Antibacterial agent delivery is of great importance in medicine and dentistry since the bacterial infections are still one of the major reasons for hospitalization and mortality. Despite of the development of technique and pharmacy, more antimicrobial agents are optimized and utilized to treat infections, and their action of principal is better understood which lay a foundation for developing strategies for infection treatment. Over the last decades, many delivery systems have been established to deliver bacterial agents and maintain a sustained activity against them. However, the bacteria are always developing and finding a way to defend themselves. A more responsive antibacterial agent delivery system, which can release the active substances on demand to match the stages of diseases, is highly desirable. Therefore, it motivates us to carry out the work to develop a multifunctional delivery system for antibacterial particle formulation and encapsulation based on the layer-by-layer self-assembly technique and electrospinning, to manipulate the release with external triggers, such as near-infrared (NIR) light and alternating magnetic field (AMF). Strategically, two different kinds of antibacterial agents, chlorhexidine and doxycycline, were studied. Chlorhexidine was fabricated into spherical particles and functionalized with both gold and magnetite nanoparticles, and doxycycline was encapsulated within microcapsules which were also functionalized with magnetite nanoparticles. Their release kinetics and possibilities to trigger the release with either a NIR light or AMF was explored. The first two chapters of the thesis give a general introduction and literature review on the current use of antibacterial agents and the problems concerned, strategies already developed for antibacterial agent delivery, and the potential triggers to induce a smart release. In chapter 3, a brief description of materials and methods, and instruments is presented. Chapter 4 is about chlorhexidine particle formulation. Firstly, particulation of chlorhexidine and mechanism of 4 spherical interconnected structure formation was explored, and then the chlorhexidine particles are encapsulated either by LbL assembly or spray-drying. The chlorhexidine spheres were also functionalized with gold nanorods and Fe3O4 nanoparticles to achieve NIR light and magnetic field manipulated release, and the effect of nanoparticles on the formation of chlorhexidine spheres was also studied. When the chlorhexidine particles were incorporated into electrospun fibers, a sustained antibacterial activity was demonstrated. Chapter 5 is about the delivery of doxycycline to cells with microcapsules and the sustained intracellular doxycycline activity was demonstrated via EGFP expression when the cells were engineered with a tetracycline regulated gene expression system. Intracellular triggered release and upregulation of EGFP expression was achieved by an AMF. The results successfully demonstrated the possibility of chlorhexidine and doxycycline delivery and NIR light/AMF triggered release, which is promising for a future application in medicine and dentistry.

Objective To evaluate the cytotoxic and antibacterial effect of Bixa orellana L. (B. orellana) (achiote) methanol extract against Streptococcus mutans (ATCC 25175) (S. mutans) and Streptococcus sanguinis (ATCC 10556) (S. sanguinis). Methods Two methanol extracts of B. orellana were prepared in vitro, from the seeds and leaves. The antibacterial activity of extracts against S. mutans and S. sanguinis was evaluated using the cup-plate agar diffusion method. The minimum inhibitory concentration (MIC) was determined using the microdilution method and the cytotoxic activity was determinated by using the cell line MDCK. Results A stronger antibacterial effect was observed with the leaves methanolic extract with an inhibition zone of (19.97 ± 1.31) mm against S. mutans and (19.97 ± 1.26) mm against S. sanguinis. The methanolic extract of the seeds had an activity of (15.11 ± 1.03) mm and (16.15 ± 2.15) mm against S. mutans and S. sanguinis, respectively. The MIC of the leaf and the seed extracts against S. sanguinis was 62.5 and 125 μg/mL, respectively, and the MIC of the leaf extract against S. mutans was 62.5 μg/mL, and for the seed extract it was 31.25 μg/mL. The 50% cytotoxic concentration was 366.45 and 325.05 μg/mL for the leaves and seeds extracts, respectively. Conclusions The experimental findings demonstrated the antibacterial effect of the methanolic extract of B. orellana (achiote) on S. mutans and S. sanguinis. The extract of this plant is cytotoxic at high concentrations. / Peer review

Diseases caused by pathogenic bacteria continue to be major health concerns. For example, it is estimated that in the year 2000 typhoid fever caused over 21,000,000 illnesses and ~200,000 deaths (Crump et al., 2004). The disease is caused by S. typhi, a closely-related serotype of S. typhiumurium, the salmonella strain in which BipA was first identified. The CDC estimated that in 2013, multidrug resistant bacteria caused over 2 million infections in the United States, ending in more than 23,000 deaths (CDC, 2013). This number is set to rise as more bacteria become resilient to the collection of conventional antibiotics. The increasing number of multidrug resistant bacterial strains necessitates the development of new antimicrobial drugs. BipA is an attractive target for drug research. As mentioned in Section 2.5.2, BipA is ubiquitous in eubacteria and lower eukaryotes such as protozoa, but is absent from higher-order eukaryotes such as humans. Because the protein is essential for bacterial survival, BipA presents a major vulnerability of pathogenic bacteria. A drug targeting the protein itself or its interactions to the ribosome will disable only the bacteria, but have no effect on the eukaryotic host. A comprehensive model of BipA bound to the 70S ribosome will provide unparalleled insight into BipA's binding site and its mechanism. Toward this goal, cryo-EM techniques were employed to visualize the binding site of BipA on the 70S ribosome, characterize its interactions with the ribosome, and elucidate its mechanism on the ribosome. An X-ray structure of isolated BipA-GMPPNP was elucidated, by collaborators, and used for further molecular modeling of the protein to reveal possible atomic interactions between BipA and 70S ribosome. Additional biochemical studies were performed to fully characterize the specific ribosomal complex that optimizes binding of the factor. Together, the cryo-EM reconstruction, the BipA X-ray structure, the subsequent molecular modeling, and the additional biochemical studies provide a comprehensive model for BipA binding. Over the last years, the introduction of new automated algorithms for particle selection (AutoPicker) and classification (RELION) for the cryo-EM technique has revolutionized the workflow of the entire imaging and reconstruction process. The BipA dataset was primed to be used as a test bed for these algorithms and classification technique, respectively. Using old and new techniques to process the dataset allows a discussion of how the single particle reconstruction process can be vastly improved, with greater automation and efficiency.

Submitted by Sandro Camargo (sandro.camargo@unipampa.edu.br) on 2015-05-08T03:06:29Z No. of bitstreams: 1 127110046.pdf: 2895137 bytes, checksum: edf417bbba917e277ef644cfa24efbf9 (MD5) / Made available in DSpace on 2015-05-08T03:06:29Z (GMT). No. of bitstreams: 1 127110046.pdf: 2895137 bytes, checksum: edf417bbba917e277ef644cfa24efbf9 (MD5) Previous issue date: 2014-07-21 / As chalconas são compostos de origem vegetal e apresentam estrutura química simples que é amplamente utilizada como molécula protótipo para a obtenção de novos compostos bioativos através de modificações estruturais nos seus anéis aromáticos. Este fato é importante uma vez que a Sociedade Americana de Doenças Infecciosas lançou a iniciativa “10 x 20”, ou seja, o rápido desenvolvimento de 10 novos antibióticos até o ano de 2020. O objetivo deste estudo é a obtenção e determinação das propriedades físico-químicas e biológicas de chalconas com atividade antibacteriana potencial. As moléculas foram planejadas utilizando-se a metodologia de bioisosterismo clássico de valência, propondo a alteração da hidroxila presente na posição 4 do anel A da Licochalcona pelo grupo amino; no anel B houve a introdução de grupos nas posições para e meta, com graus variáveis de efeito eletrônico, de acordo com o emprego do Diagrama de Craig. As aminochalconas substituídas foram sintetizadas pela reação de Claissen-Schimdt, com quantidades equimolares de 4- aminoacetofenona e benzaldeídos variados e purificadas via cromatografia em coluna ou recristalização para posterior caracterização por espectroscopia de infravermelho e ressonância magnética nuclear. A avaliação da atividade antibacteriana e sinérgica foi realizada através do método de microdiluição em caldo descrito pelo Clinical and Laboratory Standards Institute e Teste de checkerboard frente a cepas referências de bactérias gram- positivas e gram-negativas (Staphylococcus aureus ATCC 29213 e Escherichia coli ATCC 25922), além de uma cepa S. aureus resistente à meticilina (N315). Foi utilizado o programa computacional Spartan ́08 for Windows para determinação da estrutura química em 3D e cálculo dos valores das energias de HOMO e LUMO. O estudo teórico das propriedades físico-químicas foi realizado pelos programas Chem3D Ultra, Molinspiration e MarvinSketch 6.2. Para o estudo teórico da toxicidade e dos perfis de druglikeness e drugscore empregou-se o programa Osiris Property Explorer. Os compostos 1, 3 e 7 apresentaram os menores valores de CIM quando ensaiados frente a ambas cepas de S. aureus, o que sugere que o mecanismo de resistência bacteriana não afeta a atividade desempenhada pelas 4-aminochalconas. Os compostos 5 e 8 foram os mais ativos frente a cepas E. coli. Não houve interação significativa entre as 4-aminochalconas e os antibióticos testados (oxacilina e cloranfenicol). Através da avaliação das relações estrutura-atividade, verificou-se que o composto 8 apresentou vários parâmetros que podem justificar sua ação frente a bactérias gram-negativas, como peso molecular elevado e menores valores de cLogP e LogD 7.4 . Resultados superiores à levofloxacina foram também evidenciados quando calculado seu potencial de druglikeness e de drugscore. Todos os compostos apresentaram alto risco teórico para o efeito mutagênico e baixo perfil toxicológico para o efeito irritante. Em relação aos efeitos no sistema reprodutor, apenas o composto 1 apresentou um risco médio, e para os compostos 2 e 3 foi descrito alto risco para o efeito tumorogênico. Mesmo apresentando uma atividade antibacteriana baixa, estas moléculas podem vir a delinear modificações estruturais com o intuito de aumento da ação biológica, visto que todas enquadram-se dentro dos parâmetros delineados na Regra dos Cinco e, devido a isso, são promissoras a apresentar uma boa biodisponibilidade oral. / Chalcones are vegetal origin compounds and they present a simple chemical structure which is widely used as a molecule prototype in order to obtain new bioactive compounds by structural changes in their aromatic rings. Such fact is important as the American Society of Infectious Diseases has released the “10 x 20” initiative, meaning the fast development of 10 new antibiotics up to 2020. This study aims to obtain and establish chalcone’s physical- chemical and biological properties with potential antibacterial activity. The molecules were designed by the classic bioisosterismo classic methodology, proposing the modification of the hydroxyl existent in licochalcone’s position 4 of ring A in the amino group, in ring B groups were introduced in para and meta positions, with variable degrees of electronical effects, according to the usage of Craig ́s Diagram. The replaced aminochalcones were synthesized by the Claissen-Schimdt reaction with equimolar quantities of 4-aminoacetophenone and varied benzaldehydes and purified by column chromatography or recrystallization for further characterization by infrared spectroscopy and nuclear magnetic resonance. The evaluation of the antibacterial and synergic activity was made through Microdilution Methods described by the Clinical and Laboratory Standards Institute and checkerboard test against reference strains of gram-positive and gram-negative (Staphylococcus aureus ATCC 29213 and Escherichia coli ATCC 25922), plus S. aureus meticillin resistant (N315). Spartan’08 for Windows was the computational program used to establish the 3D chemical structure and the HOMO’s and LUMO’s energy amount figures. The theoretical study of Physical-chemical properties was accomplished by Chem3D Ultra, Molinspiration and MarvinSketch 6.2 programs. In order to theoretically study both toxicity and profile of druglikeness and drugscore, Osiris Property Explorer program was used. The compounds 1, 3 and 7 showed the smallest CIM values when against both S. aureus strains, which suggests that the bacterial resistance mechanism doesn’t affect the 4-aminochalcones activity. Compounds 5 and 8 were more actives against strains E. coli. There was no significant interaction among the 4- aminochalconas and the antibiotics tested (oxacilin and chloramphenicol). Throughout the structural-activity relation evaluation, it was verified that compound 8 presented several parameters that may justify its action facing Gram-negative bacteria, such as the growth of its molecular weight and decreased values of cLogP and LogD7,4 . Levofloxacin superior results were also certified when its potential of druglikeness and drugscore were figured. All the compounds have presented a high theoretical risk to the mutagenic effect and low toxicological profile to the irritant effect. Relating to the effects in the reproductive system, only compound 1 has presented a mid-risk and compound 2 and 3 high risk to tumorigenic effect. Even though these molecules have showed low antibacterial activity, they might outline changes in their structure aiming the biological action growth as they all fit the Rule of Five parameters and as a result, they tend toward a good oral bioavailability.

Submitted by Marcos Anselmo (marcos.anselmo@unipampa.edu.br) on 2016-09-22T14:22:35Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Carmem Lucia dos Santos Machado.pdf: 2830040 bytes, checksum: e257722fc7ff4b644add8fc45c9be5cd (MD5) / Approved for entry into archive by Marcos Anselmo (marcos.anselmo@unipampa.edu.br) on 2016-09-22T14:22:57Z (GMT) No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Carmem Lucia dos Santos Machado.pdf: 2830040 bytes, checksum: e257722fc7ff4b644add8fc45c9be5cd (MD5) / Made available in DSpace on 2016-09-22T14:22:57Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Carmem Lucia dos Santos Machado.pdf: 2830040 bytes, checksum: e257722fc7ff4b644add8fc45c9be5cd (MD5) Previous issue date: 2016-06-24 / A apocinina é uma acetofenona que apresenta estrutura pequena e possui diversas atividades biológicas. Sua estrutura pode ser oxidada em certas condições químicas ou biológicas e resultar na formação do seu dímero, a diapocinina. As chalconas são compostos que contém em sua estrutura básica a porção 1,3-diarilpro-2-em-1-ona e se caracterizam por apresentar ampla variedade de atividades biológicas, como atividade antioxidante e antibacteriana. Estudos demostram que modificações estruturais nos anéis aromáticos das chalconas resultam na obtenção de compostos com atividade aceptora de radicais e antibacterianas. Neste trabalho foram realizadas a síntese de chalconas derivadas da apocinina e diapocinina, a avaliação da atividade antioxidante e antibacteriana. Adicionalmente foram realizados estudos de modelagem molecular, determinação de propriedades físico-químicas e a avaliação da toxicidade in silico, com a finalidade de se obter compostos que possam ser utilizadas como protótipos a novos fármacos. As chalconas derivadas da apocinina e da diapocinina, com substituintes 4-OCH3, 3,4-OCH3, e 3,4,5-OCH3 ligados ao anel B, e não substituída (H), foram sintetizadas através da condensação de Claisen-Schmidth, dos benzaldeídos com a apocinina e diapocinina, em meio básico, temperatura ambiente, por 24 horas. Ao final de cada reação, os compostos foram analisados e purificados por recristalização ou em cromatografia em coluna. Os compostos foram caracterizados por CCD, ponto de fusão, espectroscopia de infravermelho (IV), e por 1H e 13C RMN. A análise de capacidade aceptora de radicais das moléculas sintetizadas, foram realizadas através do emprego do método colorimétrico de análise do DPPH e ABTS, utilizando como padrão BHT e Trolox, respectivamente, nas concentrações 6,75, 12,5, 25, 50, 100, 200 e 400 μM. A avaliação da atividade antibacteriana dos compostos obtidos foi realizada através do método de difusão ágar-cilindro e leitura de halos de inibição, onde foram testadas as cepas de Staphylococcus aureus ATCC 25923 (bactéria Gram positiva) e Escherichia coli ATCC 25922 (bactéria Gram negativa), utilizando como padrão o antibiótico cloranfenicol. A análise de toxidade in silico, como a genotoxidade, mutagenicidade, efeitos irritante e sobre sistema reprodutor, foi realizado através do programa computacional Osiris Property Explorer. Os compostos apresentaram atividade aceptora do radical DPPH e ABTS na faixa de concentração de 400 a 12,5μM, sendo superiores ao padrão de BHT e Trolox. No ensaio de avaliação da atividade antibacteriana todos os compostos apresentaram atividade antibacteriana frente à cepa Staphylococcus aureus, sendo, no entanto, inferior ao padrão cloranfenicol. Não foi observada atividade contra a cepa Escherichia coli. A partir da análise da toxicidade in silico observou-se que somente os compostos que tinham grupamentos OCH3 substituídos em posição para, apresentaram risco médio e alto para efeito irritante e tóxico para o sistema reprodutor. Os demais compostos apresentaram risco teórico baixo de desenvolver os efeitos tóxicos citados. Diante dos resultados obtidos, observa-se que as chalconas derivadas da apocinina e diapocinina se apresentam como compostos promissores para o estudo e o desenvolvimento de agentes com atividade antioxidante, com ação antibacteriana e baixa toxicidade teórica, sendo assim demonstrando potenciais agentes para se utilizar na terapêutica. / The apocynin is an acetophenone which has little structure and has several biological activities. Its structure can be oxidized in certain chemical or biological conditions and result in the formation of the dimer, the diapocinina. The chalcones are compounds containing in its basic structure portion 1,3-diaryl-pro-2-en-1-one and are characterized by having variety of biological activities such as antibacterial and antioxidant activity. Studies show that structural modifications on the aromatic rings of chalcones result in obtaining the compounds with acceptor radical and antibacterial activity. In this work were performed chalcones derived synthesis of apocynin and diapocinina, the evaluation of antioxidant and antimicrobial activity. It was also performed molecular modeling studies, determination of physicochemical properties and toxicity evaluation in silico, in order to obtain compounds which can be used as prototypes of new drugs. The derivatives of the chalcones and apocynin diapocinina, substituents 4-OCH3, 3,4-OCH3, and 3,4,5-OCH3 attached to B ring, unsubstituted (H) were synthesized by Claisen-Schmidth condensation, of benzaldehydes with apocynin and diapocinina, in basic medium at room temperature for 24 hours. After each reaction, the compounds were analyzed and purified by recrystallization or column chromatography. The compounds were characterized by TLC, melting point, infrared spectroscopy (IR) and 1H and 13C NMR. The acceptor capacity analysis of radical synthesized molecules were carried out by employing the colorimetric method of analysis of DPPH and ABTS using standard as BHT and Trolox, respectively, at concentrations 6.75, 12.5, 25, 50, 100 200 and 400 uM. The evaluation of the antibacterial activity of the obtained compounds was performed by the method of agar-cylinder diffusion and reading inhibition zones, where Staphylococcus aureus ATCC 25923 (Gram positive bacterium) and Escherichia coli ATCC 25922 (Gram negative bacteria) were tested using as standard antibiotic chloramphenicol. The analysis of in silico toxicity, such as genotoxicity, mutagenicity, and irritating effects on reproductive system, was performed using the computer program Osiris Property Explorer. The compounds showed acceptor activity of DPPH and ABTS radical in the concentration range of 400 to 12,5μM, being superior to the standard of BHT and Trolox. The evaluation of the antibacterial activity test all compounds showed antibacterial activity against Staphylococcus aureus strain being, however, lower than the standard chloramphenicol. There was no activity against Escherichia coli strain. From the analysis of in silico toxicity it was observed that only compounds which had OCH3 groups substituted in the para position, exhibited medium and high risk of irritating and toxic effect on the reproductive system. Other compounds showed low theoretical risk of developing toxic effects reported. Considering the results, it is observed that the derivatives of apocynin and diapocinina chalcones present as promising compounds for the study and development of agents having antioxidant activity, antibacterial action and low theoretical toxicity, thus demonstrating potential agents for use in therapy.

Thesis (M.Sc. (Biochemistry)) --University of Limpopo, 2013 / This study was carried out to investigate antioxidant and antibacterial properties of 9 indigenous medicinal plants, viz., Euclea undulata (mogweregwere), Momordica balsamia (mogapu badimo), sefapa badimo, Senecio asperulus (makgonatšohle), Stiburus alopecuroides (mošalašuping), serolana, Euphorbia inaequilatera (kgama-maswana), mokgagapitsi and Clerodendrum glabrum (mohlokohloko) and to further isolate compounds that relate to these properties. Four extracting solvents with varying polarities viz. n-hexane, dichloromethane, acetone and methanol were used to extract the bioactive compounds from the ground powdered plant materials. The TLC plates, developed in three solvent systems viz., benzene, ethanol and ammonia (BEA, 18:10:0.2, v/v/v); ethyl acetate, methanol and water (EMW, 10:1.35:1, v/v/v) and chloroform, ethyl acetate and formic acid (CEF, 10:8:2, v/v/v), were visualised using DPPH, vanillin-sulphuric acid, visible light at 366 nm, UV light at 254 nm and bioautography for the presence of potential antioxidant and antibacterial compounds. The results of the screening process showed that only four plants possessed antioxidant compound(s) while six plants had antibacterial activity against Staphylococcus aureus. Euclea undulata “MKK” was observed to possess both antibacterial and antioxidant active compounds. Two antioxidant active compounds were isolated from two plants, viz., Euphorbia inaequilatera and Dicerocaryum senecioides. Solvent-solvent extraction, column chromatography and preparative TLC were used to further isolate and characterise target compounds. The antioxidant active compounds were found to separate well under EMW, an indication that the compounds are polar and intermediate-polar. The NMR spectra of the compound isolated from the D. senecioides revealed that the compound is a stilbenoid. For the first time, we report that the anti-inflammatory, antioxidant and antiproliferation properties of the D. senecioides reported by other studies performed in this laboratory could be due to this isolated stilbenoid compound. However, further studies are still necessary to confirm this assertion.

Philosophiae Doctor - PhD / The field of nanotechnology continues to offer solutions for biotechnologists whose target is to improve the quality of life by finding new therapies to combat diseases. Gold nanoparticles (AuNPs) have been showing great potentials in many biomedical applications. The antibacterial activity of the AuNPs presents a therapeutic option for conditions caused by bacterial infections such as chronic wounds. Also, these versatile particles can offer solutions in the treatments of infectious diseases and can also be exploited as “smart” vehicles to carry drugs, such as antibiotics, for improved efficiency. Moreover, the anti-inflammatory activity of AuNPs makes them useful in the management of prolonged inflammation caused by bacterial infections. The synthesis of AuNPs can be achieved by variety of physical and chemical methods that have been successfully applied in labs and industry. Nonetheless, the drawbacks of these “conventional” methods in terms of high cost, adverse health side effects and incompatibility with the ecosystem cannot be overlooked. Thus, new safer and more cost-effective protocols have been reported for the synthesis of AuNPs. Plants have provided alternate synthesis methods in which the reducing capabilities of the phytochemicals, found in the aqueous plant extracts, can be used to chemically synthesize AuNPs from gold precursors. The biosynthesis and characterization of AuNPs from the phytochemicals of several South African plants is investigated in this study. The study also reports the optimization of the AuNPs biosynthesis by varying reaction conditions such as temperature and plant extracts’ concentrations. Furthermore, the study highlights the wound healing activity of the AuNPs synthesized from selected plants by investigating their antibacterial activity on bacterial strains known to cause chronic wounds. The ability of these AuNPs to carry ampicillin in order to enhance the antibacterial activity is also described herein. The cytotoxicity of the biosynthesized AuNPs was evaluated on human normal fibroblasts cells (KMST-6). Additionally, the immunomodulatory effect of the biosynthesized AuNPs on the cytokines production from macrophages and Natural Killer (NK) cells was examined. The study was successful to produce biocompatible and safe AuNPs synthesized from the tested aqueous plant extracts. The resulted AuNPs showed different physicochemical properties by varying the reaction conditions. The AuNPs exhibited antibacterial activity against several Gram-positive and Gram-negative bacteria. Also, ampicillin was successfully loaded on the biosynthesized AuNPs, which led to the formation of more antibacterial active conjugated AuNPs compared to the free AuNPs. The green synthesized AuNPs were also found to have anti-inflammatory responses as shown by the reduction of pro-inflammatory cytokines from immune cells. In vitro assays showed that the biogenic AuNPs were not toxic to KMST-6 cells. Overall, the data suggest that plant extracts produce biologically safe AuNPs with antibacterial and anti-inflammatory activities that can be exploited in the treatment of chronic wounds and in the management of chronic inflammation.

Self-stabilizing polyacrylate nanoparticle emulsions were previously investigated in the Turos laboratory, and provided a new model for delivering antibiotics via encapsulation or covalent binding of the desired bioactive compound within the polymer nanoparticles. The method used the in water, free radical emulsion polymerization of butyl acrylate/styrene mixture to form the polymer chain stabilized with a surfactant. Current research in this dissertation further explores the versatility of related nanoparticle emulsion systems. Chapter 2 provides an overview of the loading of certain therapeutic drugs, such as 5-aminosalicylic acid and derivatives thereof, for the treatment of irritable bowel syndrome. Chapter 3 explores homo-polymer nanoparticle emulsions composed of menthyl acrylate as the monomer. Thereby obviating the need for a copolymer emulsion polymerization. The homo(menthyl acrylate) nanoparticle emulsion provided greater stability compared to the previous copolymer models. The resulting homopolymer emulsion exhibited a decrease in cytotoxicity, and a 400% increase for loading of penicillin G. Chapter 4 explores novel polyacrylamide nanoparticle emulsion using only N-acrylated ciprofloxacin to form a homo-polymer polyacrylate nanoparticle emulsion, thereby requiring no additional co-monomers. The resulting emulsion has a relatively low cytotoxicity with similar bioactivity to free ciprofloxacin.

Carissa lanceolata (conkerberry) is a perennial woody shrub used in traditional medicine by indigenous communities in Western Australia, the Northern Territory and Queensland for various medical conditions such as toothache, respiratory infections and the cleaning of sores, which all strongly indicate an antibacterial activity. A literature review revealed that the wood of this plant possesses significant antibacterial activity, which was found to be related to the presence of eudesmane type sesquiterpenes. C. edulis and C. carandus are frequently used in other traditional systems of medicine in different parts of the world, and thus have also been investigated for bioactive compounds and pharmacological properties. Some of these were found to be in line with the main findings of this work. Carissa lanceolata root was shown to exhibit significant antibacterial activity against both Gram negative and Gram positive organisms. A micro-broth dilution assay was performed on 96-well plates using resazurin as an indicator for microbial growth of Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis and Staphylococcus aureus. Bioassays carried out in this work showed that crude extracts of root bark and wood, particularly their polar constituents were more active against the four strains of bacteria tested. / Chemical investigation of the root bark revealed that it contains a volatile oil, which was isolated by steam distillation as well as solid phase micro extraction. It was found to consist of a single compound, which was identified as 2'-hydroxy acetophenone. The identity of this compound was confirmed by GC/MS and 1H NMR spectroscopy. Furthermore, the eudesmane-type sesquiterpene, carissone, was isolated from the root bark DCM and root wood hexane extracts. Its chemical identity was confirmed by IR, 1 [superscript] H and 13 [superscript] C NMR spectroscopy. The lignan, carinol, on the other hand, was isolated from the moderately polar fractions of the root wood MeOH extract. The obtained IR and 1 [superscript] H NMR data as well as Rf values all correspond to the literature. Two other yet unidentified compounds were isolated, but further studies into their chemistry and antibacterial activity were not possible in this current study. The antibacterial activity of the isolated compounds was considerable, with 2'-hydroxy acetophenone exhibiting the strongest effect, followed by carinol and then carissone.

Microbial contamination of contact lenses is a significant risk factor leading to adverse responses. Adhesion of microorganisms to a contact lens is the first step in a series of events that leads to contact lens-related infections or inflammation. Recently, some of the non-steroidal anti-inflammatory drugs (NSAIDs) have been shown to have the ability to interfere with microbial biofilm formation. In this project, antibacterial properties of commonly used NSAIDs (salicylic acid, sodium diclofenac and ketorolac) were assessed and characterised using biological assays and molecular biological techniques. Salicylic acid, ketorolac and diclofenac reduced adhesion of a range of bacterial species isolated from corneal infection and inflammatory events to contact lenses in a dose-dependent manner. Salicylic acid also decreased the adhesion of Pseudomonas aeruginosa and Staphylococcus epidermidis to human corneal epithelial cells in a dose-dependent manner. Results further demonstrated that NSAIDs had a significant impact on the production of virulence factors such as Type IV pili mediated (twitching) motility, flagella mediated swimming, elastase, protease IV and alkaline protease and affected the production of acylated homoserine lactones of P. aeruginosa. Salicylic acid and ketorolac affect the expression of P. aeruginosa outer membrane proteins. In the presence of the salicylic acid and ketorolac more than 85% of all detectable outer membrane proteins changed and most were down-regulated. Moreover, in the presence of salicylic acid at least five gene products, including Na+ - translocating NADH (Nrq1), choline dehydrogenase (CHDH), a hypothetical protein of unknown function, a gene product with no similarity to any known sequence in the database and a sequence similar to 23S rRNA of P. aeruginosa, were down-regulated. The results of this study clearly demonstrated that NSAIDs have a significant impact on virulence factors and the expression of acylated homoserine lactones by P. aeruginosa. This thesis has illustrated the potential of NSAIDs for preventing bacterial contamination of contact lenses by ocular pathogens and highlights the potential for NSAIDs as antibacterial agents. Therefore, this class of compound should be investigated further for their therapeutic efficacy in vivo.