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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- 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.
- 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.