Departamento de Ciências Médicas
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Percorrer Departamento de Ciências Médicas por Domínios Científicos e Tecnológicos (FOS) "Ciências Médicas::Ciências da Saúde::Biomedicina"
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- Characterization of Effector-Memory CD8+ T cells and their Association with Human Cognitive FunctionPublication . Esgalhado, André João Gabriel ; Arosa, Fernando Aguilar; Uhrberg, MarkusHuman effector-memory CD8+ T cells consist of highly differentiated cells that differ in the expression of the tyrosine phosphatase isoform CD45RA, being designated as CD8+ TEM and CD8+ TEMRA cells. These highly heterogeneous and polyfunctional cells possess cytotoxic, regulatory, and suppressive features, and are capable of migrating to non-lymphoid tissues and organs, including the brain under certain conditions. Expansions of CD8+ TEM and CD8+ TEMRA cells have been described in chronic inflammatory diseases, tumors and viral infections, as well as in healthy elderly individuals, including centenarians. Over the past few decades, the role of CD8+ T cells, particularly CD8+ TEMRA cells, has been the subject of several studies in the context of aging, cognition, and neurodegeneration, where they have been generally regarded as detrimental to the central nervous system (CNS), though recent investigations have challenged this view. These highly differentiated CD8+ T cells are known to arise through TCR-dependent and TCR-independent mechanisms, such as cytokine-driven proliferation via interleukin (IL)-15. Intriguingly, chronic antigenic stimulation has been shown to drive the generation of CD8+ T cells expressing low levels of the CD8β chain, though antigen-independent mechanisms remain poorly understood. Herein, we have performed a comprehensive characterization of peripheral blood mononuclear cells (PBMC) as well as of human leukocyte antigen (HLA) molecules in a cohort of elderly volunteers differing in their cognitive status. A detailed analysis of the level of expression of CD45RA in the CD8+ TEMRA compartment revealed the presence of two distinct populations: CD8+ TEMRAlow and CD8+ TEMRAhigh cells. Notably, CD8+ TEMRAhigh cells formed a well-defined and sharply delineated population that was significantly expanded in cognitively impaired volunteers, whereas cognitively unimpaired volunteers were enriched in CD8+ TEMRAlow cells. Further analysis of CD8α and CD8β expression also identified the existence of two distinct CD8+ T cells subsets based on the expression of CD8β: CD8αβlow and CD8αβhigh T cells, with the former being more prevalent among cognitively unimpaired individuals. Moreover, stimulation with PMA and Ionomycin revealed significantly increased IFN-γ production by CD4+ T cells from cognitively impaired elderly. Noteworthy, all but one of the volunteers studied were cytomegalovirus (CMV) seropositive. Finally, a higher prevalence of the HLA-B8 serotype, which belongs to the ancestral haplotype HLA-A1, Cw7, B8, DR3, DQ2, was found among the cognitively impaired elderly. Additionally, we assessed the impact of IL-15 on the cell surface expression of CD8β using CFSE-labeled purified human naïve CD8+ T cells cultured for 12 days. IL-15 induced a robust proliferation and differentiation, resulting in a cell cycle-dependent down-modulation of CD8β from the cell surface, while CD8α expression remained stable or increased slightly. This led to the generation of CD8αβlow and CD8αβ– (i.e., CD8αα) T cells. In contrast, IL-2 and IL-7 alone were unable to replicate this effect. Determination of mRNA levels for CD8α and CD8β isoforms by qPCR revealed that IL-15 promoted a significant decrease in mRNA levels of the CD8β M-4 isoform, while increasing the levels of the M-1 and M-2 isoforms alongside with CD8α. Remarkably, analysis of the level of the tyrosine kinase Lck showed a significant increase in CD8+ T cell blasts after culture of CD8+ T cells with IL-15, when compared to CD8+ T cells at the beginning of the culture. Our findings show an association with certain CD8+ T cell subsets that is compatible with a protective role in cognition and neurodegenerative diseases by identifying novel markers that define discrete subsets of highly differentiated CD8+ T cells expanded in cognitively unimpaired elderly individuals and identify IL-15 as a factor involved in the generation of these subsets. In-depth phenotypic, functional, and transcriptomic characterization of ex vivo and in vitro obtained CD8+ T cell subset is warranted to further elucidate their unique functional properties.
- Effect of white tea on the reproductive function of diabetic or prediabetic individualsPublication . Dias, Tânia Isabel Rodrigues Amaral; Oliveira, Pedro Fontes; Silva, Branca Maria Cardoso Monteiro da; Casal, SusanaThe prevalence of diabetes mellitus (DM) has been increasing in young men worldwide. Patients with DM have a dysfunction on insulin secretion and/or insulin action, resulting in hyperglycemia. Insulin dysregulation affects glucose homeostasis in the body, having a deleterious impact on male fertility. Sertoli cells (SCs) are essential for the maintenance of male reproductive potential as they provide the physical and nutritional support that allows the differentiation of germ cells into fully competent spermatozoa. SCs function highly relies on glucose metabolism, which is their preferred substrate. Many diabetic patients are subfertile or infertile due to altered SCs function, impaired spermatogenesis and poor sperm quality. The primary strategy to counteract DM and its complications includes nutritional changes and physical activity. However, these interventions are usually unsuccessful alone and need to be complemented with medication. Still, the efficacy of conventional drugs is limited, they are expensive and have several secondary effects. In recent years, natural products, including tea and its components, demonstrated promising antioxidant and antidiabetic properties. Besides, the treatment of prediabetes can be an effective approach as it can occur up to 10 years before the progression of the disease to a more severe state. In this research project, we aimed to unravel the effects of white tea (WTEA) on male reproductive function and its protective role against reproductive dysfunctions induced by prediabetes. Further, we aimed to investigate if the effects of WTEA are due to a combined effect of all WTEA components or to a predominant effect of one of its most bioactive components. WTEA is the rarest and less studied type of tea, but it presents a potent antioxidant potential due to its high catechin content. Through proton nuclear magnetic resonance (1H-NMR), we verified that this type of tea is particularly rich in caffeine, epigallocatechin gallate (EGCG) and L-theanine. Using an in vitro model of rat SCs or human SCs (hSCs) we evaluated the effects of WTEA extract (0.5 mg/mL) and its main bioactive compounds (caffeine, EGCG and L-theanine) on cells metabolism, mitochondrial functionality and oxidative profile. WTEA extract modulated rat SCs metabolism and stimulated the production of lactate, which is essential for germ cells survival. Supplementation of hSCs culture media with 50 μM of caffeine, EGCG, or L-theanine for 24 hours induced alterations in hSCs metabolism that are important for the improvement of male reproductive potential. It resulted in an increase or maintenance of lactate production, showing a protective role against oxidative damages. However, at 50 μM, these compounds also induced some alterations in hSCs proliferation and mitochondrial functionality that may compromise hSCs function. The WTEA extract (0.5 mg/mL) showed a better improvement on SCs, highlighting the importance of the combined effect of all the tea components for its beneficial effect. We also conducted ex vivo studies using rat epididymal spermatozoa to evaluate the potential of WTEA extract (0.5 mg/mL or 1 mg/mL) and the three selected components as an additive for a sperm storage medium at room temperature for short periods. In these studies, the concentrations of caffeine (71 μg/mL) EGCG (82 μg/mL) and L-theanine (19 μg/mL), were selected based on their concentration in the WTEA extract. Sperm viability decreases over time in refrigerated samples, thus in certain cases the short-term storage at room temperature can be advantageous, such as for samples transport or assisted reproductive technology. These studies allowed us to conclude that the WTEA extract (especially at 1 mg/mL) was a better additive to the sperm storage medium at room temperature than each of the components alone, as it kept spermatozoa viability for 3 days equivalently to values obtained at the collection time. Although the combination of the three selected components together showed a higher improvement in spermatozoa viability, it also stimulated protein oxidation, supporting the beneficial combined effect of all the components constituting the WTEA extract. This led us to study the in vivo effect of a regular consumption of WTEA on the reproductive function of a rat model of prediabetes. The prediabetic rats showed alterations in the testicular and epididymal metabolism, resulting in poor sperm quality. WTEA ingestion by prediabetic rats for two months prevented many of the metabolic dysfunctions induced by the disease in the testis and epididymis, resulting in the improvement of sperm motility and viability. Our results indicate that WTEA regular consumption can be a cost-effective strategy to improve prediabetes-induced reproductive dysfunctions, paving the way for WTEA to be used for the development of new antioxidant therapies for the improvement of male fertility.
- G-quadruplex aptamers for cervical cancer therapyPublication . Carvalho, Josué Leandro de Oliveira e; Cruz, Carla Patrícia Alves Freire Madeira; Queiroz, João António de Sampaio Rodrigues; Salgado, Gilmar FernandesAnnually, more than 500,000 new cervical cancer cases are diagnosed worldwide with a 60% mortality rate. In Portugal, about 750 new cases are annually diagnosed which in 2018 resulted in 340 deaths. Human papillomaviruses (HPVs) are the principal cause for the development of cervical cancer. High-risk HPV types 16 and 18 are responsible for 70% of all cases. Persistent HPV infection leads to the hyperproliferation of the infected cells by downregulating epithelial differentiation, epidermal development, and innate immune responses. The oncogenic potential of high-risk HPV types is attributed to the expression of oncoproteins, E6 and E7, which inactivate tumour suppressor p53 and members of retinoblastoma family, leading to carcinoma development. Despite recent progresses in screening and HPV vaccination that reduced cervical cancer incidence, treatment options for women with persistent HPV infection or in its latent form are still missing. Furthermore, the available therapeutic options for cervical cancer have limited effectiveness and specificity, causing great pain to patients, and do not benefit from the research addressing the host-HPV interaction patterns during malignant progression. The genome of HPV is divided into 3 regions: a long control region (LCR) that contains cis-elements necessary for the replication and transcription of viral DNA; the early region, composed of six open reading frames (ORFs) labelled E1−E7, and a late region with two ORFs coding for capsid structural proteins L1 and L2. Recently, several guanine-rich regions have been found in the genome of ten HPV types (both high- and low-risk), and the potential of the sequences to fold into stable G-quadruplex (G4) structures was demonstrated. These guanine-rich regions are present in LCR, E1, E4 regions and L2 protein coding sequence, which are involved in transcription, replication, and viral proteins production. Hence, targeting these G-rich regions of the HPV genome could serve as a potential antiviral strategy, using small ligands that can stabilize the viral G4 regions and regulate gene expression. Nucleolin is overexpressed in the surface of cervical cancer cells and was shown to be involved in the activation of HPV18 oncogene transcription in cervical cancer and to be related with HPV16 genome stability maintenance. Nucleolin is present in the nucleus, cytoplasm, and cell surface. The roles of cell-surface nucleolin in tumorigenesis, angiogenesis, and cancer signalling pathways have also been described. Therefore, nucleolin is an attractive target for the development of targeted therapies for both cervical cancer and HPV infection given its high abundance, its multifaceted influence on oncogenesis and virulence of HPV, and its selective presence on the plasma membrane of cancer cells, but not on untransformed, normal cells. AS1411 is a nucleolin-targeted G4 aptamer with antitumoral effect in many cancers that has also been extensively used for the tumour-specific delivery of both therapeutic and imaging agents. This thesis innovates by proposing the development of aptamer-based carriers of antiviral/anticancer G4 ligands as a new therapeutic strategy for HPV infection and cervical cancer. Firstly, several G4-specific ligands belonging to different aromatic scaffolds such as indole, phenanthroline and acridine, were synthesized and characterized for their ability to bind G4 structures, either found in the cancer cell genome (oncogene promoter and telomeric G4s) or the HPV genome. Their effect in inhibiting key G4-dependent processes was demonstrated by their potential to inhibit Pif1 helicase and Taq polymerase enzymes, and to downregulate c-MYC oncogene expression. The ligands demonstrated interesting antitumoral effects in cervical cancer cell lines, particularly ligand C8, which was also found to inhibit HPV18 replication and encapsidation in organotypic raft cultures, with resulting antiproliferative effects in infected tissue. Having established the ability of the ligands as anticancer/antiviral agents, the development of an aptamer-based delivery system was envisioned. AS1411 aptamer and its derivatives AT11, AT11-L0 and AT11-B0 were used to selectively convey the G4 ligands to HPV-positive cervical cancer cells, with a simple non-covalent conjugation strategy and mediated by cell-surface nucleolin recognition. This strategy proved to be efficient at accumulating the ligands in cervical cancer cells, improving their antitumoral effect while limiting their off-target toxicity towards normal cells. Interestingly, because the used aptamers are also G4-forming sequences, the presence of the G4 ligands improved the aptamers’ properties such as cellular penetration and serum nuclease resistance, without affecting nucleolin recognition. Finally, advanced and intricate drug delivery systems were developed aiming at enhancing the clinical applicability of the anticancer/antiviral agents. AS1411-decorated nanoaggregates were synthesized to encapsulate the G4 ligands, allowing their sustained and targeted release within HPV-positive cells in precancerous and cancerous stages. The nanoaggregates presented small sizes (nanoscale), negative surface charge, stability, and a suitable drug release profile. The presence of AS1411 on the nanoaggregates surface as a cancer-specific guiding agent promoted the specific accumulation of the G4 ligands in cervical cancer cells which inhibited their growth, with little effect on non-malignant normal cells. The nanoaggregates were also able to penetrate cervix tissue biopsies of patients with HPV infection in precancerous stages, demonstrating the potential of this system to treat HPV infection in its latent stage, preventing oncogenesis and cervical cancer progression. The administration route of the drug-loaded nanosystems was also addressed. The C8-loaded nanoaggregates were formulated as a gel for local application in the female genital tract, specifically tissue with precancerous lesions and/or HPV infection. Preliminary ex vivo permeation studies using porcine vaginal tissue demonstrated the efficacy of the formulation in penetrating and accumulating the nanosystems in the target cells. Overall, the research performed throughout this doctoral thesis defined improvements in the design of G4-specific compounds with dual anticancer/antiviral effects and led to the discovery of potential aptamer-based delivery systems for G4 ligands. The original results obtained during the past years provide important knowledge in the applicability of G4 nucleic acids for the development of specific and effective therapeutics against HPV virulence and oncogenesis. Furthermore, the encouraging pre-clinical evaluation performed on the newly developed nanomedicines demonstrated the need of continuing the research initiated by this project, particularly by pursuing the in vivo studies to verify the clinical applicability of these systems. In the future, a similar strategy could be extended to the treatment of other types of cancers (for instance HPV-induced oropharyngeal cancers) and other viral infections such as HIV, hepatitis virus and SARS-CoV-2.
- Regucalcin as one of the guardians of the male reproductive functionPublication . Silva, Ana Manuela dos Santos; Baptista, Cláudio Jorge Maia; Correia, Sara Carina de LimaMale reproductive function is highly sensitive to extrinsic testicular damage or intrinsic modifications, which consequently affects spermatogenesis leading to declined sperm quality and compromised fertility. These reproductive modifications can represent a collateral effect, for example from an oncological therapy, or a natural consequence of living, such as ageing. Currently, due to early diagnostic and high survival rate, young cancer patients are living longer. Nevertheless, approaches to successfully restrain the undesirable secondary effects of oncological therapies are still missing. Furthermore, until recently, the decline in male reproductive function with the advance of age was frequently neglected. Lately, given the trend of modern society to delay the conception of a biological child, the decrease in sperm quality in older subjects has been drawing attention from the scientific community. Indeed, these problems are emerging topics on male fertility. Oxidative stress (OS) is known to be for itself associated with male infertility cases. Also, OS is implicated in radiotherapy and ageing. In this doctoral thesis, we aimed to disclose the potential of regucalcin (RGN) protein to counteract the damaging effects of testicular radiotherapy as well as to attenuate the ageing-associated changes in male reproductive tract. In recent years, RGN protein has been showing to be an important player in spermatogenesis and male fertility, managing the suppression of pro-oxidant and chemical apoptotic stimulus in seminiferous tubules. RGN is a protein widely expressed in testicular cells, and its biological function seems to be vast. Moreover, RGN is also known as Senescence Marker Protein-30 (SMP-30) as a result of its decreased expression with ageing. Recently, high levels of RGN prevented ageing-associated changes in the prostate. Using an in vivo approach, we studied the role of RGN by comparing transgenic rats overexpressing RGN (Tg-RGN) with their wild-type (Wt) counterparts. Ten weeks after rats were subjected to radiotherapy, we verified that RGN was able to mitigate radiation-induced testicular damage because Tg-RGN animals presented less affected sperm parameters as well as lower rate of testicular apoptosis in comparison with Wt rats. Moreover, Tg-RGN animals also presented unaffected sperm parameters in spite of the ageing concomitantly with lower OS levels. Interestingly, we detected an enhanced expression of RGN in irradiated testis as well as in senescent sperm both in Wt and Tg-RGN rats, reinforcing the involvement of RGN in response to stimuli or modifications that can negatively impact male reproduction. Overall, our results are strong evidence about the beneficial role of RGN as a guardian of male reproductive function, suggesting that RGN protein has great potential to be included in strategies to improve, protect, and/or recover male fertility in men undergoing oncological treatment or with advanced age who still intend to have biological descent.
- Study of neurophysiological responses associated with the application of magnetic fields to the brainPublication . Pinto, Nuno Filipe Cardoso; Patto, Maria da Assunção Morais e Cunha Vaz; Gama, Jorge Manuel dos ReisTranscranial magnetic stimulation (TMS) is a non-invasive diagnostic and therapeutic technique used to stimulate the brain in several neurological and psychiatric diseases, even though the main bases underlying its action are not fully understood. Theta Burst Stimulation (TBS), a patterned form of repetitive TMS, has been assuming particular importance due to its faster application. Research of TBS effects on some higher cortical functions such as cognition after stimulation of the prefrontal cortex (PFC), or its possible influence in some less studied cortical regions (as the temporal cortex) has been limited and revealed inconsistent results. One of the problems assessing the cognitive TBS after-effects relates to the use of multiple evaluation methods, with different sensitivities. In this matter, the use of neurophysiology studies such as the auditory P300, a cognitive evoked potential, may be of particular importance. To date, studies addressing the association between auditory P300 and TBS are scarce, and some contradictory results were found. The study of other higher cognitive domains such as creativity is even rarer, but it may be relevant given that part of the neural networks involved in creative processing are associated with the PFC. The effect of TMS over the PFC, studying the modulation of functions mediated by the autonomic nervous system has also been reported, but there is still a significant disagreement between the rare studies performed. So far, the extent of the modulatory effects associated with TBS at the sensory level is still poorly known, and research with TBS over the auditory cortex, despite showing some positive results, remains inconclusive, with some reports of sound hypersensitivity after sessions with higher intensity stimulation. It should also be noted that a significant part of the knowledge about the effects of TBS derives from studies in patients, with dysfunctional neuronal networks or hemispheric lesions, which add challenges to the search for scientific evidence in healthy individuals. Given the uncertainties that remain regarding the extent of the neuromodulatory effects of TBS, the primary objective of this thesis focused on increasing the scientific knowledge related to the use of TBS in the healthy brain. Therefore, we intended to study the neurophysiological responses (such as auditory P300), the functional responses (such as auditory thresholds), and the physiological responses (such as cerebral oximetry and blood pressure) associated with the application of TBS in the prefrontal and temporal cortices. All studies used a target population of healthy young adults, with an average age of approximately 23 years, and similar education. TBS was performed accordingly to the 600-pulse paradigm described by Huang et al. (continuous and intermittent). Sham-controlled, double-blind intervention protocols were used, with random distribution by the respective groups. The main objective of the study in chapter III was to evaluate the effect of TBS on the dorsolateral prefrontal cortex (DLPFC) of both cerebral hemispheres in cognitive processing. The objective was to assess if the auditory P300 would be influenced by the stimulation type. Results revealed that the mean P300 peak latency after TBS decreased only after leftward iTBS. A significant delay in P300 latency was originated from both right and left cTBS. Amplitude response did not change significantly. The results covered in chapter IV derived from the use of TBS on the left DLPFC, studying the possibility of a relationship between the post-TBS auditory P300 and the post-TBS neuropsychological tests: Trail Making Test (TMT) and the Stroop Test of Words and Colours. Results revealed that cTBS led to a delay of the P300, also significantly influencing the expected performance on Stroop C and Stroop Interference when compared to the groups submitted to iTBS and sham stimulation. No significant results were found in the TMT tests for any type of TBS stimulation. In Chapter V, we studied the cerebral oximetry using Near Infra-Red Spectroscopy, blood pressure, and heart rate, after applying TBS to the right and left DLPFC. We found a significant reduction in oximetry in the left frontal region after ipsilateral cTBS and a significant decrease in systolic blood pressure after cTBS to the right DLPFC. Chapter VI covered the evaluation of the effects of TBS over the left temporal cortex, specifically studying the auditory thresholds in the ear closest to the coil. Results showed no major side effects after iTBS, cTBS, or sham stimulation. It was also found that iTBS led to lower hearing thresholds, especially when comparing the iTBS and sham groups at 500Hz and between the iTBS and cTBS groups at 4000Hz. Chapter VII addresses a patent concerning the technique and possible use of iTBS as a method to influence creative processing. After iTBS over the right DLPFC, results of an adapted selection of the Torrance Tests of Creative Thinking suggest that divergent thinking, originality and fluency improved significantly compared to the sham group. An integrative analysis of the results shows that TBS seems to effectively influence the underlying cortical neurons and cortico-subcortical networks. The findings thus support the existence of a trans-synaptic effect advocated initially for the classic repetitive TMS, which after the publication of our research can continue to be extended with greater confidence to TBS protocols. Our results also support the most consensual theory about the modulatory effects of the two main forms of TBS – intermittent (excitatory) and continuous (inhibitory) – particularly on the prefrontal and temporal cortices. The effects of TBS seem to be intrinsically correlated with the hemispheric lateralization and this may be related to the specific functions or dominance of each hemisphere and the specific stimulated cortical regions. The combined results of this investigation also seem to suggest that the inhibition induced by cTBS seems more effective when compared to the excitatory effect of iTBS, which seemed stronger in the left hemisphere. After all our research with TBS in more than one cortical region, we can infer that this is a safe technique, with rare and incipient side effects. The encouraging results after using iTBS in the auditory cortex opens new perspectives regarding future implementations of the technique and should be replicated in patients, particularly with mild sensorineural hearing loss, in order to assess whether this stimulation protocol can be a valid therapeutic technique in these cases. We also conclude that the techniques used to study TBS-related effects, as the P300 or the NIRS, can be very useful in the future, as an attempt to identify the effectiveness of the therapeutic use of TBS protocols, possibly allowing to adapt and modify the idealized interventions, leading to a personalized patient intervention. Our findings provide relevant information, necessary to increase the technical and scientific credibility required for achieving a more comprehensive and reliable clinical use of TBS. This is crucial at a time when transcranial magnetic stimulation use as an off-label therapy for numerous neurological and psychiatric diseases grows unregulated, and the patient best interests must be defended.
- Taste receptors in the Choroid Plexus are functional and regulated by sex hormonesPublication . Tomás, Joana Filipa Melfe; Gonçalves, Isabel Maria Theriaga Mendes Varanda; Santos, Cecília Reis Alves dosThe choroid plexuses (CPs) are highly vascularized structures constituted by a single layer of epithelial cells that project into the brain ventricles. The CPs are the main site of cerebrospinal fluid (CSF) production and constitute the Blood-CSF Barrier (BCSFB), holding high relevance in the surveillance of the CSF chemical composition. These structures contribute for the synthesis of biological compounds essential for the functioning and protection of the central nervous system (CNS) against neurotoxic insults. The expression of taste signalling pathway components in the CPs and its regulation by sex hormones was previously determined in a cDNA microarray study previously performed by our group. Moreover, the taste signalling pathway was determined as one as the top five pathways regulated by female sex hormones. The ectopic expression of sweet, umami and bitter taste signalling in extra oral organs have been extensively studied. In these organs, the taste receptors seems to behave as sensors to assess the composition of body fluids. The expression of the taste molecular machinery and its putative regulation by sex hormones in the CP raised the hypothesis that the taste signalling pathway could be one of the mechanisms involved in the monitoring of the chemical composition of blood and CSF at the BCSFB that my differ with gender. Considering this, we aimed to evaluate the presence and the functionality of the taste signalling pathway, as well as its regulation by the female sex hormones 17β-estradiol (E2) and progesterone (P4) in rat CP. In the first study, the presence and functionality of the taste signalling pathway was assessed. Transcripts for the taste-related genes Tas1r1, Tas1r2, Tas1r3, Tas2r109, Tas2r144, Gustducin, Plcb2, Ip3R3 and TrpM5 were found in CPs from adult Wistar rats. The expression of Tas1r1, Tas1r2, Tas2r144, Gustducin, Plcb2 and TrpM5 proteins was confirmed by Western blot, immunohistochemistry and immunocytochemistry. As umami and sweet receptors are heterodimeric receptors, we performed double labelling immunofluorescence, that showed the co-expression of T1R1 and T1R3 proteins that form the umami receptor, as well as, the coexpression of T1R2 and T1R3 proteins that form the sweet receptor. Having established the cellular localization of the taste machinery in CP epithelial cells (CPEC) we further evaluated the subcellular expression of taste proteins. For that, CPs were double labelled with antibodies for each of the taste-related proteins studied and a fluorescent marker of glycosylated surfaceexpressed proteins, revealing that taste-related proteins are located in the plasma membrane. After confirming the presence of the taste pathway molecular machinery, we proceed with functional assays. Considering that most of toxic/noxious compounds are bitter compounds that may exist in the CSF, we turned our attention to the bitter taste signalling pathway. Thus, to evaluate the functionality of the bitter pathway in primary cultures of CPEC we performed single cell calcium imaging assays using D-Salicin as the bitter stimulus. We observed an increase in intracellular Ca2+ evoked by D-Salicin that was diminished in the presence of the bitter taste receptors (T2Rs) blocker Probenecid, suggesting that T2R in the CPs are capable of sensing bitter compounds in the CSF and/or blood. An analysis in silico of our previous cDNA microarray data revealed that the decline of hormone levels in female rats upon ovariectomy clearly induced an up-regulation of the T2Rs Tas2r109, Tas2r124, Tas2r134, and Tas2r144, and the downstream effector molecules Plcb2 and Trpm5. Moreover, Tas2r109 and Tas2r144 were differentially expressed between female and male, showing a higher expression in males. This data led us to the second study of these thesis where the regulation of the taste pathway by female sex hormones was analyzed. For that, we compared the expression of taste-related genes in the CPs of sham and ovariectomized female Wistar rats and in CPs explants from newborn rats incubated with different concentrations of E2 and/or P4. Our results confirmed the cDNA microarray data, corroborating the regulation of taste-related genes by E2 and P4. The bitter receptors Tas2r109, Tas2r144, and the tasterelated genes Plcb2 and Trpm5 were down-regulated by ovarian hormones both in vivo and ex vivo. Functional implications of female sex hormones regulation was assessed, by single cell Ca2+ imaging, with the bitter compound, Denatonium Benzoate (DB), which is a known ligand of Tas2r144. Single cell Ca2+ imaging was performed in the immortalized CP epithelial cell line Z310 incubated with E2 and/or P4 in the presence of the respective hormone receptor blocker (fulvestrant or mifepristone, respectively). Intracellular Ca2+ variation, observed by single cell calcium imaging, was diminished in the presence of female sex hormones. However, while E2 effects were mediated via the nuclear E2 receptor, P4 effects were not abolished by the blocker of nuclear P4 receptor. Knocking-down Tas2r144 with a specific siRNA effectively reduced the Ca2+ response to the bitter compound DB, in a similar manner to E2 and P4, suggesting that female sex hormones down-regulated the responses of CPEC to chemical stimuli by reducing Tas2r144. In summary, our results confirmed and characterized the presence and functionality of the taste signalling machinery in CPs showing its regulation by female sex hormones. These results suggest that the taste signalling pathway may be one of the mechanisms by which the CP surveys the chemical composition of the CSF and elicit responses to modulate and maintain brain homeostasis. The achievements reached with this work will contribute to a better understanding of the mechanisms underlying the sensor/protective role of CPs in the CNS.