Ciencias Exactas y Ciencias de la Salud
Permanent URI for this collectionhttps://hdl.handle.net/11285/551014
Pertenecen a esta colección Tesis y Trabajos de grado de los Doctorados correspondientes a las Escuelas de Ingeniería y Ciencias así como a Medicina y Ciencias de la Salud.
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- Production of secondary metabolites in environmental Pseudomonas aeruginosa by genetic engineering tools(Instituto Tecnológico y de Estudios Superiores de Monterrey, 2025-07-24) Salazar García, Luis Mauricio; Licona Cassani, Cuauhtémoc; emipsanchez; Ponce Noyola, Patricia; Soberón Chávez, Gloria; Orellana Montecino, Camila; Torres Acosta, Mario A.; Villalobos Escobedo, José Manuel; Escuela de Ingeniería y Ciencias; Campus MonterreyLa Biotecnología, a manera personal, consiste en la aplicación de microorganismos como un medio para la obtención de productos útiles y con ello satisfacer una necesidad. Para tal fin se necesitan herramientas eficaces para favorecer a que estos organismos nos apoyen a resolver esas necesidades. Este trabajo se enfoca al establecimiento de una técnica de edición genética que apoye al desarrollo y estudio de microorganismos. Dentro de los microorganismos con mayor versatilidad se encuentran las bacterias, en donde aquí se usaron como modelo de estudio dos cepas de Pseudomonas. Dentro de la colección del laboratorio contamos con una cepa aislada en el Golfo de México en un derrame petrolero. Esta cepa se identificó y se denominó como P. aeruginosa IGLPR01. Debido a su contexto ambiental, este microorganismo tiene la capacidad de usar hidrocarburos como fuente de carbono, por lo que se visualizó como un candidato de interés para realizar pruebas y estudios de biorremediación. Además de estudiar y caracterizar esta cepa, se propuso el reto de hacer edición genética para aumentar aún más esas capacidades que la hicieron crecer en condiciones extremas. Esto, como se detallará más adelante, representó un fuerte reto ya que los microorganismos ambientales no son tan dóciles como lo representan los microorganismos modelo que encontramos a diario en los laboratorios de investigación. En esta primera parte, se enfocó en poder montar una técnica de edición efectiva que permitió la generación de una mutante en el gen rpoS para la producción de Piocianina. Este compuesto es naturalmente producido por P. aeruginosa IGLPR01, y la mutante lo sobre produce dando un fenotipo de coloración verdosa en el medio de cultivo. Junto con esto, observamos y cuantificamos la producción de otros metabolitos importantes como son los ramnolípidos; además de aplicar el sobrenadante a pruebas de emulsificación sobre gasolina como simulación rápida de un proceso de biorremediación. Una vez teniendo esta técnica de edición decidimos aplicarla para aportar al estudio del género Pseudomonas. Este género es de especial interés ya que la Organización de las Naciones Unidad ubica a Pseudomonas aeruginosa como un microorganismo que representa una amenaza a la salud pública por su resistencia a los antibióticos y ser causa de infecciones nosocomiales. Debido a que el gen rpoS es un factor de respuesta a estrés, y ya que teníamos la estrategia de edición exitosa dirigida hacia este gen, decidimos probar si funcionaría en una cepa patógena de humano. Seleccionamos la cepa P. aeruginosa PA14, una cepa modelo para el estudio de procesos infecciosos en humanos. Una vez obtenida la mutante, decidimos realizar análisis de transcriptómica y metabolómica para evaluar el mecanismo de respuesta a estrés que está relacionado con el gen rpoS. Así, en la presente tesis, se presenta como se logró establecer una técnica de edición en una cepa de P. aeruginosa ambiental, junto con la detección y cuantificación de metabolitos de interés; además de la aplicación de la metodología a la cepa patógena P. aeruginosa PA14 para contribuir en la descripción de los mecanismos de respuesta a estrés. Finalmente, esperamos que este trabajo sirva como base para motivar a la edición genética de microorganismos ambientales, ya que muchos de los problemas actuales podrían resolverse al observar a la naturaleza como fuente inspiradora de soluciones.
- Discovery and comprehensive evaluation of new strategies for the postharvest biocontrol of anthracnose in avocado(Instituto Tecnológico y de Estudios Superiores de Monterrey, 2025-06-10) Gallardo Camarena, Marco Vinicio; Licona Cassani, Cuauhtémoc; emimmayorquin; Reverchon, Frédérique; Méndez Bravo, Alfonso; Villalobos Escobedo, José Manuel; School of Engineering and Sciences; Campus Monterrey; Torres Acosta, Mario AntonioAnthracnose, caused mainly by Colletotrichum spp, is the leading postharvest disease affecting avocado (Persea americana), contributing to significant losses in global production. As concerns grow over the environmental impact and diminishing efficacy of synthetic fungicides, this study aimed to identify and evaluate microbial antagonists as sustainable biocontrol alternatives. Microorganisms were isolated from two primary sources: extreme environments in Cuatro Ciénegas, known for their oligotrophic and selective conditions; and various niches of the avocado ecosystem, including rhizosphere, carposphere, bark, and nectar. A total of 30 actinobacteria, 78 filamentous fungi, and other 13 bacterial isolates were screened in vitro against Colletotrichum spp., with promising candidates further evaluated through in vivo inhibition assays. Among the strains tested, Kosakonia cowanii VG1, isolated from the avocado carposphere, exhibited strong antifungal activity across assays. Genome analysis revealed biosynthetic gene clusters encoding siderophores and azole-containing RiPPs, which may underlie its inhibitory effects. The techno-economic evaluation showed a competitive production cost of $0.11 per dose, highlighting its scalability potential. Actinobacterial isolate Streptomyces nanshensis CC402A also demonstrated an effective in vitro and in vivo inhibition, with a low MIC and MFC of the crude extract. This isolate presents gene clusters for known antifungal metabolites, including valinomycin and HSAF-like compounds. This work supports the integration of prospecting in environments with strong ecological selective pressures, genomic mining, and economic modeling to discover and validate new microbial biocontrol agents.
- Exploring the chemical diversity of environmental actinobacteria using genome mining and synthetic biology(Instituto Tecnológico y de Estudios Superiores de Monterrey, 2025-06) González Salazar, Luz Angela; Licona Cassani, Cuauhtémoc; emipsanchez; Pacheco Moscoa, Adriana; De la Torre Zavala, Susana; Rodríguez López, Carlos; School of Engineering and Sciences; Campus Monterrey; Cruz Morales, PabloNatural products derived from biosynthetic gene clusters (BGCs) in microorganisms are vital for medicine, agriculture, and industry. However, rediscovering known compounds and the resource-intensive nature of drug development delays the discovery of novel compounds. This study aimed to address these challenges by integrating genome mining and synthetic biology to explore the chemical diversity of actinobacteria from the oligotrophic environment of Cuatro Ciénegas, Mexico. In the first chapter of this work, we evaluate the genomic and biosynthetic potential of the environmental strains Lentzea sp. CC55 and Actinokineospora sp. PR83 isolated from Cuatro Ciénegas environment. Comparative genomics revealed open pan-genomes comprising 568 and 965 unique genes, respectively. BGC similarity networks identified unique clusters, including terpenes, RiPPs, NRPS, and polyketides. Both strains demonstrated antimicrobial and cytotoxic activities. Lentzea sp. CC55 culture showed cytotoxic activity only in liquid cultures while Actinokineospora sp. PR83 presented activity against B. subtilis for solid media. The Biosynthetic Novelty Index (BiNI) confirmed these strains as high-priority candidates for novel natural product discovery. In the second chapter, we applied the BiNI index in our collection of strains from Cuatro Ciénegas to select the candidates with the highest novelty. From the selected candidates we standardized cloning strategies for BGC expression in heterologous hosts. Using Golden Gate assembly and CRISPR/Cas9-based DNA assembly, genes were domesticated, assembled into transcriptional units, and transferred to Streptomyces albidoflavus UO-FLAV-004. Challenges with toxic fragments and incomplete assemblies were resolved using optimized host strains, resulting in the successful conjugation of core modules and the initiation of heterologous expression. In the third chapter, we explore different tools of metabolomics for the detection of metabolites produced by engineered strains. For NRPS the prediction indicates a theoretical mass: 495 Da. The RiPP corresponds to a lanthipeptide type I with a theoretical mass of 2318.9 Da. Molecular network analysis and dereplication only identified compounds produced intrinsically encoded by the host. This research presents a genomic-guide pipeline for exploring the biosynthetic potential of Cuatro Ciénegas actinobacteria, providing insights into the BGC prioritization and application of synthetic biology techniques to improve the intricate pipelines for new chemical diversity in Natural Products research.
- Design and characterization of a biosensor for the detection of codon-readthrough inducers(Instituto Tecnológico y de Estudios Superiores de Monterrey, 2025-02-28) Trejo Alarcón, Luisa María; Licona Cassani, Cuauhtémoc; emipsanchez; Utrilla Carreri, José; García Echauri, Sergio A.; Rodríguez López, Carlos Eduardo; García García, Jorge Donato; Escuela de Ingeniería y Ciencias; Campus Monterrey; Cruz Morales, PabloLas enfermedades raras afectan a menos del 0.05% de la población mundial y se atribuyen en gran medida a desórdenes genéticos que generan proteínas incompletas. Los inductores de codon-readthrough (CR), como algunos aminoglucósidos (AGs), han sido evaluados como terapias potenciales debido a su capacidad para restaurar la traducción de genes afectados. En este trabajo, desarrollamos un biosensor eficiente basado en levaduras para la detección de moléculas con actividad CR. Construimos una cepa de Saccharomyces cerevisiae con un plásmido conteniendo el gen reportero de luminiscencia nluc modificado para incluir un codón de terminación prematura (PTC), lo que lo hace no funcional. La actividad CR, inducida por AGs comerciales como G418, gentamicina, tobramicina y paromomicina, restauran la funcionalidad del gen, generando una señal de luminiscencia en ensayos in vivo en microplaca. El sistema fue aplicado al análisis de 91 cepas de actinobacterias aisladas, identificando un potencial inductor de CR, proveniente de la cepa Streptomyces sp. CR101A. A través de minería genómica, fraccionamiento por Cromatografía Líquida de Ultra Alta Resolución (UPLC) y espectrometría de masas (MS), se identificó un clúster de genes biosintéticos (BGC) como responsable de la producción de una molécula con un anillo ciclitol. Actualmente, se trabaja con herramientas de biología sintética como CRISPR/Cas9 para la generación de mutantes y expresión heteróloga, para confirmar la implicación del BGC detectado en la producción del inductor de CR.
- Insights from colostrum multi-omics: exploring bioactive co-occurrence patterns in the context of maternal obesity(Instituto Tecnológico y de Estudios Superiores de Monterrey, 2024-06-13) Gámez Valdez, July Stephany; Licona Cassani, Cuauhtémoc; emimmayorquin; Brunck, Marion Emilie Genevieve; Lara Díaz, Víctor Javier; Gutiérrez García, Karina; Mancilla Herrera, Ismael; Escuela de Ingeniería y Ciencias; Campus MonterreyBreastmilk is a complex and dynamic fluid that supplies hydration, nutrients, and bioactives essential for the optimal growth and development of neonates. Breastmilk bioactives play pivotal roles in providing passive immunity against pathogens, fostering tolerance to nascent microbiota, and facilitating the maturation of the intestinal system. Maternal obesity, a significant health concern, has been linked to changes in breastmilk composition. Understanding the intricate dynamics of bioactive compounds in breastmilk. While previous research has often focused on analyzing individual breastmilk constituents separately, there is a growing recognition of the need for a more holistic approach that considers the systemic nature of breast milk. Specifically, investigating the interactions among different bioactives, such as microbiota and metabolites, is crucial for comprehending the overall composition and function of breastmilk. Colostrum, serving as the initial seed for neonatal gut microbiota establishment, holds particular significance in this context. Nonetheless, our current comprehension of how maternal obesity impacts breastmilk bioactives through a systems biology perspective is limited. This doctoral thesis aims to bridge this gap by examining the influence of maternal obesity on the co-occurrence patterns of colostrum bioactives, including microbiota, metabolites, and pro-inflammatory cytokines. Through collaboration with the Hospital Regional de Alta Especialidad Materno Infantil, we obtained two cohorts of volunteers from Monterrey, comprising 30 and 48 participants, respectively, who generously provided colostrum samples. These cohorts were sampled at different times, one from May to December 2019 and the other from October 2020 to July 2022. Samples were categorized into two groups based on BMI, classified according to the World Health Organization as normal weight (≤ 24.9 kg/m2) or obesity (≥ 30 kg/m2). Using co-occurrence networks and multi-omics approaches, specifically 16S-microbiota profiling, and untargeted metabolomics, we explored the compositional and structural changes within the colostrum ecosystem in the context of maternal obesity. Our study reveals that maternal obesity induces alterations in the ecosystem structure of colostrum. While normal weight conditions showed more positive correlations between pro-inflammatory cytokines and Proteobacteria members, such as Neisseriaceae, Pasteurellaceae, and Enterobacteriaceae, these correlations were absent in obesity samples. Microbiota diversity remained stable, with subtle shifts like reduced Proteobacteria abundance observed in colostrum from women with obesity. Furthermore, metabolite profile, particularly 15-HEDE, a lipid mediator involved in pro-inflammatory response, vasodilation and hyperpermeability, exhibited reduced abundances in response to maternal obesity, also show intricate associations with inflammatory-related cytokines (IL-12p70 and IL-10) and microbial taxa (Beijerinckiaceae and Burkholderiaceae). Additionally, our study highlights the importance of considering neonate sex bias in breastmilk microbiota research, as it influences the prevalence of specific bacteria such as Streptococcaceae, Xanthobacteraceae, and Burkholderiaceae. These findings underscore the connection between alterations in the bacterial community, metabolites, and inflammatory markerswithin the colostrum environment in the context of maternal obesity. Further research is necessary to elucidate the interactions between the host and bioactives in colostrum and their broader implications concerning maternal obesity.
- Pan-genomic epidemiology of tuberculosis: dnraveling the genetic diversity of mycobacterium tuberculosis clinical isolates(Instituto Tecnológico y de Estudios Superiores de Monterrey, 2023-11-06) Mejía Ponce, Paulina Mayell; Licona Cassani, Cuauhtémoc; emimmayorquin; Zenteno Cuevas, Roberto; Sélem Mojica, Nelly; Treviño Alvarado, Víctor Manuel; Enciso Moreno, José Antonio; Rodríguez López, Carlos Eduardo; Escuela de Ingeniería y Ciencias; Campus MonterreyGenomic epidemiology has significantly advanced our understanding of tuberculosis (TB) by providing standards to define sub-lineages, drug resistance patterns, and transmission groups (TGs). However, this approach has its limitations, primarily its reliance on a single reference genome, which fails to capture valuable genetic information present in diverse Mycobacterium tuberculosis complex (MTBC) lineages. Moreover, the underrepresentation of specific geographical regions, notably Mexico, in defining these standards has hindered the identification of region-specific disease markers. In this study, we embark on a comprehensive exploration of the genetic diversity of MTBC across regions, sub-lineages, and TGs, marking a shift from genomic epidemiology to pan-genomic epidemiology in TB research. Our research unfolds across three distinct sections, each addressing a critical facet of our overarching project. First, we contribute to an extended dataset of Mycobacterium tuberculosis (Mtb) clinical isolates from TB patients in various Mexican states, utilizing a whole-genome sequencing (WGS)-based strategy (Chapter 2). We illustrate the distribution of sub-lineages and drug resistance patterns in the country, revealing significant disparities between phenotypic and genotypic drug resistance profiles. In the subsequent section (Chapter 3), we present the groundbreaking discovery of TGs across different Mexican states and identify the primary risk factors associated with these groups. These findings underscore the urgency of enhancing national TB surveillance and monitoring in Mexico, ultimately aiding in the effective management of the disease. In the third and final section of our study (Chapter 4), we broaden our perspective, transitioning from a genomic to a pan-genomic approach. We compile the most extensive MTBC pangenome to date, encompassing 7,034 Mtb genomes from diverse global locations. This comprehensive analysis deciphers the structure of the Mtb pangenome and uncovers specific genes associated with unique traits. Our study addresses the intricate landscape of TB, providing novel insights into genetic diversity, transmission dynamics, and innovative approaches to comprehending this persistent global health challenge
- Functional mining of snake venom metalloproteinases for the development of chronic wound therapeutics(Instituto Tecnológico y de Estudios Superiores de Monterrey, 2021-10) Meléndez Martínez, David Leonardo; MELENDEZ MARTINEZ, DAVID LEONARDO; 548216; Licona Cassani, Cuauhtémoc; emijzarate, emipsanchez; Mayolo Deloisa, Karla; Borja Jiménez, Juan Miguel; Plisson, Fabien; Escuela de Ingenieria y Ciencias; Campus Monterrey; Aguilar Yáñez, José ManuelChronic wounds such as skin burns, diabetic and varicose ulcers are a health problem in Mexico, generating the loss of millions of dollars in annual expenses. From the currently available treatments in the market, active wound treatments are the best cost-benefit option to treat this pathology and its market is in a rapid growth. Nowadays, several proteinases such as papain, bromelain or bacterial collagenases have been employed to enhance the wound healing process. Nevertheless, the use of these proteinases demonstrated poor results in patients. For this reason, other proteinase sources with more similarity to matrix metalloproteinases, plasmin and thrombin have been explored. Thus, this dissertation presents a characterization of snake venom metalloproteinases (SVMPs) and snake venom serine proteinases (SVSPs) from the Mexican rattlesnakes Crotalus atrox, C. molossus nigrescens, C. ornatus and C. scutulatus scutulatus; including the development of an aqueous two phase systems (ATPS) method for the selective recovery of SVMPs and phospholipases A2 (PLA2) from rattlesnake venom. More importantly, the generation of a phylogenetic-based tool that can be used to discriminate among hemorrhagic and non-hemorrhagic SVMPs. The results demonstrated that the SVMPs and SVSPs contained in the Mexican rattlesnakes are capable to degrade collagen derivatives (gelatin), fibrin, fibrinogen and generate fibrin clots. Three ATPS were capable to selectively recover: 1) PLA2 (PEG 400 g mol-1, VR 1, TLL 25%, and pH 7.0), 2) P-III SVMPs (PEG 400 g mol-1, VR 1, TLL 15%, pH 7.0, and NaCl 1%) and 3) SVMPs (PEG 400 g mol-1, VR 1, TLL 25, and pH 8.5). Lastly, specificity loop flexibility is involved in the hemorrhagic activity of the SVMPs. Using this data to trim the SVMP sequences in the phylogeny-based analysis, a hemorrhagic phenotype clustering was observed. Together, all these results contribute to improve in the venom inspired drug discovery, demonstrating that the rattlesnake venom toxins, particularly the SVMPs have the potential as an active wound treatment for chronic wounds.