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.
Browse
Search Results
- Rational Metabolic Engineering strategies design for the co-production of pyocyanin and rhamnolipids in Pseudomonas aeruginosa IGPR1 for the bioremediation of oil-polluted water bodies(Instituto Tecnológico y de Estudios Superiores de Monterrey, 2022-06-10) Damas Ramos, Luis Caleb; Licona Cassani, Cuauhtémoc; emimmayorquin; Escalante Lozada, José Adelfo; González García, Ricardo Axayácatl; Escuela de Ingeniería y Ciencias; Campus MonterreyFossil fuels are a central part of life, most human activities are powered by them. However, their increasing demand and manipulation suppose a constant threat of accidental release to the environment. The consequences of oil spills impact the environmental, social, and economic spheres of life, generating biodiversity, health, and monetary losses. Among oil remediation techniques, bioremediation is the most promising strategy to mitigate oil spills negative effects. Nonetheless, the implementation of bioremediation strategies is slow and uncertain as successful bioremediation efforts require deep comprehension of the strains used. The study and application of environmental strain Pseudomonas aeruginosa IGPR1 to bioremediation brings the opportunity to design rational metabolic engineering strategies to improve oil emulsification. IGPR1 is a metabolically flexible strain that produces pyocyanin and rhamnolipids, two metabolites involved in the emulsification and degradation of oil. Here we describe the characterization of IGPR1 strain through the metabolic reconstruction of its genome and the implementation of classic and novel metabolic engineering strategies such as homologous recombination and the use of CRISPR/Cas9-APOBEC1 coupled with ribozymes-mediated maturation of sgRNAs to induce premature stop codons and generate doble knockouts. This is the first study describing the CRISPR/Cas9-APOBEC1 system with ribozymes, furthermore, iCDC1103 is the first metabolic reconstruction of a P. aeruginosa environmental strain with bioremediation applications. This work sets the foundations for future research with this complex yet industrially interesting strain.
- Design of methods to monitor and evaluate Clostridium chauvoei under industrial settings for generating a fermentation map(Instituto Tecnológico y de Estudios Superiores de Monterrey, 2022-06-01) Cepeda Pérez, Daniela; Licona Cassani, Cuauhtémoc; puemcuervo, emipsanchez; Mendoza, Uri; Mayolo Deloisa, Karla; Orellana Montecino, Camila; Torres Acosta, Mario Antonio; School of Engineering and Sciences; Campus MonterreyBlackleg is a disease that affects cattle caused by the anaerobic and spore-forming bacteria Clostridium chauvoei. It possesses several virulence factors that act in synergy to cause the typical lesions. Flagellin (FliC) is a cell surface antigen that provides protective immunity to vaccinated animals against C. chauvoei infection. It is related to virulence and pathogenicity, thus, demonstrating a relationship between flagellar antigen quantity and final vaccine efficacy for blackleg disease, as demonstrated in several immunoassays. Clostridium chauvoei toxin A (CctA) is a pore-forming toxin secreted to the supernatant. It causes lysis by perforating the cell membrane, and it is known to be the main virulence factor of the pathogen. Other proteins such as sialidase and hyaluronidase are considered the main virulence factors of Clostridium chauvoei. Because blackleg is of economic importance, and as a disease that advances rapidly, vaccination has been available as a prevention method. However, since its development, the vaccine remains as a whole-cell formalin-inactivated culture due to the lack of knowledge of specific conditions to generate enough antigens to induce protection. Additionally, there is a variability of batch-to-batch immunogenic properties and final efficacy, which may be explained by the antigen’s quantity variation throughout the process. Although final batch testing is currently applied in the industry, there are no methods to monitor the fermentation dynamics and consistency of the overall production. Consistency can be achieved by setting parameters that constitute a product profile that satisfies final product requirements. The expression and purification of a recombinant FliC allowed the development of an indirect ELISA to measure the flagellin antigen quantity of Clostridium chauvoei fermentation samples. While the assay is not validated to predict the potency and final efficacy of the vaccine, it can be used as part of a battery of methods to provide a more characterized product profile to monitor trends and dynamics in the production. In addition, a rapid micro-plate bioactivity assay was developed to measure the hemolysis percentage caused by the supernatant of samples of C. chauvoei. The assays represent valuable methods to generate data and monitor the fermentation dynamics of C. chauvoei cultures under industrial conditions to achieve production consistency.
- Expression of immunogenic fraction of Apx toxins of Actinobacillus pleuropneumoniae for the design of a quantitative assay(Instituto Tecnológico y de Estudios Superiores de Monterrey, 2021-12-03) Mora Gálvez, Liliana Monserrath; MORA GALVEZ, LILIANA MONSERRATH; 800888; Licona Cassani, Cuauhtémoc; puemcuervo, emipsanchez; Torres Acosta, Mario Antonio; Mayolo Deloisa, Karla Patricia; School of Engineering and Sciences; Campus MonterreyPorcine pleuropneumonia is a highly contagious disease for pigs worldwide, and its presence represents a large economic loss. Actinobacillus pleuropneumoniae (App) is the etiological agent of porcine pleuropneumonia. Its virulence is correlated with the presence of four exotoxins, known as Apx toxins (Apx I, Apx II, Apx III, and Apx IV). 19 serotypes of App are recognized until today, and commercially available vaccines cannot ensure total protection against all, thus the relevance of new subunit vaccines to be developed and introduced into the market. To test vaccine efficiency, validation with quantitative immunoassays is required. Enzyme-linked immunosorbent assays (ELISAs) are the preferred technique to validate efficiency, but the available test only helps identify the serotype present, not specific antigen and immune reactions. Thus, an Apx antigen-specific ELISA should be developed to measure immune responses to each specific Apx toxin independently, and contribute to developing new treatments for App. Here, we generated recombinant fragments of Apx toxins IA, IIA, and IIIA, using Escherichia coli BL21 DE3 as expression host. This fragments were selected after an homology analysis to define the specific immunogenic regions for each toxin. The recombinant fragments were purified by affinity chromatography in an ÄKTA Avant purification system. Dot blot, SDS-PAGE, and Western blot techniques were used to validate the purification process from the obtained recombinant fragments. Lastly, a growth kinetics characterization of five serotypes was carried on in three different culture media. The results obtained confirm that the over-expression of the recombinant proteins of interest was possible using the pET-28b(+) plasmid with the insert to express in E. coli BL21 DE3. Further, affinity chromatography was a good purification process, allowing purification percentages around 90%. Lastly, that the five selected serotypes of App were able to grow in Brain Heart Infusion media supplemented with 5% (w/v) yeast extract, Tryptic Soy Broth supplemented with 5% (w/v) yeast extract and an Animal-free component media. Our results contribute for the development of analytical methods much needed in the veterinary industry.
- Study of chemical diversity in Streptomyces venezuelae isolated from contrasting mexican environments using genome mining and bioactivity analysis(Instituto Tecnológico y de Estudios Superiores de Monterrey, 2021-11-25) Pérez García, Viridiana; Licona Cassani, Cuauhtémoc; puemcuervo; De la Torre Zavala, Susana; Verdel Aranda, Karina; School of Engineering and Sciences; Campus Monterrey; Aguilar Yáñez, Jose ManuelMicrobial natural products (NPs) have historically been considered one of the most important sources of chemical diversity at an industrial and pharmaceutical level. Many of these molecules are generated through secondary metabolic pathways, and their biosynthesis is mediated by ecological interactions such as defense, competition, and interactions with the environment. The genus Streptomyces represents one of the main groups of producers of specialized metabolites. Previous research in this genus has revealed that even strains of the same species can differ dramatically in the genes that code for these metabolites, indicating a lot of chemical variety to be discovered. Therefore, this work aimed to compare the chemical diversity at the level of biosynthetic genes clusters (BGCs) present in strains of the same species, Streptomyces venezuelae, isolated from two contrasting habitats: a highly oligotrophic environment and a nutrient-rich environment. It is hypothesized that these factors may influence the distribution and content of clusters and the generation of chemicals linked with them. Furthermore, genome mining research revealed a large diversity of BGCs from varied chemical families. These findings open the door to further research into the chemical diversity of Actinobacteria isolated from different geographical locations in the quest for bioactive molecules.
- Development of a screening method for identifying potential aminoglycosides producers from a collection of environmental Actinobacteria(Instituto Tecnológico y de Estudios Superiores de Monterrey, 2020-12-02) González Salazar, Luz Ángela; Licona Cassani, Cuauhtémoc; puelquio/tolmquevedo; Pacheco Moscoa, Adriana; De la Torre Zavala, Susana; Cruz Morales, Pablo; Escuela de Ingeniería y Ciencias; Campus MonterreyNature represents an important source of molecules with relevant applications in agriculture, industrial and health. Although many efforts have been performed in the identification of new natural products, target, and efficient strategies to select NPs producers are still needed. Previous studies have shown that the intrinsic resistance of Actinobacteria that produce antimicrobial agents could be used as a systematic approach for the detection antibiotics producers. Therefore, the aim of this work was to test different resistance, molecular and bioinformatic analysis as selection criteria to identify possible candidates to produce aminoglycoside antibiotics. This is the first approach developed for this class of antibiotics. we generated a collection of environmental strains and standardized some conditions using model strain producers. We could standardize the procedure for molecular screening in model producers, but we did not find evidence of aminoglycoside producers for the environmental collection, this fact supported by resistance and bioinformatic analysis. According to this, an improved strategy was proposed combining field-directed sampling, an improved molecular screening, as well as the selection of bioinformatic tools with high detection sensibility to AGs BGCs. Additionally, a huge diversity of BGCs from other chemical families was observed according to the genome mining analysis. These results represent an opportunity to continue exploring the chemical diversity in Actinobacteria isolated from unique regions that can be studied as well as being the inspiration for the development of new screening protocols.
- Compositional analysis of taxa shaping microbial community in colostrum samples from individuals with gestational diabetes mellitus and obesity(Instituto Tecnológico y de Estudios Superiores de Monterrey, 2020-06-20) Gámez Valdez, July Stephany; LICONA CASSANI, CUAUHTEMOC; 176857; Licona Cassani, Cuauhtémoc; RR/puemcuervo; Brunck, Marion; Lara Díaz, Víctor Javier; García Mazcorro, José Francisco; Escuela de Ingeniería y Ciencias; Campus MonterreyDespués del nacimiento, las bacterias colonizan rápidamente el sistema gastrointestinal humano. Los vínculos entre el intestino materno y microbiota de la leche materna han demostrado que los hábitos maternos y más aún, ciertas condiciones de salud, repercuten en el sistema inmune gastrointestinal de los recién nacidos. La obesidad y la diabetes mellitus gestacional (DMG) son dos problemas de salud importantes durante el embarazo que provocan variaciones en la composición bacteriana de la leche materna y amenazan el establecimiento de la microbiota intestinal del recién nacido y la estimulación de las células inmunes. México es el segundo país con la mayor prevalencia de obesidad femenina y una incidencia de DMG 1.39 veces superior al promedio mundial. En el presente trabajo de tesis, analizamos la diversidad bacteriana e identificamos cambios en la microbiota del calostro de individuos mexicanos con obesidad y DMG. A través de una colaboración con la Escuela de Medicina y Ciencias de la Salud (Tec Salud) y el Hospital Regional de Alta Especialidad Materno Infantil, obtuvimos 43 muestras de calostro de madres mexicanas residentes de Monterrey y el área metropolitana. Las muestras se clasificaron en grupos de estudio según su índice de masa corporal (IMC), estado de salud y se subdividieron por género. Se identificaron un total de 1,675 ASVs, siendo dominadas por Pseudomonas, Gemellales, Ralstonia, Herbaspirillum, Streptococcus y Enterobacteriaceae. Utilizando un modelo linear general, obtuvimos que la condición patológica materna y el género del infante influyen en la diversidad microbiológica del calostro, siendo los subgrupos con DMG los más diversos. Se observó una mayor abundancia relativa de Firmicutes y una disminución de Bacteroidetes en los subgrupos con obesidad y DMG. Mismo comportamiento fue observado para Pseudomonas, Gemellales y Enterobacteriaceae. El género Prevotella tuvo mayor presencia en los subgrupos con DMG. Nuestro trabajo representa el primer paso para dilucidar la composición de bacterias de la leche materna de individuos afectados por DMG, así como también para identificar los taxones clave relacionados con la DMG y la obesidad.
- Design and characterization of a biosensor for the detection of aminoglycoside compounds with codon-readthrough activity.(Instituto Tecnológico y de Estudios Superiores de Monterrey) Trejo Alarcón, Luisa María; LICONA CASSANI, CUAUHTEMOC; 176857; Licona Cassani, Cuauhtémoc; emipsanchez; Aguilar Yáñez, José Manuel; Utrilla Carreri, José; Biotecnología; Campus Monterrey; Cruz Morales, PabloLas enfermedades raras o huérfanas se presentan en 5 de cada 10,000 individuos y son atribuidas en su gran mayoría a desordenes genéticos (80%); mientras que el resto son cánceres poco frecuentes, malformaciones congénitas e infecciosas, enfermedades autoinmunitarias y metabólicas; las cuales representan un problema de salud mundial ya que, al ser tan diversas entre sí, el desarrollo de fármacos para su tratamiento es escaso (solo el 5% tienen tratamiento) y aproximadamente 350 millones de la población mundial presenta afecciones de este tipo. La Distrofia Muscular de Duchenne (DMD) entra dentro de esta clasificación, al ser una enfermedad genética hereditaria, que produce un codón prematuro de terminación (PTC) en el gen que codifica para la producción de la proteína distrofina; en los años 1990s se descubrió que los aminoglucósidos (AGs) tienen potencial terapéutico para este tipo de enfermedades, ya que propician el fenómeno conocido como codon-readthrough (CR), provocando la restauración del gen afectado; sin embargo, su uso durante largos periodos de tiempo provoca ototoxicidad y nefrotoxicidad. Debido a lo anterior surgió la necesidad de descubrir nuevos productos naturales con actividad de aminoglucósido, provenientes principalmente de Actinomicetos. Actualmente las estrategias de detección de nuevos metabolitos comprenden el aislamiento, cultivo y extracción utilizando diferentes medios de cultivo; métodos de screening para detectar su bioactividad y el uso de herramientas bioinformáticas para la identificación de clusters de genes de biosíntesis (BGCs); sin embargo son procesos muy largos, costosos y carecen de especificidad al no estar encaminados a la detección de procesos celulares especializados; por lo que la industria farmacéutica ha dejado de invertir en su investigación y desarrollo. El presente trabajo describe a detalle el desarrollo un sistema de screening eficiente y mejorado con respecto a los a existentes, para la detección de moléculas con actividad de aminoglucósidos basado en CR; mediante la generación de un biosensor dentro de la levadura Saccharomyces cerevisiae utilizando un arreglo de biología sintética compuesto por los genes reporteros de crecimiento URA3 y luminiscencia Nluc.