Tesis doctorales y Tesinas
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- 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.
- 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.
- 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.
- Unravelling the role of SIRT2 in Alzheimer’s disease(Universidad de Navarra, 2023-11-16) Sola-Sevilla, N. (Noemi); Puerta, E. (Elena); Solas, M. (Maite)Sirtuin 2 (SIRT2) has been proposed to have a central role on ageing, inflammation, and age-related pathologies such as AD. Recent studies propose the pharmacological inhibition of SIRT2 as a therapeutic strategy for several neurodegenerative diseases; nevertheless, its specific role is not well understood which varies depending on the organs, cell types and contexts analyzed. In attempt to understand the role of SIRT2 in ageing, the specific isoform 3 of SIRT2 (SIRT2.3) was overexpressed in mice hippocampus. Although, SIRT2.3 overexpression was not enough to accelerate ageing in a model with normal pattern of ageing, it promoted neuroinflammation and worsened age-related cognitive decline in the SAMP8 strain with an accelerated ageing phenotype. In this context, we postulated that its pharmacological inhibition could be a pharmacological strategy for the treatment of AD. Indeed, SIRT2 inhibition improved learning and memory deficits and reduced amyloid pathology and neuroinflammation; however, it increased peripheral inflammation. These peripheral deleterious effects were confirmed when the blood-brain barrier-impermeable SIRT2 inhibitor AGK-2 was administered. In this scenario where a beneficial effect of central SIRT2 inhibition while a deleterious peripheral effect was observed, we hypothesized that the specific deletion of SIRT2 in microglial cells could be the best therapeutic strategy to minimize the possible adverse effects. Upon an acute inflammatory insult, microglial SIRT2 deficiency reduced the inflammatory response. However, microglial SIRT2 seems to be essential in a chronic inflammatory context, such as AD, since its deletion increased mortality, worsened cognition, and impaired amyloid pathology in APP PS1 mouse model. These results highlight the relevance of further investigate the specific functions of SIRT2 in each cell type which is essential to maximize its potential as pharmacological target not only for AD but also for other neurodegenerative diseases.
- Nanomedicines for the Treatment of Pediatric Neuroblastoma(Universidad de Navarra, 2023-11-09) El-Moukhtari, S.H. (Souhaila H.); Blanco-Prieto, M.J. (María José); Couvreur, P. (Patrick)Pediatric cancer is still a major challenge to our society. In Spain, more than 1000 cases are diagnosed each year. While there has been clear progress in survival rates over the last 50 years, therapeutic protocols for childhood cancers still face issues related to high toxicity and incorrect dosing regimens. In this context, nanotechnology has shown significant ability to reduce the toxicity of anticancer compounds. Improving the therapeutic index of cytostatic drugs makes this approach an alternative to conventional chemotherapy. However, the lack of nanomedicines specifically designed for pediatric cancer underscore the urgent need to ensure the proper translation of nanomedicines for children. Among pediatric cancers, embryonal nervous system tumors such as neuroblastoma arise due to disruption of normal early developmental pathways and develop in very young children, usually under 2 years old. This disease only affects around 100 children each year, making it a rare disease. Neuroblastoma exhibit significant genetic heterogeneity and varying clinical outcomes. Nevertheless, some genetic abnormalities, such as MYCN amplification, are found in 30% of cases and are associated with high-risk neuroblastoma and poorer prognosis. For these patients, common treatments are still inefficient or lead to severe long-term consequences that affect these children at adulthood. Additionally, other factors, including the tumor microenvironment, can significantly influence tumor aggressiveness, impacting treatment responses and patient survival. In this context, the use of lipid nanoparticles, which are considered safe, scalable, easy to produce and suitable for oral administration present an interesting approach to enhance the therapeutic outlook of neuroblastoma patients. This project was centered on the use of very small size particles made of solid lipids (100-200 nanometers) that act as platforms for delivering the antitumor drug, with the aim of improving its therapeutic index and the quality of life of neuroblastoma patients. The primary objective was to develop new therapeutic nanosystems that can be orally administered and potentially offer effective therapy. This involved the delivery of not only a cytostatic drug currently used for neuroblastoma treatment (etoposide), but also a promising antitumor drug (edelfosine) both combined after their vectorization in lipid nanocarriers. First of all, patient neuroblastoma tumors and samples extracted from high-risk neuroblastoma preclinical models were analyzed showing the role of tumor microenvironment elements in the stiffness of the extracellular matrix. These elements play a major role in aggressiveness and tumor progression. Subsequently lipid nanomedicines were developed and thoroughly characterized. In vitro evaluation in cells revealed that nanomedicines could enhance the drugs effectiveness while sparing non-cancerous cells. This was further corroborated in vivo using Caenorhabditis elegans. These studies provided evidence of the efficacy of the proposed lipid nanomedicines in overcoming the gastrointestinal barrier and reducing the toxicity of etoposide. Further in vivo evaluation in mice demonstrated that nanoencapsulation was a good strategy for the oral administration of anticancer drugs and facilitated lymphatic system engagement. Overall, when tested in a neuroblastoma mouse model, the nanomedicines proved to be an effective strategy that maintained the efficacy of anticancer drugs while significantly reducing toxicities. However, the combination of nanomedicines did not exhibit any improvements in terms of antitumor efficacy when compared to single treatments. Nevertheless, the combination of nanomedicines avoided the emergence of lung metastases when compared to single nanomedicines treatment.
- Synthesis and Preclinical Development of novel Organoselenium compounds as Multi-Target and Bioactive Agents(Universidad de Navarra, 2023-08-02) Astráin-Redín, N. (Nora); Sanmartin-Grijalba, C. (Carmen)The aim of the present Ph.D. thesis entitled "Synthesis and preclinical development of novel organoselenium compounds as multi-target and bioactive agents" is the development of new active Se-containing molecules and, in this way, to expand the knowledge on the design and molecular biology of organoselenium derivatives mainly in the field of cancer treatment and prevention. The results obtained during the experimental work are divided into four thematic blocks (Part I, II, III, and IV), and comprise one hundred and twelve novel Se-containing derivatives starting from the design, synthesis, purification, and structural characterization to their preliminary evaluation as theraputic agents. All the compounds have been assessed as anticancer molecules, but certain compounds have been also evaluated as acetylcholinestare (AChE) inhibitors in the field of Alzheimer's disease (AD). Part I, entitled "Natural and synthetic allylic derivatives", includes Chapter 1 which contains a literature review with future perspectives on the allyl moiety, which is present in a great variety of natural sources, and its role in the design of novel anticancer drugs. Based on Chapter 1, different series of molecules, combining Se with the allylic entity (among other entities), have been designed as future anticancer agents and are collected in the following parts. Parts II, III, and IV are divided according to the functional group in which Se is found in the molecule. Thus, Part II collects selenoesters (Chapters 2-5), Part III includes acylselenoureas (Chapter 6), and Part IV contains 1,2,4-diselenazolidines (Chapter 7). Part II is further divided into 3 sections: Part II.1, Part II.2, and Part II.3. Part II.1 is entitled "First Generation", since it presents the first approach in the development of allylic selenolester derivatives (Chapters 2 and 3). In Chapter 2, the preliminary anticancer activity and the inhibition of AChE are studied. Then, Chapter 3 presents the evaluation as cancer-associated carbonic anhydrase (CA) isoforms IX and XII inhibitors. Part II.2 comprises the "Second Generation" of allylic selenolester derivatives and the molecules developed therein are symmetrical with two-substituted selenolester functionalities such as their sulfur analogs, the allyl disulfides, present in garlic (Chapter 4). Besides, their biological evaluation as potent chemotherapeutic agents is reported. Finally, Part II.3 includes molecules that in addition to having a functional selenoester group also have a benzo[c][1,2,5]selenadiazole moiety and it also reports their preliminary cytotoxic activity (Chapter 5). Part III presents the acylslenourea derivatives containing the allyl, propargyl, or propyl chain. These derivatives have been evaluated as dual radical scavenging and anticancer agents (Chapter 6). Part IV includes a series of 1,2,4-diselenazolidines that were formed as by-products during the synthesis of acylselenoureas. This is a very novel Se-structure that has not been evaluated as a chemotherapeutic agent to date (Chapter 7).
- Impact of astrocytic GLUT1 ablation on glucose homeostasis and cognition(Universidad de Navarra, 2022-01-24) García-Ardanaz, C. (Carlos); Ramirez, M.J. (María Javier); Solas, M. (Maite)Glucose transporter 1 (GLUT1) is the main transporter mediating glucose access from the blood to the brain. In fact, homozygous ablation of GLUT1 selectively in brain vascular endothelial cells is ultimately incompatible with life, and its heterozygous ablation in these cells prompts a severe breakdown of both brain glucose metabolism and cognitive abilities. Astrocytes keep an intimate connection with both brain blood vessels and synaptic terminals, and are considered an essential source of blood-borne glucose or its metabolites to neurons. Nonetheless, the necessity of the main astrocyte glucose transporter, i.e. GLUT1, for brain glucose metabolism has not been defined. Unexpectedly, we found that brain glucose utilization was paradoxically augmented in mice featuring astrocyte-specific GLUT1 ablation (GLUT1GFAP mice). These mice also exhibited improved systemic glucose metabolism with increased glucose tolerance and glucose-stimulated insulin secretion, as well as upgraded brown adipose tissue activity. Obese GLUT1GFAP animals especially benefited from these phenotypic features, which resulted in rendering them metabolically healthier than obese control animals. Noteworthy, GLUT1GFAP mice did not present alterations regarding spatial memory performance, and even slightly outperformed control mice regarding recognition memory capacities. Mechanistically, we observed that GLUT1-ablated astrocytes exhibited increased insulin receptor-dependent ATP release, and that both astrocyte-specific insulin signaling and brain purinergic signaling are essential for the improved systemic glucose homeostasis and cognitive maintenance shown by GLUT1GFAP mice. Furthermore, it is known that Alzheimer s disease (AD) patients feature decreased GLUT1 levels, and that GLUT1 ablation in brain vascular endothelial cells accelerates the progression of an AD-like pathology in an AD mouse model. Moved by the above-mentioned findings, we induced astrocyte-specific GLUT1 ablation in the AD mouse model APP/PS1 (APP/PS1-GLUT1GFAP mice). Strikingly, we found that APP/PS1-GLUT1GFAP mice showed a decreased mortality rate, as well as markedly improved cognitive performance, in comparison to APP/PS1 mice. Importantly, chemogenetic perturbation of astrocytic Ca2+ activity totally abrogated the cognitive improvement elicited by astrocytic GLUT1 ablation. Collectively, we demonstrate that astrocytic GLUT1 is central to the regulation of brain energetics, yet its ablation triggers a reprogramming of brain metabolism sufficient to sustain brain energy requirements, systemic glucose homeostasis and cognitive function.
- Toxicological evaluation of mycotoxin mixtures: development of in vitro testing strategies and in vivo assessment of a binary mixture(Universidad de Navarra, 2023-01-16) Alonso-Jáuregui, M. (María); Vettorazzi, A. (Ariane); Gonzalez-Peñas, E. (Elena)Mixtures assessment is a key challenge in the field of mycotoxins. The general aim of this work was to contribute to the current toxicological evaluation of mycotoxin mixtures with a special focus on genotoxicity. Firstly, the current evidence about the combined toxicity among aflatoxins and ochratoxin A was gathered. These mycotoxins were selected due to their individual toxicity and the probable human co-exposure. Inconclusive results were observed among the articles due to the diversity of experimental designs and methodologies employed for assessing the type of interaction. As there is a lack of tools for substance prioritization, we developed an in silico-in vitro strategy. The endpoint selected was genotoxicity as the main concern associated with mycotoxins is derived from the long-term exposure. The strategy allowed us to classify 12 mycotoxins considering their genotoxic potential, concretely, point mutations. The mycotoxins of the strategy were selected considering the toxicity potential and occurrence data. Two steps were applied. Firstly, two in silico tools were used. DEREK Nexus® is an expert knowledge tool commercially available commonly used in the pharmaceutical industry. VEGA QSAR© is a statistical based and publicly available tool that requires an expert knowledge on chemistry and modelling for its use. In phase 2, the in vitro SOS/umu test in absence and presence of liver S9 was used. It is a medium-throughput assay with high concordance with the Ames test; the gold standard test for gene point mutations. However, a high amount of time and test item is required. Moreover, this test also enables us to characterize the 12 mycotoxins using a kidney S9 fraction for in vitro bioactivation. We validated the test in these conditions with the negative and positive controls. Expected results were obtained with positive controls (4-nitroquinoline-N-oxide and 2-aminoanthracene) without metabolic activation or with liver S9, but a potent concentration-dependent effect with 4-nitroquinoline-N-oxide and no activity of 2-aminoanthracene with kidney S9 were observed. It showed to be a reproducible assay for clear negative and positive compounds. However, for some of the mycotoxins classified as equivocal a higher variability was observed with kidney S9. The SOS/umu test is normally performed only once. But if equivocal results are obtained is advisable to repeat it and to be used with complementary assays. Aflatoxin B1 and sterigmatocystin resulted in positive genotoxicity outcomes in silico and in vitro with liver S9. Moreover, both are part of the same biosynthetic route. Therefore, an in vivo assessment of the binary mixture was carried out in male Wistar rats. A single oral dose of AFB1 (0.25 mg/kg) and STER (20 mg/kg) alone or in combination did not cause histopathological alterations in kidney or liver. A slight DNA damage (DNA strand breaks) was detected in liver of AFB1 treated animals, as well as a slight increase in the Fpg-sensitive sites in liver of animals treated with the mixture. The exposure to the individual toxins or the mixture did not increase the % of micronuclei in the bone marrow. The absence of genotoxicity observed in this study might be due to the vehicle (corn oil) and the rat strain (Wistar) used, as the levels of mycotoxins reached in target organs were x1.5 times lower than in previous studies using the same AFB1 dose dissolved in NaHC03 and administered to F344 rats. The lack of genotoxic response avoided to discuss the interaction in more detail. However, the plasmatic and tissular levels in the mixture of both mycotoxins were higher than in the samples of animals treated with the mycotoxins alone. This could point a possible toxicokinetic interaction of both mycotoxins at level of metabolism.
- Role of HDAC5 and SIRT2 in depression and clinical efficacy of antidepressants(Universidad de Navarra, 2023-01-16) Cortés-Erice, M. (María); Tordera, R.M. (Rosa María)Personalised medicine proposes the use of peripheral biomarkers of major depression (MD) to optimize the treatment and medical care to each person from the first day. The epigenetic hypothesis of depression proposes that MD is linked to remodelation of chromatin leading to neuronal plasticity failure in limbic brain regions. Among epigenetic mechanisms, histone posttranslational modifications such as histone acetylation could influence neuroplasticity. Among the members of the histone deacetylase (HDAC) superfamily, our laboratory has identified two HDAC enzymes, HDAC5 and SIRT2, as oppositely regulated by stress and antidepressant drug treatment in the brain of animal models. Importantly, both HDAC5 and SIRT2 exert a repressing action on the expression of BDNF, a gene with a leading role in neuroplasticity and that is downregulated in MD. In line with preclinical studies, peripheral blood mononuclear cells or prefrontal cortex tissue from depressed patients show an increase of HDAC5 and SIRT2. The general aim of this thesis is to explore in monocytes or T-cells whether HDAC5 and SIRT2, as well as the expression of genes linked to their function could be epigenetic peripheral biomarkers of MD and/or antidepressant therapy. A team of psychiatrists from different hospitals and health centres from Pamplona (Navarra, Spain) recruited 56 MD patients scoring for their Montgomery-Åsberg scale (MADRS≥20) and age and sex matched healthy controls (MADRS<7). Blood (20 mL, heparine tubes) was obtained from each participant and monocytes (classic, intermediate and non-classic) and T-cells (CD3+) were isolated by fluorescence activated cell sorter. HDAC5 and SIRT2 immunofluorescences were carried out and their distribution between cytoplasm and nucleus was calculated by confocal microscopy. In addition, classic monocytes and T-cells were incubated in vitro with the adrenoceptor agonist naphazoline and phosphorylation of HDAC5 at serine 498 was measured by immunofluorescence. Gene expression studies by qPCR included HDAC5 and SIRT2 as well as other genes linked to their function related to brain plasticity (BDNF), and other functions such as inflammation (KLF2, eNOS). In T-cells, the expression of some regulatory markers was also studied (FOXP3, PD-1, CTLA-4). Subsequently, full-length RNAseq of classic monocytes was applied to a selected sample of 9 MD patients and 9 healthy controls. Both in monocytes and T-cells of MD patients HDAC5 mRNA expression was upregulated and HDAC5 cytoplasm/nucleus ratio was decreased. Moreover, a negative correlation between MADRS score and HDAC5 cytoplasm/nucleus ratio was observed. In addition, SIRT2 cytoplasm/nucleus ratio was decreased in responder MD patients. Thus, in MD patients, these enzymes might accumulate in the nucleus of these cells. In T-cells of MD patients, BDNF and KLF2 mRNA expression were downregulated matching with the expected repressing action of nuclear HDAC5 and SIRT2. In monocytes, ADRB2 mRNA, gene encoding β2 adrenoceptor, was upregulated Interestingly, a significant negative correlation between ADRB2 mRNA expression and HDAC5 cytoplasm/nucleus ratio in monocytes of MD patients. Longitudinal studies showed a persistent BDNF mRNA downregulation in monocytes of non-responder MD patients. Finally, RNA sequencing of monocytes from highly TRD patients revealed alterations in biological functions that suggest an inflammatory profile in these cells. In this study, we have provided evidence of the existence of peripheral epigenetic biomarkers associated to MD and that might influence the activity of immune system. Moreover, BDNF expression in T-cells appears for the first time as a peripheral biomarker of MD. Future studies should explore whether restoring these biomarkers up to healthy values would contribute to clinical response in MD patients.
- Design and synthesis of new selenoderivatives with biomedical applications(Universidad de Navarra, 2023-01-13) Ramos-Inza, S. (Sandra); Sanmartin-Grijalba, C. (Carmen); Plano-Amatriain, D. (Daniel)The purpose of this Ph.D. project is the development of novel therapeutic agents incorporating the selenium (Se) atom into the structure of small molecules, thus expanding further knowledge about the design and molecular biology of Se-containing drugs. Se is an essential trace element that exerts multiple and complex effects on human health. This element is crucial for human well-being largely due to its involvement in several physiological functions such as protection against oxidative stress or immune response through its incorporation into selenoproteins. Additionally, Se supplementation and adequate maintenance levels of this micronutrient in the organism have been generally related to the prevention of certain pathologies such as cancer, diabetes, neurological disorders, and infections, among others. Accordingly, a plethora of natural and synthetic Se-containing compounds have been recently studied and explored for their therapeutic potential in the treatment of cancer, leishmaniasis, and other diseases. In this context, we designed four series of compounds in which Se was included in a variety of chemical entities, including acylselenourea, selenocyanate, diselenide, and selenide. Different organic nuclei, ferrocene, and NSAIDs as parent molecules that are suitable to be chemically modified by the incorporation of Se into their structures were also considered in our design. Thus, a total of eighty-three novel selenoderivatives were synthesized and purified. Additionally, their preliminary assessment as therapeutic agents were studied in both in vitro and in vivo assays. The results compiled in this Ph.D. project confirm our initial hypothesis of designing selenoderivatives that may confer biomedical properties and provide new strategies and insights for further developing new small molecules containing Se with therapeutic activity. Our results also reinforce the importance of the chemical form of this chalcogen atom in the structure of the organic scaffold, since it is directly linked to the metabolism process and therefore the biological effect of the resulting selenoderivative. The combination of Se entities with other relevant structural features including the incorporation of biologically active frameworks led to the discovery of potent anticancer or even multitarget drugs with additional antileishmanial and antibacterial activities. This Ph.D. memory is expected to provide a basis for future drug development and stimulate further research of new Se-containing compounds with biomedical applications.