Doctor of Philosophy / Department of Biology / Kristin Michel / Malaria remains a major human vector-borne disease, greatly contributing to global human morbidity and mortality. Control of mosquito vectors that transmit malaria continues to be dependent on the widespread application of chemical insecticides through indoor residual spraying and insecticide treated bed nets. However, resistance to these insecticides is spreading within many mosquito populations, adding an ever-increasing urgency to the development of alternative vector control measures. The mosquito immune system is a potential novel target for such alternative measures, as the immune response initiated in these insects during infection with vector-borne disease agents is a key determinant of vector competence and, thus, contributes to a species’ vectorial capacity. These immune responses, additionally, interact with and respond to parasitic or symbiotic biocontrol agents employed to kill or manipulate infection outcome with vector-borne disease agents. Entomopathogenic fungi, including Beauveria bassiana, have been considered as an alternative vector control measure, functioning as biopesticides. The Toll pathway is a key antifungal immune pathway in insects that impacts an insect’s ability to survive fungal infections. A better understanding of Toll signaling immune function and evolution in anophelines, both vector and nonvector, can thus help to improve future biocontrol methods of important vector mosquitoes like Anopheles gambiae. In this dissertation, I report the use of B. bassiana strain I93-825 in An. gambiae to analyze the impact of Toll pathway modulation on mosquito survival. Mosquito survivorship was strongly affected by B. bassiana exposure dose by several measured parameters including median survival, longevity, and hazard. Modulation of Toll signaling, by way of knockdown by RNA interference, revealed a dose-dependent trade-off between immune activation state and survivorship in An. gambiae. To better determine the full Toll immune signaling repertoire in mosquitoes, I annotated and describe the evolutionary history of intracellular Toll pathway members and Toll-like receptors (TLRs) within 21 mosquito genomes. The intracellular signaling pathway is conserved with 1:1 orthology, and evolutionary rates across different intracellular pathway members vary widely as compared to the conserved protein core of these mosquito species. In contrast, TLRs evolved largely by duplication events within certain anopheline lineages, most dramatically in the An. gambiae complex, where six TOLL1/5 paralogs likely possess different ligand binding specificities. Thus, these TLRs should be prioritized for experimental analyses of TLR immune function in An. gambiae. Taken together, the work in this dissertation identifies Toll pathway modulation as a potential resistance mechanism that could impact malaria biocontrol strategies and provides a foundation for future detailed studies of Toll pathway function in important mosquito vector species.

Toll plaza operation is a critical component of roadway operations throughout the United States, as tolls provide both revenue for expansion and opportunity for demand management. Originally cash or physical currency based, tolling has morphed to meet the twentieth century demand in terms of throughput and efficiency in the form of electronic toll collection. Electronic tolling has introduced a new form of driver decision making at toll plazas due to the additional payment choice. Despite the user convenience these facilities provide to consumers, this form of collection has not come without safety and operational concerns. Confusion at the toll plaza, unsafe merging maneuvers, and the unexpected behavior has actually increased certain crash patterns at toll plazas in some electronic tolling facilities. Building upon existing research, further work was completed to quantify the related impacts of electronic toll collection on traffic operations through a microsimulation model, and static evaluation study. While in Massachusetts overall toll plaza crashes are a minimal portion of 200,000 crashes each year in the Commonwealth at less than 0.1 percent of all crashes some toll plazas have higher crash rates than the state wide urban interstate average. Interchange 14 in Weston, Massachusetts had the highest crash rate among state toll plazas. Rear-end and same direction sideswipe collisions accounted for the highest crash numbers between the years 2010 and 2012. Microsimulation of various lane configurations derived from static evaluation feedback on driver decision making created six alternate configurations. Current plaza configuration was verified by the validated VISSIM microsimulation model to be the highest performing in terms of efficiency. A lane configuration with grouped payment lanes provided the best overall performance for alternatives with less than 1 percent difference from the current West Springfield interchange configuration. Static evaluation and microsimulation results pointed to increased efficiency and safety benefits with combination lanes. Additionally, drivers tended to avoid following heavy vehicles through plaza lanes. Motorists were willing to make up to 3 lane changes to avoid queues and may avoid combination lanes as an electronic toll customer if they anticipate a greater delay than an adjacent dedicated electronic lane. Recommendations for future research include: 1) further microsimulation modeling to examine traffic flow and safety impacts at toll plazas under varying traffic conditions and demand with open road tolling lanes strategies; and 2) developing enhancements to VISSIM to address parameter limitations associated with discrete choice modeling at toll plazas.

published_or_final_version / Transport Policy and Planning / Master / Master of Arts

A great deal of research has been conducted on Central Florida toll roads to better understand the characteristics of the tolling operation. In this thesis, the development and calibration of a toll plaza simulation models will be analyzed using two simulation programs varying mostly in their modeling theory. The two models utilized are, SHAKER, a deterministic queuing model for vehicles utilizing toll collection facilities, and VISSIM, a globally popular stochastic simulation software. The benefits of simulation models leads to the purpose of this thesis, which is to examine the effectiveness of two toll modeling programs that are similar in purpose but vary in approach and methodology. Both SHAKER and VISSIM toll plaza models have the potential to work as a tool that can estimate the maximum throughput and capacity of toll plazas. Major operational benefits resulting from developing these models are to simulate and evaluate how traffic conditions will change when demand increases, when and if queues increase when a lane is closed due to maintenance or construction, the impact of constructing additional lanes, or determining whether or not the best lane type configuration is currently implemented. To effectively calibrate any model available site data must be used to compare simulation results to for model validity. In an effort to correctly calibrate the SHAKER toll plaza tool and VISSIM model, an extensive field collection procedure was conducted at four Florida Turnpike operated toll facilities located in Central Florida. Each site differed from the others in terms of number of lanes, lane configuration, toll base fee, highway location, traffic demand, and vehicle percentage. The sites chosen for data collection were: the Lake Jesup Mainline Plaza along the Seminole Expressway (SR-417), the Beachline West Expressway Toll Plaza along the SR-528, the Daniel Webster Western Beltway Plaza along SR-429, and the Leesburg Toll Plaza along the Florida Turnpike Mainline SR-91. Upon completion of calibration of the two simulation models it is determined that each of the two software are successful in modeling toll plaza capacity and queuing. As expected, each simulation model does possess benefits over the other in terms of set up time, analysis reporting time, and practicality of results. The SHAKER model setup takes mere seconds in order to create a network and input vehicle, another few seconds to calibrate driving parameters, and roughly 10 additional seconds to report analysis. Conversely, setting up the VISSIM model, even for the most experienced user, can take several hours and the report analysis time can take several more hours as it is dependant on the number of required simulation runs and complexity of the network. VISSIM is most beneficial by the fact that its modeling allows for driver variability while SHAKER assumes equilibrium amongst lane choice and queuing. This creates a more realistic condition to observed traffic patterns. Even though differences are prevalent, it is important that in each simulation model the capacity is accurately simulated and each can be used to benefit operational situations related to toll plaza traffic conditions.

published_or_final_version / Public Administration / Master / Master of Public Administration

Ingeniero Civil en Biotecnología / Enfermedades como la sepsis, artritis reumatoide, tendinitis, entre otras, están ligadas a respuestas pro-inflamatorias anómalas. En ellas participan citoquinas pro-inflamatorias y factores de transcripción activados a partir de la vía de señalización mediada por receptores Tipo Toll y proteínas adaptadoras afínes. Estos receptores inician una cascada de señalización a partir de su homodimerización al ser inducidos por señales microbianas. Luego prosigue el reclutamiento de una serie de adaptadores proteicos que terminan finalmente con la transcripción de factores y producción de citoquinas proinflamatorias. En particular, este proyecto se enmarca en el estudio de las vías de señalización en las cuales está involucrado el receptor TLR4. Las enfermedades mencionadas pueden ser tratadas mediante el uso de fármacos, pero su uso resulta poco efectivo en etapas medio-avanzadas de la enfermedad y la presencia de efectos secundarios serios como osteoporosis, obesidad centrípeta, hipertensión, diabetes mellitus, entre otros. En vista de esto se plantea que la descripción de las interacciones del sistema mediado por TLR4 puede permitir diseñar nuevos tratamientos para estas enfermedades caracterizadas por respuestas inflamatorias anómalas. En función de lo anterior, se define el objetivo general de este trabajo que corresponde al estudio y reconstrucción in silico del acoplamiento e interacciones proteína-proteína producidas en la vía pro-inflamatoria activada por TLR4 y proteínas adaptadoras MAL, TRAM y MyD88. Por medio del uso de algoritmos de acoplamiento y herramientas de dinámica molecular se estudia el acoplamiento de los dominios TIR del homodímero de TLR4 y los adaptadores MAL, MyD88 y TRAM. Las interacciones propuestas son caracterizadas determinándose: área de contacto, tipos de interacciones y energía libre de los complejos. De manera adicional, se comparó los resultados con otros modelos presentados en publicaciones anteriores (Nuñez et al. (2007), Basith et al. (2011)). Los resultados obtenidos muestran que los adaptadores MAL y TRAM interactúan con el receptor TLR4 mediante la unión a la nueva superficie formada por la homodimerización de los dominios TIR de TLR4. En ambos casos, se observa que tanto MAL como TRAM se acoplan al receptor TLR4 orientando frente a frente sus lazos BB, lo que confirma la relevancia de este sector en las interacciones de esta vía. Otra zona importante de interacción corresponde al lazo CD del adaptador MAL y TRAM, el que según los resultados interactuaría con la Hélice B de TLR4. Por su parte, el estudio del complejo TLR4-MAL-MyD88, muestra que el adaptador MyD88 se une preferentemente a la nueva superficie que se produce a partir de la interacción TLR4-MAL. A partir de la estimación de la energía libre de unión vía MM-PBSA, se demuestra que los contactos y el acoplamiento de los modelos propuestos muestran estructuras de mayor estabilidad termodinámica que las presentadas por Nuñez (2007). Los resultados demuestran que zonas como los lazos BB, CD, hélice A y C son transversalmente relevantes dentro de la cadena de señalización y que presentan características generalizables al tipo de interacciones que se produce en esta vía mediada por el receptor TLR4. Finalmente, se destaca que la metodología planteada en este trabajo se transforma en un procedimiento novedoso y no descrito en trabajos anteriores relacionados a la descripción de interacciones vía TLR. Más aún, el conjunto de herramientas utilizadas permiten modelar las interacciones, probar hipótesis de interacción, brindar explicación a la acción de mutantes y encontrar sectores potenciales de interacción en cualquier tipo de sistema proteína-proteína.

Memoria para optar al Título Profesional de Químico Farmacéutico / Los fibroblastos cardíacos (FC), han sido denominados células centinelas del corazón, pues responden de manera variada y compleja ante cualquier elemento externo o interno a fin de mantener la homeostasis. Esta respuesta se lleva a cabo mediante una maquinaria conformada por una amplia red de receptores, entre ellos los receptores tipotoll y, en particular, el TLR4. La activación de este receptor ha mostrado mediar respuestas inflamatorias y fibróticas tanto en FC, como en otras células; en donde la expresión de proteínas de adhesión y reclutamiento de células del sistema inmune componen un eje vital en estos procesos. Por tanto se buscó determinar si la activación de TLR4 en el FC de ratas adultas genera una mayor adhesión de monocitos a través de un aumento en los niveles de E-selectina. La activación de TLR4 por LPS en FC, indujo un aumento en la expresión de Eselectina, situación que es revertida con el pre tratamiento con TAK-242 (inhibidor de TLR4). Adicionalmente, la activación de TLR4 generó un aumento en la adhesión de monocitos sobre los FC, mientras que la inhibición de TLR4 y las vías transduccionales ERK1/2, PI3K/Akt y NF-κB previo al estímulo con LPS, revirtió el aumento de adhesión de monocitos. De igual forma, el bloqueo de E-selectina, mediante el uso de un anticuerpo bloqueante, también revirtió el aumento de la adhesión, dando luces de que esta proteína de adhesión es fundamental para el reclutamiento de monocitos y por tanto para mediar la respuesta inflamatoria orquestada por el FC / Cardiac fibroblasts (CF) have been considered as sentinel cells, since they respond in a complex and diverse manner to external and internal stimulus, in orden to maintein homeostasis of the heart. This is mediated through a wide number of receptor, among then Toll-Like receptors, an particulary TLR4, play a critical role in cardiac inflammation. TLR4 activation has been involved in the regulation of inflammatory and fibrotic response both in CF and other cells; adhesion molecules and recruiment of immune cells are key elements of these process. Therefore we sought to evaluate whether TLR4 activation in CF obteined from adults rats increased monocyte adhesion trough increased levels of E-selectin. TLR4 activation by LPS in CF, induce increased in E-selectin expression, which was abolished when CF were pre treatment with TAK-242 (TLR4 inhibitor). In adittion, TLR4 activation increased monocyte adhesion to CF, whereas the inhibition of this receptor and releated signaling pathway ERK1/2, PI3K/Akt and NF-κB, previous LPS exposure reverse monocyte adhesion. Similarly, E-selectin blockade, by the use of blocking antibody, also blocked monocyte adhesion, which a yeast the importance of this adhesion molecule in monocyte recruitment and inflammatory response orchestrated by CF

Toll-like receptor 4 (TLR4) is an important vertebrate innate immune receptor. TLR4 recognizes both endogenous and exogenous danger signals to trigger an NF-kB dependent inflammatory response. While exogenous danger signal recognition is an essential part of pathogen response by the innate immune system, endogenous danger signal recognition by TLR4 can lead to chronic and pathological inflammation. Understanding the differences in recognition of these two types of danger signals would allow for independent modulation of pathogen and host triggered inflammatory response through TLR4. Here, we examine the evolution of activation of TLR4 by two agonists, pathogen-derived lipopolysaccharide and host-produced S100A9. We show that these two types of signals evolved earlier than previously thought. We identified TLR4 cofactors MD-2 and CD14 in amphibians and fish, and validated that zebrafish TLR4 can recognize LPS. By contrast, we find that S100 activation evolved in the ancestor of amniotes. We identified an ortholog of S100A9 in birds and reptiles capable of activating TLR4. Using comparative immunology, we found that the requirements for LPS and S100A9 activation are different. In addition to our evolutionary studies, we used molecular approaches to probe if zinc binding to S100A9 is necessary for TLR4 activation. We found that activation of TLR4 by S100A9 occurs even in the absence of zinc. Finally, we describe how our evolutionary approach led to mechanistic hypotheses regarding TLR4 activation by both LPS and S100A9. This has led to ongoing projects in the Harms lab. This dissertation includes previously published and unpublished co-authored material. / 2021-04-30

Resumo: Não disponível / Abstract: Acute chorioamnionitis is a response to microbial infection of the amniotic fluid. The innate immune system constitutes the host's first line of defense against pathogens and, in this regard, Toll-like receptors (TLRs) are important regulators of such nonspecific response. However, the expression of these receptors in chorioamniotic membranes in pregnancies complicated by chorioamnionitis has not been well established. Objective:The purpose of this study was to examine the localization of TLR-1, TLR-2, TLR-4 and TLR-6 in fetal membranes and determine whether histologic chorioamnionitis is associated with changes in gene expression of these receptors. One hundred and fifteen chorioamniotic membranes were included in the study. They were collected at the Obstetrics Service of the Botucatu Medical School, São Paulo State University, UNESP, from pregnant women with preterm delivery or term delivery with or without labor. Both groups were stratified on the basis of the presence of histologic chorioamnionitis. Fragments of the chorioamniotic membranes were sent for histopathologic analysis in order to confirm histologic chorioamnionitis. Other membranes fragments measuring 1cm2 were placed into RNA later and submitted to total RNA extraction. After RNA extraction, the samples with concentration between 0.02 and 0.2 g/L of RNA were submitted to cDNA collection for later use in quantifying the expression of TLR-1, TLR-2, TLR-4 and TLR-6 by the real-time PCR technique using the TaqMan® Gene Expression Assays System. All membranes analyzed expressed TLR-1 and TLR-4, whereas 99.1% expressed TLR-2 and 77.4% expressed TLR-6. TLR-1 and TLR-2 expression were statistically higher in the membranes of preterm pregnancies in the presence of chorioamnionitis as compared with preterm membranes in the absence of the inflammatory infiltrate. Among the membranes of term pregnancies, there was... (Complete abstract click electronic access below) / Orientador: Márcia Guimarães da Silva / Coorientador: José Carlos Peraçoli / Banca: Angela Maria Victoriano de Campos Soares / Banca: Rosiane Mattar / Mestre

The aim of this work is to describe and evaluate the project of road charging in London. The theoretical part deals the issue of transport in urban conditions, its basic types, problems and possible approaches to tackling it. It also includes a description of the electronic toll systems as tools used to manage traffic in the city. The practical part deals with the use of video detection technology for road charging in London. It includes the preparation and implementation of the project, its basic principles of operation and adjustments made during the period of its operation. Followed by evaluation of the project in terms of impact on traffic, business and air quality in London. At the end of the work there are some recommendations for possible implementation of a similar charging system in terms of the city of Prague.