Ciencias Exactas y Ciencias de la Salud
Permanent URI for this collectionhttps://hdl.handle.net/11285/551039
Pertenecen a esta colección Tesis y Trabajos de grado de las Maestrías correspondientes a las Escuelas de Ingeniería y Ciencias así como a Medicina y Ciencias de la Salud.
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- Development of an aptazyme-driven toehold switch platform for colorimetric detection of SARS-CoV-2 N gene conserved regions(Instituto Tecnológico y de Estudios Superiores de Monterrey, 2024) Esquivel Ortiz, Karla Mariel; Antonio Pérez, Aurora; emipsanchez; Benítez Cardoza, Claudia Guadalupe; School of Engineering and Sciences; Campus Estado de México; Torres Huerta, Ana LauraIn response to the urgent need for advanced diagnostic tools highlighted by the COVID-19 pandemic, this thesis project aimed to develop a novel, label-free colorimetric diagnostic platform using parallel G-quadruplex/hemin-aptazymes for detecting the SARS-CoV-2 virus. This work, rooted in designing these aptazymes in-silico that specifically target conserved regions of the SARS-CoV-2 N gene, namely TV1 and TV2, and to experimentally evaluate their potential as highly specific bioreceptors for biosensing technologies. First, extensive in-silico analyses were carried out to identify the potential targets and to evaluate the aptazyme sequence and interaction design. Multiple Sequence Alignment analyses helped us to identify SARS-CoV-2 N gene regions with high conservation across betacoronaviruses and SARS-CoV-2 variants present in Mexico, which would serve us as stable targets for our diagnostic platform. The designed aptazymes and identified target sequences were then studied for their secondary structure and stability at different working parameters using the mFold software. Tertiary structures were retrieved using RNAComposer, 3DNA, and PyMol software to evaluate structural changes at different working parameters. Finally, molecular docking simulations were carried out using PyDock DNA online server to predict the aptamer-target interactions. These initial studies determined the experimental working parameters in terms of ionic concentrations and provided an understanding of the molecules individually and in complex. Characterization techniques included Dynamic Light Scattering (DLS) to understand the size change behavior of the molecules, both individually and in complex; UV-Vis Spectroscopy to assess the aptazymes’ catalytic activity, analytical sensitivity and specificity, and capability of producing a colorimetric response when in complex; and Circular Dichroism (CD) to gain insights into the structural changes of the aptazymes alone and in complex. UV-Vis spectroscopy analyses revealed that aptazymes were non-specific to their targets, consistently demonstrating a colorimetric shift within the first five minutes of reaction. CD showed that the predominant structure observed after complex formation was the B-form hairpin topology for all aptazymes. These structural changes explained the non-specific results observed in UV-Vis spectroscopy. Consequently, the overallaptazyme sequence design should be refined. Following our aptazyme toehold design, increasing the length of the oehold sequence might improve the formation of a parallel G-quadruplex structure instead of a B-form hairpin. Further experiments or refinements are recommended to better characterize TV2 aptazyme which appeared to be a more promising andidate than TV1, based on its greater stability, fewer structural conformation, higher specificity, and lower limit of detection.
- SARS-CoV-2 facemask-based diagnostics derived from pooled samples(Instituto Tecnológico y de Estudios Superiores de Monterrey, 2022-12-04) Limón Mazo, Liliana María; Mahlknecht, Jürgen; emimmayorquin; Trujillo de Santiago, Grissel; School of Engineering and Sciences; Campus Monterrey; Moisés Álvarez, MarioThe emergence of SARS-CoV-2 and the resulting COVID-19 pandemic has caused a tremendous impact on lives and economies. During the outbreak, clinical testing via reverse-transcription polymerase chain reaction (RT-PCR) was implemented to establish a surveillance system for COVID-19. However, clinical testing reached soon its testing capacity and many people remain undiagnosed due to the relatively long incubation time of 14 days, increasing the risk of spreading the virus. Thus, alternative testing tools need to be developed to enable accessible, inexpensive, and reliable surveillance of virus transmission in communities. In this study, a proof-of-concept for SARS-CoV-2 diagnosis based on pooled wastewater samples from used facemasks was developed as a testing alternative. For the development of this test, a methodology for purification of SARS-CoV-2 synthetic DNA with silica nanoparticles was adapted, and different viral RNA concentrations were successfully evaluated with loop-mediated isothermal amplification (LAMP), nested PCR, and spectrophotometry using Nanodrop. Consistent, positive results were obtained with all the concentrations evaluated, obtaining amplification bands of 200 bp and mean viral loads between 42 - 275 ng/uL. This technology can serve as a solid epidemiological tool to be applied in future epidemiologic health threats to the world population.
- Tiempo de resolución de cetoacidosis diabética en pacientes con COVID-19 como factor de riesgo para mortalidad(Instituto Tecnológico y de Estudios Superiores de Monterrey, 2022-01-10) García Vega, Alberto; Sánchez Ávila, Juan Francisco; emipsanchez; Pérez Alba, Eduardo; Garza Salinas, Sergio; Escuela de Medicina y Ciencias de la Salud; Campus Monterrey; Castillo Castro, CarolinaIntroducción: Durante la pandemia de COVID 19 se observó un aumento de la mortalidad en pacientes diabéticos que presentaron una cetoacidosis diabética asociada a la infección por SARS CoV2. Los pacientes con mayor edad, raza negra e hispana fueron los que presentaron una mayor mortalidad. El tiempo de resolución del estado de cetoacidosis diabética asociada a infección por SARS CoV2 fue mayor en comparación a otra causa detonante de CAD. Objetivo: El objetivo de este estudio es determinar sí un tiempo de resolución mayor a 36 horas del estado de cetoacidosis diabética aumenta la mortalidad en pacientes hispanos infectados por SARS-CoV2. Materiales y Métodos: Este es un estudio casos y controles, retrospectivo, analítico, comparativo y observacional en el cual se determinó si un tiempo de resolución mayor a 36 horas del estado de cetoacidosis diabética impacta en la mortalidad en pacientes hispanos con infecciónn por SARS-CoV2. Resultados: Se incluyeron 59 pacientes (edad promedio: 47 [DE, ±15.98] años, hombres: 33 [55.9 %]). Se observó mortalidad en 18 pacientes (30.5%). Los pacientes con infección por SARS CoV2 y cetoacidosis diabética que tuvieron una resolución menor a 36 horas presentaron un OR para mortalidad de 4.06 (IC 95% 1.21-13.5, p <0.05). En el análisis multivariado no se encontró diferencia significativa con un OR 3.59 ([0.95-13.54], p=0.059). La gravedad de la infección por SARS CoV2 al ingreso se identificó como un factor de riesgo de mortalidad con un OR 9.64 [(2.38-39], p<0.05). Conclusión: El principal factor de riesgo para mortalidad es la gravedad de la infección por SARS CoV2. En este estudio observamos en el análisis univariado que un menor tiempo de resolución de CAD tiene un mayor riesgo de mortalidad en los pacientes infectados por SARS CoV2, sin embargo, no encontramos tal diferencia en el análisis multivariado.
- Development of a nanostructured membrane-based passive sampler as a tool for the monitoring of viral pathogens through wastewater-based epidemiology(Instituto Tecnológico y de Estudios Superiores de Monterrey, 2022) Jiménez Rodríguez, Mildred Gissel; PARRA SALDIVAR, ROBERTO; 2035753; Parra Saldívar, Roberto; emipsanchez; Oyervides Muñoz, Mariel Araceli; Coronado Apodaca, Karina Guadalupe; Martínez Ruiz, Manuel; School of Engineering and Sciences; Campus Monterrey; Sosa Hernández, Juan EduardoInfectious disease outbreaks are a global burden that is continuously increasing. Classic epidemiological approaches to identify infected individuals and prevent the spread of the diseases usually include individual clinical screenings, sentinel surveillance, and the analysis of surveys, hospital data, and mortality and morbidity rates. However, these methods have failed to detect early warnings of future outbreaks and are resource-dependent. Thus, wastewater-based epidemiology (WBE) has been used as a novel community-wide monitoring tool capable of providing comprehensive real-time data on the public and environmental health status, which can ultimately contribute to a faster adoption of public health interventions. Efficient spatial and temporal sampling is critical for the success of WBE, however, current sampling methods mainly consist of grab samples, which can lead to the over- or underestimation of pathogen concentration. Passive samplers are a promising alternative to improve WBE since they can be used for prolonged periods to obtain more representative information of the real conditions in the area. Advances in nanotechnology have also helped to overcome the efficiency of recovery yields for membrane-based methods. WBE monitoring was employed at Tecnologico de Monterrey to assess the prevalence of COVID-19 risk throughout its community.Therefore, this research project aimed to develop a nanostructured polymeric membrane-based passive sampler to monitor SARS-CoV-2’s RNA through WBE. The scope of the study was to determine the membrane’s morphology, its stability, and RNA adsorption kinetics, which were evaluated to establish a WBE passive sampling protocol to detect and quantify the viral load in wastewater and study the trends in the circulation. Different formulations were explored to determine the influence of the selected membrane components in its morphology and RNA adsorption capacity. Three suitable candidates were selected to further pursue as potential stand-alone detection devices. RNA adsorption was achieved within 0.5 h and 1 h of magnetic stirring in previously spiked water samples with two of the selected samplers. However, the obtained values were very low and cannot be used to conclude that the passive samplers work as per their intended use. Nonetheless, they are an indication of potential opportunity areas within such formulations.
- Development of a pseudotyped lentivirus-based assay to measure neutralizing antibody activity against SARS-CoV-2 in Mexico(Instituto Tecnológico y de Estudios Superiores de Monterrey, 2021-06-09) Cruz Cárdenas, José Antonio; Brunck, Marion Emilie Genevieve; puelquio; Palomares Aguilera, Laura Alicia; Licona Cassani, Cuauhtémoc; Guitierrez Mayret, Michelle; Escuela de Ingenieria y Ciencias; Campus MonterreyThe SARS-CoV-2 virus is responsible for the COVID-19 pandemic declared by the WHO in March 2020, which has caused more than 172 million confirmed cases and 3.69 million deaths worldwide to date. Infection with SARS-CoV-2 leads to the development of antibodies in patients. The presence of neutralizing antibodies protects against SARS-CoV-2 infections and is an essential parameter that confirms the success of vaccination. The titration of neutralizing antibodies by classical methods is not trivial since it requires the use of replicative virus, which implies a high risk of infection and requires facilities certified with the BSL-3 biosafety level. Mexico is one of the countries most affected by SARS-CoV-2 and there are not enough facilities to carry out effective immunity monitoring. Here, this thesis presents the results of a multi-institutional national collaboration in the design of a non-replicative pseudovirus that expresses the SARS-CoV-2 spike protein on its surface, which is applied in the development of a method for quantifying SARS-CoV neutralizing antibodies. The development of this assay will facilitate the characterization and monitoring of humoral immunity against SARS-CoV-2, and can be easily modified to monitor immunity against emerging variants in the country. To the best of our knowledge, this work presents the first report of measuring neutralizing antibody to SARS-CoV-2 in the Mexican population using a pseudovirus system.