotic resistance genes in the environment is reported. The project investigated how environmental parameters may affect the rate of dissemination of ARGs and in what extent the resistance level of the population is changed after mutation and conjugation event. The chemostat reactors were built to simulate surface water receiving wastewater effluent. Two E. coli strains, CV601 and J53, were used as donor and recipient cells, respectively. First, CV601 cells received plasmids from an activated sludge sample through filter mating and became resistant to tetracycline. Then, J53 cells (i.e., to simulate river microbes) were established in chemostats before CV601 cells (i.e., to simulate wastewater microbes) were introduced. Two nutrient levels (3× and 10× diluted Muller Hinton broth) and two growth rates (0.15 and 0.45 hr-1) were included. Results show that regardless the nutrient level, the conjugation frequency was 10-2 and 10-6 for the high and low growth rate, respectively. The minimum inhibitory concentration (MIC) of the recipient cells increased from 2 to 64-128 mg/L after the conjugation test, which was 2 to 4 fold lower than donor for the same plasmid. In addition, recipient cells grown under 0.45 hr-1 and 1/10 MHB showed MIC increased from 2 to 8 mg/L in mutation experiment. Whole genome sequencing was conducted to 1) prove the presence of tetracycline resistant plasmid in donor; 2) Transferability of the plasmid to recipient cells; 3) and the emergence of mutants in recipient cells. The results will allow us to better model and predict the proliferation of ARGs in the environment. Advisor: Xu Li

Enterococcus faecalis is a commensal member of the gastrointestinal tract of animals including humans but is also an opportunistic pathogen and a major cause of healthcare-associated infections. Its pathogenicity is thought to arise in immunocompromised people and after infection, treatment is difficult due to antibiotic resistance. E. faecalis is particularly good at transferring antibiotic resistance by mechanisms like conjugation and conjugative transfer of plasmids can occur at a high frequency without antibiotic selection. Conjugative plasmid pCF10 encodes tetracycline resistance and transfer between E. faecalis cells is facilitated by cell-to-cell communication. This signaling triggers expression of genes from pCF10 that encode for transfer machinery. The response to signaling is robust and has been extensively studied at the population level. However, it has recently become apparent that there is response variation. Understanding the mechanisms that underlie variation in response initiation is important to preventing transfer. Studies presented in this dissertation adapt fluorescence in situ Hybridization Chain Reaction (HCR) for single cell analysis of transcripts and explore questions about the pCF10 conjugation system that would not have otherwise been possible. In chapter 3, variation in the signaling response was assessed and the response was shown to be very heterogenous. When the level of signal is low, (like what might occur naturally), a minority of cells respond. Although stochasticity in the system may give rise to such heterogeneity, work in chapter 4 investigates the response impact of a few specific mechanistic players (PrgX, C, and I). Changing the levels of these components was shown to change the outcome. Lastly, single cell analysis was used in chapter 5 to assess the expression of genes required for conjugative transfer. These results show that the few responding cells commit to expression of all the genes encoding for production of the conjugation machinery. Overall, these results suggest that the pCF10 system is evolutionarily tuned for specific levels of each component and poised to have response variation for a population of cells. Thus, a small percent of cells can respond and since the majority of responding cells are able to conjugate, plasmid transfer is highly efficient. These results also exemplify how small differences in two cells can precipitate different responses in otherwise identical cells exposed to very similar conditions. Information about variation in the initiation of the signaling response required for pCF10 transfer is important to understanding the general biology of gene transfer among bacteria. In the future, this information will be important for successful design of effective interventions to the transfer of genes conferring antibiotic resistance.

Mesoporous silica nanoparticles (MSNPs) possess large surface areas and ample pore space that can be readily modified with specific functional groups for targeted binding of bioactive materials to be transported through cellular barriers. Engineered silica nanoparticles (ESNP) have been used extensively to deliver bio-active materials to target intracellular sites, including as non-viral vectors for nucleic acid (DNA/RNA) delivery such as for siRNA induced interference. The reverse process guided by the same principles is called “nanoharvesting”, where valuable biomolecules are carried out and separated from living and functioning organisms using nano-carriers. This dissertation focuses on ESNP design principles for both applications. To investigate the bioactive materials loading, the adsorption of antioxidant flavonoids was investigated on titania (TiO2) functionalized MSNPs (mean particle diameter ~170 nm). The amount of flavonoid adsorbed onto particle surface was a strong function of active group (TiO2) grafting and a 100-fold increase in the adsorption capacity was observed relative to nonporous particles with similar TiO2 coverage. Active flavonoid was released from the particle surface using citric acid-mediated ligand displacement. Afterwards, nanoharvesting of flavonoids from plant hairy roots is demonstrated using ESNP in which TiO2 and amine functional groups are used as specific binding sites and positive surface charge source, respectively. Isolation of therapeutics was confirmed by increased pharmacological activity of the particles. After nanoharvesting, roots are found to be viable and capable of therapeutic re-synthesis. In order to identify the underlying nanoparticle uptake mechanism, TiO2 content of the plant roots was quantified with exposure to nanoparticles. Temperature (4 or 23 °C) dependent particle recovery, in which time dependent release of ESNP from plant cells showed a similar trend, indicated an energy independent process (passive transport). To achieve the selective separation and nanoharvesting of higher value therapeutics, amine functionalized MSNPs were conjugated with specific functional oligopeptides using a hetero-bifunctional linker. Fluorescence spectroscopy was used to confirm and determine binding efficiency using fluorescently attached peptides. Binding of targeted compounds was confirmed by solution depletion using liquid chromatography–mass spectrometry. The conjugation strategy is generalizable and applicable to harvest the pharmaceuticals produced in plants by selecting a specific oligopeptide that mimic the appropriate binding sites. For related gene delivery applications, the thermodynamic interaction of amine functionalized MSNPs with double-stranded (ds) RNA was investigated by isothermal titration calorimetry (ITC). The heat of interaction was significantly different for particles with larger pore size (3.2 and 7.6 nm) compared to that of small pore particles (1.6 nm) and nonporous particles. Interaction of dsRNA also depended on molecular length, as longer RNA (282 base pair) was unable to load into 1.6 nm particles, consistent with previous confocal microscopy observations. Calculated thermodynamic parameters (enthalpy, entropy and free energy of interaction) are essential to design pore size dependent dsRNA loading, protection and delivery using MSNP carriers. While seemingly diverse, the highly tunable nature of ESNP and their interactions with cells are broadly applicable, and enable facile nano-harvesting and delivery based on a continuous uptake-expulsion mechanism.

The novel PosiDAF process that uses cationic-polymer modified bubbles has been suggested as an alternative to conventional dissolved air flotation for the separation of algae. However, a cationic PosiDAF effluent compared to an anionic influent as detected by charge measurements indicated that effluent contained high polymer residuals and was undesirable. To prevent this, prior research investigated stronger polymer-bubble adhesion by developing hydrophobically modified polymers (HMPs) of poly(dimethylaminoethyl methacrylate) (PDMAEMA). However, while bench scale tests using the HMPs were successful, commercially available poly(diallyldimethylammonium chloride) (PDADMAC) outperformed the HMPs in pilot scale, suggesting that PDADMAC has a more suitable polymer backbone. Moreover, algal organic matter (AOM) released by cells, particularly biopolymers, was observed to influence cell separation. Further research is required to investigate alternate polymers and to determine more precisely the underlying mechanisms governing polymer-bubble-AOM interactions in PosiDAF. In this study, PDADMAC was modified with various aromatic and aliphatic pendant groups to generate several HMPs. Select HMPs of PDADMAC and previously investigated PDMAEMA were compared to evaluate polymer-bubble attachment and PosiDAF performance to separate algae and cyanobacteria. The composition of AOM, particularly biopolymers from each strain tested was characterised and their influence examined by conducting experiments with various AOM, protein and carbohydrate concentrations. The results showed that HMP coated bubbles had lower surface tensions and consequently, anionic effluents and strong polymer-bubble adhesion. Concurrently, cell separation was either comparable, or slightly better between HMPs. However, separation effectiveness varied for several algae, indicating that AOM impacted separation. Moreover, cell separation of the strains increased to > 95% when exudates from the best separated strain were added. On bulk and molecular characterisation of the cultures, the best separated strain was found to be biopolymer rich in comparison to the other strains. Hence, proteins and carbohydrates were dosed to study their influence and were observed to either depress or enhance the flotation depending on their character. It was concluded that the interplay of biopolymers with polymer-bubble-cell was responsible for the variations in cell removal observed across several strains. Overall, with a well-defined AOM character and low polymer residual in effluent, PosiDAF has been demonstrated as a robust and sustainable process.

Antibiotics have been an extremely important weapon in the fight against bacterial infections for over half a century. However, excessive use of antibiotics has led to increased frequencies of resistance among bacteria. Antibiotic resistance is an inevitable outcome of natural selection as organisms undergo random mutations to escape lethal selective pressure. Many of these resistant bacteria can also transfer their genetic material to other bacteria through direct cell-cell contact via conjugation, further facilitating the spread of resistance. The human gastrointestinal tract, replete with a high density of bacteria and often exposed to antibiotics, provides an ideal environment for antibiotic resistance genes to arise and propagate through bacterial populations. Enterococcus faecalis, a commensal bacterium of the human intestinal tract, has emerged as a major cause of healthcare-associated infections. Treatment of these infections has become increasingly difficult with the emergence of E. faecalis strains that are resistant to multiple major classes of antibiotics. The organism’s ability to acquire and transfer resistance genes and virulence determinants through conjugative plasmids poses a serious clinical concern. Here we present our study on conjugation of a tetracycline-resistance plasmid pCF10 which is regulated by intercellular communication using two antagonistic signaling peptides. An inducer peptide produced by the plasmid-free recipient cells functions as a “mate-sensing” signal and triggers the conjugative plasmid transfer in donors. The donors encode an inhibitor peptide on the plasmid which represses conjugation and functions as a "self-sensing" signal, reducing the response to the inducer in a density-dependent fashion. This form of dual signaling-controlled conjugation was also found to be prevalent across other pheromone-responsive plasmids, including pAD1 and pAM373. Though the donors calibrate their conjugation response in accordance with the relative abundance of donors and recipients, plasmid transfer can occur under otherwise unfavorable conditions, such as low inducing pheromone and high inhibitor concentrations. To better understand this apparent inconsistency, we formulated a stochastic mathematical model that integrates intracellular molecular regulation of conjugation and interactions between donors and recipients through the signaling peptides. Kinetic parameters for the model were estimated from literature and augmented by experimental RNA-Seq data and binding constant measurements. Simulations of the stochastic model and single-cell analysis using transcript quantification by HCR-FISH and GFP reporter fusions revealed distinct subpopulations of rapid responders under unfavorable conditions for plasmid transfer. We developed a series of fluorescent reporters to track the uninduced/induced donors, recipients, and uninduced/induced transconjugants in real-time using confocal microscopy and flow cytometry. We are further developing a microfluidic gut model which allow for co-culturing of human and bacteria cells in an in vivo-simulated microenvironment. This system will be used to model the in vivo biology of conjugation and gain a better mechanistic understanding of the community balance between the microbial inhabitants of the GI tract. A better understanding of the bacterial signaling mechanisms in vivo and the downstream effects on microbiome community balance may help us identify alternate strategies to prevent the spread of antibiotic resistance.

Enterococcus faecalis contains mobile genetic elements that can rapidly spread antibiotic resistance and virulence genes through its population by conjugation. The chromosomally encoded pheromone cCF10 (LVTLVFV) induces conjugative transfer of E. faecalis plasmid pCF10. Pheromone inducible plasmids have evolved a highly specific and sensitive response to pheromone to allow their host (donor) cell to sense recipients while minimizing expression of the conjugation genes in the absence of recipients. The pCF10 PrgY protein reduces production of endogenous pheromone by donor cells to prevent self-induction. Recent data suggested that PrgY shares significant homology to the eukaryotic metalloprotease TIKI that has been shown to cleave the amino terminus of mature Wnt proteins, thereby regulates the Wnt signaling. Comparative modeling of PrgY active site revealed that PrgY and TIKI share key conserved residues in the metal-binding catalytic core as well as overall secondary structure. Based on the structural similarity between PrgY and TIKI, we hypothesized that PrgY reduces endogenous pheromone production in donor cells by specifically binding and degrading cCF10 as it is secreted across the cytoplasmic membrane. To test the working model, affinity chromatography and Surface Plasmon Resonance were used to demonstrate the direct interaction between PrgY and cCF10, and their binding affinities. The results of this work revealed that PrgY directly interacts with cCF10. Strong binding between cCF10 and PrgY was observed, and the binding can be saturated at a level comparable to the calculated theoretical maximum assuming 1:1 binding. Mass spectrometry was used to identify possible degradation products of cCF10 in the culture supernatant from a strain expressing PrgY. Peptide LVTL was uniquely identified in the donor culture supernatant expressing PrgY. This suggests that PrgY cleaved cCF10 after the second leucine, and released the degraded peptide fragments LVTL and VFV that do not have any pheromone activity. The cumulative results of this research provide important insights into the molecular mechanism of PrgY, and advance our understanding on the function of each of the PrgY family members found in a diverse range of species.

ABSTRACT OF THE DISSERTATIONEngineering and Virology of the Cowpea Mosaic Virus and Flock House Virus,Positive Sense Icosahedral RNA VirusesbyJamie Patrick PhelpsDoctor of Philosophy in ChemistryUniversity of California, San Diego, 2017Professor John Johnson, ChairProfessor Tim Baker, Co-Chair Small positive sense icosahedral RNA viruses include polio, rhinovirus, dengue, Norwalk virus, cowpea mosaic virus, and flock house virus. Cowpea mosaic virus (CPMV) and flock house virus (FHV) are excellent model viral systems for studying and engineering these viruses. Engineering BSA conjugated to CPMV attenuated 99% of the virus through 10,000-fold serial dilutions and conjugating RNase safely inactivated the virus. RNase activity was inhibited 26-fold. Engineering additional cysteine mutations to virus particles adds functional reactive potential. Reducing agents stabilize this reactive potential while protecting the proteins from unwanted aggregation. Mercaptopyridine based molecules are oxidizing agents that stabilize proteins by capping reactive thiols forming stable aqueous disulfide bonds that can be reduced as needed with traditional reducing agents. Stably bound mercaptopyridine or 2- mercaptopyridine-3-carboxylic acid was visualized with gel shifts in SDS-PAGE analysis. The fixed positioning of metal nanoparticles on functional virus particles produced plasmonic nanoclusters with resonances at visible and infrared wavelengths. The metal cluster arrangement was controlled with placement of cysteine on the virus and VLPs. The fully assembled NCs gave rise to a 10-fold surface averaged enhancement of the resonance signal, which was consistent with calculated simulations. The viral coat protein plays a role in almost every step of the life cycle of the virus. The stability of the capsid is crucial for protecting the genome from physical, chemical, and enzymatic damages. Magnetic tweezer forces were applied to the coat protein to understand the mechanical stability of the virus. Unfolding of individual coat proteins occurred over the entire force range while maintaining their attachment to the surrounding capsid proteins. The emergence of new viruses into humans costs billions of dollars of medical relief. It is vital to understand the mechanisms of cross species viral transmission. The virology of cross species FHV infection in stressed mammalian cells was studied. A new approach of analyzing cross species infection unveiled a new organelle association structure, various forms of organelle malformation, multiple virus assemblies, and a more realistic understanding of the range of phenotypes in viral infection.

Filamentous marine cyanobacteria are known to produce a variety of structurally diverse and biologically active secondary metabolites, many of which are being studied for a wide spectrum of applications, ranging from biofuels to pharmaceuticals. The development of a stable heterologous expression host for cyanobacterial biosynthetic pathways would alleviate current bottlenecks in natural products research, such as the low yield of many of these potentially valuable compounds from field-collected extracts. Additionally, it would allow for a deeper investigation of the expression and genetic regulation of these complex pathways while providing a platform in which to investigate pathways from strains for which no genetic tools may yet be developed. To date, there are no known model filamentous cyanobacterial strains of marine origin. In this study we demonstrate the first known transformation and expression of a self-replicating broad-host-range vector and a knockout vector, carrying two reporter genes, in a filamentous marine cyanobacterium.

Autophagy is a highly conserved pathway in which an autophagosome envelops cytoplasmic cargo and delivers it to the lysosome for degradation in order to maintain cellular homeostasis or survival in response to stress. The ATG12–ATG5-ATG16L1 complex functions as an essential regulator of autophagosome formation. We have discovered that DU145 prostate cancer (PCa) cells have a splice donor-site mutation that triggers aberrant splicing of ATG5 and leads to the proteasomal degradation of ATG12 and ATG16L1, thus completely inactivating autophagy. We demonstrate that ATG5, ATG12, and ATG16L1 are coordinately degraded when not associated with the complex and that the ATG5-ATG16L1 interaction is essential for preventing ubiquitination and turnover, thereby facilitating ATG12 conjugation. We also show that this interaction can be disrupted through alternative ATG5 splicing and by ATG5 genetic mutations that have been identified in human tumors. Meta-analysis of available mRNA expression data indicates that ATG5 is significantly downregulated in PCa. We confirmed previous reports that found prostate cancers have frequent deletions of the 6q21 locus containing ATG5. However, mRNA expression of neighboring genes is largely unaffected, indicating ATG5 can also be selectively downregulated though other mechanisms. Together, this suggests that ATG5 functions as a tumor suppressor gene that can be inactivated by a variety of different mechanisms. ATG5 is more significantly underexpressed than many established PCa tumor suppressor genes and is also underexpressed in PCa metastases compared to primary tumors. This implies that ATG5 is also a tumor suppressor in advanced PCa. ATG5 re-expression in ATG5-deficient DU145 PCa cells resulted in dramatic suppression of xenograft tumor growth, indicating that ATG5 is a functional PCa tumor suppressor gene. Therefore, autophagy may actually be tumor suppressive at both early and late stages of prostate tumorigenesis, which suggests that autophagy inhibition may be counterproductive for the treatment of advanced prostate cancers.

Protein therapeutics offer numerous advantages over small molecule drugs and are rapidly becoming one of the most prominent classes of therapeutics. Unfortunately, they are delivered almost exclusively by injection due to biological obstacles preventing high bioavailability via the oral route. In this work, numerous approaches to overcoming these barriers are explored. PH-Responsive poly(itaconic acid-co-N-vinylpyrrolidone) (P(IA-co-NVP)) hydrogels were synthesized, and the effects of monomer ratios, crosslinking density, microparticle size, protein size, and loading conditions were systematically evaluated using in vitro tests. P(IA-co-NVP) hydrogels demonstrated up to 69% greater equilibrium swelling at neutral conditions than previously-studied poly(methacrylic acid-co-N-vinylpyrrolidone) hydrogels and a 10-fold improvement in time-sensitive swelling experiments. Furthermore, P(IA-co-NVP) hydrogel microparticles demonstrated up to a 2.7-fold improvement in delivery of salmon calcitonin (sCT) compared to methacrylic acid-based systems, with a formulation comprised of a 1:2 ratio of itaconic acid to N-vinylpyrrolidone demonstrating the greatest delivery capability. Vast improvement in delivery capability was achieved using reduced ionic strength conditions during drug loading. Use of a 1.50 mM PBS buffer during loading yielded an 83-fold improvement in delivery of sCT compared to a standard 150 mM buffer. With this improvement, a daily dose of sCT could be provided using P(IA-co-NVP) microparticles in one standard-sized gel capsule. P(IA-co-NVP) was also tested with larger proteins urokinase and Rituxan. Crosslinking density provided a facile method for tuning hydrogels to accommodate a wide range of protein sizes. The effects of protein PEGylation were also explored. PEGylated sCT displayed lower release from P(IA-co-NVP) microparticles, but displayed increased apparent permeability across a Caco-2 monolayer by two orders of magnitude. Therefore, PEG-containing systems could yield high bioavailability of orally delivered proteins. Finally, a modified SELEX protocol for cellular selection of transcellular transport-initiating aptamers was developed and used to identify aptamer sequences showing enhanced intestinal perfusion. Over three selection cycles, the selected aptamer library showed significant increases in absorption, and from an initial library of 1.1 trillion sequences, 5-10 sequences were selected that demonstrated up to 10-fold amplification compared to the naïve library. These sequences could provide a means of overcoming the significant final barrier of intestinal absorption.

Abstract: Bacterial conjugation is the process of plasmid DNA transfer from a donor cell to a recipient cell. This process is mediated in F-like plasmids by proteins expressed by the tra operon. The relaxosome forms at oriT (origin of transfer), where the nicking and unwinding of a single-stranded copy of the plasmid begins, and the transferosome forms a transmembrane pore through which the DNA is transferred. TraM is a tetrameric relaxosomal protein which binds to 3 sites at oriT –sbmA, sbmB, and sbmC. TraD is an inner membrane protein of the transferosome that is homologous to FtsK/SpoIIIE hexameric ATPases. The interaction between the C-terminal tail of TraM and TraD is essential for high conjugation efficiency. The structural basis of this interaction is revealed by the crystal structure of F TraM58-127 in complex with TraD711-717. Electrostatic complementarity is a key feature of TraM-TraD interaction, which includes the TraM K99-TraD D715 and TraM R110-TraD F717 C-terminal carboxylate interactions. An additional feature is the fit of the phenyl side chain of F717 into a hydrophobic pocket. The importance of the TraD C-terminal tail for binding to TraM was tested with a pulldown assay comparing TraD constructs with and without a C-terminal truncation of 8 residues. In vivo assays confirmed the role of the C-terminal tail and its individual residues in conjugation. TraM interacts with sbmA in a highly plasmid-specific manner. The basis of this is revealed by the crystal structure of pED208 TraM in complex with sbmA. The structure shows that the N-terminal domain of TraM is a dimeric ribbon-helix-helix fold which recognizes the DNA bases which make up the binding motif. Two tetramers are bound to sbmA on opposite faces of the DNA without protein-protein contact, confirming the TraM-sbmA binding stoichiometry obtained from various biophysical methods. The cooperative nature of TraM binding to sbmA is therefore entirely through DNA distortions observed in the crystal structure, which include underwinding and kinking. Efforts to determine the structural basis of F TraM-sbmA interaction were undertaken but no diffraction-quality crystals were obtained.

Enterococcus faecalis has recently emerged as an important nosocomial pathogen. Pathogenicity of these organisms depends greatly on a few important aspect of enterococcal physiology. The ability of enterococci to form biofilms greatly enhances their virulence. Their innate resistance to many antibiotics and their ability to transfer these resistance genes through conjugation heightens their threat to human health. The work described in this thesis attempts to explain the roles of biofilm growth, conjugation, and cell communication in E. faecalis. To examine the role of biofilm growth on the E. faecalis transcriptome, RNAseq analysis was undertaken. We found that over 100 genes were measurably upregulated during biofilm growth while approximately 26 genes were downregulated. These data gives us important insights into the biology of enterococcal biofilms. In clinical settings, biofilms are likely locations for antibiotic resistance transfer events involving nosocomial pathogens such as E. faecalis. Conjugation is an important mode of horizontal gene transfer in bacteria, enhancing the spread of antibiotic resistance. In this work, I demonstrate that growth in biofilms alters the induction of conjugation by a sex pheromone in E. faecalis. Mathematical modeling suggested that a higher plasmid copy number in biofilm cells would enhance a switch-like behavior in the pheromone response of donor cells with a delayed, but increased response to the mating signal. Alterations in plasmid copy number and a bimodal response to induction of conjugation in populations of plasmid-containing donor cells were both observed in biofilms, consistent with the predictions of the model. The pheromone system may have evolved such that donor cells in biofilms are only induced to transfer when they are in extremely close proximity to potential recipients in the biofilm community. These results have important implications for development of chemotherapeutic agents to block resistance transfer and treat biofilm-related clinical infections.

The frequency of S. aureus infection and subsequent biofilm formation associated with vascular catheterization has been increasing in recent years and often begins as a local colonization at the site of the catheter insertion. Antimicrobial enzymes and peptides, which are effective against a broad range of pathogens and low rates of resistance, have attracted attention as promising alternative candidates in treatment of infections caused by antibiotic resistant bacteria. The use of nanoparticles as carriers for enzymes, in addition to their size, charge, high surface area per volume etc. offers targeted delivery of enzymes to pathogenic bacteria. We proposed to use nanoparticles as surfaces for targeted delivery of antibacterial enzymes and as `surrogate' surface coatings on indwelling central venous catheters (CVCs) to inhibit bacterial colonization and subsequent biofilm formation. It was shown that nanoparticle charge can be used to enhance delivery and increase bactericidal activity of an antimicrobial enzyme, lysozyme. In the case of bacterial lysis assay with a Gram-positive bacterium Micrococcus lysodeikticus, activity of lysozyme conjugated to positively charged nanoparticles was approximately twice as high as that of free lysozyme. This was believed to occur through charge-directed targeting of enzyme-nanoparticle conjugates to negatively charged bacterial cell walls through enhanced electrostatic interactions. In a clinically more relevant model, we studied antimicrobial activity of lysostaphin against S. aureus for both lysostaphin-coated and lysostaphin-antibody coated nanoparticle conjugates at different enzyme: antibody: nanoparticle ratios. At the highest antibody loading, bacterial lysis rates for antibody-lysostaphin-coated samples were significantly higher than for plain lysostaphin-coated samples and free enzyme due to multiple-ligand directed targeting of antibody molecules to bacterial cell walls (p We also performed in vitro experiments to evaluate the inhibition of bacterial colonies adhering to a surface. Bacterial infections by S. aureus strains are among the most common postoperative complications in surgical hernia repair with synthetic polymer meshes. Colony counting data from the broth count (model for bacteria in wound fluid) and wash count (model for colonized bacteria) for the enzyme-coated samples showed significantly decreased number of colony forming units (CFU) when compared to uncoated samples (p< 0.05). A pilot in vivo study showed a dose-dependent anti-S. aureus efficacy of lysostaphin-coated meshes in a rat model. Finally, we observed that that coating of nanoparticles overall did not significantly improve binding yield, leaching, durability and antibiofilm activity of enzymes adsorbed on catheter segments (p>0.05). Alternatives to coating catheter surfaces using covalent chemistry through functional groups on nanoparticles either directly or through appropriate crosslinkers could result in significantly higher enzyme loadings, better stability and long term durability for future applications. The approach developed here is universal and can potentially be used for treatment of other medical device-associated infections. Moreover, use of antibacterial enzyme-NP conjugates can eventually be expanded for intravenous administration, which will further broaden its range of application.

The ability of sub 100 nm nanoparticles to target and modulate the biology of cells will enable major advancements in cellular imaging and therapy in cancer and atherosclerosis. A key challenge is to load an extremely high degree of targeting, imaging, and therapeutic functionality into small, yet stable particles. A general mechanism is presented for thin autocatalytic growth on nanoparticle substrates (TAGS), as demonstrated for a homologous series of < 5 nm textured Au coatings on < 42 nm iron oxide cluster cores. Very low Au supersaturation levels are utilized to prevent commonly encountered excessive autocatalytic growth that otherwise produce thick shells. The degree of separation of nucleation to form the seeds from growth is utilized to control the morphology and uniformity of the thin Au coatings. The thin and asymmetric Au shells produce strong near infrared (NIR) absorbance with a cross section of ~10⁻¹⁴ m², whereas the high magnetic content per particles provides strong r2 spin-spin magnetic relaxivity of 200 mM⁻¹s⁻¹. TAGS may be generalized to a wide variety of substrates and high energy coatings to form core-shell nanoparticles of interest in a variety of applications as diverse as catalysis and bionanotechnology. High uptake of the nanoclusters by macrophages is facilitated by the dextran coating, producing intense NIR contrast both in cell culture and an in vivo rabbit model of atherosclerosis. A novel conjugation technique further allows covalent binding of anti-epidermal growth factor receptor (EGFR) monoclonal antibody (Ab) to the nanoclusters for highly selective targeting to EGFR over expressing cancer cells. AlexaFluor 488 tagged Ab nanocluster conjugates were prepared to correlate the number of conjugated Abs with the hydrodynamic diameter. The high targeting efficacy was evaluated by dark field reflectance imaging and atomic absorbance spectrometry (AAS). Colocalization of the nanoparticles by dual mode in-vitro imaging with dark field and fluorescence microscopy demonstrates the Abs remained attached to the Au surfaces. The extremely high curvature of the Au shells with features below 5 nm influence the spacing and orientations of the Abs on the surface, which has the potential to have a marked effect on biological pathways within cells. These targeted small multifunctional nanoclusters may solve some key molecular imaging challenges for cancer and atherosclerosis.

This study outlines the development of Portuguese and Spanish verbal morphology from Latin in the context of the Hispanic branch of Romance, with a focus on the conjugational classes, whose number has been reduced to only three in this branch of Western Romance. It is innovative in approaching the topic as a study of sequential productive grammars in an Item and Process type framework. We have found evidence to indicate that in addition to regular phonological change, morphological restructuring and language contact each played an important role in the reclassification of the Latin verb classes II-IV into the verb classes of the Hispano-Romance daughter languages. The historical data studied have been collected from published secondary sources of manuscript and dialectological data and supplemented with data from searchable electronic corpora.

Flat dendrimers, consisting of a hexavalent aromatic core and rigid ethynyl units locked in place by ether connections were developed based upon the divergent synthetic method. Alternating functional groups were adopted on each site of the hexa-substituted benzene, in order to avoid undesired cyclization pathways. The flat structures of conjugated dendrimers would allow investigation on the discotic liquid crystal properties. In addition, these ethylnyl dendrimers are expected to show directed energy and electron transfer with a highly conjugated system, and thus are effective in the preparation of photoreactive materials such as electronic sensors or light harvesting materials. Conjugated polycyclic aromatic hydrocarbons, consisting of naphthalene, anthracene, pyrene, and phenanthrene groups connected via 1,3,5-triethynylbenzene cores, were synthesized. These molecules exhibited luminescence properties and the π-complexation with a mercury trifunctional lewis acid are expected to enhance the phosphorescence in the presence of the heavy metal due to the spin-orbit coupling. Besides, owing to the presence of heavy metal atom in the Au (I) complexes linked by s-bonded triethynyltriphenylene luminophore, the phosphorescence occurs from a metal-centered emission. The conjugated organic luminophores have been developed to produce excellent quantum efficiencies, brightness, and long lifetimes.

Most foods can be considered as ecosystems containing various microorganisms including pathogenic, spoilage, commensal microbes and fermentation starter cultures. Microbial biofilm ecosystems also form on the surfaces of processing equipment. The interactions among microbes and between microbes and various surfaces play an important role in the persistence and the prevalence of these microbes in the food environment. Diversity of food matrices adds complexity to and directly shapes the composition of the microbiota in these ecosystems. Proper identification and quantification of microbes, evaluation of potential risks in the food ecosystems, and characterization of the physical and molecular events involved in ecosystems, including biofilm development, are among the primary tasks for food microbiologists. The objectives of this study are to develop a rapid detection system for foodborne microorganisms using molecular approaches, to characterize component(s) involved in biofilm development, and to examine the contribution of commensal organisms in ecosystem development and horizontal gene transfer.A real-time PCR system was developed to rapidly detect Alicyclobacillus spp. and Listeria monocytogenes in food. Detection of less than 10 bacterial cells per reaction was achieved within 4-7 hours. CluA, a surface protein related to cell clumping, was found to be an important component in the development of Lactococcus lactis biofilm. Biofilm attributes can be disseminated within the ecosystem by conjugation, and a lactococcal strain carrying the intrinsic high frequency gene transfer mechanism could increase the transfer of pAMβ1 by 10,000-fold. The study demonstrated, for the first time, that conjugation facilitated biofilm formation in Gram-positive bacteria, and that commensal organisms not only served as a gene pool but also as enhancers facilitating horizontal gene transfer, including the dissemination of the drug resistance genes. Finally, our data showed that commensal organisms, such as Pseudomonas , enhanced mixed-culture biofilm formation involving other microorganisms such as Staphylococcus , suggesting the contribution of commensal organisms in pathogen persistence.The rapid detection system developed in this study, has direct applications in both food industry and basic scientific research. Results from this study advanced the knowledge in biofilm development and gene transfer mechanisms.

pAM373 is a conjugative Enterococcus faecalis plasmid that encodes a response to a peptide sex pheromone secreted by plasmid-free enterococci. It was of interest because a related activity was produced by Staphylococcus aureus and Streptococcus gordonii. Because of clinical concern relating to the possible mobilization of vancomycin resistance determinants from enterococci into pathogens such as S. aureus, further characterization of pAM373 was initiated. Mating experiments demonstrated that pAM373 can transfer into S. aureus, and that when fused with an appropriate plasmid, a cointegrate is formed that can establish in this species. Complete nucleotide sequencing showed that pAM373 is 36,679 by in size and contains 48 open reading frames, 22 of which have homologues in the enterococcal plasmids pAD1, pPD1 or the conjugative transposon Tn916. Transposon insertions in a region corresponding to more than half of the plasmid affected the mating response. While homology at the amino acid level is weaker than among equivalent proteins from other pheromone plasmids, the overall organization of the pheromone response region appears conserved. It contains a gene encoding a pheromone-binding surface protein (TraC), and determinants with negative regulatory activity (e.g. traA) that control expression of downstream structural genes and conjugation functions. The precursor peptide of the inhibitor iAM373 was identified; its gene is located upstream of a transcription terminator t1. Unique features of pAM373 include the lack of determinants equivalent to traB (reduces endogenous pheromone) and sea1 (prevents futile conjugation between homologous donors) of pAD1. It was shown that pAM373 does not utilize an entry exclusion system. The gene for aggregation substance (asa373) differs from other aggregation substances, which generally are highly conserved; its predicted protein is smaller and aggregation does not require the presence of phosphate and divalent canons. Transcriptional analyses indicated that pheromone-inducible transcription from the iam373 promoter is crucial to the regulation of the pheromone response. Induction increased the level of transcripts extending up to t1 and initiated expression of downstream determinants. Finally, a counter-transcript (mD) was identified that is highly transcribed in uninduced cells and disappears after induction. It may reduce transcription from the iam373 promoter in the absence of pheromone.