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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- Development and optimization of a flexible enzyme-based platform for the colorimetric quantification of metabolic disease-related salivary biomarkers(Instituto Tecnológico y de Estudios Superiores de Monterrey, 2022-04) Ornelas Gonzalez, Alonso; RITO PALOMARES, MARCO ANTONIO; 9659; Rito Palomares, Marco Antonio; emipsanchez; Martagón Rosado, Alexandro José; Mayolo Deloisa, Karla Patricia; Richard Coale, Wilson; Escuela de Medicina y Ciencias de la Salud; Campus Monterrey; González González, Mirna AlejandraEarly detection aims at timely treatment to improve patient's fate. To fulfill this, the identification and validation of appropriate biomarkers, as well as the development of rapid, simple, sensitive and low-cost methodologies are crucial. These factors would enable the identification of biomarkers in rural and remote locations or where sophisticated equipment and highly trained personnel are not available. Among the current techniques for this purpose, enzyme-based colorimetric platforms stand out as a great alternative due to their properties including low cost, simplicity, flexibility, specificity, and adjustable sensitivity. These platforms are the core of diagnostic kits and point-of-care devices that fully comply with the aforementioned characteristics. Saliva is an emerging biofluid that contains multiple useful molecules for the non-invasive diagnosis of diseases. Its collection is carried out through a simple and painless process that either does not require qualified personnel or even by self-sampling. Therefore, saliva stands out as an alternative to blood due to its great potential for diagnostic purposes. The first stage of this work aims to develop and optimize a multi-enzymatic platform for the colorimetric quantification of salivary glucose. The methodology tests two different dyes to compare the results and demonstrate the versatility of the system for glucose quantification in both buffer conditions and human saliva samples in which concentrations are up to 100 times lower than those found in blood. The second stage of this study focused on demonstrating the flexibility of the multi-enzyme platform. This was performed by modifying simple parameters such as pH buffer, incubation time, as well as the concentration of the enzymes and 3,3’,5,5’-tetramethylbenzidine (TMB). These modifications enabled the adaptation and optimization of this platform for the detection and quantification of clinically relevant biomolecules, such as galactose, uric acid and 1,5-anhydroglucitol in buffer conditions. Overall, results suggest that this platform is useful for measuring the proposed biomarkers at concentrations found in different biofluids, both conventional (blood and urine) and unconventional (saliva, sweat and tears). Thus, this work is the basis of a platform that could be further adapted for the quantification of other clinically relevant biomolecules. However, more studies are required to demonstrate its correct operation in terms of accuracy, precision, and specificity with the biofluids.
- Development of novel polymer-protein conjugates and characterization of chromatographic supports(Instituto Tecnológico y de Estudios Superiores de Monterrey, 2021-06-10) Sánchez Trasviña, Calef; SANCHEZ TRASVIÑA, CALEF; 655893; Rito Palomares, Marco Antonio; emipsanchez; Aguilar Jiménez, Oscar Alejandro; Zavala Arcos, Judith; Chuck Hernández, Cristina Elizabeth; School of Engineering and Sciences; Campus Monterrey; Mayolo Deloisa, Karla PatriciaProtein versatility has positioned them as a high-value biotechnological product. Among protein applications, its use as a therapeutic agent is highlighted. However, some therapeutic proteins need a modification process to increase their pharmacokinetic properties. During the protein modification process, the purification step, mainly performed by chromatography, represents a critical stage in ensuring the safety of modified therapeutics proteins. Being chromatography the main purification method, it is mandatory to fully understand the effect of all its operational variables on the separation performance. This work presents a deep analysis of chromatographic strategies used to purify modified therapeutic proteins commercially available. Furthermore, an alternative protein modification process is presented using N-(2-hydroxypropyl) methacrylamide (HPMA) polymer and its purification by chromatography. Besides, the development and characterization of PEGylated monoliths as an alternative to purify PEGylated proteins is performed. Finally, the characterization of core-shell particles being used as chromatographic support is developed. The results showed that the selection of purification strategies of commercial modified proteins depends on physicochemical properties of both protein and attached molecule and is highly dependent on the matrix where the protein is recovered. HPMA copolymers can be conjugated with Ribonuclease A (RNase A) under non-demanding conditions (PO4-3 buffer 50 mM pH 5.1 + 20 mM NaBH3CN). The new conjugates showed higher hydrophobic behavior than the native protein being this feature exploited by hydrophobic interaction chromatography (using 1.5 M (NH4)2SO4) to separate the conjugates from the unreacted protein. On the other hand, PEGylated monoliths can separate PEGylated RNase A and even isoforms when large polyethylene glycol molecules (>20 kDa) are attached to the monolith. Lastly, the CaptoTM Core 700 resin structural (50.4 nm pore size, 4.18 µm shell thickness, and 90.7 µm particle size) and adsorptive properties allowed modeling and adsorption prediction of two model proteins. In combination, all these results represent new knowledge in the polymer-protein technology and chromatography areas that proportionate guidelines to purify a wide range of molecules such as native, recombinant, and modified proteins efficiently.
- Sustained release of antiretroviral therapy from nanochannel delivery implant for HIV pre-exposure prophylaxis and treatment(Instituto Tecnológico y de Estudios Superiores de Monterrey, 2021-04-08) Pons Faudoa, Fernanda Paola; PONS FAUDOA, FERNANDA PAOLA; 781417; Rito Palomares, Marco Antonio; emipsanchez; Anderson, Peter L.; Castorena Torres, Fabiola; González González, Mirna Alejandra; School of Engineering and Sciences; Campus Monterrey; Grattoni, AlessandroHIV-1 pre-exposure prophylaxis (PrEP) adherence and implementation challenges, including pill fatigue and limited medical access, have motivated research into long-acting (LA) strategies such as formulations or controlled release implants to obviate user-dependent dosing. Focusing on a single-drug regimen permits maximal drug loading and prolongs treatment periods. The nanofluidic implant consists of a drug reservoir and a drug-releasing nanochannel membrane. The pharmacokinetics of two different antiretrovirals (ARV), cabotegravir (CAB) and tenofovir alafenamide fumarate (TAF) released from nanofluidic implants were assessed in separate studies. In the first study, 2-hydroxypropyl-β-cyclodextrin enhanced cabotegravir release in vitro and in vivo in Sprague Dawley rats. Nanofluidic implants loaded with 2-hydroxypropyl-β-cyclodextrin (βCAB) maintained clinically relevant plasma CAB concentrations 2 times above the preventive clinical threshold for 3 months. Additionally, sustained CAB release achieved drug penetration into tissues relevant to HIV-1 transmission. In the second study, the first-ever preventive assessment of LA ARV implant and foremost of TAF was conducted. Nonhuman primates (NHP) were subcutaneously implanted with a nanofluidic device loaded with TAF. Pharmacokinetics in HIV-1 target cells, peripheral blood mononuclear cells (PBMC) were monitored for 4 months, achieving median tenofovir diphosphate (TFV-DP) concentrations of 390 fmol/106 PBMC. Importantly, NHP were exposed to rectal simian HIV (SHIV) challenges after TFV-DP concentrations were above clinically protective levels. Constant and sustained administration of TAF from the nanofluidic implant demonstrated partial protection, with a protective efficacy of 67%. Furthermore, the nanofluidic implant exhibited a foreign-body inflammatory response categorized as slight reaction. In addition, the nanofluidic implant was assessed as an HIV-1 treatment platform. Upon SHIV infection, the control NHP cohort from this study was transferred to a TAF treatment study. The viral load reduction from subcutaneous TAF monotherapy was assessed for a month in treatment naïve NHP. Continuous TAF release exhibited a first-phase viral load decay of -1.14 ± 0.81 log10 copies/mL. Notably, TAF delivered at a lower dose from the nanofluidic implant had similar effects to oral TAF. Thus, demonstrating potential as a platform for LA ARV therapy. In conclusion, the nanofluidic device shows promise as an HIV PrEP and treatment delivery platform that addresses poor patient adherence.
- Novel bioengineering strategies for the recovery and purification of PEGylated lysozyme conjugates: in situ ATPS and affinity chromatography(Instituto Tecnológico y de Estudios Superiores de Monterrey, 2017-07-13) Mejía Manzano, Luis Alberto; Mejía Manzano, Luis Alberto; 375359; Rito Palomares, Marco Antonio; Mayolo Deloisa, Karla Patricia; González Valdez, José Guillermo; Asenjo de Leuze, Juan A.; Parra Saldívar, RobertoPEGylation is the modification of therapeutic proteins with polyethylene glycol (PEG) with the goal of improving their bioavailability and effectivity in the organism. During the PEGylation process, proteins with different degrees of PEGylation and positi