Facultad de Farmacia y Nutrición - Tesis Doctorales y Tesinas
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- Mechanisms of the neurodegenerative effect produced by ochratoxin A(2024-11-27) Serrano-Civantos, M. (María); Vettorazzi, A. (Ariane); Lopez-de-Cerain, A. (Adela)The hypothesis of the key role of environmental factors on the development of Parkinson’s Disease (PD) has gained importance in recent years. Thus, some lines of research have set their focus on environmental neurotoxins, such as mycotoxins. Low doses of the mycotoxin ochratoxin A (OTA) have been reported to cause neurotoxicity. Indeed, previous in vivo studies with Balb/c mice demonstrated that OTA treatment p.o. (0.21 and 0.5 mg/kg of body weight) induced motor and dopaminergic (DA) alterations, associated with the phosphorylation of a-syn, at both intestinal and brain levels. These alterations were reported 6 months after the end of the OTA treatment. In vitro, it was observed that subtoxic doses of OTA caused a decrease in the levels of LAMP-2A, a protein involved in a-syn degradation, as well as an increase of a-syn levels, and its half-life, when treating intestinal and neuronal cell models with 100 and 200 nM OTA for 72 h. With this in mind, the present work is first focused on a description of the current available strategies for the assessment of the neurotoxic potential of a substance, as well as analysing if said strategies covers the endpoints of PD. To achieve this, the Organisation for Economic Cooperation and Development (OECD) test guidelines (TG) for the testing of chemicals and the European Food Safety Authority (EFSA) publications concerning neurotoxicity were reviewed. Among the different OECD TGs, only TG 424 collects methods for the specific evaluation of adult neurotoxicity (ANT). Most available strategies for the evaluation of neurotoxicity are centred on effects observable immediately after the exposure, yet there are not described strategies for the evaluation of long-term neurotoxic potential of a compound. Therefore, both EFSA and OECD organisms recognise the need for the development of on an in vitro test battery for the evaluation of neurodegeneration. A systematic search of scientific articles studying OTA neurotoxic effects was then carried out, analysing those articles according to the Adverse Outcome Pathway (AOP) of PD. Authors agree that in vitro and in vivo exposure to OTA causes mitochondrial dysfunction, impaired proteostasis, degeneration of DA neurons and neuroinflammation. However, further research might be needed to provide more information about OTA neurodegenerative potential and deepening in the elaboration of its neurotoxic profile. On another hand, this project aimed, through in vitro and in vivo studies, to unravel the timeline of the dysregulation of cellular processes, caused by OTA, that could be leading to mechanistical features observed in PD, as well as studying if these alterations are occurring at an intestinal level. To achieve this, proliferating and differentiated neuroblastoma cells, SH-SY5Y overexpressing wild type human a-syn (WT a-syn SH-SY5Y), were exposed to subtoxic concentrations of OTA (100 and 200 nM), for 24 h, 48 h and 72 h, and Balb/c mice were orally treated with 0.21 mg OTA/kg b.w. daily for 28 days, and sacrificed immediately after the end of the treatment, and 3 and 6 months after the end of the treatment (AET). In vitro, the effects of OTA treatment over a-SYN mRNA and protein levels were studied, as well as over the levels and/or activities of some molecules (LAMP-2A, LC3, p62, GBA, HEX, ß-GALACT, TFEB) involved in different a-syn degradation routes (chaperon-mediated autophagy (CMA) and macroautophagy). In vivo, the influence of OTA over a-syn and LAMP-2A expression was studied at both brain and intestinal level, as well as the presence of phosphorylated a-syn (p-syn) aggregates in midbrain. Effects of OTA over the nigrostriatal DA pathway were also assessed. Our results showed that OTA induced a time-dependent CMA dysfunction by decreasing the levels of LAMP-2A, in both proliferating and differentiated SH-SY5Y cells, starting at 24 h, and being worsen after longer OTA exposures (48 h and 72 h). A similar outcome was also observed in vivo, at brain and intestinal levels, being detected a reduction in LAMP-2A protein levels immediately after the OTA treatment, remaining downregulated over time (3- and 6-months AET timepoint). No alterations were detected in a-syn mRNA and protein levels both, in vitro and in vivo, but progressive apparition over time of p-syn aggregates in midbrain was observed in animals exposed to the mycotoxin. Also, a significant decrease in the total DA cell number was observed 3 months AET. Therefore, putting all this evidence together, we could state that exposure to subtoxic doses of OTA first causes malfunction of CMA at brain and intestinal levels (observable immediately AET and maintained throughout time), which promotes a-syn aggregation and this, in turn, leads to DA cell death (both detectable from 3 months AET). We also demonstrated, in vitro, that, when exposing SH-SY5Y cells to subtoxic concentrations of OTA, in presence and absence of bafilomycin A1 (BAF), an inhibitor of the autophagosomelysosome fusion, LC3 and p62 protein levels were increased, being this effect more pronounced in the presence of BAF. This suggested that exposure to OTA causes an increase in the formation of autophagosomes, indicating an enhancement of the macroautophagy route, possibly as a compensatory mechanism to face the CMA impairment.
- Deciphering the impact of astrocytic insulin receptor depletion on Alzheimer's disease pathology(2024-09-27) Ezkurdia-Lasarte, A. (Amaia); Solas, M. (Maite); Ramírez-Gil, M. (María)--
- Mechanistic modelling of immuno-modulators in oncology drug development(Universidad de Navarra, 2024-01-09) Sancho-Araiz, A. (Aymara); Mangas-Sanjuan, V. (Víctor); Fernández-de-Trocóniz, J.I. (José Ignacio)Over the past decade, the development of immunotherapy has become one of the most exciting breakthroughs in cancer research. However, from the discovery phase to late-stage clinical testing and regulatory approval, challenges in the development of immuno-oncology (IO) drugs are multi-fold and complex. In the preclinical setting, some of these challenges include: the broad number of potential drug targets, the expanding array of immuno-modulatory molecules, the emergence of exploratory biomarkers, and the unleashed potential of combining different treatments. In the clinical setting, the qualification of predictive surrogate biomarkers of treatment efficacy or outcome, and the corresponding optimization of IO trial design have become major obstacles. All these challenges necessitate the development of quantitative, mechanistic-oriented systems models incorporating key biological and pathophysiological aspects of IO and the pharmacokinetics of immuno-modulators. This thesis focuses on the development of model-based approaches in the area of IO, including pharmacokinetics (PK), pharmacodynamics (PD), and disease progression models, using data from different stages of the drug development process. These models aim to characterize the tumor growth behavior and its interaction with the immune system in order to better understand the efficacy of multiple immunotherapies and therefore support IO drug development. The first section of the thesis, the Introduction, provides a general overview of the mechanisms involved in the interplay between cancer cells and the immune system, and highlights the most promising immunotherapies. Additionally, this section summarizes the main concepts of Pharmacometrics and Systems Pharmacology (PSP), and introduces the different modeling strategies that will be further discussed along the thesis. Chapter 1 first reviews the recently approved immunotherapies and the multiple targets for these treatment approaches. Subsequently, the different mathematical approaches applied to IO and integrating the cancer immunity cycle are presented, grouped in top-down, middle-out and bottom-up. Chapter 2 introduces a semi-mechanistic model incorporating key processes of the cancer immunity cycle to account for the pharmacodynamic effects of three different immunotherapeutic agents in monotherapy or combination in cold tumors. Chapter 3 investigates the non-monotonic tumor growth dynamics in untreated animals and proposes a mechanistic framework able to describe this particular behavior and untangling the possible underlying mechanisms. Chapter 4 describes the development of a physiologically-based model for oncolytic viral therapy. The model accounts for viral kinetics, distribution to the tumor, viral dynamics at tumor level, and the elicited response after two different routes of administration. Chapter 5 further expands the model developed in Chapter 4 to include the pharmacokinetics of pembrolizumab and describe the efficacy of both drugs administered in combination. Finally, the General Discussion integrates and highlights the most relevant aspects of the five chapters, to end with the Conclusions where a summary of the most main findings of the thesis are presented.
- Impact of a personalized nutritional strategy on the dietary pattern of children and potential interplay with nutritional and health status(Universidad de Navarra, 2024-01-09) Andueza-Pacheco, N. (Naroa); Cuervo, M. (Marta); Navas-Carretero, S. (Santiago)La dieta desempeña un papel crucial en la salud humana y la prevención de enfermedades. Las dietas inadecuadas se han asociado con un gran número de enfermedades no transmisibles, como la obesidad, la diabetes o el síndrome metabólico. En este sentido, la infancia es un periodo clave, ya que es en esta época cuando comienzan a establecerse y consolidarse los hábitos alimentarios. Sin embargo, en las últimas décadas se ha observado un deterioro de los hábitos alimentarios de los jóvenes. A pesar de los esfuerzos para mejorar la dieta de los niños mediante intervenciones nutricionales, su impacto ha sido modesto y con una evidencia limitada. En este contexto, el objetivo principal de este proyecto de investigación fue evaluar la eficacia de la nueva estrategia nutricional diseñada para mejorar el patrón alimentario de niños de 6 a 12 años, así como valorar su efectividad sobre diferentes variables relacionadas con la salud. Para ello, se llevó a cabo un ensayo aleatorizado, controlado y paralelo de 2 meses de duración. En ambos grupos de estudio, ALINFA y control, la intervención dietética se basó en la dieta Mediterránea, con la diferencia de que el grupo ALINFA recibió una intervención intensiva basada en el seguimiento de una dieta que incluía productos saludables, comidas preparadas y recetas saludables diseñadas específicamente para niños, mientras que el grupo control solo recibió recomendaciones dietéticas. La intervención nutricional ALINFA demostró ser una estrategia útil capaz de mejorar la calidad de la dieta de los niños, cambiando de la necesidad de mejora el patrón dietético a una dieta de calidad óptima, según el índice KIDMED. El patrón dietético mejoró en términos de perfil dietético, así como de consumo de grupos de alimentos. A su vez, la mejora en la calidad de la dieta condujo a una mejora en el estado nutricional, disminuyendo la puntuación z del IMC, la circunferencia de la cintura y la masa grasa, reduciéndose así el riesgo de obesidad y cardiometabólico. Además, estos cambios condujeron a una reducción de los niveles de leptina. Los análisis de adherencia mostraron una alta tasa de adherencia a la dieta ALINFA, lo que da fiabilidad a los resultados observados. No obstante, los datos mostraron que, independientemente del grado de adherencia, todos los participantes se beneficiaron de la intervención. La calidad de la dieta inicial resultó ser el principal factor predictivo de la adherencia a la intervención. Además, los cambios en la calidad de la dieta produjeron beneficios en la microbiota fecal, con un aumento de la diversidad y riqueza microbiana y un cambio en la composición de la microbiota fecal hacia un perfil más saludable. Este cambio en la composición de la microbiota fecal se debió principalmente a un aumento de los filos Bacteroidetes y Firmicutes y de ciertos taxones bacterianos beneficiosos del filo Protebacteria. Los principales componentes dietéticos implicados fueron el aumento de la ingesta de fibra y el cambio en el tipo de grasa consumida hacia un perfil saludable. Finalmente, los cambios dietéticos producidos tras la intervención se reflejaron en las modificaciones en el perfil metabolómico urinario, siendo los principales elementos dietéticos que produjeron este cambio la cantidad y el tipo de proteína consumida y el tipo de grasa consumida. Además, dos metabolitos, NRibosilhistidina (derivado de la histidina) y Suberoil-L-carnitina (acilcarnitina), se correlacionaron con la calidad de la dieta, siendo potenciales nuevos biomarcadores de patrones dietéticos saludables en niños. En conclusión, todos estos parámetros analizados ponen de manifiesto la eficacia de la estrategia ALINFA. Además, se identificaron los elementos clave de la eficacia para esta población, provisión de alimentos saludables e implicación de toda la familia en el cambio de salud.
- Deciphering the connection between aging, insulin resistance and cognitive decline in Alzheimer's disease: role of TMAO as linking mechanism(Universidad de Navarra, 2021-12-21) Janeiro-Arenas, M.H. (Manuel Humberto); Ramirez, M.J. (María Javier); Solas, M. (Maite)Insulin resistance and neurodegenerative diseases such as Alzheimer s disease are considered part of the main threats to health in old age. In light of the high numbers of overweight/obese and diabetic individuals, there is a clear need to better understand the pathophysiological mechanisms underpinning insulin resistance/obesity and the accompanying impact on cognitive function. The aim of the present study was to investigate the missing link between aging, insulin resistance and cognitive decline as aging and insulin resistance are both risk factors for Alzheimer s disease. TMA is a gut metabolite which proceeds from the bacterial synthesis of substrates such as L-carnitine and choline. TMA is then rapidly further oxidized by hepatic flavin monooxygenases FMO3 and FMO1 to form TMAO. At first, TMAO was thought to be a waste product of choline metabolism without action in our organism, but nowadays, there is emerging evidence linking TMAO to atherosclerosis, systemic inflammation, type 2 diabetes mellitus and even neuropathologies. Plasma TMAO levels show wide inter- and intra-individual variations. These levels are influenced by several factors but the main factor influencing TMAO levels is aging. Some studies performed in human and rats have revealed that plasma TMAO levels are closely related to aging showing increasing levels with age. In this context, we have investigated if TMAO could be the link between metabolic diseases and cognitive deficiencies. In vitro studies showed that TMAO was able to raise the differentiation of mature adipocytes from preadipocytes, increase expression of pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) from macrophages and neurons and reduce expression of tight junction proteins in blood brain barrier cells. Moreover, TMAO was able to activate microglia and raise the expression of the pro-inflammatory marker CD16/CD32, increasing aberrantly myelin phagocytosis. SAMP8 a senescence accelerated mouse model was used to study the effect of aging (the main risk factor for AD) in peripheral inflammation and neuroinflammation. Studies were performed at three different ages: 2-months-old SAMP8 (young mice), 6-months-old SAMP8 (adult mice) and 10-month-old SAMP8 (old mice). Aging altered peripheral insulin sensitivity and glucose homeostasis in SAMP8 mice but not brain insulin signaling. Moreover, aging induced cognitive deficiencies and promoted peripheral and central inflammation. Finally, aging also induced gut dysbiosis, showing reduced diversity and changes in gut microbiota composition with enterotypes that could be associated to higher TMAO levels. This fact was further contrasted using LC-SM/SM what showed greater TMAO levels in serum of SAMP8 mice. Brain TMAO levels also increased with age in mice and humans. Treatment with 3,3-dimethyl-1-butanol (DMB), which is a choline TMA lyase enzyme inhibitor that decreases TMAO serum levels, restored peripheral inflammation reducing fat adipose tissue and reversing insulin and glucose alterations. Moreover, DMB also restored neuroinflammation decreasing expression of pro-inflammatory cytokines in hippocampus, reducing gliosis and restoring GFAP levels back to normal. Finally, SAMP8 performance in behavioral test was improved after DMB treatment ameliorating and restoring cognitive dysfunction.
- Pediococcus acidilactici CECT 9879 (pA1c®) as a probiotic for the prevention of type 2 diabetes mellitus and related comorbidities. In vivo evaluation and mechanism of action(Universidad de Navarra, 2023-12-14) Yavorov-Dayliev, D. (Deyan); Milagro-Yoldi, F.I. (Fermín Ignacio); Aranaz-Oroz, P. (Paula)Gut microbiota is a unique microbiological community consisting of different microorganisms (mostly bacteria) that reside in the human intestine. Host immune and inflammatory homeostasis can be directly affected by these microorganisms. Indeed, gut microbiota imbalance, called dysbiosis, has been correlated with the occurrence of numerous disorders. Therefore, gut microbiota modulation has emerged as a promising strategy to prevent and manage the development of different metabolic diseases, including type 2 diabetes mellitus (T2DM), insulin resistance (IR) and obesity. In turn, probiotics have emerged as an alternative therapy, as microbiota-modulating agents, to ameliorate T2DM and T2DM-related disturbances, including IR, obesity, metabolic syndrome (MS), hepatic steatosis and inflammation. In this regard, the present thesis aims to study in-depth the normoglycemic and antiobesogenic activities of the probiotic Pediococcus acidilactici CECT9879 (pA1c®) in different in vivo models, to elucidate its mechanism of action, and the impact that exerts on gut microbiota. In the first Chapter, the normoglycemic and lipid-lowering activities of the probiotic pA1c®, and the determination of its mechanism of action were demonstrated in the Caenorhabditis elegans (C. elegans) in vivo model. In Chapter 2, through a screening conducted in C. elegans, the most normoglycemic synbiotics (between pA1c® and other bioactive compounds) were selected (being pA1c®+chromium picolinate (PC)+ Oatàß-glucans (BGC) the most active), and their mechanisms of action were described. Moreover, the normoglycemic efficacy of the selected synbiotics, as well as their microbiota-modulating effect by 16s metagenomics were evaluated in male mice. In Chapter 3, the activity of pA1c® against T2DM-related comorbidities independent of glucose metabolism, including inflammation, hypercholesterolemia, obesity and dyslipidemia, as well as its gut microbiota-shaping potential by 16s metagenomics were reported in rats. In chapter 4, the role of pA1c® and heat-inactivated pA1c® (pA1c® HI) in the regulation of glucose metabolism and in the modulation of the gut microbiota by shot-gun sequencing was shown in diet-induced gestational diabetes mellitus (GDM)-mice and in T2DM-female-mice. It was demonstrated that supplementation with pA1c®, alone and in combination with PC and BGC, together with the pA1c® HI significantly enhance the carbohydrate and lipid metabolism of C. elegans, mice and rats, based on hyperglycemia reduction, blood glucose regulation, adiposity and hepatic steatosis attenuation and IR alleviation. The different mechanistic studies carried out in this work demonstrate that these effects are mainly mediated by regulating the insulin signaling pathway, and by activating the peroxisomal- and mitochondrial-fatty acid (FA) Oatàß-glucans oxidation process and reducing lipogenesis in all in vivo models. The anti-diabetic properties of pA1c® have been accompanied by the reduction of the visceral adiposity by the inhibition of the hepatic lipogenesis, the decrease of the intrahepatic triglycerides (TGs), the enhancement of the cholesterol metabolism and the reduction of pro-inflammatory cytokines. Lastly, metagenomic analyses demonstrated that the probiotic was able to colonize the colon in all pA1c®-treated animals, settle in the intestine and revert the HFS diet-induced dysbiosis, through the increase in T2DM beneficial bacteria, the decrease in T2DM negative bacteria, the increase in the microbial Oatàß-glucans-diversity, the promotion of SCFA producing-bacteria and the competition of Pediococcus acidilactici with obesity and dyslipidemia-associated bacteria, such as species of the genus Streptococcus. Thus, pA1c® could be considered as a potential probiotic strain for the prevention and management of T2DM and GDM and its related complications such as MS, obesity, IR, chronic inflammation, hypercholesterolemia, hepatic steatosis and gut microbiota dysbiosis.
- Role of plant microRNAs in altered physiological features of obesity: effects on inflammation and metabolism(Universidad de Navarra, 2023-12-14) Díez-Sainz, E. (Ester); Lorente-Cebrian, S. (Silvia); Milagro-Yoldi, F.I. (Fermín Ignacio)Obesity is a chronic metabolic disease that requires the development of effective therapeutic strategies to be easily implemented worldwide. Plant-based food interventions are promising therapeutic approaches for the management of obesity and associated comorbidities such as fatty liver. In this sense, plant microRNAs (miRNAs) have emerged as key bioactive molecules in cross-kingdom communication and could be involved in the beneficial effects of plants on human health. The present work aimed to evaluate the effect of plant miRNAs on the modulation of physiological features altered during obesity, particularly on inflammation and metabolism, with a special focus on the evaluation of gene expression changes. This research was conducted using in vitro cell culture models of macrophages, hepatocytes, and adipocytes, and human faecal and serum samples from volunteers subjected to an acute intake of plant-based foods. In Chapter 1, we identified by Next Generation Sequencing (NGS) a myriad of miRNAs in plant food products. It was found that miR156e, miR159 and miR16262, selected to estimate the general bioavailability of plant miRNAs, resisted degradation during cooking processes. Moreover, we demonstrated that an acute consumption of plant products increased the amount of these miRNAs in the human gut, although they were not detected in serum. In Chapter 2, we demonstrated that plant miR482f and miR482c-5p downregulated the expression of inflammation-related human genes (CLEC7A and NFAM1, and TLR6 respectively), some of them had been previously found to be dysregulated during obesity (CLEC7A and TLR6), in THP-1 monocytes differentiated into macrophages. In addition, these plant miRNAs had an impact on the gene expression profile of anti- and pro-inflammatory cytokines (i.e., IL10, TNF, and IL1B), and preliminary results suggested that they could enhance IL-10 protein secretion levels. In addition, miR482f and miR482c-5p were found to be present in edible plants, resist degradation during cooking processes, and were bioavailable in the human gut, but not serum. In Chapters 3 and 4, we unveiled that plant miR8126-3p, miR8126-5p and miR6262 inhibited the expression of metabolic-related human genes, which have been associated with obesity pathophysiology (QKI, MAPKAPK2, and RXRA, respectively) and had an impact of the expression profile of key metabolic genes (i.e., PPARA and SREBF1), in an in vitro model of lipid accumulation (HepG2 hepatocytes treated with free fatty acids). In addition, plant miR8126 isoforms attenuated lipid accumulation increase in hepatocytes. Plant miR6262 also downregulated the expression of the predicted target RXRA in hMADS adipocytes differentiated into brown-like adipocytes and modulated the expression of genes related to metabolism and thermogenesis (CIDEA, CPT1M, and PLIN1). However, we did not find a biological function for plant miR6262 neither in hepatocytes nor adipocytes. Overall, the results of the present thesis show that plant miRNAs are cross-kingdom gene expression regulators with the capacity to modulate the expression of mammalian (human) genes dysregulated during obesity and improve the inflammatory and metabolic gene expression profile. Moreover, this work shows that the bioavailability of plant miRNAs in humans could be achieved through acute plant intake, but eventually restricted to the gut levels, suggesting that other strategies should be developed to promote their absorption and potentially reach peripheral tissues and organs. This work lays the foundations for further studies exploring the potential role of miR482f, miR482c-5p, miR8126 (-3p and -5p), and miR6262 as an underlying mechanism by which plant foods influence obesity outcome in humans, and thus, their therapeutic potential to restore metabolic and inflammatory homeostasis in obesity.
- Exploring novel Se-compounds as promising therapeutical option for leishmaniasis management(Universidad de Navarra, 2023-12-14) Henriquez-Figuereo, A. (Andreina); Sanmartin-Grijalba, C. (Carmen); Plano-Amatriain, D. (Daniel)Globally, more than one billion people are affected by neglected tropical diseases (NTDs). The control, treatment, and elimination of NTDs, which mainly affect socio-economically disadvantaged regions, is one of the crucial obstacles for these countries to achieve health equity1 . Leishmaniasis is a NTD and the second leading cause of death from parasites in the world. Between 700,000 and 1 million new cases are reported worldwide each year. Treatment remains a challenge and still relies on compounds with toxic side effects. More than 20 parasite species cause leishmaniasis, making difficult the use of the same treatment regimens in multiple regions. There is no universal treatment for leishmaniasis2,3 . For many decades, most patients have been treated with intravenous or intramuscular injections of antimonials as first-line treatment. The use of antimonials is associated with life-threatening side effects, including damage to the heart, liver, and pancreas. In addition, treatments vary between regions and depend on the form of leishmaniasis, the causative parasite, the immune status of the patient and the local availability of therapy. Therefore, new treatments for leishmaniasis are urgently needed4 . Selenium (Se) is a metalloid of the chalcogen group. Se is among the essential trace elements because of its key role in cellular and thyroid metabolism, fertility, immune function, protection against oxidative damage and other vital functions5 . Se is acquired through the diet in two main forms: selenocysteine, found mostly in animal foods, and selenomethionine, found in plant products. Dietary Se-compounds differ in their metabolism and ability to produce different metabolites. The biological activity of Se-compounds is exerted via their metabolites. Therefore, the routes by which each compound is metabolized, and the relative abundance of each metabolite are related to their efficacy in the prevention and treatment of different diseases6 . Se plays a key role in the immune response against leishmaniasis, a disease caused by the Leishmania parasite7 . Low Se levels are associated with more severe forms of the disease, due to reduced antioxidant enzyme activity and increased oxidative stress. This suggests that Se is important in the pathophysiology of leishmaniasis, and its deficiency may aggravate Leishmania infection8 . The scientific literature is not abundant in relation to the development of new organic compounds derived from Se for the treatment of leishmaniasis. Studies have shown the antioxidant, antiviral, and anticancer properties of Se, it has been incorporated into antimicrobial nanomaterials for the treatment of Leishmania strains, with promising results, so it can be considered as an attractive novel therapeutic agent. As a consequence of the problems shown by drugs for the treatment of leishmaniasis, research into newly synthesized compounds that improve these limitations is necessary. For this reason, the aim of this Memory entitled: "Exploring novel se-compounds as promising therapeutical option for leishmaniasis management", is to provide an advance in the knowledge of a type of structures that can contribute as promising therapeutic agents for this disease. Our research group has more than 10 years of experience in the synthesis and biological evaluation of new synthesized Se-derivatives, which presented superior levels of activity and selectivity than reference drugs currently used in clinic. The chemical compounds presented in this Ph.D. project are newly synthesized and have been developed by means of a rational design and starting from economically accessible reagents. The structure of these compounds is characterized by their simplicity, being compounds mostly of low molecular weight, with molecular symmetry, and the presence of at least one Se atom in different functional groups. In some cases, the sulfur (S) analog has been synthesized, with the aim of evaluating the importance of the Se atom in the biological activity of the compounds. The results obtained during the development of this research project were grouped into Chapters I, II, and III.
- Strategies for the development of food products oriented to people with dysphagia(Universidad de Navarra, 2023-12-14) Giura, L.M. (Larisa-Mihaela); Astiasarán, I. (Iciar); Ansorena-Artieda, D. (Diana)Foods developed for people with dysphagia should have specific rheological/textural characteristics and high nutritional value in order to avoid aspiration, chocking or malnutrition problems in these patients. The objective of this project was to design and develop appropriate food products for people with swallowing difficulties. Various protein-enriched vegetable purees and one cacao dessert were developed. To improve nutritional aspects of these foods, different protein sources and microalgae were used. For the texture optimization, gum-based hydrocolloids (guar gum, tara gum, xanthan gum, and carboxymethylcellulose) and high-pressure processing (HPP) were applied. All the developed products showed a shear thinning behavior with apparent viscosity values meeting the target value (>1750 mPa.s). Moreover, tan 948; values were between 0 and 1, meeting the weak gel structure which is needed in dysphagia foods. The effect of freezing on rheological/textural properties of ten formulations with protein (casein and pea protein) and hydrocolloids was investigated. Vegetable purees enriched with casein exhibit better post-freezing rheological stability compared to pea protein-enriched ones. Comparative research demonstrates that xanthan gum exhibits greater stability compared to other hydrocolloids in frozen vegetable purees. Regarding HPP treatment, a weak gel structure was formed in the cocoa dessert rich in protein becoming from liquid to semisolid, giving to the product an adequate texture for dysphagia. Additionally, to compare the use of hydrocolloid (xanthan gum) and HPP technology the rheological/textural properties of a vegetable puree with lentil protein were analyzed. The results revealed that HPP treatment and XG are good strategies for developing dysphagia foods. While HPP treatment gave rise to a clean label product with an extended shelf-life, higher G and firmness, samples with xanthan gum showed higher elasticity, have a reduced cost and are accessible to a large part of the industry. To improve the antioxidant capacity and mineral content of dysphagia foods, three types of Chlorella vulgaris were incorporated in a lentil protein enriched vegetable puree obtaining a high nutritional product with minimal impact on rheological/textural characteristics, and 3D printable.
- Albumin based-nanoparticles as a platform for oral and intravenous delivery of monoclonal antibodies(Universidad de Navarra, 2023-11-30) Pangua-Irigaray, C. (Cristina); Espuelas, S. (Socorro); Irache, J.M. (Juan Manuel)This research was centred on the hypothesis that the use of albumin serum nanoparticles (NP) decorated with various pharmaceutical excipients, such as polyethylene glycol and dextrans, has the potential to significantly enhance the delivery of therapeutic proteins via both oral and intravenous routes. For oral administration due to the challenge of this route for therapeutic proteins, we propose that encapsulating bevacizumab (used as model of therapeutic protein) within PEG-coated albumin NP, with the inclusion of permeation enhancers, can facilitate the delivery of the monoclonal antibody to the epithelial surface. This encapsulation approach is expected to enhance the oral bioavailability of bevacizumab. For intravenous administration, to enhance the accumulation of monoclonal antibodies within tumor we are persuaded that encapsulating therapeutic proteins in dextran-coated albumin NP may promote their accumulation in the tumor mass leading to enhance its efficacy while minimizing potential side effects. Albumin NP (containing either DS or DOCU) were successfully prepared by desolvation and, then, coated with poly(ethylene glycol) 35,000 (PEG). Diffusion mucus studies showed increased diffusivity for DS-NP-P while in vivo fluorescence imaging in rats demonstrated the ability of PEG-coated NP to reach the intestinal surface. In C. elegans FT63, DS and DOCU, free or encapsulated, disrupted the integrity of the intestinal epithelium, without affecting the overall survival of the worms. A hydrophobic ion pairing complex between bevacizumab and DS or DOCU were successfully formed being the complex formation efficiency higher for the B-DOCU. The bevacizumab HIP complexes were successfully encapsulated in albumin NP, particularly those based on DS. The release of bevacizumab from NP was characterized by an important burst effect in SGF; although in SIF, NP containing the bevacizumab complexes displayed lower released rates. Finally, the PK study of the oral administration of B-DS-NP-P allowed to confirm an oral bioavailability of bevacizumab up to 3.7%. Albumin NP coated with dextran 40,000 (DEX) were prepared by a desolvation method. The encapsulation of bevacizumab in DEX-coated NP produced an encapsulation efficiency higher than 80%. The pharmacokinetic profile of B-NP-DEX was characterized by a rapid increase in the plasma levels and a plateau for 23 hours, the AUC was lower than free bevacizumab. The effect of bevacizumab either free or encapsulated in NP (B-NP-DEX) was evaluated in a xenograft model of colorectal cancer. B-NP-DEX caused a reduction in tumor growth of approximately 40% compared to the control and free bevacizumab. Levels of the monoclonal antibody in tumor were 2.5-times higher in animals treated with B-NP-DEX. VEGF expression in tumor showed a significant reduction in mice treated with B-NP-DEX. The effect of bevacizumab encapsulated in DEX-coated NP (B-NP-DEX) plus intravenous PTX was evaluated in an in vivo model of colorectal cancer. B-NP-DEX+PTX exhibited a higher reduction rate in tumor growth. Regarding proliferation by ki67 and VEGF levels a significant reduction was observed for B-NP-DEX+PTX. The effect of IV bevacizumab plus oral paclitaxel NP was also evaluated. Some similar outcomes with free BEVA+PTX had been observed considering the different routes of administration of PTX (IV vs oral). These outcomes might open an alternative route for paclitaxel administration upgrading the existing treatments. This research provides valuable insights into the use of albumin nanoparticles coated with different excipients to improve the delivery of therapeutic proteins, with the potential to enhance their bioavailability and efficacy in the treatment of cancer.