Evaluación de la vacuna multicomponente TcVac1 contra la enfermedad de Chagas utilizando un protocolo de electroporación intradérmica

Tesis de Doctorado de WAEL HEGAZY HASSAN MOUSTAFATrypanosoma cruzi (T. cruzi), un protozoario hemoflagelado, es el agente etiológico de la enfermedad de Chagas. Ésta es la causa más común de muertes relacionadas insuficiencia cardiaca congestiva entre los adultos jóvenes en las áreas endémicas de Sudamérica, Centroamérica y México. También se ha convertido en un problema de salud importante en los Estados Unidos y Europa debido a la migración a gran escala de latinoamericanos en las últimas décadas. Los esfuerzos para el desarrollo de vacunas contra la infección por T. cruzi han aumentado en los últimos años. Nuestro grupo de trabajo ha diseñado una serie de vacunas (TcVac) compuestas por antígenos de membrana anclados a la GPI derivados de T. cruzi. Se ha demostrado que las vacunas TcVac aumentan las respuestas inmunitarias humorales y celulares y proporcionan un control significativo (pero no completo) de la infección experimental en ratones y perros. En el presente documento, nos propusimos probar dos protocolos de inmunización para la entrega de la vacuna de ADN (TcVac1) compuesta de antígenos TcG2 y TcG4 en un modelo de ratón BALB/c. En un primer ensayo se comparó la protección conferida por la vacuna TcVac1 administrada vía intramuscular (IM) contra el protocolo de vacunación electroporación/intradérmica (IDE). Se vacunaron cuatro veces, con quince días de diferencia a doce ratones BALB/c por grupo. Dos semanas después de la última inmunización, se sacrificó a la mitad de los animales (n=6) de cada tratamiento, para la evaluación de la eficacia de la vacuna en la pre-infección y la segunda mitad (n=6) se sacrificaron 60 días después de la infección con trypomastigotes cepa Sylvio X10/4 de T. cruzi, para ser evaluados durante la fase aguda de la infección. La respuesta inmune humoral se evaluó mediante la evaluación de los subtipos de IgG anti-TcG2 y anti-TcG4 utilizando un ensayo de ELISA. La respuesta inmunitaria celular se evaluó mediante un ensayo de proliferación de linfocitos. Por último, se evaluaron los aspectos clínicos y morfopatológicos para todos los animales experimentales. Nuestros resultados demostraron que al comparar la entrega IDE TcVac1 frente a la entrega IM, el primero indujo un nivel significativamente mayor de respuesta de anticuerpos específicos por III antígeno (IgG2a+IgG2b>IgG1) y proliferación de linfocitos, que se expandió en respuesta al desafío de infección. La evaluación histológica después de la infección por desafío mostró infiltración de células inflamatorias (macrófagos y linfocitos) en el corazón y el tejido esquelético de todos los ratones infectados. Sin embargo, el mayor aumento de infiltrados inflamatorios se observó en ratones TcVac1_IDE/Tc en comparación con TcVac1_IM/Tc o ratones no vacunados/infectados. La extensión del infiltrado inflamatorio tisular se asoció directamente con el control de los nidos de amastigotes intracelulares en ratones vacunados/infectados (contra a los no vacunados/infectados). Nuestros resultados sugieren que la administración de IDE mejora la eficacia protectora de la vacuna TcVac1 contra la infección por T. cruzi en ratones en comparación con la administración IM de la vacuna. Posteriormente en un segundo experimento, se probó la eficacia de diferentes dosis vacunales utilizando la modificación a la vacuna eliminando los plásmidos que codifican para la interleucina-12 y factor estimulante de colonias granulocitos-macrófagos (GM-CSF). En este ensayo, se probaron cinco dosis diferentes (1, 5, 10, 15 y 20ug) de la vacuna para determinar la dosis óptima recomendada para prevenir la infección por T. cruzi con aplicación intradérmica/electroporación de la vacuna. En este experimento se utilizaron cuarenta y dos ratones hembra, cepa CD1, subdivididos en 7 tratamientos diferentes (n=6/grupo). Después de la vacunación, los ratones fueron desafiados con cepa Ninoa/MHOM/MX/1994 de T. cruzi (500 trypomastigotes/ratón, intraperitonealmente). Se analizaron parámetros tales como parasitemia, prueba de supervivencia, prueba serológica (ELISA) y análisis histopatológicos para evaluar la eficacia de la protección de la vacuna y determinar la dosis óptima. Los resultados demostraron que el grupo vacunado con 10 μg de dosis de ADN plasmídico indujeron los mejores niveles de inmunoglobulinas específicos, la parasitemia más baja y el mayor control del nidos de parásitos en cardiomiocitos y tejido muscular esquelético. Se confirmó que la aplicación intradérmica en conjunto con la electroporación es un método eficaz para la administración de vacunas con ADN, ya que mejora la inmunogenicidad del plásmido entregado, y reduce el tiempo y el costo de preparación de la vacuna, se minimiza la dosis de la vacuna, el número de IV refuerzos de la vacuna se reduce a una sola aplicación y la vacuna no requiere el uso de adyuvantes. La patología es una disciplina que depende crucialmente de la interpretación de imágenes para el diagnóstico y pronóstico de enfermedades. Tradicionalmente las técnicas de interpretación se basaron en gran medida en el análisis subjetivo de los especímenes, con un acuerdo variable entre los observadores. La interpretación moderna hace uso tanto del análisis molecular, como de la cuantificación objetiva de a través de microscopía auxiliada por sistemas de digitalización óptica. La dificultad para que el sistema ocular/cerebro humano analice objetivamente un objeto dentro de una escena, independientemente de su información contextual (por ejemplo, la percepción de un objeto dado puede ser muy diferente según su contexto), lo que promueve la necesidad de un ayuda microscopía computarizada para su uso en la patología molecular. De ahí que nuestro tercer experimento se enfocó en el desarrollo de un sistema digital de análisis de imágenes histopatológicas. En este tercer ensayo se utilizaron técnicas computarizadas de análisis digital de imágenes histopatológicas (técnicas de interpretación histológica basadas en el análisis de imágenes, cuantificación molecular de marcadores mixtos en estudios colorimétricos) enfocado en la detección y conteo de los infiltrados linfocitarios, conteo de cardiomiocitos y la determinación de hipertrofia cardiaca en ratones infectados con cepa Sylvio X10/4 de T. cruzi y la comparación de los resultados obtenidos con los resultados histológicos basados en observación y análisis visual realizado con tres patólogos en un ensayo doble ciego. Se usaron ratones BALB/c (n=48), los cuales fueron vacunados con TcVac1 vía intradérmica con electroporación, los ratones fueron sacrificados al día 60 post-infección y el análisis histopatológico se llevó a cabo como sigue; A) Se examinaron al menos cinco secciones por tejido (ventrículo derecho, ventrículo izquierdo y septum) para cada ratón para detectar la presencia de inflamación aguda y nidos de parásitos (magnificación: 400X y 100X), el parasitismo tisular se evaluó contando los nidos de amastigotes presentes en 100 campos microscópicos en cada uno de los corazones analizados. Del mismo modo, el V infiltrado inflamatorio se visualizó en >200 campos microscópicos de las secciones de tejido del corazón, tres patólogos analizaron en un estudio doble ciego las muestras, y se registraron los resultados finales del consenso. B) los tejidos fueron procesados en cortes 4 μm, teñidos con HE y analizados en las mismas secciones mencionados anteriormente. Los tejidos fueron observados bajo microscopio óptico y se llevó a cabo el análisis colorimétrico a través del software Image-Pro Plus 5.1. para todas las muestras. Finalmente, la comparación de los datos obtenidos de ambas técnicas fueron comparados. Los resultados mostraron la eficacia del análisis computarizado y la precisión de los datos obtenidos, donde había una diferencia estadísticamente significativa (P˂0.05) entre los grupos vacunados y no vacunados. la conclusión del presente experimento fue la obtención de datos cuantitativos para los estudios histopatológicos sin la necesidad de usar otras técnicas más costosas como inmunohistoquímica, FISH, ISH (silver-based), immunocitoquìmica, expresión de proteínas marcadas, etc. y evadir el margen de error humano en la recopilación y procesamiento de datos.UAEM con número de registro 3326/2012C, y al Consejo Nacional de Ciencia y Tecnología (CONACYT No. 156701), bajo la responsabilidad del Dr. Juan Carlos Vázquez Chagoyán Título: Correlación de las concentraciones séricas de troponina I con los cambios clínicos cardiovasculares en perros infectados con Trypanosoma cruzi en fase crónica Clave: 4518/2018/C

Optimization of a New Array Noise Tool; Analysis & Interpretation of Case Studies of Down- Hole Leak Detection

This thesis is concerned with the optimization and analysis of new array noise tools. Various lab and field case studies are analyzed to test the capabilities and determine the optimum operating conditions of the new tool for leak detection purposes. The purpose of this study is to optimize the logging procedure of real-time array noise tool, selecting adequate gain parameters and frequency band in the processing phase, and to analyze the data acquired through multiple labs and field tests. The study methodology involved the following steps: 1. Performing lab and field tests for the tool in a range of different scenarios. 2. Carrying out multiple real-life case studies and provide interpretation using commercial software. The gain setting was optimized to obtain the best results through logging and adding additional auxiliary logging accessories was proven to enhance the acquisition process. Moreover, implementing proper logging procedure that suits the tool capabilities aid the analysis process and the objective was met successfully. The tool was tested and proved consistent results, which can be commercialized and used as real-time noise tool. The new array noise tool overcomes the limitations of the memory noise tool in which it will save both time and money on the oil companies and will be able to provide in situ answers for leak detection purposes

PROTECCION INMUNE CONTRA Trypanosoma cruzi INDUCIDO POR LA VACUNA TCVAC1 MODELO MURINO DE USANDO EL PROTOCOL DE ELECTROPORACIÓN INTRADÉRMICA

Trypanosoma cruzi, a parasitic protozoan, is the etiologic agent of Chagas disease. Chagas disease is the most common cause of congestive heart failure related deaths among young adults in the endemic areas of South and Central America and Mexico. It has also become an important health issue in the United States and Europe due to large scale migration of Latin Americans over the last few decades. No vaccines are currently available until now. In this study, we tested the vaccine efficacy of two antigen candidates against T. cruzi infection and disease in a mouse model. The use of TcVac1 (TcG2, TcG4, T cruzi antigen encoding plasmids, interleukin-12 [IL-12] and granulocyte-macrophage colony-stimulating factor [GMCSF] encoding plasmids as genetic adjuvants) anti T. cruzi candidate vaccine injected intramuscularly has been previously reported in mice with very encouraging results. Here we evaluated the comparative protection conferred by TcVac1 when administrated intramuscular (IM) versus an intradermal/electroporation (IDE) vaccination protocol. Twelve BALB/c mice per group were vaccinated four times fifteen days apart. Half the animals (n=6) from each treatment were sacrificed two weeks after the last immunization for pre-infection vaccine efficacy evaluation, and the second half (n=6) was sacrificed 60 days post-infection (dpi) with T.cruzi Trypomastigotes (Sylvio X10/4 strain). Immune response was assessed through anti-TcG2 and TcG4 T. cruzi antigens.TcVac1 induced a strong IgG response (IgG2b>IgG1) that was significantly expanded post-infection, and moved to a nearly balanced IgG2b/IgG1 response in chronic phase. High IgG titers with IgG2 predominance in response to T. cruzi infection specific serum antibodies with an Enzyme Linked Immunosorbent Assay (ELISA) and lymphocyte activation against the studied antigens was evaluated through a lymphocyte proliferation assay. We found that IDE induced significantly larger surges of IgG antibodies including subtypes IgG1, IgG2a and IgG2b, during the pre- and post-infection periods for the two antigens used in the experiment. The ratio of antibodies IgG2b/IgG1 was >1 for TcG2 antigen in the pre-infection period in both administration routes. However for the TcG4 antigen the ratios were opposite for animals belonging to different administration routes1 for IM. During the post infection period for both treatments IgG2b/IgG1 ratio was always <1. Suggesting, as previously reported that a switch from Th1 to Th2 type immune response occurs in vaccinated/infected animals. During the Lymphocyte proliferation assays we observed that both antigens were able to induce lymphocyte proliferation during the pre-infection period. However, we observed that animals from the IDE group induced more proliferation than IM mice group when TcG4 was used to activate the cells, which was also observed during the post-infection phase of the experiment. No animals died due to infection, vaccinated mice appeared to have healthier status than the control animals

Robust Design of Variation-Sensitive Digital Circuits

The nano-age has already begun, where typical feature dimensions are smaller than 100nm. The operating frequency is expected to increase up to 12 GHz, and a single chip will contain over 12 billion transistors in 2020, as given by the International Technology Roadmap for Semiconductors (ITRS) initiative. ITRS also predicts that the scaling of CMOS devices and process technology, as it is known today, will become much more difficult as the industry advances towards the 16nm technology node and further. This aggressive scaling of CMOS technology has pushed the devices to their physical limits. Design goals are governed by several factors other than power, performance and area such as process variations, radiation induced soft errors, and aging degradation mechanisms. These new design challenges have a strong impact on the parametric yield of nanometer digital circuits and also result in functional yield losses in variation-sensitive digital circuits such as Static Random Access Memory (SRAM) and flip-flops. Moreover, sub-threshold SRAM and flip-flops circuits, which are aggravated by the strong demand for lower power consumption, show larger sensitivity to these challenges which reduces their robustness and yield. Accordingly, it is not surprising that the ITRS considers variability and reliability as the most challenging obstacles for nanometer digital circuits robust design. Soft errors are considered one of the main reliability and robustness concerns in SRAM arrays in sub-100nm technologies due to low operating voltage, small node capacitance, and high packing density. The SRAM arrays soft errors immunity is also affected by process variations. We develop statistical design-oriented soft errors immunity variations models for super-threshold and sub-threshold SRAM cells accounting for die-to-die variations and within-die variations. This work provides new design insights and highlights the important design knobs that can be used to reduce the SRAM cells soft errors immunity variations. The developed models are scalable, bias dependent, and only require the knowledge of easily measurable parameters. This makes them useful in early design exploration, circuit optimization as well as technology prediction. The derived models are verified using Monte Carlo SPICE simulations, referring to an industrial hardware-calibrated 65nm CMOS technology. The demand for higher performance leads to very deep pipelining which means that hundreds of thousands of flip-flops are required to control the data flow under strict timing constraints. A violation of the timing constraints at a flip-flop can result in latching incorrect data causing the overall system to malfunction. In addition, the flip-flops power dissipation represents a considerable fraction of the total power dissipation. Sub-threshold flip-flops are considered the most energy efficient solution for low power applications in which, performance is of secondary importance. Accordingly, statistical gate sizing is conducted to different flip-flops topologies for timing yield improvement of super-threshold flip-flops and power yield improvement of sub-threshold flip-flops. Following that, a comparative analysis between these flip-flops topologies considering the required overhead for yield improvement is performed. This comparative analysis provides useful recommendations that help flip-flops designers on selecting the best flip-flops topology that satisfies their system specifications while taking the process variations impact and robustness requirements into account. Adaptive Body Bias (ABB) allows the tuning of the transistor threshold voltage, Vt, by controlling the transistor body voltage. A forward body bias reduces Vt, increasing the device speed at the expense of increased leakage power. Alternatively, a reverse body bias increases Vt, reducing the leakage power but slowing the device. Therefore, the impact of process variations is mitigated by speeding up slow and less leaky devices or slowing down devices that are fast and highly leaky. Practically, the implementation of the ABB is desirable to bias each device in a design independently, to mitigate within-die variations. However, supplying so many separate voltages inside a die results in a large area overhead. On the other hand, using the same body bias for all devices on the same die limits its capability to compensate for within-die variations. Thus, the granularity level of the ABB scheme is a trade-off between the within-die variations compensation capability and the associated area overhead. This work introduces new ABB circuits that exhibit lower area overhead by a factor of 143X than that of previous ABB circuits. In addition, these ABB circuits are resolution free since no digital-to-analog converters or analog-to-digital converters are required on their implementations. These ABB circuits are adopted to high performance critical paths, emulating a real microprocessor architecture, for process variations compensation and also adopted to SRAM arrays, for Negative Bias Temperature Instability (NBTI) aging and process variations compensation. The effectiveness of the new ABB circuits is verified by post layout simulation results and test chip measurements using triple-well 65nm CMOS technology. The highly capacitive nodes of wide fan-in dynamic circuits and SRAM bitlines limit the performance of these circuits. In addition, process variations mitigation by statistical gate sizing increases this capacitance further and fails in achieving the target yield improvement. We propose new negative capacitance circuits that reduce the overall parasitic capacitance of these highly capacitive nodes. These negative capacitance circuits are adopted to wide fan-in dynamic circuits for timing yield improvement up to 99.87% and to SRAM arrays for read access yield improvement up to 100%. The area and power overheads of these new negative capacitance circuits are amortized over the large die area of the microprocessor and the SRAM array. The effectiveness of the new negative capacitance circuits is verified by post layout simulation results and test chip measurements using 65nm CMOS technology

Estimation of post-harvest losses of Manfalouty pomegranate fruits

Weight loss considered one of the main causes of quality loss in pomegranate fruits during chain marketing. Therefore, this study was conducted on Manfalouty pomegranate (Punica granatum L.) in a private orchard in El Badary, Assiut Governorate, Egypt in 2017 and 2018 to define the various causes of losses during chain handing and estimate it. The fruits harvested at three periods early (September) mid (October) and late season (November). The total losses at harvest were 5.94%, 9.30% and 23.50% for early, mid and late season, respectively. The main cause of losses is due to cracked and infected pests. The total loss of fruits during chain marketing was highest in retail market in comparison with wholesale during early, mid and late season. The main causes of losses due to weight loss and shrinkage fruits. According to data dealing with storage pomegranate fruits at 5±1°C and relative humidity 85-90%, the highest fruit losses found in the third month and this losses due to fruit weight loss and internal chilling injury (brown discoloration) so the storage life of fruit should be two months. DOI: http://dx.doi.org/10.5281/zenodo.405122

Improving optical access, sampling speed, and resolution for in vivo multiphoton microscopy

Multiphoton microscopy is a powerful optical imaging modality renowned for its non-invasive nature and relatively affordable characteristics. In particular, it has found its niche in neuroimaging due to its ability to probe in vivo biological processes in scattering brain tissue approaching millimeter depths with cellular resolution. However, the brain is a large and complex organ, and in order to fully understand its heterogeneous architecture and associated functional roles, several distal regions must be imaged simultaneously. Moreover, due to the critical implications of organelle features in various macroscale processes, whole-brain imaging at subcellular resolution scales presents itself as one of the outstanding challenges faced by the neuroscientific community today. Primarily, this research aims to expand the depth, field-of-view, and temporal throughput of multiphoton microscopy to enable large volume imaging of microvasculature at greater acquisition speeds. To accomplish this, we combine multi-faceted efforts focused on the engineering and development of advanced multiphoton microscopy techniques and technologies. This includes the characterization of novel contrast agents, the optimization of scan system optics, and the integration of high-repetition rate lasers with a resonant galvanometer. In addition, we develop a two-color imaging system capable of enhancing excitation efficiency, improving signal-to- background ratio, and further extending imaging depth. Finally, we present a novel application for two-color non-degenerate mode mixing to effectively circumvent the diffraction-limited nature of optical resolution and enable subcellular imaging. Collectively, these efforts advance the state-of-the art of multiphoton microscopy for routine cerebrovascular and neuroimaging.Biomedical Engineerin

The Impact of Training Programs for Faculty Members' Skills Development: A Field Study of Najran University

This research aimed to assess the impact of training programs at the Deanship of Development and Quality-Najran University (DDQ-NU) on the development of faculty members' skills from their perspective according to variables gender, faculty, academic rank, and years of experience. Researchers used a descriptive method and designed a questionnaire composed of 42 items distributed on four domains: (job performance, group performance, participant satisfaction, and participant knowledge gained). The sample of the study consisted of 175 faculty members at Najran University who responded to the questionnaire. The most important results that there is a positive impact of training programs in DDQ-NU on improving faculty members' skills. Keywords: Evaluation, Faculty members, Human development, Saudi universities, Training Program

Physiological factors affecting the production of an antimicrobial substance by Streptomyces violatus in batch cultures

Egyptian Journal of Biology Vol.3 2001: 1-1

Proposed Hybrid Power System for Short Route Ferries

A fractional fuel consumption saving as well as pollution cuts will be a thinking pattern or a key concern in modern ship designs. Recent advances in technology for solar cells and photovoltaic (PV) modules have resulted in solar power being a cost-effective fuel reduction alternative for this objective. This paper is intended to provide a hybrid solar diesel power system for short-run ferries. This work proposes and emphasizes the energy efficiency, cost efficiency and minimal environmental impact of hybrid-powered ferries with solar diesel. The proposed system has been studied on the example of passenger-car ferry connecting the two banks of the Suez Canal at Port Said city - Egypt. Economic and environmental analyses have been conducted to determine and measure the advantages of the proposed system. The results show an economically viable and environmentally sustainable system if it were treated as a long-term investment. Compared to the equivalent diesel generator system, this system reduces exhaust emissions by about 375 tonnes per year. The fuel cost savings achieved are also significant