A leptospirose é considerada a zoonose mais difundida do mundo, assim como uma doença reemergente. Esta enfermidade, causada por bactérias patogênicas do gênero Leptospira, possui altas taxas de infecção em países em desenvolvimento, ocasionando graves prejuízos econômicos e de saúde pública. Até o momento, não existem vacinas humanas licenciadas contra leptospirose. Após o seqüenciamento do genoma de três espécies de Leptospiras vários genes foram apontados como candidatos vacinais promissores. Uma categoria importante de genes candidatos são aqueles com possível atividade hemolítica. Neste trabalho, clonamos e expressamos diversas proteínas com possível atuação hemolítica. As proteínas recombinantes obtidas, no entanto, não exibiram atividade hemolítica. Uma destas proteínas, TlyC, foi investigada quanto à sua capacidade de interagir com os componentes da matriz extracelular (MEC). Os resultados obtidos indicam que TlyC liga-se com alta afinidade a diversos componentes da MEC, e que esta proteína é capaz de inibir competitivamente a adesão de Leptospiras à um material biológico que se assemelha à MEC. A transcrição e expressão destas proteínas foi detectada em cultura de Leptospira. Algumas das proteínas recombinantes foram utilizadas em um desafio animal contra leptospirose, mas nenhum delas foi protetora. Concluímos que estas proteínas não parecem ser bons candidatos vacinais e que TlyC é uma proteína que interage com componentes da MEC. / Leptospirosis is considered the most disseminated zoonosis of the world, and also a reemerging disease. This disease, caused by pathogenic bacteria of the genus Leptospira, has high rates of infection in developing countries, leading to severe economic and medical costs. There is not a licensed vaccine against leptospirosis for human use. After the genome sequencing of three species of leptospires, several genes were pointed to be promising vaccinal candidates. An important category of these candidates are those with putative hemolytic activity. In this work, we cloned and expressed some proteins with putative hemolytic activity. The recombinant proteins obtained, however, did not show hemolytic activity. One of these proteins, TlyC, was investigated with regard to its possible ability to interact to extracellular matrix (ECM) components. The results obtained indicate that TlyC binds with high affinity to several ECM components and that this protein can inhibit the Leptospira bind to a biological material that ressambles the ECM. The transcription and expression of these proteins were detected in leptospires cultures. Some of the recombinant proteins were used in an animal challenge against leptospirosis, but none of them were protective. We conclude that these proteins do not seem to be good vaccine candidates and that TlyC is a protein that interacts with the ECM and its components.

Single polymer passage through geometrically confined regions is ubiquitous in biology. Recent technological advances have made the direct study of its dynamics possible. We studied the capture of DNA molecules by the electroosmotic flow of a nanopore induced by its surface charge under an applied electric field. We showed theoretically that the DNA molecules underwent coil-stretch transitions at a critical radius around the nanopore and the transition assisted the polymer passage through the pore. To understand how a polymer worms through a narrow channel, we investigated the translocation dynamics of a Gaussian chain between two compartments connected with a cylindrical channel. The number of segments inside the channel changed throughout the translocation process according to the overall free energy of the chain. We found a change in the entropic driving force near the end of the process due to the partitioning of the chain end into the channel rather than the initial compartment. We also developed a theory to account for the electrophoretic mobility of DNA molecules passing through periodic confined regions. We showed that the decrease in the translocation time with the molecular weight was due to the propensity of hairpin entries into the confined regions. To further explore the dynamics of polymer translocation through nanopores, we performed experimental studies of sodium polystyrene sulfonate translocation through α-hemolysin protein nanopores. By changing the polymer-pore interaction using different pH conditions, we identified the physical origins of the three most common event types. We showed that increasing the polymer-pore attraction increased the probability of successful translocation. Motivated by understanding the dynamics of a polymer in a crowded environment, we investigated the dynamics of a chain inside a one dimensional array of periodic cavities. In our theory, the chain occupied different number of cavities according to its confinement free energy which consisted of entropic and excluded volume parts. By assuming that the chain moved cooperatively, the diffusion constant exhibited Rouse dynamics. Finally, we performed computer simulations of a chain inside a spherical cavity. We found that the confinement effect was best described by the hard sphere chain model. We further studied the escape dynamics of the chain out of the cavity through a small hole. The equilibrium condition of the chain during the escape was discussed.

<p>Urinary tract infections (UTIs) are typically caused by bacteria that colonize different regions of the urinary tract, mainly the bladder and the kidney. Approximately 25% of women that suffer from UTIs experience a recurrent infection within 6 months of the initial bout, making UTIs a serious economic burden resulting in more than 10 million hospital visits and $3.5 billion in healthcare costs in the United States alone. Type-1 fimbriated Uropathogenic E. coli (UPEC) is the major causative agent of UTIs, accounting for almost 90 % of bacterial UTIs. The unique ability of UPEC to bind and invade the superficial bladder epithelium allows the bacteria to persist inside epithelial niches and survive antibiotic treatment. Persistent, intracellular UPEC are retained in the bladder epithelium for long periods, making them a source of recurrent UTIs. Hence, the ability of UPEC to persist in the bladder is a matter of major health and economic concern, making studies exploring the underlying mechanism of UPEC persistence highly relevant. </p><p>In my thesis, I will describe how intracellular Uropathogenic E.coli (UPEC) evade host defense mechanisms in the superficial bladder epithelium. I will also describe some of the unique traits of persistent UPEC and explore strategies to induce their clearance from the bladder. I have discovered that the UPEC virulence factor Alpha-hemolysin (HlyA) plays a key role in the survival and persistence of UPEC in the superficial bladder epithelium. In-vitro and in-vivo studies comparing intracellular survival of wild type (WT) and hemolysin deficient UPEC suggested that HlyA is vital for UPEC persistence in the superficial bladder epithelium. Further in-vitro studies revealed that hemolysin helped UPEC persist intracellularly by evading the bacterial expulsion actions of the bladder cells and remarkably, this virulence factor also helped bacteria avoid t degradation in lysosomes. </p><p>To elucidate the mechanistic basis for how hemolysin promotes UPEC persistence in the urothelium, we initially focused on how hemolysin facilitates the evasion of UPEC expulsion from bladder cells. We found that upon entry, UPEC were encased in “exocytic vesicles” but as a result of HlyA expression these bacteria escaped these vesicles and entered the cytosol. Consequently, these bacteria were able to avoid expulsion by the cellular export machinery. </p><p>Since bacteria found in the cytosol of host cells are typically recognized by the cellular autophagy pathway and transported to the lysosomes where they are degraded, we explored why this was not the case here. We observed that although cytosolic HlyA expressing UPEC were recognized and encased by the autophagy system and transported to lysosomes, the bacteria appeared to avoid degradation in these normally degradative compartments. A closer examination of the bacteria containing lysosomes revealed that they lacked V-ATPase. V-ATPase is a well-known proton pump essential for the acidification of mammalian intracellular degradative compartments, allowing for the proper functioning of degradative proteases. The absence of V-ATPase appeared to be due to hemolysin mediated alteration of the bladder cell F-actin network. From these studies, it is clear that UPEC hemolysin facilitates UPEC persistence in the superficial bladder epithelium by helping bacteria avoid expulsion by the exocytic machinery of the cell and at the same time enabling the bacteria avoid degradation when the bacteria are shuttled into the lysosomes. </p><p>Interestingly even though UPEC appear to avoid elimination from the bladder cell their ability to multiple in bladder cells seem limited.. Indeed, our in-vitro and in-vivo experiments reveal that UPEC survive in superficial bladder epithelium for extended periods of time without a significantly change in CFU numbers. Indeed, we observed these bacteria appeared quiescent in nature. This observation was supported by the observation that UPEC genetically unable to enter a quiescence phase exhibited limited ability to persist in bladder cells in vitro and in vivo, in the mouse bladder. </p><p>The studies elucidated in this thesis reveal how UPEC toxin, Alpha-hemolysin plays a significant role in promoting UPEC persistence via the modulation of the vesicular compartmentalization of UPEC at two different stages of the infection in the superficial bladder epithelium. These results highlight the importance of UPEC Alpha-hemolysin as an essential determinant of UPEC persistence in the urinary bladder.</p> / Dissertation

Single-molecule detection has provided insights into how molecules behave. Without the averaging effect of ensemble measurements, the stochastic behaviour of single molecules can be observed and intermediate steps in multistep transformations can be clearly detected. The single-molecule reactants range from small molecules (e.g. propene) to proteins of several tens of kDa (e.g. myosin). One single-molecule detection technique is single-channel electrical recording. This approach is based on the measurement of the transmembrane ionic current flowing through a nanoscale transmembrane pore under an applied potential. In this thesis, the protein α-hemolysin was employed as a nanoreactor. α-Hemolysin is a toxin secreted by Staphylococcus aureus. Its transmembrane pore (~100 Å in length and ≥14 Å in diameter) allows ions, water and small molecules to pass through its lumen. Under an applied potential, chemical changes in reactants attached to the internal wall of the pore modulate the flow of ions, leading to changes in the transmembrane ionic current. Analysis of this current provides information about the reaction kinetics and mechanisms. Chapter 1 – Single-Molecule Chemistry and α-Hemolysin is an introductory chapter that is divided into two parts. Section 1.1 provides an overview of the different techniques for the detection of chemical reactions at the single-molecule level. Section 1.2 gives a brief review of the protein pore α-hemolysin, including its structure, properties and various applications. Chapter 2 – S-Nitrosothiol Chemistry applies cysteine-containing α-hemolysins to study the biologically relevant chemistry of S-nitrosothiols (RSNO). RSNO are important molecules involved in cell signalling, which control physiological processes such as vasodilation and bronchodilation. Three reactions, namely transnitrosation (the transfer of the ‘NO’ group from RSNO to a thiol), S-thiolation (the formation of a disulfide from RSNO and thiol) and S-sulfonation (the generation of an S-sulfonate (RSSO₃⁻) from RSNO and sulfite ion), were investigated at the single-molecule level. The pH-dependency of the two competing reactions (transnitrosation and S-thiolation), the lifetime of the proposed transnitrosation intermediate, and nature of the chemical reaction between RSNO and sulfite (a bronchoconstrictor) were determined. Chapter 3 – Silver(I)-thiolate and cadmium(II)-thiolate complexes describes the kinetics of the formation and breakdown of these two metal-thiolate complexes. Ag⁺ and Cd²⁺ are commonly used in probing the membrane topology and gating properties of ion channels using the scanning cysteine accessibility method (SCAM). The binding of two Ag⁺ ions per thiol group and the stepwise build-up and dissociation of Cd²⁺-glutathione complexes were unambiguously characterized. Chapter 4 – Copper(II)-Catalyzed Diels-Alder Reactions reports the attempt to carry out copper(II)-catalyzed Diels-Alder reactions inside an engineered α-hemolysin. An iminodiacetate ligand was covalently attached within the lumen of the α-hemolysin pore. This ligand chelates Cu²⁺ ion, which can bind bidentate dienophiles and activate them towards Diels-Alder reaction with dienes. However, due to the ‘slow’ reaction rate of the Diels-Alder reaction (rate constant ~10⁻¹ M⁻¹s⁻) relative to the time-scale of the single-molecule experiment, we failed to observed chemical conversion at the single-molecule level. Nevertheless, the engineered metal-binding α-hemolysin may be useful for sensing molecules bearing metal-coordinating groups.

Metallic quantum clusters are stable structures that can exhibit many useful magnetic, chemical, and optical properties. Developing clusters for specific applications requires accurate methods for characterizing their physical and chemical properties. Most cluster characterization methods are ensemble-based measurements that can only measure the average values of the cluster properties. Single cluster measurements improve upon this by yielding information about the distribution of cluster parameters. This investigation describes the initial results on a new approach to detecting and characterizing individual gold nanoclusters (Au25(SG)18) in an aqueous solution with nanopore-based resistive pulse sensing. We also present a new application where the clusters are shown to increase the mean residence time of polyethylene glycol (PEG) molecules within an alpha hemolysin (αHL) nanopore. The effect appears over a range of PEG sizes and ionic strengths. This increases the resolution of the peaks in the single molecule mass spectrometry (SMMS) current blockade distribution and suggests a means for reducing the ionic strength of the nanopore solute in the SMMS protocol.

EPEC atípicas (EPECa) são isoladas de surtos diarréicos em crianças, adultos e animais em países industrializados e em desenvolvimento. Hemolisinas são toxinas que atuam sobre a membrana de eritrócitos e de diversos tipos celulares. Tal grupo de toxinas é reconhecido como importante fator de virulência envolvido na patogênese bacteriana, por isso o objetivo foi avaliar a expressão de enterohemolisina em isolados de EPECa e analisar características fenotípicas como interferência do meio de cultivo e adesão a componentes de matriz extracelular. Observou-se prevalência de 5,4% de isolados enterohemolíticos, e variações na expressão da toxina foram associadas à concentrações de lactose e caseína nos meios de cultivo. Além disso, verificou-se forte associação entre produção de enterohemolisina e ligação à fibronectina celular. / Atypical EPEC (aEPEC) are isolated from diarrheal outbreaks in children, adults and animals in industrialized and developing countries. Hemolysins are toxins that act on the red cell membrane and various cell types. This group of toxins is recognized as an important virulence factor involved in the bacterial pathogenesis. The aim of this study was the evaluation of the enterohemolysin expression in aEPEC isolates as interference of the culture media and adhesion to components of extracellular matrix. The prevalence of enterohemolytic isolates was 5.4%, and variations in toxin expression were associated with lactose and casein concentrations in culture media. Moreover, it was found association between production of enterohemolysin and adhesion to cellular fibronectin.

Wong, Sze Ki. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 87-106). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / Abstract in Chinese --- p.iv / Table of contents --- p.V / List of figures --- p.ix / List of abbreviations --- p.X / Chapter Chapter 1. --- General introduction --- p.1 / Chapter Chapter 2. --- Literature review --- p.6 / Chapter 2.1. --- Pathogenic mechanisms of Vibrio species in fish --- p.7 / Chapter 2.1.1. --- Introduction --- p.7 / Chapter 2.1.2. --- Adhesion --- p.7 / Chapter 2.1.3. --- Invasion --- p.8 / Chapter 2.1.4. --- Proliferation --- p.9 / Chapter 2.2. --- Vibrio virulence factors --- p.12 / Chapter 2.2.1. --- Introduction --- p.12 / Chapter 2.2.2. --- Hemolysin --- p.12 / Chapter 2.2.3. --- Protease --- p.14 / Chapter 2.2.4. --- Siderophore --- p.15 / Chapter 2.2.5. --- Lipopolysaccharide --- p.15 / Chapter 2.3. --- General apoptotic pathways --- p.17 / Chapter 2.3.1. --- Introduction --- p.17 / Chapter 2.3.2. --- Extrinsic apoptotic pathway --- p.17 / Chapter 2.3.2.1. --- Death receptor signaling apoptosis --- p.17 / Chapter 2.3.2.1.1. --- Fas signaling pathway --- p.18 / Chapter 2.3.2.1.2. --- TNF-R1 signaling pathway --- p.19 / Chapter 2.3.2.1.3. --- TRAIL receptors signaling pathway --- p.20 / Chapter 2.3.2.2. --- Growth factor receptor signaling apoptosis --- p.21 / Chapter 2.3.3. --- Intrinsic apoptotic pathway --- p.21 / Chapter 2.3.3.1. --- Mitochondrial apoptotic pathway --- p.21 / Chapter 2.3.3.1.1. --- Cyto c --- p.22 / Chapter 2.3.3.1.2. --- Smac/DIABLO --- p.22 / Chapter 2.3.3.1.3. --- Omi/HtrA2 --- p.22 / Chapter 2.3.3.1.4. --- AIF and endo G --- p.23 / Chapter 2.3.3.1.5. --- Bcl-2 family --- p.23 / Chapter 2.3.3.1.6. --- Mitochondrial membrane permeabilization (MMP) --- p.23 / Chapter 2.3.3.2. --- p53-regulated apoptotic pathway --- p.24 / Chapter 2.3.3.3. --- Endoplasmic reticulum (ER) stress-induced apoptotic pathway --- p.25 / Chapter 2.4. --- Membrane vesiculation in erythrocytes --- p.26 / Chapter 2.4.1. --- Introduction --- p.26 / Chapter 2.4.2. --- Induction of vesiculation --- p.26 / Chapter 2.4.3. --- Contents of vesicles --- p.28 / Chapter 2.4.4. --- Mechanisms involved during vesiculation --- p.29 / Chapter 2.4.5. --- Correlation between apoptosis and membrane vesiculation in erythrocytes --- p.31 / Chapter 2.4.6. --- Reasons for vesiculation --- p.31 / Chapter Chapter 3. --- "Induction of apoptosis by Vibrio alginolyticus thermolabile hemolysin (TLH) in blood cells of silver sea bream, Sparus sarba" --- p.33 / Chapter 3.1. --- Abstract --- p.34 / Chapter 3.2. --- Introduction --- p.34 / Chapter 3.3. --- Materials and methods --- p.36 / Chapter 3.3.1. --- Experimental fish --- p.36 / Chapter 3.3.2. --- Whole blood preparation --- p.37 / Chapter 3.3.3. --- Preparation of V. alginolyticus TLH --- p.37 / Chapter 3.3.4. --- "Caspase-3, -8, -9/6 fluorescent assay" --- p.38 / Chapter 3.3.5. --- TUNEL assay --- p.39 / Chapter 3.3.6. --- Apoptotic DNA ladder assay --- p.40 / Chapter 3.3.7. --- Statistical analysis --- p.41 / Chapter 3.4. --- Results --- p.42 / Chapter 3.4.1. --- "Increase of caspase-3, -8, -9/6 activities" --- p.42 / Chapter 3.4.2. --- Detection of DNA fragmentation by TUNEL assay --- p.44 / Chapter 3.4.3. --- Detection of DNA fragmentation by apoptotic DNA ladder assay --- p.44 / Chapter 3.5. --- Discussion --- p.46 / Chapter Chapter 4. --- "Occurrence of membrane vesiculation, apoptosis and post-apoptotic necrosis after exposure to Vibrio alginolyticus thermolabile hemolysin (TLH) in erythrocytes of silver sea bream, Sparus sarba" --- p.51 / Chapter 4.1. --- Abstract --- p.52 / Chapter 4.2. --- Introduction --- p.52 / Chapter 4.3. --- Materials and methods --- p.54 / Chapter 4.3.1. --- Experimental fish --- p.54 / Chapter 4.3.2. --- Whole blood preparation --- p.54 / Chapter 4.3.3. --- Preparation of V. alginolyticus TLH --- p.55 / Chapter 4.3.4. --- Light microscopy --- p.55 / Chapter 4.3.5. --- Transmission electron microscopy (TEM) --- p.56 / Chapter 4.3.6. --- Measurement of membrane vesiculation - acetylcholinesterase (AChE) assay --- p.56 / Chapter 4.3.7. --- Measurement of necrosis - hemoglobin colorimetric assay --- p.57 / Chapter 4.3.8. --- Apoptotic DNA ladder assay --- p.58 / Chapter 4.3.9. --- Flow cytometry --- p.59 / Chapter 4.3.10. --- Statistical analysis --- p.59 / Chapter 4.4. --- Results --- p.60 / Chapter 4.4.1. --- Ultrastructural changes in red blood cells after exposure to TLH --- p.60 / Chapter 4.4.2. --- Changes of cell population in size and granularity after exposure of TLH --- p.67 / Chapter 4.4.3. --- Effect of TLH dosage on necrosis and DNA fragmentation --- p.72 / Chapter 4.4.4. --- "Occurrence of membrane vesiculation, necrosis and DNA fragmentation in cells exposed to TLH" --- p.72 / Chapter 4.5. --- Discussion --- p.76 / Chapter Chapter 5. --- General conclusions --- p.82 / References --- p.87

Leung, Ho Wai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 136-146). / Abstracts in English and Chinese. / THESIS COMMITTEE --- p.II / ACKNOWLEDGEMENT --- p.III / ABSTRACT --- p.IV / CHINESE ABSTRACT --- p.VI / TABLE OF CONTENT --- p.VII / OVERVIEW OF THIS PROJECT --- p.1 / Chapter SECTION 1: --- Purification and Characterization of hemagglutinins from French bean and mottled kidney bean / Chapter Chapter 1 --- INTRODUCTION / Chapter 1.1 --- General Introduction --- p.2 / Chapter 1.2 --- Physiological functions of plant lectins --- p.6 / Chapter 1.3 --- Physiological functions of animal lectins --- p.9 / Chapter 1.4 --- Biological functions of lectins --- p.12 / Chapter 1.5 --- Clinical and research applications of lectins --- p.16 / Chapter 1.6 --- Legume lectins --- p.17 / Chapter 1.7 --- Isolation and purification of lectins --- p.19 / Chapter 1.8 --- Objectives of the present study --- p.21 / Chapter Chapter 2 --- MATERIALS AND METHODS / Chapter 2.1 --- Chemicals --- p.22 / Chapter 2.2 --- Assay of hemagglutinating activity --- p.24 / Chapter 2.3 --- Purification protocol --- p.26 / Chapter 2.4 --- Assay of saccharide inhibition of hemagglutination --- p.28 / Chapter 2.5 --- Assay of pH stability --- p.28 / Chapter 2.6 --- Molecular mass determination and N-terminal sequence determination --- p.28 / Chapter 2.7 --- Assay of mitogenic activity --- p.29 / Chapter 2.8 --- Assay of antiproliferative activity --- p.30 / Chapter 2.9 --- Assay for antifungal activity --- p.30 / Chapter 2.10 --- Assay of HIV-1 reverse transcriptase inhibitory activity --- p.31 / Chapter 2.11 --- Assay of stability towards trypsin and chymotrypsin --- p.31 / Chapter 2.12 --- Assay of nitric oxide production --- p.32 / Chapter 2.13 --- Assay ofHIV-1 integrase --- p.32 / Chapter Chapter 3 --- EXPERIMENTAL RESULTS / Chapter 3.1 --- Purification scheme --- p.35 / Chapter 3.2 --- Size determination and N-terminal sequencing --- p.36 / Chapter 3.3 --- Temperature stability assay --- p.37 / Chapter 3.4 --- pH stability assay --- p.37 / Chapter 3.5 --- Saccharides inhibition of hemagglutination --- p.37 / Chapter 3.6 --- Stability towards Trypsin and Chymotrypsin --- p.38 / Chapter 3.7 --- Anti-proliferative activity --- p.38 / Chapter 3.8 --- HTV-1 reverse transcriptase inhibition --- p.39 / Chapter 3.9 --- Mitogenic activity --- p.39 / Chapter 3.10 --- Nitric oxide production --- p.39 / Chapter 3.11 --- HIV-1 integrase --- p.39 / Chapter 3.12 --- Defensin --- p.40 / Chapter Chapter 4 --- DISCUSSION / Chapter 4.1 --- Purification scheme --- p.68 / Chapter 4.2 --- Sequence comparison --- p.69 / Chapter 4.3 --- Physical Stability of the hemagglutinins --- p.70 / Chapter 4.4 --- Protease Stability --- p.71 / Chapter 4.5 --- Sugar Specificity Assay --- p.72 / Chapter 4.6 --- Anti-proliferative Aactivity toward Cancer Cells --- p.73 / Chapter 4.7 --- HTV-1 reverse trancriptase and H̐ơþV integrase inhibition --- p.74 / Chapter 4.8 --- Mitogenic activity --- p.75 / Chapter 4.9 --- Antifungal protein --- p.76 / Chapter Chapter 5 --- CONCLUSION --- p.78 / Chapter SECTION 2: --- Purification and Characterization of flammulolysin from mushroom Flαmmulinα velutipes / Chapter Chapter 1 --- INTRODUCTION / Chapter 1.1 --- General Introduction --- p.79 / Chapter 1.2 --- Mechanisms of hemolysis --- p.80 / Chapter 1.3 --- Biological role of hemolysins --- p.80 / Chapter 1.4 --- Mushroom hemolysin --- p.82 / Chapter 1.5 --- Applications of hemolysins --- p.83 / Chapter 1.6 --- Objectives of the present study --- p.83 / Chapter Chapter 2 --- MATERIALS AND METHODS --- p.84 / Chapter Chapter 3 --- EXPERIMENTAL RESULTS / Chapter 3.1 --- Purification and sequence determination --- p.90 / Chapter 3.2 --- Effect of sugars and salts on hemolysin --- p.90 / Chapter 3.3 --- Effect of Temperature and pH on hemolysin --- p.91 / Chapter 3.4 --- Effect of Proteases on hemolysin --- p.91 / Chapter 3.5 --- Effect of osmotic protection on hemolysin --- p.91 / Chapter 3.6 --- Effect of hemolysin on tumor cells --- p.91 / Chapter 3.7 --- Effect of hemolysin on spleen cells --- p.92 / Chapter 3.8 --- Effect of hemolysin on bacterial growth --- p.92 / Chapter 3.9 --- Effect of hemolysin on fungal growth --- p.92 / Chapter Chapter 4 --- DISCUSSION / Chapter 4.1 --- Purification and sequence comparison of hemolysin --- p.103 / Chapter 4.2 --- Sugar and Salts inhibition --- p.104 / Chapter 4.3 --- Temperature stability --- p.105 / Chapter 4.4 --- pH stability --- p.106 / Chapter 4.5 --- Protease stability --- p.106 / Chapter 4.6 --- Osmotic Protection --- p.106 / Chapter 4.7 --- Anti-tumour activity of the hemolysin --- p.107 / Chapter 4.8 --- Anti-fungal activity --- p.108 / Chapter Chapter 5 --- CONCLUSION --- p.109 / Chapter SECTION 3: --- Purification and Characterization of antifungal peptide from buckwheat seeds Fagopyrum esculentum / Chapter Chapter 1 --- INTRODUCTION / Chapter 1.1 --- Plant antiftmgal proteins --- p.110 / Chapter 1.2 --- Classification of antifungal proteins --- p.110 / Chapter 1.3 --- Distribution of antifungal proteins in plants --- p.111 / Chapter 1.4 --- Mechanisms of antifungal activity --- p.111 / Chapter 1.5 --- Future Perspectives of Antifungal proteins --- p.112 / Chapter 1.6 --- Antifungal peptide from Buckwheat --- p.112 / Chapter 1 .7 --- Objectives of the present study --- p.113 / Chapter Chapter 2 --- MATERIALS AND METHODS --- p.114 / Chapter Chapter 3 --- EXPERIMENTAL RESULTS / Chapter 3.1 --- Purification and sequence determination --- p.118 / Chapter 3.2 --- Effect on anti-fungal activity --- p.118 / Chapter 3.3 --- Effect of temperature and pH on antifungal activity --- p.118 / Chapter 3.4 --- Effect of the antifungal peptide on tumor cells --- p.119 / Chapter 3.5 --- Effect of antifungal peptide on HIV-1 Reverse transcriptase Activity --- p.119 / Chapter 3.6 --- Effect of antifungal peptide on spleen cells and NO Production --- p.119 / Chapter Chapter 4 --- DISCUSSION / Chapter 4.1 --- Purification scheme and N-terminal sequence --- p.130 / Chapter 4.2 --- Antifungal Activity --- p.131 / Chapter 4.3 --- Physical stability --- p.131 / Chapter 4.4 --- Anti-proliferative activity toward cancer cells --- p.131 / Chapter 4.5 --- HTV-1 Reverse Transcriptase Inhibitory activity --- p.132 / Chapter 4.6 --- Mitogenic activity and nitric oxide production --- p.132 / Chapter Chapter 5 --- CONCLUSION --- p.133 / OVERALL CONCLUSION --- p.134 / REFERENCES --- p.136

In nanopore analysis, peptides and proteins can be detected by the change in current when single molecules interact with an α-hemolysin pore embedded in a lipid membrane. Studies into the effects of fluorenylmethoxycarbonyl (Fmoc), acetylation or proline modification to negatively charged α-helical peptides showed that Fmoc peptides give more translocations than acetylated peptides. The addition of a proline in the middle of an acetylated peptide further reduces the number of translocations compared to Fmoc. The effect of peptide conformation on translocation or intercalation was studied with small α-helical and β-sheet hairpins. The capped β-hairpin increased translocations compared to the uncapped. The Fmoc-α-helical hairpin, containing a disulfide link, displayed both bumping and translocations whereas in the unlinked peptide the proportion of translocations was greater. Prion diseases arise from the misfolding and aggregation of the normal cellular prion protein. Nanopore analysis of prion peptides with α-helical and β-strand sequences show changes to the event parameters that help distinguish them. The interaction of bovine prion protein (bPrP), with α-hemolysin showed both bumping (type-I) and intercalation/translocation (type-II) events. There are several lines of evidence that indicate these type-II events with a blockade current of -65 pA for bPrP, represent translocations. Nanopore analysis showed that about 37% events were translocations. The interaction of metal ions with bPrP showed that Cu(II) or Zn(II) reduced translocations. Surprisingly, Mn(II) caused an increase in translocation events to about 64%. Complex formation between antibodies and prion peptides and proteins can be detected by nanopore analysis. The PrP/antibody complex is too large to translocate whereas the event parameters for unbound molecules are unchanged. In principle, a nanopore can detect a single molecule; thus, this work represents the first step towards the development of a prion detector. The nanopore will provide the sensitivity and the antibodies will provide the specificity to distinguish between PrPC and PrPSc. Also, the prion N- and C-terminal signal peptides interact with bPrP changing the event parameters, relating to a new mechanism. Finally, the folding intermediates of bPrP at 0.86 M Gdn-HCl suggests that the protein unfolds and then refolds into a different conformation with event parameters similar to those of bPrP.

In nanopore analysis, peptides and proteins can be detected by the change in current when single molecules interact with an α-hemolysin pore embedded in a lipid membrane. Studies into the effects of fluorenylmethoxycarbonyl (Fmoc), acetylation or proline modification to negatively charged α-helical peptides showed that Fmoc peptides give more translocations than acetylated peptides. The addition of a proline in the middle of an acetylated peptide further reduces the number of translocations compared to Fmoc. The effect of peptide conformation on translocation or intercalation was studied with small α-helical and β-sheet hairpins. The capped β-hairpin increased translocations compared to the uncapped. The Fmoc-α-helical hairpin, containing a disulfide link, displayed both bumping and translocations whereas in the unlinked peptide the proportion of translocations was greater. Prion diseases arise from the misfolding and aggregation of the normal cellular prion protein. Nanopore analysis of prion peptides with α-helical and β-strand sequences show changes to the event parameters that help distinguish them. The interaction of bovine prion protein (bPrP), with α-hemolysin showed both bumping (type-I) and intercalation/translocation (type-II) events. There are several lines of evidence that indicate these type-II events with a blockade current of -65 pA for bPrP, represent translocations. Nanopore analysis showed that about 37% events were translocations. The interaction of metal ions with bPrP showed that Cu(II) or Zn(II) reduced translocations. Surprisingly, Mn(II) caused an increase in translocation events to about 64%. Complex formation between antibodies and prion peptides and proteins can be detected by nanopore analysis. The PrP/antibody complex is too large to translocate whereas the event parameters for unbound molecules are unchanged. In principle, a nanopore can detect a single molecule; thus, this work represents the first step towards the development of a prion detector. The nanopore will provide the sensitivity and the antibodies will provide the specificity to distinguish between PrPC and PrPSc. Also, the prion N- and C-terminal signal peptides interact with bPrP changing the event parameters, relating to a new mechanism. Finally, the folding intermediates of bPrP at 0.86 M Gdn-HCl suggests that the protein unfolds and then refolds into a different conformation with event parameters similar to those of bPrP.