Plasma membrane derived microvesicles (MV) are nanoscale particles released from cells both constitutively and in response to stimuli including stress, apoptosis and oncogenic transformation. Due to their mechanism of biogenesis, the majority of MV expose phosphatidylserine (PS) on their surface and as such can be identified by staining with annexin V (AxV). First observed nearly 40 years ago as coagulant ‘dust’ originating from activated platelets, MV were initially studied for their role in thrombosis. In more recent years it has become apparent that MV release is increased in several diseases including cancer; this, in conjunction with their ability to carry cargo such as proteins and nucleic acid species, strongly implicates them in disease pathology. Given their small size it is considered likely that MV are able to travel to distal sites within the body allowing the widespread dissemination of effects otherwise not achievable by their parent cells. In the context of malignancy, the contribution of MV is especially important in that MV have been demonstrated to have roles in oncogenic transformation, promotion of tumour growth and increasing metastatic potential. Although clearly important in pathogenesis, their small size makes qualitative and quantitative analysis extremely difficult. Furthermore, the study of MV has been greatly hampered by a lack of standardised protocols for their isolation and as such the majority of studies have been in vitro. In line with this, the relevance of observed effects to in vivo systems is often questioned; given the high quantities of MV used in in vitro systems, the question of whether these concentrations bear any relevance in vivo remain to be answered. We hypothesise that the high rates of apoptosis observed in many tumours, most notably in the high grade B cell malignancy, Non-Hodgkin’s lymphoma (NHL), provides an environment whereby MV are continually released into the surrounding milieu allowing for an amplification of effects. As apoptosis has been previously implicated in promoting tumourigenesis we propose that this is extended to include MV released from apoptotic tumour cells (aMV). Given the numerous technical challenges involved in MV research, initial studies involved identifying the limitations of the instruments available for MV analysis. Preliminary experiments identified considerable resolution issues with the older style EPICS XL flow cytometer (Beckman Coulter) and so a newer flow cytometer, The Attune™ (Thermo Fisher), capable of higher resolution was utilised for the remainder of the project. Despite this improvement, flow cytometry was demonstrated to be less effective at quantifying MV than nanoparticle tracking analysis (NTA). As the fluorescent capacity of NTA is still in its infancy, it was used in concert with flow cytometry in order to quantify and phenotype MV as accurately as possible. As there is currently no concensus on an optimal method of MV isolation subsequent studies focused on determining a method of MV isolation that was appropriate for our experimental system. To this end, centrifugation, filtration and antibody coated magnetic bead-based methods were all tested and their limitations identified. In terms of bead-based isolation strategies, the generation of AxV, protein S, gla domain and gas 6 fusion proteins was attempted with the intention to conjugate to magnetic beads and provide a novel means to isolate aMV. Unfortunately this aspect of the project was ultimately abandoned due to time constraints and although commerically available antibody coated beads were tested for their ability to isolate MV, later co-culture experiments demonstrated that the beads had off target effects that were deleterious to cells. As a result, centrifugation and filtration methods were next researched and validated extensively. TEM analysis of MV morphology identified damage likely induced by the high-speed centrifugation of a fragile apoptotic cell population. As such, a protocol combining low speed centrifugation and filtration was designed and validated by several methods including TEM and staining with AxV. The surface levels of parent cell markers (CD19 and CD20) and apoptosis associated proteins were compared in aMV and vMV (MV released from viable tumour cells) and results demonstrated that B cell surface markers were off loaded into MV to a greater extent following apoptosis. Additional phenotypic studies extended previous work from the group demonstrating the presence of apoptotic cell associated molecular patterns (ACAMPs) capable of binding a panel of antibodies to LPS. Flow cytometry results confirmed the presence of ACAMPs on aMV and results from co-culture experiments with CD14 positive and negative cells suggested that unlike recognition of LPS, binding via ACAMPs was not CD14 dependent. The protein and nucleic acid content of MV was also studied and interestingly, results demonstrated significantly increased quantities of DNA and RNA in aMV compared to vMV. Furthermore, aMV were also shown to contain the matrix metalloproteinases, MMP2 and MMP12 alluding to a role for aMV in angiogenesis. The final stage of the project was focused on determining the roles of aMV in the tumour microenvironment and effects relating to cell growth, cell cycle and angiogenesis were studied and compared to vMV. Results showed that both aMV conditioned supernatant and aMV concentrated by the centrifugation were able to significantly increase the growth of the parent cell population. Further studies using DAPI staining to determine the cell cycle status of cells co-cultured with aMV demonstrated an increase in DNA synthesis and cell division upon incubation with aMV. An in vitro angiogenesis assay was designed to determine any pro-angiogenic capabilities of aMV given the earlier results demonstrating the presence of MMPs. These results provided some of the most interesting findings of the project and showed that aMV were able to increase the angiogenic potential of human endothelial cells (HUVECs); an effect that was shown to be greatly reduced following storage at either 4 or - 80°C. These results demonstrated that aMV possess factors capable of manipulating the tumour microenvironment to favour disease progression and that previously described pro-tumour functions of MV are increased as a result of apoptosis. These findings have implications both in terms of extending the previously described hallmarks of cancer and also when designing a course of therapy whereby in some instances the generation of large amounts of apoptosis may in fact serve to promote regeneration of the tumour cell population.

Le virus de l'hépatite B (VHB) est un virus enveloppé composé d'un ADN partiellement double brin (ADNrc) contenu dans une capside icosahédrique. Le VHB est responsable d'infections aiguës et chroniques. VHB est non cytopathique mais l’inflammation chronique entraîne une fibrose hépatique, une cirrhose et un carcinome hépatocellulaire. Le VHB se réplique via un intermédiaire à ARN. La transcription nécessite que l'ADNrc soit convertit en un ADN circulaire clos de manière covalente (ADNccc). Cet ADNccc sert de matrice pour la transcription de l'ARN prégénomique (ARNpg), qui est spécifiquement encapsidé grâce aux interactions entre la polymérase virale, l'ARNpg et la protéine core (Cp) qui forme la capside. La polymérase rétrotranscrit l'ARNpg en ADN monocaténaire puis en ADNrc, conduisant à des matrices de capside matures (MatC). Cp avec 185 aa contient un domaine N-terminal structuré, et un domaine C-terminal (CTD) flexible. Le CTD comprend deux signaux de localisation nucléaire (NLS) et un domaine de liaison avec l’importin β (IBB). Le CTD est orienté vers l'intérieur de la capside de part son interaction avec les acides nucléiques simples brins tandis qu'il est exposé vers l'extérieur dans les capsides vides (EmpC) et les MatC. De plus Cp étant surexprimée, cela conduit à l'assemblage des EmpC. Le VHB doit délivrer son génome dans le noyau des cellules infectées pour sa réplication. Le transport nucléaire est médié par la capside qui interagit avec les récepteurs d'import. L’équipe a démontré préalablement que ce transport a besoin des récepteurs Importin α (Imp.α) et Importin β (Imp.β) en induisant le transport des capsides au panier nucléaire où elle est stoppée par l'interaction avec la nucléoporine 153 (Nup153). Nous avons démontré que l’Imp.α, mais pas l'Imp.β, se lie aux MatC suggérant que seule la partie du CTD qui contient les NLS est exposée à l’extérieur des MatC. En comparaison, nous avons analysé les EmpC en collaboration avec Adam Zlotnick (Université d'Indiana, États-Unis) et démontré que les EmpC sont capables de lier directement l'Imp.β. Cette interaction qui est plus forte que l’interaction avec l'Imp.α s'effectue via la reconnaissance du domaine IBB du CTD, ce qui implique une exposition totale du CTD à l'extérieur de la capside. Nous avons aussi montré que la liaison avec l'Imp.β à des concentrations très élevées fournit des forces de déstabilisation menant au désassemblage des EmpC. La libération du génome dans le panier nucléaire implique que l’interaction entre les MatC et Nup153 participe au désassemblage de la capside. Afin de valider cette hypothèse, nous avons exposé des MatC dont le génome est radiomarqué avec un fragment de Nup153 contenant le domaine clé, montré pour interagir avec la capside, en présence de nucléases. Nous avons mis en évidence qu'en présence de ce fragment, les MatC restent stables. Cela suggère la nécessité de facteurs cellulaires additionnels pour le désassemblage des MatC. Cette conclusion est conforme avec nos résultats sur noyaux isolés, dans lesquels nous avons observé une localisation nucléaire des capsides laissant supposer que les facteurs cellulaires nécessaires au désassemblage des MatC sont nucléaires. Afin d'étudier plus en détail l'étape de désassemblage et la libération du génome viral, nous avons mis au point un système permettant de suivre en temps réel le devenir du génome viral. / The Hepatitis B Virus (HBV) is an enveloped virus containing a partially double stranded DNA genome (rcDNA). HBV causes acute and chronic infections. HBV is not cytotoxic but chronic inflammation leads to liver fibrosis, cirrhosis and hepatocellular carcinoma. HBV replicates via an RNA intermediate, which is transcribed from a covalently closed circular form of the viral DNA (cccDNA). This pregenomic RNA is specifically encapsidated into the capsid by interaction with the viral polymerase, which also interacts with the core protein (Cp), forming the capsid. The polymerase retrotranscribes the pregenomic RNA into single stranded DNA and subsequently partially double stranded DNA resulting in mature capsids (MatC). Cp is an 185 aa long polypeptide comprising a N-terminal assembly domain, and a flexible C-terminal domain (CTD). The CTD includes two overlapping nuclear localization signals (NLS) of eight aa and an Importin ß Binding Domain (IBB) of 34 aa. The CTD is fixed in the interior of the capsid by interacting with single stranded nucleic acids but translocates to the exterior in MatC and empty capsids (EmpC). Cp is over expressed leading to assembly of EmpC. The virus has to deliver its genome into the nucleus of infected cells for replication. Nuclear transport is mediated by the capsid that interacts with nuclear import receptors. The group has recently shown that MatC need either both, importin  (Imp.) and importin ß (Imp.ß), or Imp.ß alone for transport of the capsids into the nuclear basket. In this structure where genome liberation likely occurs, the transport of the capsid is arrested by interaction between the capsid and the nucleoporin Nup153. In the thesis we demonstrate that MatC binds to Imp.α but not Imp.ß, suggesting that only the part of the CTD, which contains the NLSs is exposed on capsids’ surface. In collaboration with the Adam Zlotnick (Indiana University, U.S.A.) we showed that EmpC, in contrast, bind Imp.β directly without Imp.α acting as an adaptor. This interaction, which is stronger than the one of Imp. occurs needs IBB exposure, meaning that the entire CTD becomes externalized. Furthermore, exposure to very high Imp.ß concentration led to EmpC destabilization. The genome release within the nuclear basket implies that Nup153 is involved in genome liberation from MatC. To verify this hypothesis we used MatC with a radioactively labeled genome, which were exposed to the capsid binding-Nup153 fragment. Investigating the accessibility of the genome to nucleases we found that the Nup153 fragment had no impact on capsids stability, suggesting the need of cellular factors driving disassembly. This conclusion is in agreement with our observation that MatC added to isolated nuclei resulted in nuclear capsid entry, which requires disassembly. To further study the disassembly step and the consequent release of the viral genome, we developed a system to directly visualize the viral genome allowing investigations of genome uncoating in real time. The system is based on the cooperative binding of a fluorescent fusion protein between the bacterial protein OR with GFP to a double stranded DNA sequence called Anch. Using this model we showed that infection of OR-GFP-expressing hepatoma cells with HBV containing a modified Anch genome allowed monitoring genome release into the nucleus. In future, this system may help identifying factors involved in genome release and repair and to decipher their molecular interactions.

The problem of jail overcrowding has forced corrections officials and jail administrators to examine ways in which to better manage available jail space. Pretrial release and detention policies have been a target of this examination as pretrial defendants typically account for 50% of a jail's population. Standards for pretrial release exist, but their administration varies by jurisdiction. The impact of jail overcrowding on pretrial release policies has been to decrease the time available to render a decision. Recent efforts to standardize pretrial release standards in Oregon have not addressed the issue of expediency. The current study examines pretrial misconduct (failure to appear in court and rearrest) with regard to information that is available to jail personnel and release office personnel at the time of arrest, with the specific intent to develop a predictive model of pretrial misconduct that will function as an initial risk assessment. Six hundred defendants arrested in Washington County, Oregon during 1991 served as subjects. The results indicated that 90.9% of all defendants arrested are released pending trial/ and that 22.7% of those released engaged in pretrial misconduct. The results of the loglinear model-building indicated that the variables prior failure-to-appears/ employment, and age were the best predictors of pretrial misconduct. The construction sample (n = 395) accurately predicted 94.5% of the observed pretrial misconduct compared to 90.7% for the validation sample (n = 150). The loglinear analysis yielded 16 typologies (based on the variables included in the model) by which defendants could be ranked as to their risk of pretrial misconduct. Spearman Rank Order coefficents for the construction and validation samples were .847 and .626 respectively. Data were also collected on detained subjects. A Chi-Square test using detained with released ?Ubjects by typology indicated that the categories are not independent (p < .01). Further examination indicated that the detained subjects did represent higher risks of pretrial misconduct as estimated by the typologies. The results also indicated that defendants currently on probation or parole were more likely to detained than other defendants. The results do not reject the assumptions by Sturz {1962), whose Manhattan Bail Project is the basis for pretrial release, that persons with strong ties to the community may pose the least risk of pretrial misconduct. The results also found sex and ethnic differences with regard to pretrial misconduct. The sex differences may have been confounded by age and crime type; however, the ethnic differences may reflect a systemic inability to communicate with Hispanic offenders.

Crops grown in sandy soils require frequent irrigation. As a result, nitrogen (N) fertilizers. such as ammonium sulfate((NH4)2SO4), are leached from the rooting zone of crops. This loss of N increases N fertilizer use and the potential for nitrate (NO3-) contamination of water. Ammonium-loaded clinoptilolite (NH4+-Cp) may reduce this N leaching, increase N fertilizer use-efficiency, and prevent NO3- contamination of water while sustaining normal crop growth. The potential of NH4+-Cp as a N fertilizer was assessed in three leaching experiments without plants and two leaching experiments with plants. Pots containing rounded quartz sand were amended with (NH4)2SO4 and one of three NH4+-Cp size fractions: small ( Finally, in two leaching studies, pots containing the sandy soil were planted with sweet corn and grown for 35 d and 42 d, respectively. No differences were found among N sources in corn relative growth rates, leaf area ratios, and net assimilation rates, even though the corn plants that were fertilized with NH4+-Cp assimilated significantly more N than the (NH4)2SO4-fertilized plants. The pots fertilized with NH5+-Cp leached In the greenhouse, NH4+-Cp is a slow-release fertilizer that will reduce N leaching while maintaining normal plant growth. However, field studies are needed to confirm the suitability of NH4+-Cp as a slow-release fertilizer under field conditions.

The capacity of a calcareous desert soil, Thiokol silt loam, to retain natural , as well as added , orthophosphate-P was measured by equilibrium adsorption employing a batch technique and evaluated using the two-slope Langmuir adsorption isotherm. From these data, corrected to account for indigenous soil P, a hypothesis was formulated as to the nature of retention of P by the soil, including the identification of two interfacial reactions involving P, and a value calculated for the adsorption maximum as defined by the Langmuir isotherm equation f o r P with soil at each of two soil depths and t h ree constant temperatures within the range of biological activity . The initial reaction was considered to be surface adsorption , where phosphate ions interact with the clayand lime mineral surfaces at definite sites. The activity of the second mechanism was identified as adsorption, and in addition, the heterogeneous nucleation of metal phosphates on the lime mineral surfaces. In addition to these quantitative studies, the flux of P in the soil was also investigated for the same soil and temperatures by means of kinetic experiments conducted to identify the nature (mechanisms) and measure the rates and release maxima of indigenous P release from the soil. These experiments were carried out using an anion-exchange resin as an infinite sink for P, again applying a batch technique. Ultimately, the release of indigenous P from the soil under saturated conditions was attributed to three simultaneous first-order reactions. 'll1e rate constants of the three reactions were found to be of orders of 10-4, 10-5, and 10-6 (1/sec), and did not vary significantly with soil depth or ternperature. The three reactions above were identified as dissolution of poorly crystalline or amorphous calcium phosphates, the desorption of surface site adsorbed or labile P, and the slow dissolution of calcium hydroxyapatite, respectively.

An abnormally high number of explosion quakes were noted during the monitoring effort for the 2007 eruption of Pavlof Volcano on the Alaskan Peninsula. In this study we manually counted the explosion quakes from their characteristic ground-coupled air waves. This study makes an effort at better quantifying the number of explosion quakes and how the characteristic ground-coupled air waves are affected by wind direction and wind speed. Additionally this study investigates how the ground coupled air waves might be used in a monitoring or analysis effort by calculating energy release and gas mass release. Over 3.2x104 quakes were recorded. It was found that wind direction affects the travel time of the air wave by up to 0.7 seconds depending on station location and wind direction. Wind direction and speed, however, are demonstrated not to cause an appreciable difference in ground-coupled air wave frequencies or amplitude ratios. The energy release from the explosions is calculated to be 3.04x1011 J. and the total gas mass (assuming 100% water) released was 729 metric tons. These values are compared to other volcanoes in the literature and found to be somewhat lower. Nevertheless, the tracking of explosion quakes has the potential to become a valuable member of the seismic monitoring arsenal.

Surfaces submerged in marine environments rapidly get colonized by marine organisms, a process known as biofouling. Fouling costs maritime industries billions of dollars annually. The most common methods of combating marine biofouling are toxin containing antifouling coatings which often have detrimental non-target environmental effects. These effects and proposed bans on harmful substances in antifouling coatings, mandates development of more environmentally friendly antifouling technologies. Of these, foul-release coatings, which minimize attachment and adhesion of fouling organisms (rather than killing them) are promising alternatives. Here I explored the utility of petroleum waxes as novel antifouling/foul-release coatings. I first investigated the responses of propagules (larvae or spores) of six common fouling organisms to wax coatings in the laboratory. A wide variation in the response of these different organisms, and in the different types of response (settlement, adhesion, etc.) by the same organism, was observed, but the most inhibitory coatings were those made from microcrystalline wax and silicone oil. However, in field trials in Sydney Harbour, paraffin waxes had the strongest antifouling performance, with activity up to one year (the trial duration). These waxes also had strong foul-release effects, with fouling that did attach mostly removed by a low pressure water jet. Composition of fouling communities on paraffin waxes differed significantly from other waxes or controls, with little or no hard fouling organisms (barnacles, bivalves) on paraffin. The mechanisms of antifouling and foul-release actions of paraffin waxes appear to be due to changes in surface properties. The surfaces of the paraffin waxes changed noticeably after 4 - 8 weeks immersion in the sea or in seawater aquaria. Antibiotic treatments showed that this change in surface appearance was due to biological (microbial) activity. Bacteria appear to remove the amorphous phase from the surface of the paraffin waxes, revealing an underlying crystalline phase, which is less affected by bacterial action. I suggest that these crystals form a microstructured ?bed of nails? of crystals of varying shapes and sizes which inhibit settlement and reduce adhesion strength of those organisms which do settle.

半導體產業在近二十年來一直在我國經濟發展舞台上扮演積極重要的角色，其績效自然是研究者欲探索之重點。在管理會計頗有作用的作業制成本制度，其提供不同於以往傳統成本制度之資訊，在在替全球知名企業創造更具優勢的管理績效。本研究之重心即在：以深入訪談及田野實證研究方式探討半導體公司導入作業制成本制度後，所發生的績效變化、變化時間與相關資訊，用實證資料檢視作業制成本制度與公司實際經營績效之間的關係，為作業制成本制度在企業之成效作一較為完整且具實務運用的描述，進而提供後續推行作業制成本制度之研究價值，並給予實務界導入作業制成本制度之相關資訊，以作為我國企業推動作業制成本管理制度之參考。 　　本研究以田野實證之資料為主，並以迴歸方式驗證作業制成本資訊釋出前後對企業生產成本與品質之影響，實證結果顯示成本會隨資訊釋出而降低，品質則不會有所變化，表示作業制成本制度確實可以幫助管理者從事成本抑減之工作；也得到品質不會在短期內改善之結論，暗示作業制成本制度之財務績效先於品質績效，資訊使用者之熟練程度差異可能影響績效之出現與否。 　　基於研究所得結論，本研究建議個案公司可以將成本下降之經驗擴散至全廠區，藉由新制度教育員工成本與獲利觀念，並針對員工之使用感想修正個案公司之作業制成本制度。同時也建議未來的研究者，對品質與作業制成本制度甚至成本之關係，作更進一步之研究。 / Using field study method, this study focuses on the relationship between the manufacturing performance of the semi-conductor industry and activity-based costing system information. The analysis suggests that with better decision based on activity and cost driver information, the actvity-based costing information will reduce manufacturing costs while leave quality unchanged. 　　In conclusion, empirical results reveal that manufacturing costs actually decrease with the release of activity-based costing information, while manufacturing quality remains the same. The finding shows that the release of activity-based costing information can reduce the manufacturing costs, but there may not be an immediate effect of quality improvement, suggesting that cost can actually be improved by the activity-based costing system, and quality may need more time and skill toward activity-based costing to change.

<p>Silver was used for its antimicrobial effect by the ancient Greeks, long before the existence of microorganisms were first suspected. Nowadays a wide range of antimicrobial dressings containing silver, either incorporated within or applied on the dressings, are available for clinical use. This type of dressings is designed to provide the antimicrobial activity of silver in a more convenient application.</p><p>The aim with this master thesis was to evaluate if silver release and antimicrobial effect of nine silver containing dressings are dependent on the test medium and if there is any relation between silver release and antimicrobial effect.</p><p>Release of silver and antimicrobial effect was evaluated by using a 6-well co-culture system, with inoculated test medium in the wells and dressing pieces in the culture well inserts. Three different test media with increased complexity and nutrient value were inoculated with either</p><p>Results show that release of silver depends on the test fluid used; for phosphate buffered saline (PBS), the silver concentration was as most 1.2 ppm, but for a complex media containing calf serum (SWF), it varied from 9 ppm to 134 ppm. The viable counts in PBS were reduced by at least 3 log units for all dressings and bacteria, whereas in SWF there were no reduction and instead growth was observed. In general, a high release resulted in less bacterial growth. Results also indicated that kinetics of silver release affect the antimicrobial effect. It is likely to assume that it is important for a dressing to release silver quickly.</p><p>It has previously not been possible to correlate silver release of wound care dressings and antimicrobial effect, since the two factors have been measured in different test systems and in different media. Since both factors depend on test medium and method used, it is shown in the present study that it is important to use relevant test medium for in-vitro evaluation. When measuring silver release and antimicrobial effect in the same test system, a relation is found.</p>

<p>New smart drugs demand new smart drug delivery systems and also new smart analysis methods for the drug delivery process and material characterization. This thesis contributes to the field by introducing a new electrodynamic approach for studying the drug diffusion proc-esses as well as the formation of a new type of drug delivery systems, the so called mesoporous nano excipients.</p><p>Drug diffusion processes from different pharmaceutical materials were examined. The transport of charged drug substances was investigated by electrodynamic methods; either as a release process governed by diffusion using the alternating ionic current method or by applying a voltage, sinusoidal or dc, to force the drug ions to move in an electric field.</p><p>Temperature-dependent drug release from microcrystalline cellulose tablets was examined in order to extract information about the diffu-sion process. Percolation theory was also employed to binary mixtures of an insoluble and electrically insulating matrix material together with a soluble and ionic conducting drug. Further, dielectric spectros-copy was proven to be a powerful method for examining the state of vesicle formation of drug and surfactant molecules in a carbopol gel. Finally, a new potential class of pharmaceutical materials were exam-ined, namely the AMS-n mesoporous materials, showing that the al-ternating ionic current method is powerful both in the study of the synthesis of and in the release process from these. </p>