Platelet exocytosis is essential for hemostasis and for many of its sequelae. Platelets release numerous bioactive molecules stored in their granules enabling them to exert a wide range of effects on the vascular microenvironment. Are these granule cargo released thematically in a context-specific pattern or via a stochastic, kinetically-controlled process? My work describes platelet exocytosis using a systematic examination of platelet secretion kinetics. Platelets were stimulated for increasing times with different agonists (i.e. thrombin, PAR1-agonist, PAR4-agonist, and convulxin) and micro-ELISA arrays were used to quantify the release of 28 distinct α-granule cargo molecules. Agonist potency directly correlated with the speed and extent of release. PAR4-agonist induced slower release of fewer molecules while thrombin rapidly induced the greatest release. Cargo with opposing actions (e.g. pro- and anti-angiogenic) had similar release profiles, suggesting limited thematic response to specific agonists. From the release time-course data, rate constants were calculated and used to probe for underlying patterns. Probability density function and operator variance analyses were consistent with three classes of release events, differing in their rates. The distribution of cargo into these three classes was heterogeneous suggesting that platelet secretion is a stochastic process potentially controlled by several factors such as cargo solubility, granule shape, and/or granule-plasma membrane fusion routes. Sphingosine 1 phosphate (S1P) is a bioactive lipid that is stored in platelets. S1P is essential for embryonic development, vascular integrity, and inflammation. Platelets are an abundant source of S1P due to the absence of the enzymes that degrade it. Platelets release S1P upon stimulation. My work attempts to determine how this bioactive lipid is released from platelets. Washed platelets were stimulated with agonists for defined periods of time and the supernatant and pellet fractions were separated by centrifugation. Lipids were separated by liquid phase extraction and S1P was quantified with a triple quadrapole mass spectrometer. A carrier molecule (BSA) is required to detect release of S1P. Further, there is a dose-dependent increase in total S1P with increasing BSA. S1P release shows characteristics similar to other platelet granule cargo e.g. platelet factor IV (PF4). Platelets from Unc13-d Jinx mice and VAMP8-/- mice, which are secretion-deficient (dense granule, alpha granule and lysosome), were utilized to understand the process of S1P release. S1P release was more affected in Unc13-d Jinx mice mirroring their dense granule secretion defect. Fluorescence microscopy and sub-cellular fractionation were used to examine localization of S1P in platelets. S1P was observed to be enriched in a granule population. These studies indicate the existence of two pools of S1P, a readily extractable agranular pool, sensitive to BSA, and a granular pool that requires the secretion machinery for release. The secretion machinery of platelets in addition to being involved in the release of normal granule cargo is thus proved to be involved in the release of bioactive lipid molecules like S1P.

Au cours de l'endocytose, les cargos de membrane plasmique (PM) sont internalisés puis dirigés vers l'endosome précoce (EE), les corps multivésiculaires (MVBs), puis le lysosome/vacuole pour dégradation. Le ciblage des protéines vers les zones invaginées des MVBs requiert l'action des complexes ESCRTs. Chez la levure comme chez les mammifères, les mutants déficients pour ces complexes présentent un compartiment endosomal anormal (Cl E) et une accumulation à la PM de diverses protéines. Nous avons montré que chez S. cerevisiae la stabilisation de la perméase à uracile (Fur4p) à la PM dans les mutants ESCRTs résulte de son recyclage vers la PM après internalisation. Fur4p ne traverse pas les compartiments Golgiens lors de son recyclage depuis le compartiment Cl E. Cette voie de recyclage est distincte de celle empruntée par la v-SNARE Snc1p. Fur4p est également capable de recycler depuis l'EE et suit alors la même voie de recyclage que Snc1p. Ceci suggère une complexité inattendue des voies de recyclage chez la levure.<br />Nous nous sommes aussi intéressés à Irs4p et Tax4p, 2 protéines à domaine EH, un domaine trouvé jusqu'alors dans des protéines impliquées dans l'endocytose ou le recyclage. Irs4p et Tax4p ne sont pas impliquées dans ces processus mais interviennent de façon redondante au cours de l'autophagie, un transport catabolique vésiculaire de fractions de cytoplasme ou d'organelles vers le lysosome/vacuole. Irs4p et Tax4p interagissent physiquement avec la machinerie d'autophagie et sont partiellement localisées à la structure Pré-autophagosomale, d'où émanent les vésicules d'autophagie. Ces résultats étendent donc la panoplie des fonctions des protéines à domaine EH.

The world's shipping lanes are an area of intense focus in the Global War on Terror. Every day millions of tons of cargo are shipped through thousands of ports. Most cargo is harmless, however, some ships carry the weapons and human operators of terrorist organizations. To prevent the spread of weapons and terror suspects on the sea lanes, the cargo, passengers, and crew of these vessels must be subject to a level of scrutiny that is orders of magnitude greater than current efforts. The ability to rapidly extend a network and provide virtual expert services to Visit, Board, Search and Seizure (VBSS) boarding teams is crucial to protecting the United States and its allies from seaborne terror attacks and infiltration. This thesis uses scenario-based experimentation to examine the methods for implementing near-real-time collaborative work spaces in a virtual environment able to support VBSS operation anywhere in the world, limited only by network connectivity. The use of collaborative tools vastly increases the amount, type, and accuracy of information that can be processed. Radiation detection or classification and biometric fusion are among the hundreds of virtual collaborative sources that can be leveraged as force multipliers to bring network centric warfare to the maritime security domain.

The term "Maritime Interdiction Operations" usually refers to Visit, Board, Search and Seizure (VBSS) operations executed today all over the world. These operations are conducted as a part of the maritime law enforcement policy of each country inside their respective territorial waters or as a part of the homeland security requirements as they are mandated today by the global war against terrorism. Very often lately, they are conducted by allied maritime forces in international waters as well. Although such operations might seem quite simple in execution, the global war against terrorism has dramatically increased their level of complexity. In the past, searching cargo ships for illegal or contraband cargo was not that complicated or that important for national security, but now, searching for non-proliferation, radiological or bio-chemical material, as well as for possible terrorists among the crew members of a ship, is a very complex operation that cannot tolerate mistakes or omissions. This thesis examines the requirements posed by a boarding team, either from the navy or the law enforcement community, on information flow from and to them, in order to enhance their situational awareness and decision making capability during Maritime Interdiction Operations. That information flow is provided by several wireless network technologies, implemented during field trials, as part of the NPS CENETIX (Center for Network Innovation and Experimentation) lab s maritime subset of experimentation. During these field trials, a wireless extension of the internet is deployed to the sea, allowing the boarding team to access information and collaborate with remotely located experts and respective operational commands, the technical aspects, the benefits and shortcomings of the utilized technologies and collaborative tools are screened against the maritime war fighter's operational requirements.

mRNA localization ensures correct spatial and temporal control of protein synthesis in the cell. Using a single molecule in vitro approach, we provide insight into the mechanisms by which localizing mRNAs are carried by molecular motors on cytoskeletal tracks to their destination. Budding yeast serves as a model system for studying the mechanisms of mRNA transport because localizing mRNAs are moved on actin tracks in the cell by a single class V myosin motor, Myo4p. Molecular motors that specialize in cargo transport are generally double-headed so that they can "walk" for many microns without dissociating, a feature known as processivity. Thus, is was surprising when Myo4p purified from yeast was shown by in vitro assays to be non-processive. The reason for its inability to move processively is that the Myo4p heavy chain does not dimerize with itself, but instead binds tightly to the adapter protein She3p to form a single-headed motor complex. The mRNA-binding adapter protein She2p links Myo4p to mRNA cargo by binding She3p. To understand the molecular mechanisms of mRNA transport in budding yeast, we fully reconstituted a messenger ribonucleoprotein (mRNP) complex from purified proteins and a localizing mRNA (ASH1) found in budding yeast. Using single molecule in vitro assays, we find that She2p recruits two Myo4p-She3p complexes, forming a processive double-headed motor complex that is stabilized by mRNA at physiological ionic strength. Thus, only in the presence of mRNA is Myo4p capable of continuous mRNA transport, an elegant mechanism that ensures that only cargo bound motors are motile. We next wished to understand if the principles of mRNA transport in budding yeast are conserved in higher eukaryotes. In Drosophila, mRNA is transported on microtubule tracks by cytoplasmic dynein, and the adapters that link the motor to localizing transcripts are well-defined. The adapter protein bicaudal D (BicD) coordinates dynein motor activity with mRNA cargo binding. The N-terminus of BicD binds dynein, and the C-terminus interacts with the mRNA-binding protein Egalitarian. Unlike mammalian dynein alone, it was recently shown that an N-terminal fragment of BicD (BicD2CC1), in combination with a large 1.2MDa multi-subunit accessory complex called dynactin, forms a complex (DDBCC1) that is activated for long processive runs. But unlike the constitutively activated BicD2CC1 fragment, the full-length BicD molecule fails to recruit dynein-dynactin because it is auto-inhibited by interactions between the N-terminal dynein binding domain and the C-terminal cargo binding domain. To understand how dynein is activated by native cargo and full-length adapters, we fully reconstituted a mRNP complex in vitro from tissue-purified dynein and dynactin, expressed full-length adapters BicD and Egalitarian, and a synthesized localizing mRNA found in Drosophila. We find that only mRNA-bound Egalitarian is capable of relieving BicD auto-inhibition for the recruitment of dynein-dynactin, and activation of mRNA transport in vitro. Thus, the presence of an mRNA cargo for activation of motor complexes is a conserved mechanism in both budding yeast and higher eukaryotes to ensure that motor activity is tightly coupled to cargo selection.

Resumé The thesis titled "Topical Issues of the Regulation of International Air Carriage" concerns itself with legal regulation germane to operating the international carriage by air. It is focused on carriage of cargo; carriage of passengers is only marginally mentioned. In the first chapter the author of the thesis defines the term of transport, transport law and compares the advantages and disadvantages of the means of transport in connection with the carriage of cargo. The second chapter of the thesis deals with the term of aviation law, historical development of the air transport and introduces the most important international organizations in the field of the civil air transport. The third chapter is dedicated to the historical development of the legal regulation in the field of the civil air transport, mainly the international legal regulation carried out by the Montreal Convention and the Warsaw Convention (with its amendments). This chapter also deals with the legal regulation of the European Union and analyses some selected EU regulations and describes national laws (mainly Civil Aviation Act). In this chapter there is also included the analysis of sources of the legal regulation and their mutual relations. It also explains the basics of the methods of regulation in the international private law -...

<p>This dissertation contributes to the rapidly growing empirical research area in the field of operations management. It contains two essays, tackling two different sets of operations management questions which are motivated by and built on field data sets from two very different industries --- air cargo logistics and retailing. </p><p>The first essay, based on the data set obtained from a world leading third-party logistics company, develops a novel and general Bayesian hierarchical learning framework for estimating customers' spillover learning, that is, customers' learning about the quality of a service (or product) from their previous experiences with similar yet not identical services. We then apply our model to the data set to study how customers' experiences from shipping on a particular route affect their future decisions about shipping not only on that route, but also on other routes serviced by the same logistics company. We find that customers indeed borrow experiences from similar but different services to update their quality beliefs that determine future purchase decisions. Also, service quality beliefs have a significant impact on their future purchasing decisions. Moreover, customers are risk averse; they are averse to not only experience variability but also belief uncertainty (i.e., customer's uncertainty about their beliefs). Finally, belief uncertainty affects customers' utilities more compared to experience variability. </p><p>The second essay is based on a data set obtained from a large Chinese supermarket chain, which contains sales as well as both wholesale and retail prices of un-packaged perishable vegetables. Recognizing the special characteristics of this particularly product category, we develop a structural estimation model in a discrete-continuous choice model framework. Building on this framework, we then study an optimization model for joint pricing and inventory management strategies of multiple products, which aims at improving the company's profit from direct sales and at the same time reducing food waste and thus improving social welfare.</p><p>Collectively, the studies in this dissertation provide useful modeling ideas, decision tools, insights, and guidance for firms to utilize vast sales and operations data to devise more effective business strategies.</p> / Dissertation

One of the significant side-effects of growing urbanization is the constantly increasing amount of freight transportation in cities. This is mainly performed by conventional vans and trucks and causes a variety of problems such as road congestion, noise nuisance and pollution. Yet delivering goods to residents is a necessity. Sustainable concepts of city distribution networks are one way of mitigating difficulties of freight services. In this paper we develop a two-echelon city distribution scheme with temporal and spatial synchronization between cargo bikes and vans. The resulting heuristic is based on a greedy randomized adaptive search procedure with path relinking. In our computational experiments we use artificial data as well as real-world data of the city of Vienna. Furthermore we compare three distribution policies. The results show the costs caused by temporal synchronization and can give companies decision-support in planning a sustainable city distribution concept.

and key words Mouse polyomavirus-derived virus-like particles composed from major capsid protein VP1 (MPyV VP1-VLPs) are interesting structures for use as a delivery system of various cargos into cells. VP1 protein self-assembles into icosahedral particles of 45 nm in diameter that are hollow highly regular nanoparticles. In this work, model small molecule cargo, Cyclodextrin-Based Bimodal Fluorescence/MRI Contrast Agent, was encapsidated into MPyV VP1-VLPs. The cargo was stably associated with VLPs and was delivered into mammalian cells using these VLPs. To prevent VLPs entrapment in endolysosomal compartments and increase the potential of VLPs applications, MPyV VP1 protein was modified by insertion of histidine-tag (6 histidine long sequence surrounded by glycine and serine) sequences into VP1 surface loop DE, because histidine modification of synthetic systems had enhancing effect on endosome escape and cargo delivery. With the use of in Bac-to-Bac® baculovirus expression system His-VP1 protein was expressed in insect cells and a variety of VP1-assemblies was obtained: long tubules and small 20nm VLPs formed from VP1 with 4 histidine-tags in DE loop, and novel VP1 nanostructure, which we named nano-jumpers, formed from VP1 with 2 histidine-tags. Nonetheless the endosome escape properties of...

O presente trabalho trata da solução para o problema da elaboração de programações de transporte do sistema de distribuição de materiais da Força Aérea Brasileira (FAB). Essas programações de transporte consistem em definir os roteiros de entrega e coleta de materiais a serem realizadas simultaneamente em cada local de entrega/coleta a partir de um centro de distribuição, considerando-se a frota de veículos homogênea. Isto é característico de um Problema de Roteirização de Veículos com Coletas e Entregas Simultâneas (PRVCES). A gestão do sistema de distribuição física da FAB considera a complexidade desse sistema e os dados relativos às demandas de transporte de carga em cada um desses locais para elaborar as programações de transporte. Essas programações são elaboradas tendo em vista os limites de capacidade dos veículos, as características físicas das cargas e as prioridades de embarque. O gestor desse sistema possui boa visibilidade das demandas de transporte, porém, devido à grande quantidade de informações disponíveis e à elevada complexidade desse sistema, é impossível elaborarem-se manualmente programações de transporte que resultem em viagens de distribuição eficientes. O PRVCES foi resolvido por meio da meta-heurística Busca Dispersa (do inglês Scatter Search) integrada com a meta-heurística Descida em Vizinhança Variável (do inglês Variable Neighborhood Descent) utilizada como método de melhoria das soluções. Os resultados superaram ou se igualaram a alguns dos obtidos por outros autores para os mesmos problemas de teste com as mesmas restrições, o que demonstra que a Busca Dispersa implementada é competitiva para solucionar o PRVCES. Quanto à aplicação na FAB, os resultados mostraram que a utilização do método de solução desenvolvido resultará em programações de transporte elaboradas em curto tempo de processamento e que estas incidirão positivamente sobre a eficiência do sistema de distribuição de materiais da FAB. / This work deals with the solution to the problem of drawing up transport schedules in the material distribution system of the Brazilian Air Force (BAF). These transport schedules consist in defining the routes for material pickup and delivery to be accomplished simultaneously in each delivery/pickup location from a distribution center, considering a homogeneous fleet of vehicles. This is characteristic of a Vehicle Routing Problem with Simultaneous Delivery and Pick-up (VRPSDP). The management of the physical distribution of BAF considers the complexity of this system and the data regarding the cargo transport demands in each one of those locations to draw up transport schedules. These schedules are drawn up regarding the capacity limits of the vehicles, the physical characteristics of the cargoes and the shipping priorities. A good visibility of transport demands in each location is available to the manager of this system, but due to the great quantity of data to deal with and the high complexity of the physical distribution system of BAF, it is impossible to draw up transport schedules that result in efficient distribution trips. The VRPSDP was solved by means of the Scatter Search meta-heuristic integrated with the Variable Neighborhood Descent meta-heuristic as the solution improvement method. The results exceeded or equaled some of those obtained by other authors using the same test problems with the same restrictions, what indicates that the implemented Scatter Search is competitive to solve the VRPSDP. As for the application in the BAF, the results showed that using the solution method developed will result in schedules drawn up in short processing time and focused on the efficiency of the material distribution system of the BAF.