Bacterial infections are a serious problem for patients with burns and other wounds. Such burn wound infection accounts for the pathogenic bacteria by colonising onto burned areas. Therefore, the need for detection and inhibition of such bacterial colonisation requires a methodology for sensing/killing pathogenic bacteria. This research project aims to design a ‗smart‘ wound dressing system which can respond to the microbiological environment of the wound via a simple colour change and will release antimicrobials only when required. Two strains of pathogenic bacteria Staphylococcus aureus (MSSA 476) and Pseudomonas aeruginosa (PAO1) were used in the study. The non-pathogenic bacterium E.coli (DH5α) was used as a control organism as it does not secrete virulence factors and therefore does not lyse membranes of vesicles. The key contributions of this thesis are outlined below. Firstly, an initial responsive nanocapsule system was studied. The fundamental work with giant unilamellar vesicles proved such a responsive system can provide antimicrobial properties when antimicrobial agents were encapsulated within the vesicles. Secondly, partially polymerised vesicles—polydiacetylene/phospholipid vesicles were then developed to improve vesicle stability. The vesicle system was optimised by varying molar concentration of diacetylene monomers (TCDA) in order to obtain relatively stable vesicles as well as sensitivity to the toxins secreted by the pathogenic strains. Measurements proved that the polydiacetylene/phospholipid vesicles can respond to pathogenic bacteria when fluorescent dye/antimcirobials were encapsulated in the vesicles. Finally, a simple prototype dressing was constructed. Plasma polymerised maleic anhydride (pp-MA) deposited onto non-woven polypropylene was shown to be a good method to stabilise vesicles via covalent bonding. Vesicle adhered to pp-MA non-woven polypropylene showed the ability to inhibit/kill the pathogenic strains, quantified by the Japanese Industry Standard assay and also gave a fluorimetric colour response in the presence of pathogenic bacteria when a fluorescent dye is encapsulated within vesicles. Other simple prototypes were also attempted by using hydrogels (gelatine and collagen) to maintain vesicle stability as well as promote tissue healing.

Neurons rely on precise spatial and temporal control of neurotransmitter release to ensure proper communication. Neurotransmission occurs when synaptic vesicles in the presynaptic compartment fuse with the plasma membrane and release their contents into the synaptic cleft, where neurotransmitters bind to receptors on the postsynaptic neuron. Synaptic vesicle pools must maintain a functional repertoire of proteins in order to efficiently release neurotransmitter. Indeed, the accumulation of old or damaged proteins on synaptic vesicle membranes is linked to synaptic dysfunction and neurodegeneration. Despite the importance of synaptic vesicle protein turnover for neuronal health, the molecular mechanisms underlying this process are unknown. In this thesis, we present work that uncovers key components that regulate synaptic vesicle degradation. Specifically, we identify a pathway that mediates the activity-dependent turnover of a subset of synaptic vesicle membrane proteins in mammalian neurons. This pathway requires the synaptic vesicle-associated GTPase Rab35, the ESCRT machinery, and synaptic vesicle protein ubiquitination. We further demonstrate that neuronal activity stimulates synaptic vesicle protein turnover by inducing Rab35 activation and binding to the ESCRT-0 component Hrs, which we have identified as a novel Rab35 effector. These actions recruit the downstream ESCRT machinery to synaptic vesicle pools, thereby initiating synaptic vesicle protein degradation via the ESCRT pathway. Interestingly, we find that not all synaptic vesicle proteins are degraded by this mechanism, suggesting that synaptic vesicles are not degraded as units, but rather that SV proteins are degraded individually or in subsets. Moreover, we find that lysine-63 ubiquitination of VAMP2 is required for its degradation, and we identify an E3 ubiquitin ligase, RNF167, that is responsible for this activity. Our findings show that RNF167 and the Rab35/ESCRT pathway facilitate the removal of specific proteins from synaptic vesicle pools, thereby maintaining presynaptic protein homeostasis. Overall, our studies provide novel mechanistic insight into the coupling of neuronal activity with synaptic vesicle protein degradation, and implicate ubiquitination as a major regulator in maintaining functional synaptic vesicle pools. These findings will facilitate future studies determining the effects of perturbations to synaptic homeostasis in neuronal dysfunction and degeneration.

This dissertation will focus on the volatile compounds released upon the burning of incense which are numerous and varied. The first part of this dissertation is the gas chromatography-mass spectral (GC-MS) analysis of burning incense collected via solid phase microextraction (SPME) with the aim of developing a library of compounds found in incense as used in the Orthodox church. The second part of this dissertation has the aim of developing a method for forming oleic acid bilayer vesicle membranes and a fluorescence spectroscopy method by which the reactivities of these vesicles can be analyzed. These reactivities include permeability, fluidity, aggregation, and fusion of the membranes. One family of the volatile compounds found in incense are the terpenes and terpenoids. The reactivity of the terpenes and terpenoids found in incense will be analyzed using the oleic acid vesicles with the hypothesis that terpenes of the same structural groups will act similarly on oleic acid vesicle membranes and these reactivities can be related to mechanistic interactions.

<p>Prostasomes are submicron-sized, membrane-bound organelles produced by the epithelial cells of the prostate and normally found in the secretion in the gland ducts. Their physiological role is in the promotion of sperm-function in human reproduction. This thesis contains four papers dealing with the production of prostasomes and some possible applications in clinical urology of the prostasome. </p><p>Paper I and II provided an ultrastructural description of the synthesis, storage and secretion of prostasomes in benign as well as in malignant tissue. Most notable were the extracellular appearances of prostasomes in metastatic lesions whereby the prostasomes become exposed to the immune system of the patient. This supported findings in earlier studies in which patients with advanced prostate cancer had elevated levels of anti-prostasome antibodies. The results of paper III reinforced the view of the prostate-unique origin of the prostasome. In particular, there were no indications in SDS-PAGE patterns or flow-cytometric studies of material from seminal vesicle secretion that it contained components that could be associated with a production of prostasomes. </p><p>Some possible clinical functions of the prostasomes were investigated in paper IV. Exposure of prostasomes to the immune system through mechanical and thermal trauma to the prostate did not induce an evident formation of anti-prostasome autoantibodies. Furthermore, the serum levels of anti-prostasome antibodies registered by assays with preparations of prostasomes from seminal plasma as antigen did not correlate with existing prostate cancer. Seminal prostasomes seemed not to function as substitute markers for prostate cancer in the test kit used. A possible explanation could be underestimated differences in antigen properties between seminal or prostate gland-derived prostasomes and prostasomes from tumor tissue.</p>

Prostasomes are submicron-sized, membrane-bound organelles produced by the epithelial cells of the prostate and normally found in the secretion in the gland ducts. Their physiological role is in the promotion of sperm-function in human reproduction. This thesis contains four papers dealing with the production of prostasomes and some possible applications in clinical urology of the prostasome. Paper I and II provided an ultrastructural description of the synthesis, storage and secretion of prostasomes in benign as well as in malignant tissue. Most notable were the extracellular appearances of prostasomes in metastatic lesions whereby the prostasomes become exposed to the immune system of the patient. This supported findings in earlier studies in which patients with advanced prostate cancer had elevated levels of anti-prostasome antibodies. The results of paper III reinforced the view of the prostate-unique origin of the prostasome. In particular, there were no indications in SDS-PAGE patterns or flow-cytometric studies of material from seminal vesicle secretion that it contained components that could be associated with a production of prostasomes. Some possible clinical functions of the prostasomes were investigated in paper IV. Exposure of prostasomes to the immune system through mechanical and thermal trauma to the prostate did not induce an evident formation of anti-prostasome autoantibodies. Furthermore, the serum levels of anti-prostasome antibodies registered by assays with preparations of prostasomes from seminal plasma as antigen did not correlate with existing prostate cancer. Seminal prostasomes seemed not to function as substitute markers for prostate cancer in the test kit used. A possible explanation could be underestimated differences in antigen properties between seminal or prostate gland-derived prostasomes and prostasomes from tumor tissue.

This thesis examines the dynamics of the formation of tethers, which are tubes of lipids 20 - 200 nm in diameter. In particular, this work investigates how the loading rate affects the observed threshold force at which a tether forms from a vesicle membrane. Tether dynamics are important to a myriad of biological processes such as signaling when white blood cells adhere to the walls of healthy and diseased blood vessels, or in the transport of intracellular material between neighboring cells. To understand the dynamics of tether formation in such systems more fully, the studies presented in this thesis focus on the dependence of the force needed to create a tether on the rate of force change. To conduct these experiments, I combined, for the first time, a microfabricated magnetic force transducer, or a microscale device that generates well-controlled and localized magnetic fields, and microaspiration, a technique to apply known tension to a lipid membrane. Using the combined global and local mechanical control of the joint system, I discovered a strong correlation between the threshold force of tether formation and the applied force ramp. An energy model, based upon that used to describe membrane rupture, characterized the observed dependencies and provided a mechanism to examine physically relevant quantities within the system. The usefulness of this combined approach was further substantiated by determining the influence of membrane modulators, including cholesterol, tension, adhesion site concentration, and phosphatidylserine, on the dependence seen between force threshold and force rates. Additionally, application of the experimental technique developed in this thesis led to the calculation of the inter-layer drag coefficient between membrane leaflets and to the first measurements of the thermal expansivity in aspirated 1-stearoy1-2-oleoyl- sn -glycero-3-phosphatidylcholine vesicles. This new tool for dynamic studies of membrane mechanics may further be extended to study how tethers form off of flowing cells or how phase regimes, induced by the presence of cholesterol, influence membrane dynamics.

In the last few decades, an increasing number of physicists specialized in soft matter, including polymers, have turned their attention to biologically relevant materials. The properties of various molecules and fibres, such as DNA, RNA, proteins, and filaments of all sorts, are studied to better understand their behaviours and functions. Self-assembled biological membranes, or lipid bilayers, are also the focus of much attention as many life processes depend on these. Small lipid bilayers vesicles dubbed liposomes are also frequently used in the pharmaceutical and cosmetic industries. In this thesis, work is presented on both the elastic properties of polymers and the response of lipid bilayer vesicles to extrusion in narrow-channels. These two areas of research may seem disconnected but they both concern deformed soft materials. The thesis contains four articles: the first presenting a fundamental study of the entropic elasticity of circular chains; the second, a simple universal description of the effect of sequence on the elasticity of linear polymers such as DNA; the third, a model of the symmetric thermophoretic stretch of a nano-confined polymer; the fourth, a model that predicts the final sizes of vesicles obtained by pressure extrusion. These articles are preceded by an extensive introduction that covers all of the essential concepts and theories necessary to understand the work that has been done.

In the last few decades, an increasing number of physicists specialized in soft matter, including polymers, have turned their attention to biologically relevant materials. The properties of various molecules and fibres, such as DNA, RNA, proteins, and filaments of all sorts, are studied to better understand their behaviours and functions. Self-assembled biological membranes, or lipid bilayers, are also the focus of much attention as many life processes depend on these. Small lipid bilayers vesicles dubbed liposomes are also frequently used in the pharmaceutical and cosmetic industries. In this thesis, work is presented on both the elastic properties of polymers and the response of lipid bilayer vesicles to extrusion in narrow-channels. These two areas of research may seem disconnected but they both concern deformed soft materials. The thesis contains four articles: the first presenting a fundamental study of the entropic elasticity of circular chains; the second, a simple universal description of the effect of sequence on the elasticity of linear polymers such as DNA; the third, a model of the symmetric thermophoretic stretch of a nano-confined polymer; the fourth, a model that predicts the final sizes of vesicles obtained by pressure extrusion. These articles are preceded by an extensive introduction that covers all of the essential concepts and theories necessary to understand the work that has been done.

Thesis (Ph. D.)--University of Hong Kong, 1989.

Bacterial communities engage in social activities, exhibiting behaviors such as communicating with small signaling molecules (quorum sensing [QS]) and building antibiotic-resistant biofilms. The opportunistic human pathogen Pseudomonas aeruginosa produces both freely diffusible QS molecules, as well as a QS molecule that is packaged or transported across cell membranes via the production of outer membrane vesicles. Despite the ubiquity of vesicle production in bacteria, the mechanism of outer membrane vesicle production has not been fully elucidated. In addition, most of our understanding of QS and biofilm formation arises from in vitro studies of bacterial communities containing large numbers of cells, often with greater than 10⁸ bacteria. However, many bacterial communities are comprised of small, densely packed aggregates of cells (≤10⁵ bacteria), and it is unclear how group behaviors and chemical interactions take place in densely packed, small populations. This dissertation has two main goals: i) to provide insights into the mechanism of bacterial membrane vesicle production, and ii) to understand how population size and the spatial distribution of cells affect cell-cell interactions and the nutritional microenvironment within a small (≤10⁵ bacteria) prokaryotic community. / text