Dissertation (Ph.D.) -- Worcester Polytechnic Institute. / Keywords: target space; STELLA; plan analysis; evacuation simulation. Includes bibliographical references.

During herpes simplex virus 1 (HSV-1) replication, newly constructed capsids escape the nucleus to undergo maturation in the cytoplasm via a process termed nuclear egress. Capsids perform nuclear egress through localized disruption of the nuclear lamina, envelopment of the inner nuclear membrane to create a perinuclear enveloped virion, and de-envelopment of the outer nuclear membrane for capsid release into the cytoplasm. Critical virial factors for this process are viral proteins pUL31 and pUL34 that interact to form heterodimers. These heterodimers form larger hexameric arrays to drive membrane budding. Through the characterization of phenotypes of UL34 point mutants, we are able to further study the underlying mechanisms of nuclear lamina disruption and nuclear budding. One such mutant, UL34(Q163A), results in impaired virus production, cell-cell spread, and an inability to disrupt lamin A/C networks. Selection for extragenic suppression of UL34(Q163A) yielded the UL31(R229L) mutation, that partially rescued the growth and spread defects of UL34(Q163A), but was unable to regain the ability to disrupt lamin A/C networks. Through this study we concluded that disruption of lamin A/C networks was not required for efficient HSV-1 replication. In order to understand the underlying mechanisms of membrane budding, the previously characterized UL34(CL13) double mutant, which results in a 100-fold reduction in virus production, a severe impairment in cell-cell spread, and an accumulation of capsid-less perinuclear vesicles was further studied. Characterization of the single mutations of UL34(CL13), UL34(R158A) and UL34(R161A) revealed that neither single mutation was responsible for spread or growth defect, but that either single mutation resulted in a promiscuous budding phenotype. Through this study, we concluded that although individual steps of the nuclear egress pathway are tightly regulated, alteration of the regulation at a single step does not grossly impact HSV-1 replication.

None

No description available.

BACKGROUND:Subspecies B1 human adenoviruses (HAdV-B1) are prevalent respiratory pathogens. Compared to their species C (HAdV-C) counterparts, relatively little work has been devoted to the characterization of their unique molecular biology. The early region 3 (E3) transcription unit is an interesting target for future efforts because of its species-specific diversity in genetic content among adenoviruses. This diversity is particularly significant for the subset of E3-encoded products that are membrane glycoproteins and may account for the distinct pathobiology of the different human adenovirus species. In order to understand the role of HAdV-B-specific genes in viral pathogenesis, we initiated the characterization of unique E3 genes. As a continuation of our efforts to define the function encoded in the highly polymorphic ORF E3-10.9K and testing the hypothesis that the E3-10.9K protein orthologs with a hydrophobic domain contribute to the efficient release of viral progeny, we generated HAdV-3 mutant viruses unable to express E3-10.9K ortholog E3-9K and examined their ability to grow, disseminate, and egress in cell culture.RESULTS:No differences were observed in the kinetics of infected cell death, and virus progeny release or in the plaque size and dissemination phenotypes between cells infected with HAdV-3 E3-9K mutants or the parental virus. The ectopic expression of E3-10.9K orthologs with a hydrophobic domain did not compromise cell viability.CONCLUSIONS:Our data show that despite the remarkable similarities with HAdV-C E3-11.6K, HAdV-B1 ORF E3-10.9K does not encode a product with a "death-like" biological activity.

"The purpose of this thesis is to demonstrate and enhance the technical basis of the procedure for evaluating lifesafety within the Building Firesafety Engineering Method (BFSEM). A framework for the analysis has been documented, but not extensively tested in a building situation. Hence, procedures to obtain the necessary input data and to evaluate that data needed to be developed. In addition, the general framework had to be tested rigorously enough to identify weaknesses. "

Plasmodium falciparum, l'agent étiologique du paludisme, est un parasite intracellulaire obligatoire du phylum des Apicomplexa, responsable de 445 000 décès par an. Le développement de Plasmodium dans les globules rouges (GRs) humains correspond à la phase symptomatique de la maladie. Il commence par la pénétration active de la cellule hôte par la forme invasive nommée mérozoïte, suivie par la multiplication du parasite dans un processus appelé schizogonie pour former 16 à 32 nouveaux mérozoïtes qui sont alors libérés des GRs (étape de sortie) et peuvent alors initier un nouveau cycle. Au cours de son développement intra-érythrocytaire de 48h, ce parasite utilise la phosphorylation réversible de protéines pour réguler les étapes d‘invasion, de schizogonie et de sortie du GR, mais nos connaissances actuelles sur la contribution des phosphatases parasitaires dans ces mécanismes demeurent très incomplètes.L'objectif de ma thèse était d’identifier et de caractériser des phosphatases potentiellement impliquées dans la sortie ou l'invasion des GRs par P. falciparum. J'ai centré mon travail sur 4 d'entre elles, à savoir PP1, PP4, PP7 et Shelph2, sur la base de leur profil d'expression transcriptionnelle tardive au cours du cycle intra-érythrocytaire, qui correspond à ces deux évènements cellulaires. La première partie de cette étude est consacrée à la caractérisation fonctionnelle de Shelph2, une phosphatase d'origine bactérienne. Par génétique inverse utilisant la stratégie CRISPR-Cas9, nous avons étiqueté le gène au locus endogène et montré que Shelph2 est stockée dans des vésicules apicales des mérozoïtes en formation. Nous avons également démontré que cette phoshpatase n’est pas essentielle pour le développement intra-érytrocytaire du parasite dans les GRs car la délétion du gène n'affecte pas les étapes d'invasion, de multiplication des parasites ou de leur sortie des GRs, ce qui suggère la possibilité d’une redondance fonctionnelle avec d'autres phosphatases parasitaires.Dans la deuxième partie de ce travail, nous avons cherché à décrire les rôles de PP1, PP4 et PP7. Les gènes codant pour ces enzymes étant décrits comme probablement essentiels, nous avons mis en place au laboratoire une stratégie de knock-down conditionnel (ribozyme glmS), avec l’idée de déstabiliser l’ARNm après auto-clivage du ribozyme lors de l’addition d‘un métabolite, ici la glucosamine. Nous avons introduit avec succès la séquence glmS en 3 'des gènes d’intérêt pour PP4 et PP7, mais nous n’avons pas observé de déplétion protéique significative lors de l’addition de glucosamine, empêchant d’utiliser ces lignées pour étudier les fonctions de PP4 et PP7. Cependant, ces lignées parasitaires modifiées ont été utilisées pour analyser la localisation subcellulaire de ces phosphatases. Comme alternative au ribozyme, nous avons utilisé une approche de knock-out inductible (iKO) basée sur une recombinase Cre dimérisable (système DiCre) qui excise des fragments d'ADN situés entre deux sites loxP. Nous avons établi deux lignées de parasites, iKO-PP7 qui n'a pas encore été caractérisée et la souche iKO-PP1. En utilisant les parasites iKO-PP1, nous avons montré que PP1 était principalement une phosphatase cytosolique majoritairement exprimée au stade schizontes. De plus, l'excision inductible du gène PP1 à deux moments différents du cycle érythrocytaire de P. falciparum nous a permis de révéler que PP1 joue deux rôles essentiels, l'un pendant la schizogonie et l'autre au moment de la sortie du parasite. A notre connaissance, ce travail représente la première description d'une phosphatase parasitaire requise pour ces étapes du développement asexué de P. falciparum. / Plasmodium falciparum, the etiologic agent of malaria, is an obligate intracellular parasite of the Apicomplexa phylum that is responsible for 445000 deaths annually. Plasmodium development in human red blood cells (RBCs) corresponds to the symptomatic phase of the disease. It starts by the active penetration of the host cell by the invasive form named merozoite, followed by the parasite multiplication in a process called schizogony to form 16-32 new merozoites that are released from the RBC (egress step) and start a new cycle. During its 48h intra-erythrocytic development, this parasite uses reversible protein phosphorylation to regulate invasion, schizogony as well as egress, but our current knowledge on the contribution of parasite phosphatases in these cellular events is still very poor. The objective of my thesis was to identify and functionally characterize phosphatases potentially involved in egress or invasion during P. falciparum RBC cycle. I focused my work on 4 of them, namely PP1, PP4, PP7 and Shelph2, on the basis of their late transcriptional expression profile during the intra-erythrocytic cycle, as this profile matches the timing of these two essential events. The first part of this study is dedicated to the functional characterization of Shelph2, a phosphatase of bacterial origin. By reverse genetics using CRISPR-Cas9 strategy, we endogenously tagged the gene, and showed that Shelph2 is stored in apical vesicles in the developing merozoites. We also demonstrated that it is dispensable for parasite RBC development, as the deletion of the gene did not affect invasion, parasite multiplication nor egress, suggesting possible functional redundancy with other parasite phosphatases.In the second part of this work, we aimed to describe the roles of PP1, PP4 and PP7. As they were described as likely essential, we set up in the laboratory a conditional knock-down strategy named the glmS ribozyme, with the idea of destabilizing the mRNA following self-cleavage of the ribozyme upon metabolite addition, here glucosamine. We successfully introduced the glmS sequence in 3’ of the genes of interest for PP4 and PP7 but we did not observe any significant protein depletion upon glucosamine addition, thus preventing us to use these lines to study PP4 and PP7 functions. Yet, these engineered parasite lines were used to analyze the subcellular localization of these phosphatases. As an alternative to the ribozyme, we used an inducible knock-out (iKO) approach based on a dimerizable Cre recombinase (DiCre system) that excises DNA fragments located between two loxP sites. We established two parasite lines, the iKO-PP7 that has not been further characterized and the iKO-PP1 strain. Using the iKO-PP1 parasites, we showed that PP1 is predominantly a cytosolic phosphatase mostly expressed during schizogony. Furthermore, the inducible excision of PP1 gene at two different time points of P. falciparum RBC cycle permitted us to reveal that PP1 plays two essential roles, one during schizogony and the other one at the time of parasite egress. This is to our knowledge the first description of a parasite phosphatase required for these developmental steps.

The effectiveness of an emergency response during an incident is often affected by the lack of information provided to the people within the situation about the current conditions. Deaths in large-scale fires are often likely to have been caused by delays in the occupants receiving relevant information on the fire and egress routes. This is why pre-movement behaviour, which is defined as the behaviour which occurs before an alarm is sounded and includes the activities which occur between the alarm sounding and the occupants beginning to move towards an exit, is believed to be generally more important to survival than the actual movement speed. It is the unpredictability and complexity of human behaviour that is the most influential factor on the success / failure of an evacuation plan. Unfortunately, evacuation plans rely on the use of purposely designed egress routes which often are not the common everyday exits. These specifically designed egress routes, which an engineer may assume will be used during an evacuation, are often ignored by occupants due to the lack of information and noticeable distinguishing features. Having occupants moving in directions away from these intended routes may result in the increasing possibility of occupants finding themselves in a dangerous situation, ultimately leading to potential loss of life. The value of a sensor-linked fire model has been demonstrated and the potential for interpretation of human behaviour shown. However, there are many challenges in representing and interpreting data on human behaviour. Within most emergency evacuation situations, occupants will often walk past emergency exits without using them and exit through the main entrance or main exit, as displayed during an evacuation experiment held in IKEA in 1996. Problems occur because occupants will rely on the familiar exits over the closest emergency exit, which could be potentially overcome by the use of an information driven evacuation system. The main function of the Information Driven Evacuation System or I.D.E.S. is to provide occupants with information on the most appropriate egress paths within a building based on the development of the fire and the movement of other occupants. The system is a combination of real-time sensor data, a prediction modelling tool and the information driven way-finding tools. However, as all three processes are independent systems, a central server will be required in order to ensure that all the different processes are speaking the same language and that the information from one system can be understood by another. of the components within the system interact with each other. The basis of the system will combine the use of sensors within a building and specific way-finding tools to give the I.D.E.S. the ability to change the information provided by the way-finding tools by having the sensors within the building interfacing with a computer server. This server will incorporate a modelling program that will have ability to assess the data gathered by the sensors, and use the servers “intelligence” (i.e. predicting capabilities) to alter the information provided by the way-finding tools. The server will also have the ability to use the sensor data to predict the development of the fire and the movement / behaviours of the occupants. The way-finding tools used within the I.D.E.S. would have the primary goal of relaying the information to the occupants within the building through the use of both audio (e.g. directional speakers) and visual (e.g. flashing lights) capabilities. Basic audio and visual tools are already used as common features of an evacuation plan [9] and include exit signage and alarm bell/sirens. The computer model used as part of the “intelligence” of the server will need to have predicative capabilities that incorporate information provided in real time. It is believed that the combination of these tools will be able to provide the occupants with the information required to evacuate the building in a safe and efficient way without causing confusion, thus reducing the possibility of stress and anxiety. However, the solution will only work if the combination of the tools, sensors and systems are able to be integrated into a central control panel that can be understood and used effectively by fire service and/or security staff. The following is the Chapter breakdown of the thesis: Chapter 1 discusses the nature of the problem that is to be addressed by the I.D.E.S. as well as the proposed solution and the overall concept of the system. Chapter 2 provides an overview of the system to be developed as part of this thesis, via a graphical overview as well discussing the current status of the system and an higher level summary. Chapters 3, 4 and 5 discusses the background research and information gathered on the current code requirements for an evacuation design, the current theories and completed research focusing on the human behaviour of occupants during an evacuation, and the development of computer modelling programs and their historical implementation. Chapter 6 focuses and describes the development of predictive modelling, the limitations that dictates the modelling process, the requirements of the I.D.E.S. modelling programme and a review of the possible programmes for their ability to be used as part of the system. Chapter 7 covers each of the three evacuation experimental series that were conducted as part of this thesis. The purpose of each of the relevant experiment is covered, the results gathered, the analysis of the research, and how the results influenced the development of the simulation methodology. Chapter 8 focuses on the development of the modelling programme, based on the experimental data gathered, and presents a feasibility study that will demonstrate how the system would work during a simulated real life evacuation based on the information gathered from the experiments using the updated CRISP program and the hypothetical installation of the system within an existing building, located in Auckland, New Zealand. The possible further evolution of the system is discussed in Chapter 10.

Tall buildings are becoming more common in the modern built environment and the method of evacuating or moving to a place of safety using the stairs is still the primary means of egress. Typically designers use tools such as computer models and hand calculations to predict the time taken for occupants to evacuate to an exit or place of safety. However, increasing trends of obesity, age and a sedentary lifestyle is raising questions about the accuracy of some of the tools. As the tools are based on case study data carried out in the 1980’s. This research compares evacuation performance of case study buildings to the predictions by Pauls’ simplified hand calculation and the EvacuatioNZ computer model. The comparison uses four multi-storey buildings from the case study data, ranging from 11 to 27 stories high. The research will also investigate the effect of how the building is represented in EvacuatioNZ on the performance of the prediction and make recommendations in best practice for further work. Results from the comparisons shows EvacuatioNZ is within 15% for total egress time of the case study data in six out of eight of the stairs. The average difference of EvacuatioNZ to the case study is 8.6%. Further comparisons of exit flow rate and descent speed show EvacuatioNZ is within 10% of the case study data in five out of eight of the stairs. Paul’s simplified hand calculation predicts a total egress time which is 6% to 38% shorter than the case study data. Modifying the equation to equalise stair entry delay improves the prediction to a difference of 0.9% to 31%. The modified equation is within 10% in five out of eight stairs. The comparisons for EvacuatioNZ indicate predictions which are generally with 10-15%. However individual performance is not investigated and this area should be fully investigated to answer concerns about contemporary occupants and their ability to descend multiple flights of stairs. Further work should include a larger range of data, particularly exploring building height and population. Given the recommendations are followed and more data becomes available for further work to support this research; EvacuatioNZ could be used as a tool for predicting evacuations in multi-storey buildings. Pauls’ hand calculation is not recommended for predictions of multi-storey evacuations without a safety factor. Differences between the prediction and case study result were improved with a modification of the equation to account for the case study stair entry times.

This study proposed a concept and a method for building egress analysis. The works of this research include: develop procedures to analyze the egress system of a plan, propose methods and a program to identify target spaces in evacuation, present a dynamic blocking viewpoint of an egress system, dedicate a way to identify the threatening room of fire origin, and create an occupant evacuation model (ESM), whose functions are: to simulate the occupant evacuation in a building in the static and the dynamic blocking situations, to record the people changes at every compartment during evacuation, and to review the clearing times of different compartments.