Vesicular membrane fusion involves the release of contents in a broad array of biological systems, such as intracellular trafficking, secretion, fertilization, and development. It is also a critical step in the infection of cells by membrane enveloped viruses such as HIV, influenza, and Ebola. SNARE proteins form the core of the fusion machinery in nearly all intracellular fusion processes. The initial complete connection between two fusing membranes is the fusion pore. There is considerable evidence that both the fusion machinery and the biophysical properties of the membranes themselves affect contents release, lipid mixing, and fusion kinetics, but the mechanisms are poorly understood. Flickering of fusion pores during exocytotic release of hormones and neurotransmitters is well documented, but without assays that use biochemically defined components and measure single pore dynamics the contributions from different influences are almost impossible to separate. This thesis examines the biophysical mechanisms by which SNAREs and lipid composition control fusion rates and fusion pore kinetics. First, we studied fusion pore flickering in vitro. We used total internal reflection fluorescence (TIRF) microscopy to quantify fusion pore dynamics in vitro and to separate the roles of SNARE proteins and lipid bilayer properties. To interpret the experimental measurements quantitatively, we developed a mathematical model to describe the diffusion of labelled lipids from a vesicle, through a flickering fusion pore, and into a supported bilayer. When small unilamellar vesicles (SUV) bearing neuronal v SNAREs fused with planar bilayers (SBL) reconstituted with cognate t SNARES, lipid transfer rates were severely reduced, suggesting that pores flickered. We developed an algorithm which included a complete description of fluorophores in the TIRF field. We accounted for the intensity decay of the evanescent TIRF wave normal to the SBL, the polarization of the evanescent TIRF wave, and any potential quenching effects. In general, the first two effects are coupled. This algorithm allowed us to measure the sizes of docked vesicles using fluorescent microscopy. From the lipid release times we used the model to compute pore openness, the fraction of the time the pore is open, which increased dramatically with cholesterol. For most lipid compositions tested SNARE mediated and non specifically nucleated pores had similar openness, suggesting that pore flickering was controlled by lipid bilayer properties. However, with physiological cholesterol levels SNAREs substantially increased the fraction of fully open pores and fusion was so accelerated that there was insufficient time to recruit t SNAREs to the fusion site, consistent with t SNAREs being pre clustered by cholesterol into functional docking and fusion platforms. Our results suggest that cholesterol opens pores directly by reducing the fusion pore bending energy, and indirectly by concentrating a number of SNAREs into individual fusion events. In the second part of the thesis, I describe my contributions to a project in which a mathematical model was developed to describe the behavior of SNAREpins connecting SUVs of different sizes to a planar membrane. It was necessary to quantify the membrane membrane and SNAREpin membrane interaction forces. By combining the well known van der Waals, electrostatic, and steric hydration membrane forces with the SNAREpin membrane electrostatic interactions I developed a complete description of the membrane forces involved in SUV-SBL fusion. We then combined the description of the interactions with experimentally measured SNARE zippering energies. We find that the predominant driving forces for membrane fusion, once the SNAREpins have completely zippered, are steric hydration forces among the SNAREpins and membranes. These forces enlarge a SNAREpin cluster, which in turns pulls the membranes together due to curvature effects.

Quiescent neural stem cells support continuous, lifelong neurogenesis in specific regions of the adult mammalian brain. The largest adult neurogenic region is the ventricular-subventricular zone (V-SVZ), which lines the entire lateral wall of the lateral ventricles. Quiescent neural stem cells (qNSCs) enter the cell cycle (activate) and give rise to new neurons during homeostasis and regeneration, suggesting they can potentially be harnessed for regenerating the brain after neurodegenerative disease, stroke, and injury. Defining the signals that regulate NSC quiescence and activation is essential to unlock their potential for regenerative medicine. NSCs residing in specific regions of the V-SVZ give rise to distinct subtypes of olfactory bulb interneurons. It is unknown whether quiescence-regulating signals map onto the regional heterogeneity of NSCs, and might thereby underlie the production of distinct interneuron subtypes. A major limitation to our understanding of the regulation of NSC quiescence has been the lack of specific markers to identify qNSCs, and prospectively purify them from their in vivo niche. Using a novel fluorescence-activated cell sorting (FACS) strategy that allows the purification of qNSCs from the adult mouse V-SVZ niche for the first time, I performed in vitro screens for quiescence-regulating signals. Unexpectedly, neurotransmitters emerged as the main class of qNSC-activating signals, including dopamine, GABA, serotonin, acetylcholine, and opioids. Local and long-range neurons that use these neurotransmitters innervate the V-SVZ in unique regional patterns, suggesting these signals map onto the regional heterogeneity of NSCs. Consistent with this hypothesis, infusions of cholinergic agonist and antagonists into the lateral ventricle resulted in regional changes in NSC proliferation. Moreover, cholinergic antagonists blocked the activation of qNSCs during regeneration, providing evidence that neurotransmitter signaling activates qNSCs in vivo. I then showed that hypothalamic Pomc-expressing neurons innervate the anterior-ventral V-SVZ and promote the activation of Nkx2.1+ qNSCs. Ablation of Pomc+ neurons resulted in decreased proliferation of NSCs in the anterior-ventral, but not anterior-dorsal, V-SVZ. Moreover, both the activity of Pomc+ neurons, and the proliferation of Nkx2.1+ NSCs in the anterior-ventral V-SVZ decreased in fasted animals, suggesting that hunger and satiety states regulation the generation of a single olfactory bulb interneuron subtype. Indeed, ablation of Pomc+ neurons resulted in a loss of the subtype of olfactory bulb interneuron that is generated by Nkx2.1+ NSCs. Together, my findings suggest that both local and long-range neurons regionally innervate the V-SVZ and mediate neural stem cell activation from the quiescent state.

Abstract not available

<p>The purpose of this study is to examine the relevant literatures about coordination mechanisms as well as study the use of coordination mechanisms by MNCs. Since many scholars have presented various models and claimed that their ideas are useful for MNCs and   subsidiary. This is due to getting a better understanding of how coordination works and what problems may occur.</p>

<p>This paper gives an overview of workflow management systems (WfMSs) and their security requirements with focus on access mechanisms. It is a descriptive paper in which we examine the state of the art of workflow systems, describe what security risks affect WfMSs in particular, and how these can be diminiuished.</p><p>WfMSs manage, illustrate and support business processes. They contribute to the performance, automation and optimization of processes, which is important in the global economy today. The security of process flows is important, since the sensitive business data need to be protected to inhibit illegal activities, such as blackmailing, imitation and fraud and to provide for good customer service.</p><p>This paper focuses on access mechanisms, because they are basic security mechanisms used by WfMSs assuring that only authorized users are provided access to data and resources. Also because of the unsecurity of the Internet, which is commonly used as infrastructure of Workflow systems, additional security mechanisms, such as PKIs, digital signatures and SSL have to be used to provide secure workflows.</p><p>Depending on the particular requirements in workflow systems, different extensional access control (AC) mechanisms have been developed to maintain security. But when it comes to commercially used WfMSs, the availability of the system is of utmost importance. It is the prerequisite for the system to be employed by companies. The problem is that there is always a trade-off between availability of the system and security. Because this trade off is generally solved in favor of availability, a major part of the developed AC mechanisms are not used in commercially used WfMS.</p><p>After the first part of this paper which is rather theoretical, we examine a commercial WfMS, namely IBM's MQ Workflow , and its security mechanisms. We show vulnerabilities of the system that could be abused by attackers. Afterwards, we show which security mechanisms, in particular, AC mechanisms are provided to secure against threats. We conclude with a summary, which highlights the difference between security concepts developed in the research area and those really implemented by the commercially used WfMS.</p>

Rapid degradation of certain short lived "timing" proteins is an effective mechanism for cells to control important regulatory pathways. The mechanisms by which regulatory proteases recognize their substrates are not well understood. Escherichia coli Lon, an energy dependent protease highly conserved in many prokaryotes and eukaryotes provides a model system to study protease/substrate interactions. RcsA, a regulator of capsule synthesis, when present in levels high enough to saturate Lon, cannot protect SulA, a cell division inhibitor, from being degraded. These observations suggest Lon recognizes its different substrates with different affinities. The different affinities of these substrates might relate to the role these substrates play in the cell: stabilization of RcsA leads to a nonlethal phenotype (capsule), while stabilization of SulA leads to lethal filamentation. To further examine protease/substrate interactions, targeted mutagenesis was employed to select for mutations in rcsA which give rise to mutant RcsA protein no longer degraded by Lon protease. Two mutants with an increased half-life in the presence of Lon were identified. Their mutations fall into the C-terminal region of RcsA, supporting the hypothesis that this region is involved in the interaction of RcsA with Lon. Stabilization of RcsA was dependent on its partner RcsB; the interaction of RcsA with RcsB is believed to protect RcsA from Lon dependent degradation. However, it was shown that rcsA expression is enhanced in the presence of RcsB, and RcsA protein cannot be detected in strains mutant for RcsB in the presence or absence of Lon. Furthermore, rcsA expression was shown to be activated by RcsA itself: rcsA::lacZ expression is low in the absence of RcsA. A conserved 25 by motif, designated "RcsA-Box" was identified in the promoter region of the rcsA and capsule (cps) genes. This motif was shown to be a likely candidate for RcsA binding: high level expression of both cps::lacZ and rcsA::lacZ fusions was shown to be dependent on the presence of the "RcsA-Box". These studies expand the understanding of the specific interactions between regulatory proteases and their targets, specifically as they relates to complex regulatory networks. / Graduation date: 1998

The purpose of this study is to examine the relevant literatures about coordination mechanisms as well as study the use of coordination mechanisms by MNCs. Since many scholars have presented various models and claimed that their ideas are useful for MNCs and   subsidiary. This is due to getting a better understanding of how coordination works and what problems may occur.

There is an increasing interest for business organizations to engage into the global inter-firm alliance nowadays. The companies are striving for accessing the opportunities created by emerging markets, diversification of the products’ offer or the access to lower cost inputs (Barnes, et al., 2010). However, along with the benefits, there are also risks that the inter-firm alliances are challenged with. Those risks are represented by various forms of opportunistic behavior, which might be further caused by business partners (Williamson, 1975). The companies that decide to engage into inter-firm alliance need to invest in the implementation of control mechanisms that will protect them against opportunistic behavior. The preventing exante mechanisms can be implemented prior to the official start of cooperation while the cause ex-post mechanisms are applied during further stage of the collaboration. However, due to the cost of these implementations, companies cannot afford employing every available control mechanism. They need to select only the ones that their benefits exceed their costs. One of the main factors that influence the selection process of control mechanisms is the level of knowledge sensitivity of a product. The main objective of this master thesis is to determine how the level of knowledge sensitivity of a product influences the selection of ex-ante control mechanisms.

Thesis (Ph. D.)--University of Chicago, Dept. of Molecular Genetics and Cell Biology, 2002. / Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--University of Chicago, Committee on Human Nutrition and Nutritional Biology, 2001. / Includes bibliographical references. Also available on the Internet.