Tesis Doctorals - Departament - Patologia i Terapèutica Experimental

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Novel pharmacological strategies for neurological conditions involving glutamate and adenosine
(Universitat de Barcelona, 2025-05-28) Sarasola Telleria, Laura I.; Ciruela Alférez, Francisco; Salort Flaquer, Glòria; Universitat de Barcelona. Departament de Patologia i Terapèutica Experimental
[eng] Neuropsychiatric disorders such as schizophrenia and bipolar disorder present alterations in dopaminergic and glutamatergic neurotransmission, which has led to the development of therapeutic strategies targeting these systems. Recently, the adenosynergic system has emerged as a key regulator of dopaminergic and glutamatergic activity, with evidence that activation of adenosine receptorA2A can modulate dopaminergic transmission and affect neuropsychiatric symptoms. In addition, the interaction between the A2A receptor and the metabotropic glutamate receptor type 5 in the striatum represents a potential therapeutic convergence point for the treatment of these disorders. In this context, nanobodies have emerged as innovative tools for modulating membrane macromolecular interactions in a highly specific manner. Unlike conventional antibodies, nanobodies are heavy-chain antibody fragments that have advantages such as their small size, high stability, and ability to access inaccessible regions of proteins. In this thesis, we explore the development and characterization of the Nb43 nanobody, designed to modulate the glutamate receptor mGlu5R, as well as its Nb43-CGS21680 conjugate, which incorporates an A2AR agonist to enhance its effect. The thesis is divided into three chapters. In the first chapter, we characterize in vitro the binding and functional effect of Nb43 and Nb43-CGS21680 in cellular systems expressing mGlu5R and A2AR. In the second chapter, we evaluate the distribution, penetration into the central nervous system and pharmacological activity of these nanobodies in murine models. Finally, in the third chapter, we performed a meta-analysis of adenosine metabolism modulators in schizophrenia and bipolar disorder, to evaluate their clinical impact and therapeutic potential. The results of the in vitro study of Nb43 showed that Nb43 selectively binds to mGlu5R in a physiological context. On the other hand, Nb43-CGS21680 showed a dual-action profile, with simultaneous modulation of mGlu5R and A2AR. Interestingly, the co-activation of these receptors resulted in a more specific modulation of intracellular activity. In vitro experiments of PLA and NanoBiT confirmed the existence of functional interactions between mGlu5R and A2AR, supporting the hypothesis that these receptors form heteromers in the neuronal membrane. To evaluate the biodistribution of Nb43 and Nb43-CGS21680, we performed in vivo imaging studies after intravenous and intranasal administration. The results indicated that Nb43 does not cross the blood-brain barrier significantly when administered intravenously. However, in intranasal administration, small concentrations were detected in the olfactory bulb, suggesting a possible route of access to the central nervous system. The behavioral effects of Nb43 and Nb43-CGS21680 were evaluated after their intracerebroventricular administration, which allowed analyzing their direct impact on the central nervous system without the limitation of the blood-brain barrier. In the locomotion test, locomotor activity CGS21680 significantly reduced, in line with previous studies suggesting its anti-dopaminergic effect. Similarly, Nb43 and Nb43-CGS21680 also decreased locomotion, although with less intensity. This effect suggests that Nb43 could be modulating dopaminergic neurotransmission through its action on mGlu5R in the striatum. In the haloperidol-induced catalepsy test, catalepsy CGS21680 significantly increased, confirming its role in dopaminergic inhibition. Interestingly, Nb43 also exacerbated catalepsy, indicating that its effect on mGlu5R could be modulating the activity of the dopaminergic pathway. Unlike CGS21680, whose effects disappeared after 24-48 hours, Nb43-CGS21680 prolonged the catalepsy effect until 48 hours after administration, suggesting a more sustained mechanism of modulation of neurotransmission. Additionally, we performed catalepsy experiments after intranasal administration of Nb43 and Nb43-CGS21680. The results showed that Nb43 had a significant impact on haloperidol-induced catalepsy, while Nb43-CGS21680 showed no effects. This suggests that Nb43 was able to reach the central nervous system through the olfactory bulb, where mGlu5R is expressed and its role in modulating locomotion and balance has been reported. On the other hand, the meta-analysis study included clinical studies that evaluated the impact of adenosine metabolism modulators, such as allopurinol, dipyridamole, pentoxifylline, and propentophylline, on the symptomatology of schizophrenia and bipolar disorder. The results indicated that allopurinol, a xanthine oxidase inhibitor that raises adenosine levels, was the most studied and showed positive effects in reducing positive and negative symptoms of schizophrenia and bipolar disorder. Dipyridamole, an adenosine reuptake inhibitor, also showed improvements in positive symptoms, although studies were limited. Propentophylline, a xanthine derivative with neuroprotective and anti-inflammatory, had more marked effects on negative symptoms and cognitive impairment. The findings of the meta-analysis reinforce the adenosynergistic hypothesis of schizophrenia and bipolar disorder, which proposes that modulation of the adenosynergistic system may influence dopaminergic and glutamatergic neurotransmission. In this sense, the results suggest that strategies aimed at enhancing adenosynergistic signaling could represent a promising therapeutic avenue. Overall, this thesis contributes to the knowledge about the role of nanobodies in the pharmacology of membrane receptors and highlights the importance of the adenosynergic system in the modulation of dopaminergic and glutamatergic neurotransmission in neuropsychiatric disorders.
Lateral Ankle Instability: From Anatomy to Surgery
(Universitat de Barcelona, 2025-04-12) Nunes, Gustavo Araujo; Dalmau-Pastor, Miki; Dalmau-Pastor, Miki; Universitat de Barcelona. Departament de Patologia i Terapèutica Experimental; Universitat de Barcelona. Departament de Patologia i Terapèutica Experimental
[eng] INTRODUCTION: Knowledge of the anatomy of the lateral ligaments of the ankle is essential for understanding their function, pathophysiology, and treatment options. Recently, new anatomical features have been described, including previously undescribed connections between these ligaments. Consequently, new concepts and anatomical techniques for the arthroscopic treatment of lesions in this area have emerged. However, these anatomical advances’ applicability and mechanical role still need to be studied. Additionally, the clinical relevance of these anatomical concepts in the surgical field requires further exploration and investigation. HYPOTHESIS: The connections between the lateral collateral ligaments of the ankle extend to the medial facet of the fibula and have a biomechanical role. Based on new anatomical concepts, arthroscopic anatomical techniques used to repair the lateral ligaments of the ankle have good clinical and functional outcomes. OBJECTIVES: This PhD thesis aims to conduct biomechanical and anatomical research on the lateral ligaments of the ankle and their connections and analyze the clinical outcomes of the arthroscopic anatomical techniques used to treat lateral ankle instability.
Modeling neuro-immune interaction in Parkinson’s disease using an iPSC-based autologous in vitro system
(Universitat de Barcelona, 2024-12-20) Baruffi, Valentina; Consiglio, Antonella; Universitat de Barcelona. Departament de Patologia i Terapèutica Experimental
[eng] Parkinson’s disease (PD) is a chronic, incurable, progressive neurodegenerative disorder pathologically characterized by intracellular aggregates of α-synuclein (α-syn) forming Lewy bodies (LBs) and the loss of dopaminergic (DA) neurons in the substantia nigra (SN). In addition to neurodegeneration, PD is associated with inflammation, both in the periphery and in the brain, suggesting that the adaptive and the innate immune systems may participate in the pathogenesis of the disease. However, the dynamic and context-dependent interaction between brain- resident microglia and CNS-recruited T lymphocytes and their impact on PD onset and/or progression is not yet fully understood. To examine these interactions, in the present study we generated functional dopaminergic neurons and microglia-like cells from induced pluripotent stem cells (iPSC) from patients with PD associated to LRRK2 mutations, the most common cause of genetic PD, along with their gene-corrected isogenic controls. We then isolated T cells from the same LRRK2 PD patient and proved their functionality. Upon co-culture, LRRK2-PD T-lymphocytes induce neurodegeneration in LRRK2-PD dopaminergic neurons, whereas LRRK2-PD microglia-like cells do not. However, dopaminergic cell death began earlier and was exacerbated when DAn were cultured with LRRK2- PD microglia-like cells and patients T cells. Using DAn differentiated from the reporter LRRK2-PD SNCA-FLAG iPSC we demonstrated that the tagged α-syn is captured and phagocytosed by both LRRK2-PD and Iso microglia-like cells. Immunofluorescence analysis revealed that unstimulated LRRK2-PD microglial-like cells exhibit increased HLA class I and II complexes compared to the isogenic counterpart. Upon α-syn stimulation, LRRK2-PD microglia like-cells displayed increased HLA class II complex within endosomal compartment. Moreover, increased formation of HLA-DR/ α-syn complexes was observed. Overall, these data demonstrate that our human neuro-immune axis PD model is a valuable tool for studying the crosstalk between innate and adaptive immune cells in Parkinson’s disease pathogenesis, with potential implications for developing future interventions.
Targeting TREX2: an innovative keratinocyte-based therapy for psoriasis and atopic dermatitis
(Universitat de Barcelona, 2024-12-19) Filgaira Enri, Ingrid; Soler Prat, Concepció; Ciruela Alférez, Francisco; Universitat de Barcelona. Departament de Patologia i Terapèutica Experimental
[spa] The fact that TREX2 is specifically expressed on KCs, upregulated in psoriatic skin and its loss leads to a decreased inflammation in mice models of the disease, points TREX2 as a candidate target for new KC-based PsO therapies. We hypothesize that the inhibition of TREX2 can be as effective as the most advanced available treatments, which mainly target the immune system, while avoiding their adverse broad immunosuppressive effects. Therefore, blocking TREX2 activity could be a promising non-immune and skin-specific approach to diminish KC-derived inflammation and avert the adverse side-effects associated to systemic treatments targeting the immune system. In this context, the global aim of the present PhD thesis was to identify, develop and characterize specific and effective TREX2 inhibitory compounds for PsO treatment and other TREX2-driven diseases and further determine their suitability and efficacy for future clinical trials. The specific goals were: 1. To identify and characterize specific, selective, potent and safe TREX2 inhibitory compounds to treat PsO. 2. To evaluate the role of TREX2 in AD and as therapeutic target in this disease. 3. To characterize the molecular mechanisms of TREX2 inhibitors.
Natural cannabinoids against Alzheimer’s disease: a role in glutamatergic regulation
(Universitat de Barcelona, 2024-12-05) Sánchez Fernández, Nuria; Aso Pérez, Ester; Universitat de Barcelona. Departament de Patologia i Terapèutica Experimental
[eng] Alzheimer's disease (AD), characterized by the loss of memory and other cognitive abilities, is the predominant form of dementia. Given the impact it has on individuals, affected families and health systems, it is considered a serious problem globally. Current treatments for this disease only minimally reduce the progression of cognitive and psychiatric symptoms, without offering a cure, which is why numerous alternative therapeutic strategies are currently being studied. In this sense, animal models are a valuable tool to understand the pathophysiological mechanisms that characterize the disease and identify suitable targets. During the development of this doctoral thesis, the APP/PS1 mouse has been used as an animal model of AD to study one of the mechanisms involved in the early stages of the disease: dysregulation of the glutamatergic system. It is characterized by an excess of the excitatory neurotransmitter glutamate, which causes excitotoxicity and neuronal damage. The endocannabinoid system (ECS) is a neuromodulatory system that is involved in numerous physiological functions, including glutamatergic transmission, which could be of interest in the context of neurodegenerative diseases such as AD. For this reason, there is a growing interest in the study of the therapeutic potential of natural cannabinoids (substances obtained from the Cannabis sativa plant) in AD and other neurodegenerative diseases, especially with regard to the combination of the two most abundant, Δ9-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD). The hypothesis of this doctoral thesis is that the combination of Δ9-THC and CBD is capable of regulating aberrant glutamatergic activity in a model of AD, thus contributing to the efficacy of this treatment as a therapy against this disease. In this context and based on previous evidence, the adenosine A2A receptor (A2AR) could contribute to the effects of Δ9-THC and CBD. To validate the working hypothesis, the following objectives were proposed: 1) To characterize the dysregulation of the glutamatergic system in the hippocampus of the APP/PS1 mouse, used as a model of AD. 2) To explore the effects of Δ9-THC and CBD on the alterations of glutamatergic activity observed in the hippocampus of APP/PS1 mice. 3) To evaluate the role of A2AR in the effects of Δ9-THC and CBD.
Ankle lateral instability: arthroscopic treatment strategy
(Universitat de Barcelona, 2024-11-28) Cordier, Guillaume; Dalmau-Pastor, Miki; Universitat de Barcelona. Departament de Patologia i Terapèutica Experimental
[eng] - INTRODUCTION: A lateral ankle sprain is the most common injury to the lower extremity. It can cause chronic ankle instability (CAI) in 20 to 30% of cases with an impact on public health. Surgical treatment is recommended in case of failure of conservative treatment to avoid functional sequelae. Until now, with open surgery, 2 surgical "gold standards" were practiced: repair with retinaculum augmentation called "Broström-Gould" and anatomical reconstruction with graft. - HYPOTHESIS: Surgical treatment has evolved a lot over the last decade with the development of arthroscopic techniques. Ligament repair or reconstruction can be performed arthroscopically. We hypothesize that arthroscopic treatments are safe and give an equivalent or better result than open surgery treatment. - OBJECTIVES: The aim of the thesis is to study the data of cohorts of patients with 2 years of follow-up later. Arthroscopic repair of the lateral ligaments of the ankle with retinaculum augmentation (arthroscopic Broström-Gould) and after endoscopic reconstruction of the lateral ligaments of the ankle. It is then possible to compare both data with the published literature for techniques.
CB(1), A(2A) and D(2) receptors balance as a target against psychotic-like symptoms associated to Alzheimer’s disease and cannabis abuse
(Universitat de Barcelona, 2024-10-31) Gómez Acero, Laura; Ciruela Alférez, Francisco; Aso Pérez, Ester; Universitat de Barcelona. Departament de Patologia i Terapèutica Experimental
[eng] Psychosis is a mental health problem characterized by a loss of contact with reality, which is considered the defining feature of schizophrenia spectrum disorders. However, psychotic symptoms may occur in other medical conditions such as neurodegenerative diseases and substance abuse or intoxication. Here, we have focused on the psychotic symptoms associated with AD and chronic use of cannabis because of two main reasons: (i) their neurobiological bases are poorly understood, what limits the development of specific and effective treatments, and (ii) epidemiology data suggest that the prevalence of both disorders will increase worldwide in the next years due to the increase in life expectancy and in cannabis consumption among the population. Thus, the overall aim of this thesis project is to provide novel insights about the neurobiological bases underlying such psychotic symptoms with the ultimate goal to identify new therapeutic targets to reduce the negative consequences of these mental problems in the quality of life of patients. The hypothesis of this study is that alterations in the interplay between DAergic, adenosinergic and endocannabinoid systems are a common neurobiological substrate for different psychotic disorders and, consequently, that reversing such unbalance between these neurotransmitter systems could lead to therapeutic consequences. To evaluate this hypothesis, the specific aims (SA) of this thesis project are: - SA1: To evaluate the age-dependent psychotic symptoms in an AD model (i.e. APP/PS1 mice). - SA2: To correlate these psychotic symptoms in APP/PS1 mice with alterations in the balance between relevant components of the DAergic, adenosinergic and endocannabinoid systems. - SA3: To test the potential therapeutic properties of targeting adenosinergic and endocannabinoid systems against the psychotic symptoms in APP/PS1 mice. 84 - SA4: To evaluate the long-lasting behavioral consequences of chronic exposure to high doses of THC during the adolescence and the adulthood. - SA5: To correlate these behavioral consequences of THC exposure in the balance between relevant components of the DAergic, adenosinergic and endocannabinoid systems. - SA6: To explore the anatomical substrates underlying these behavioral consequences of THC exposure by analyzing the functional connectivity alterations between specific brain areas in treated mice. - SA7: To investigate the differential effects of THC on the activity of DAergic neurons in the VTA depending on the age of the individuals and the role played by A2AR in such effects as a potential substrate underlying the higher vulnerability to the psychotic symptoms induced by cannabis reported in the adolescence.
Super-Cationic peptides: insights on their antimicrobial action
(Universitat de Barcelona, 2022-11-25) Pérez Guillén, Isabel; Viñas, Miquel; Sierra Ortigosa, Josep Maria; Universitat de Barcelona. Departament de Patologia i Terapèutica Experimental
[eng] Bacteria have been to the human civilization a threat as well as an ally, since the beginning of time. The introduction of antibiotics into clinics to treat infections was one of the best medical achievements ever accomplished. However, the increase of antimicrobial resistance (AMR) as well as the failure to develop new antibiotics is a cause of much concern, and is essential to create new strategies of development in order to fight this global threat. Bacterial cell membranes are a key factor in the emergence and evolution of resistant bacteria, due to their capacity to interact with other organisms, the environment or both. Therefore, the study of the physical and chemical properties of biological membranes is essential to understand their role in resistance. One path to develop new strategies and create new antimicrobial agents to address the global threat of resistant bacteria is the synthesis of new compounds as an alternative therapy, like synthetic antimicrobial peptides (AMPs). In this context, the active exploration of peptides from the new AMP family, named the Super-Cationic peptide dendrimers (SCPDs) was carried out. HYPOTHESIS: The main hypothesis of this thesis is that synthetic peptides may have antimicrobial properties against clinical bacterial strains, and may provide weapons to treat infections caused by multidrug-resistant strains. A deep research of the mechanisms underlying antimicrobial action should be carried out. OBJECTIVES: The main objective of this thesis is to determine the antimicrobial properties of synthetic peptides from a new AMP family, SCPDs, specifically the peptide G1OLO-L2OL2. The secondary objectives of this thesis are divided in three parts: First, finding the eventual target and interactions those peptides may have with the bacteria, particularly its possible interaction with the bacterial cell membrane. Second, to determine the cytotoxicity these molecules may have over eukaryotic cells. And third, to observe the effect these molecules may have on the bacteria, the cell membranes and on artificial membranes by means of atomic force microscopy (AFM).
Streptococcus pneumoniae, resistance and disease in adults in the era of conjugate vaccines
(Universitat de Barcelona, 2021-07-27) Càmara Mas, Jordi; Ardanuy Tisaire, María Carmen; Universitat de Barcelona. Departament de Patologia i Terapèutica Experimental
[eng] Streptococcus pneumoniae is an important human pathogen, being one of the main etiological agents of serious infectious diseases such as pneumonia or meningitis and other less serious like otitis media. S. pneumoniae adheres to the respiratory epithelium being part of the nasopharyngeal microbiome. This nasopharyngeal colonization is more frequent in young children (colonization percentages of around 27-65%) than in adults with a frequency of colonization around 10%. In recent years, the introduction of the pneumococcal conjugate vaccines (PCVs) for children has changed the epidemiology of pneumococcal diseases. Since vaccination by PCVs prevents the carrier status of those strains expressing serotypes included in the vaccine, their transmission is reduced and a beneficial group protection (“herd protection”) occurs in the non-vaccinated population. Three PCVs have been marketed in Spain: PCV7 (introduced in 2001), PCV10 (in 2009) and PCV13 (which replaced PCV7 in 2010). All of them were administered voluntarily until 2015, when PCV13 became part of the routine vaccination schedule for children and were subsidized by the government. The main objective of this thesis is to study the impact of the introduction of PCVs in children on invasive pneumococcal disease in adults (IPD). The works included in this thesis focus on describing the effects of the introduction of PCVs in Spain on the epidemiology of IPD in adults. Specifically, this thesis has studied the changes in the incidence of IPD, in the rates of antibiotic resistance, in the clones responsible for IPD and in the clinical characteristics of patients in the era of PCVs. The first part of this thesis shows how the introduction of PCV13 in Spain caused an early decrease in the incidence of adult IPD. The global incidence of IPD decreased by 33.9% from 2008-2009 to 2012-2013. These results were related to a decrease in the incidence of those serotypes included in PCV7 (decrease of 52.7%) and of those additional PCV13 (decrease of 55.0%), while disease caused by non-vaccine serotypes remained stable. The reduction in IPD rates was greater in the Community of Madrid, a community in which PCVs (PCV7 and later PCV13) were included in the routine childhood vaccination schedule in 2009. Regarding the additional PCV13 serotypes all decreased except serotype 3 which remained stable, possibly indicating a lower effectiveness of PCV13 in preventing IPD due to this serotype. On the other hand, a change was observed in two non-vaccine serotypes: first, a decrease in the incidence of serotype 8, probably due to the disappearance of an outbreak in young adults in the Madrid region in previous years. And, second, an increase in serotype 6C that may indicate a minor effect of the described cross-reactivity with serotype 6A (included in PCV13). Regarding antibiotic susceptibility, a non-statistically significant increase in the frequency of isolates non-susceptible to penicillin (MIC ≥ 0.12 mg/L, 22.7% vs 26.8%, p = 0.065), cefotaxime (MIC > 0.5 mg/L, 10.1% vs 12.5%, p = 0.14) and MDR isolates (13.1% vs 16.2%, p = 0.08) was observed. In spite of that, the incidence of IPD caused by these resistant isolates remained stable over time. Because we detected an increase in the rates of non-susceptibility to penicillin and cefotaxime after PCV13, in the second work we aimed to study the local impact of PCVs on the incidence of IPD due to MDR and penicillin non-susceptible strains. The period analysed (1994-2018) includes two key moments for the study of the evolution of IPD in Spain: the introduction of PCV7 and PCV13 vaccines in 2001 and 2010, respectively. In this study, we analysed changes in the incidence of IPD, antimicrobial susceptibility, evolution of clones responsible for multidrug-resistance, and clinical characteristics of patients. Our results demonstrated that PCVs have been beneficial in reducing the incidence of resistant strains (risk ratio [RR] comparing the period 1994- 2000 with the period 2016-2018: 0.70 [95% CI 0.53–0.93]) while the effect on the incidence of IPD due to susceptible strains was limited (RR 0.96 [95% CI 0.80–1.16]). Therefore, while the overall incidence of IPD in the adult population has shown a slight non-statistically significant decrease (RR 0.87 [95% CI 0.74–1.02], the rates of resistance to most antimicrobials have been reduced. With respect to genotypes associated with antimicrobial resistance, about 50% of IPD due to resistant strains in the 2016-2018 period was caused by two clones: Spain9V-ST156 (serotype 11A) and Denmark14-ST230 (serotype 24F). We also evaluated the putative coverage of two vaccines that are under development (PCV15 and PCV20), concluding that almost half of the current resistant strains express serotypes not included in these vaccines, so their efficacy with respect to antibiotic resistance may be limited. Finally, we also analysed the clinical characteristics of the patients with resistant IPD, showing that, despite not being an independent factor associated with mortality, resistant strains appear in patients with worse prognosis (advanced age, more comorbidities, nosocomial acquisition and previous antibiotic therapy) which leads to higher mortality. The last part of this thesis focuses on the study by WGS of the genetic evolution of the clone Spain9V-ST156 (PMEN3) over a period of 30 years (1987–2016). In Spain, this clone has been the most frequent among β-lactam resistant strains causing IPD and has classically been associated with serotypes 9V and 14. In our study, this clone was responsible for more than 12% of all cases of IPD in adults during the 90s. The introduction of PCV7 in 2001 (which included serotypes 9V and 14) caused a progressive decrease in the incidence of the PMEN3 clone. Despite this, the appearance of strains expressing serotype 11A (not included in PCV13) in recent years has allowed this clone to persist as a cause of IPD. The WGS study allowed us to identify 6 lineages within this clone with different prevalence over time. All lineages were derived from the original ST1569V except the last one (ST652111A) which originated from the ST8389V lineage through a capsular switching event and the acquisition of capsular type 11A. Using WGS, it was also possible to verify the existence of two different serotype 14 lineages (ST156) that independently emerged during the 1990s and that could not be easily detectable with the usual typing techniques (PFGE or MLST). Furthermore, the study showed evidence of recombination events in three chromosomal regions: the capsular operon (capsular switching) and the adjacent regions containing pbp2x and pbp1a genes, the murM region, and the region containing pbp2b–ddl genes. Then, all the chromosomal regions that had undergone changes were associated with capsular switching or β-lactam resistance, which evidenced the enormous impact of the introduction of PCVs and broad-spectrum β- lactams on the evolution of this clone. Finally, despite the genetic changes detected, no differences in the clinical characteristics of patients were found, which highlighted the importance of the PMEN3 genetic background. In this sense, the majority of strains harboured prophages with genes coding for PblB proteins, which have recently been associated to platelet aggregation and increased mortality in IPD.
Estudio de las resistencias a los fármacos y los mecanismos moleculares más frecuentes en Mycobacterium tuberculosis complex en España
(Universitat de Barcelona, 2021-05-03) Pérez Risco, Daniel; Alcaide Fernández de Vega, Fernando; Universitat de Barcelona. Departament de Patologia i Terapèutica Experimental
[spa] En España, existe una monitorización insuficiente de la tuberculosis, no incluyendo en la declaración obligatoria la notificación de algunos datos relevantes del paciente y de laboratorio. Esta infra-notificación afecta, de forma relevante, a gran parte de los datos de sensibilidad a los fármacos contra la tuberculosis. De este modo, muchos de los datos existentes proceden de estudios puntuales más o menos representativos a nivel geográfico y/o temporal. Por ello los objetivos de esta tesis son: 1) conocer en la actualidad la resistencia a los fármacos antituberculosos en múltiples áreas geográficas de España, además de analizar los mecanismos moleculares relacionados con la misma y determinar qué factores clínico-epidemiológicos pueden estar asociados a la resistencia (Estudio REMOTUBES; 2016-2018); 2) conocer y analizar cómo ha evolucionado la resistencia a los fármacos antituberculosos en España en la última década (2006-2016), y determinar qué factores clínico-epidemiológicos pueden estar implicados en la resistencia a la tuberculosis en el país; 3) evaluar la eficacia de un sistema de PCR a tiempo real ultrasensible para la detección de M. tuberculosis complex y su multirresistencia; y 4) evaluar el rendimiento diagnóstico del sistema molecular Anyplex II MTB/MDR/XDR para la detección rápida de las resistencias a los fármacos antituberculosos más importantes en M. tuberculosis complex.
Epidemiología y estudio de la dinámica poblacional en las infecciones por Staphylococcus aureus resistente a meticilina
(Universitat de Barcelona, 2023-04-27) Vázquez Sánchez, Daniel A.; Domínguez Luzón, Ma. Ángeles (María Ángeles); Camara Mas, Jordi; Universitat de Barcelona. Departament de Patologia i Terapèutica Experimental
[spa] Uno de los problemas de salud de más relevancia a nivel global es el de las infecciones por bacterias resistentes a los antibióticos. La Organización Mundial de la Salud (OMS) junto al Centro para el Control y la Prevención de Enfermedades (CDC) consideran a los patógenos resistentes a los antibióticos como una de las amenazas inminentes para la salud humana. El listado de microorganismos llamado ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter bau-mannii, Pseudomonas aeruginosa y Enterobacter spp), recoge las especies patógenas de más relevancia por su potencial de presentar resistencia a múl-tiples antibióticos. En ese sentido, S. aureus destaca por la adquisición de mecanismos de resistencia a casi todos los antibióticos empleados para su tratamiento, como los β-lactámicos, los glicopéptidos y las oxazolidinonas. MRSA a su vez, es uno de los patógenos resistentes más relevante, con una amplia distribución y asociado a infecciones hospitalarias, comunitarias e incluso ganaderas. También destaca su gran plasticidad genética, que le permite adaptarse a condiciones ambientales diversas, su capacidad patogénica y la frecuente combinación de resistencias a otros grupos de antibióticos. La accesibilidad durante la última década a técnicas de secuenciación del genoma completo de los microorganismos y la disminución de su coste económico, han cambiado la perspectiva de los estudios epidemiológicos y de la tipificación molecular. Esta tecnología ofrece la posibilidad de analizar genomas completos con un nivel de discriminación de unos pocos nucleótidos y establecer relaciones genéticas con una precisión inalcanzable por técnicas moleculares clásicas. Con una única técnica, se puede obtener una gran cantidad de información para el estudio de genes relacionados con la resistencia antibiótica, la producción de toxinas, la adherencia bacteriana y estudios de elementos móviles y fijos. Por ello, esta tesis doctoral pretende analizar la epidemiología de las infecciones por MRSA y las características clínicas de la infección bacteriémica; la dinámica poblacional de los clones de MRSA y su evolución en el tiempo y, por último, la base molecular de la resistencia a los antibióticos en MRSA, mediante la incorporación de tecnologías de secuenciación del genoma completo.
Epidemiology, population structure, and pathogenesis of opportunistic respiratory tract colonising bacteria
(Universitat de Barcelona, 2022-12-21) Carrera Salinas, Anna; Domínguez Luzón, Ma. Ángeles (María Ángeles); Martí Martí, Sara; Universitat de Barcelona. Departament de Patologia i Terapèutica Experimental
[eng] The bacterial colonisation of the respiratory tract is critical for health as it establishes a symbiotic relationship between the bacterial community and the host. Haemophilus influenzae and Staphylococcus aureus are two opportunistic microorganisms that usually colonise the respiratory tract, and while their interaction is usually asymptomatic, in some cases colonisation is the first step before the development of severe infections. From a microbiological perspective, the goals of this thesis focus on studying the epidemiology, the population structure, and the pathogenesis of these microorganisms, since changes in some of these factors may influence the development of serious diseases. We provided an update on H. influenzae disease at the Hospital Universitari de Bellvitge between 2014 and 2019, and compared its evolution to a previous period, 2008 and 2013. The overall incidence of H. influenzae disease was 2.07 cases per 100,000 population, and remained constant between the two periods. During the second period (2014-2019), the rate of ampicillin resistance increased from 10% to 17.6%, mainly due to the production of β-lactamase TEM-1. Non-typeable H. influenzae (NTHi) strains were the leading cause of invasive H. influenzae disease in both study periods (84.3% and 85.3%, respectively). NTHi population was genetically heterogeneous, and its classification into six clades (I-VI) based on the presence and absence of 17 accessory genes showed that most of them were clustered in clade V. Strains from clade V and grouped the most frequent NTHi STs (ST3, ST103, ST160), and were associated with the production of β-lactamase TEM-1. On the other hand, capsulated strains were uncommon in invasive disease, with serotype f being the most prevalent (8.8%). We analysed the population structure and the pangenome of capsulated strains, paying especial attention to serotype f, which is rising among cases of invasive disease in Europe. Capsulated genomes belonged to a small number of clonal complexes (CCs) associated with each serotype, which were highly clonal. The pangenome analysis revealed that serotype b genomes had a larger pool of genes and more accessory genes per genome than the other serotypes. The analysis of single nucleotide polymorphisms (SNPs) showed that the total number of SNPs in serotype f was significantly lower than serotypes a, b, and e, indicating low variability within CCs of serotype f. These findings support the high genetic stability of this serotype observed in the strains isolated from different countries, revealing no relationship between phylogeny and geographical origin. We also examined the genetic changes of H. influenzae during respiratory colonisation, which could contribute to the progression of chronic obstructive pulmonary disease (COPD). We studied the respiratory isolates from 15 patients with COPD and prolonged azithromycin therapy, of which, four had persistent H. influenzae colonisation, and two were persistently colonised by H. parainfluenzae. All persistent lineages isolated before treatment were azithromycin susceptible, but antibiotic pressure led to resistance development in the first months. Different resistant genetic determinants were identified in each lineage, including successive mutations in 23S rRNA, mutations in the genes encoding ribosomal proteins L4 and L22, and the acquisition of MefE and MsrD efflux pumps. Other genetic variation mainly occurred in genes associated with cell wall and inorganic ion transport and metabolism, such as licA and hgpB genes, linked to lipooligosaccharide synthesis and heme uptake, respectively. Regarding S. aureus, we compared the biofilm formation of endemic (CC5, CC8, and CC22) and sporadic (CC1, CC30, CC45, CC72, and CC398) methicillin-resistant S. aureus (MRSA) clones in our healthcare setting. The endemic clones produced more biofilms than the sporadic clones. Furthermore, allelic variant 1 of sasG, encoding the S. aureus surface protein G, was found in the most prevalent clones (CC5 and CC8), in correlation with increased biofilm formation. Thus, allelic variant 1 of sasG could be considered an important virulence factor that may favour bacterial survival. Furthermore, we also examined the genetic changes associated with intracellular invasion in patients with respiratory colonisation and subsequent bacteraemia caused by S. aureus. The bacteraemic isolates showed a significantly lower ability to invade lung epithelial cells than the respiratory isolates, and genetic analysis revealed differences in genes encoding proteins involved in the interaction of epithelial cells with the bacterial surface.
Synaptic transmission in autaptic circuits: presynaptic homeostatic plasticity and microtubule dynamics
(Universitat de Barcelona, 2022-04-22) Velasco Domínguez, Cecilia; Llobet Berenguer, Artur, 1972-; Terni, Beatrice; Universitat de Barcelona. Departament de Patologia i Terapèutica Experimental
[eng] Synapses are the contact sites where the transfer of information from the presynaptic to the postsynaptic site occurs. Correct processing of information requires that synapses continuously adapt their properties to an ever-changing environment. Here, we describe novel aspects that help understand the function of presynaptic terminals in the context of a simple neuronal network. The exuberant number of synaptic contacts formed during the development of the nervous system is selectively refined by a process of synapse elimination. In the current thesis we have exploited the action of peptide p4.2, a 20 amino acid fragment located in the C-terminus of the glial secreted protein SPARC, which promotes synapse elimination in an autaptic circuit. We found that neurons forming autaptic synapses sense and compensate for synapse elimination by activating a mechanism of presynaptic homeostatic plasticity driven by presynaptic potentiation and rapid assembly of new synaptic contacts. Both actions occur concomitantly, indicating that the formation of novel synapses is associated to an overall increase in presynaptic calcium influx. To further investigate the molecular mechanisms underlying this compensatory response, we moved our interests to the participation of microtubules in the maintenance of synaptic connectivity and synaptic strength. This question could only be addressed by gaining a better understanding of the participation of microtubules in presynaptic terminal function. By providing ultrastructural, morphological, and physiological evidence we have shown that microtubule plus-ends transiently invade presynaptic boutons and that microtubule instability is directly involved in the regulation of spontaneous neurotransmitter release probability. Microtubule polymerization is also important to postsynaptic function, indicating microtubule dynamics might be involved in forms of postsynaptic plasticity. Altogether, the development of this project has allowed to identify yet unknown mechanisms key for better understanding the cell biology of presynaptic terminals. These findings should not only be considered in the context of a simple neuronal network, chosen because of its unique experimental possibilities, but as fundamental neuronal properties. Yet, future research in complex systems are required to further validate our findings.
La senyalització purinèrgica en l'endometriosi. Paper en el teixit eutòpic i ectòpic
(Universitat de Barcelona, 2021-07-26) Trapero Candela, Carla; Martín Satué, Mireia; Universitat de Barcelona. Departament de Patologia i Terapèutica Experimental
[cat] L’endometriosi és una malaltia ginecològica, associada a un component inflamatòri crònic, caracteritzada per la presència de teixit endometrial fora de l’úter. L’etiologia de la malaltia encara és manté incerta i el seu diagnòstic sovint presenta alts retrassos, degut essencialment a la manca de tests de diagnòstic no invasius. L’adenosina trifosfat (ATP), una molècula proinflamatoria, promou i afavoreix el manteniment de l’estat inflamatori de l’endometriosi. A més, l’ATP influeix directament en les principals manifestacions clíniques de la malaltia: el dolor i la infertilitat. La senyalització purinèrgica, el grup de respostes biològiques a nucleòtids extracel·lulars com l’ATP o nucleòsids com l’adenosina, es troba alterada en moltes malalties amb un important component inflamatori,com és l’endometriosi. Previament, el nostre grup ha demostrat l’expressió de les ectonucleotidases, els enzims que regulen els nivells extracel·lulars d’ATP i adenosina, en l’endometri no patològic, amb canvis dependents d’hormones al llarg del cicle menstrual. Per una altra banda, vam determinar un increment de l’activitat nucleotidasa en el fluid contingut en endometriomes ovàrics, una presentació freqüent d’endometriosi. La nostra hipòtesi és que la senyalització purinèrgica juga un paper en l’endometriosi i que l’expressió diferencial de les ectonucleotidases pot utilitzar-se com un nou biomarcador de la malaltia. L’objectiu principal d’aquesta tesi és conèixer el paper de la senyalització purinèrgica, en particular de les ectonucleotidases, en l’endometriosi, per tal d’augmentar el coneixement sobre la fisiopatologia d’aquesta malaltia i determinar-ne possibles biomarcadors d’us diagnòstic i/o terapèutic. En la present tesi, ens hem centrat en l’estudi de l’expressió de diferents ectoenzims en el fluid contingut en endometriomes mitjançant immunoassajos, i en el teixit endometrial eutòpic i ectòpic de dones amb endometriosi mitjançant immunohistoquímica i assajos d’activitat ectonucleotidasa in situ. A més, metodològicament, s’ha optimitzat un protocol per a la detecció simultània de l’expressió proteica i l’activitat nucleotidasa. Altrament, s’ha establert un mètode d’obtenció de cultius primaris d’estroma endometrial de dones amb i sense la patologia i s’ha validat com una eina útil d’estudi de l’endometriosi. Els resultats obtinguts en l’estudi del contingut dels endometriomes han determinat els ectoenzims nucleòtid pirofosfatasa/fosfodiesterasa 1 (NPP1) i l’adenosina desaminasa (ADA) com a candidats a biomarcadors per l’endometriosi. D’altra banda, l’assoliment d’una millora metodològica en l’estudi de les ectonucleotidases ens ha permès caracteritzar l’expressió de la nucleòsid trifosfat difosfohidrolasa 2 (NTPDasa2) en el teixit endometrial humà. Hem determinat l’NTPDasa2 com a un nou marcador de cèl·lules mare mesenquimals endometrials (eMSCs) SUSD2+ i com a marcador de l’estroma basal, troballes importants per la medicina regenerativa. En el teixit endometrial ectòpic de dones amb endometriosi profunda, el subtipus més greu, hem demostrat la pèrdua d’expressió de les ectonucleotidases CD39 i CD73, principal eix d’hidròlisi de l’ATP. Així mateix, s’ha identificat l’NPP3 com a un nou marcador histopatològic de la malaltia, ja que únicament s’expressa en l’estroma endometrial eutòpic i ectòpic de les pacients amb endometriosi. Així doncs, l’expressió diferencial de les ectonucleotidases pot emprar-se com a biomarcador de la malaltia i presenta la senyalització purinèrgica com una via d’interès pel disseny de noves estratègies diagnòstiques i terapèutiques per l’endometriosi.
Adenosine receptors in Atrial Fibrillation
(Universitat de Barcelona, 2021-09-29) Godoy Marín, Héctor; Ciruela Alférez, Francisco; Universitat de Barcelona. Departament de Patologia i Terapèutica Experimental
[eng] Atrial fibrillation (AF) is the most common arrhythmia affecting 1 of every 20 persons older than 65 years and 9% of the octogenarians, and extensive research efforts have been undertaken to identify molecular, electrophysiological, and clinical mechanisms that contribute to the induction and maintenance of this arrhythmia. In spite of this, treatment of this arrhythmia remains deficient or inefficient, with frequent re-incidence of the arrhythmia after treatment because knowledge of the complex pathophysiology of the disease remains incomplete. However, dysfunctional calcium handling is gaining strength as a key contributor to the induction of cardiac arrhythmia. In the literature is described those pathological changes in adenosine A2A receptor (A2AR) expression and activation promote arrhythmogenic calcium release from the sarcoplasmic reticulum (SR) in atrial myocytes from patients with AF. Accordingly, this proposal aims to test the general working hypothesis postulating that adenosine receptors constitute a new source of therapeutical targets and biomarkers for prevention, risk-stratification, and treatment of atrial fibrillation. In the present work we analysed the A2AR expression and mRNA content from AF patients to determine its AF phenotype. Moreover, we analysed the impact of AF in peripheral tissues. Thus, A2AR expression was measured in peripheral blood mononuclear cells (PBMCs) from AF patients, finding its enhanced expression. In addition, ADA and adenosine plasma content was also altered in AF patients. Concomitantly, these results suggest a huge imbalance in adenosinergic signalling that is not hitting just cardiac tissue but also peripherical ones. Secondly, we aimed to develop new tools to use in the AF research. In our case were the HL-1 cells for in vitro assays and pigs for animal modelling. We compared the results on adenosine 1 receptor (A1R) and A2AR expression and heterodimerization of the induced AF phenotype in the two models with AF human patients. Interestingly, we found the same alterations (enhanced expression and reduced heterodimer levels) in all of them. These results point to a usefulness of these two models in the AF research. Moreover, this was the first time that heterodimer A1R-A2AR was detected in a protein:protein interaction assay. Nevertheless, further research is needed to understand the role and the impact of this imbalance during AF events. Also, we have found the same alterations in calcium handling related proteins in pig and humans with AF. We found a reduction in calsequestrin CSQ and PLB expression in both models, which probably contributes to a loss of calcium buffering in AF. In the last chapter, we aimed to develop new pharmacological approaches y precluding A2AR activation or enhancing A1R activity, which will probably restore the normal beating in the heart during the arrythmia. We successfully blocked A2AR activity by the SCH442416 full antagonist. Also, we enhanced A1R activity with CPA agonist and T62 PAM. In addition, we measured the impact on the heterodimerization in a heterologous system by transfected A1R-A2AR HEK cells. Our results shown that T-62 and SCH442416 does not affect heterodimer formation, being potentially useful in the AF treatment. Finally, we developed the same drugs from the optopharmacological point of view, in order to avoid side-off target effects. We have successfully developed the four light-sensitive compounds, which mimic the effect of the classical drugs under light conditions. This opens a new field to treat AF and other diseases. In conclusion, the present work has deepened into the AF understanding and treatment. We have revealed new insights related with AF through the ADA and Adenosine alterations found in plasma. Also, we have revealed new impacts of AF in surrounding cells that offer a new course to study or prevent and detect AF. We have additionally provided evidence for the use of relevant tools to research about AF in in vitro and animal models by using HL-1 cells and pigs. That will facilitate the future analysis of AF causes, effects, and treatment. In addition, novel findings of AF impacts have been found in the three systems through the heterodimeric A1-A2AR reduced formation and the alterations in CSQ, PLB and A1R expression. Finally, we showed new potential approaches to pharmacologically tackle this disease by antagonizing A2AR or potentiate A1R activity. In addition, we have demonstrated that the emerging field of optopharmacology could give us the chance to go further the classical pharmacology and possibly be useful for the development of light- dependent treatments in the near future.
Outer membrane: a key obstacle for new antimicrobial agents
(Universitat de Barcelona, 2021-07-23) Jorba Pedrosa, Marta; Viñas, Miquel; Universitat de Barcelona. Departament de Patologia i Terapèutica Experimental
[eng] INTRODUCTION: Antimicrobial resistance is one of the world’s major challenges in both microbiology and public health since infections caused by multidrug-resistant bacteria are reaching alarming levels. The world is currently facing a global antibiotics crisis and some new strategies need to be explored to tackle these resistant infections. The outer membrane is a differential structure of Gram-negative bacteria that works as a highly effective selective permeability barrier. Thus, the permeation through the Gram- negative cell envelope is a challenge for drug compounds to reach their targets. One path to open new antimicrobial perspectives is the chemical modification of old antimicrobial compounds that may result in optimized drugs with improved antimicrobial properties. In this context, the activity exploration of the derivatives of Microcin J25 and Trimethoprim was carried out. HYPOTHESIS: The main hypotheses of this thesis are that chemical modifications of current antibiotics may significantly contribute to overcoming the problems arising from the increase and spread of antibiotic resistance in bacteria. Moreover, the combination of these modified compounds with old-rescued antibiotics may contribute to the solution bases of the problem caused by resistance. Objectives: The main objective of this thesis is to determine the antimicrobial properties of chemically modified drug compounds. The secondary objectives of this thesis are divided in two parts: Microcin J25: Study of the antimicrobial activity of the modified Microcin J25 as well as determination of its toxicity. Trimethoprim: Study of the antimicrobial activity and cytotoxicity of the Trimethoprim derivatives, exploration of the effect of the new derivatives on the Dihydrofolate Reductase (DHFR) enzyme and start a computational approach to decipher the intimal mechanisms of action. METHODOLOGY: The antimicrobial activity of the derived compounds was explored against planktonic bacteria (studying the Minimum Inhibitory Concentration and the FIC index) and against sessile bacteria (exploring the Minimum Biofilm Eradication Concentration and the Biofilm Prevention Concentration). The growth curves of several microorganisms in contact with these compounds and in combination with colistin were also studied. The cytotoxicity of all the compounds was tested. An enzymatic assay with E. coli DHFR as well as a docking modelling were carried out to investigate the mechanism of action of TMP derivatives. RESULTS: With respect to Microcin MccJ25, it has been detected that the chemical modification of the compound resulted in a new peptide without antimicrobial activity. It acted synergistically with sublethal concentrations of colistin. When referring to Trimethoprim (TMP), some of the new derivatives showed antibacterial similar to that of TMP. Moreover, almost all the new TMP-like compounds acted synergistically with SMX. P. aeruginosa PAO1 was fully resistant to TMP and all its derivatives as well as to the combination of TMP-SMX. The combination of TMP, TMP- like molecules and SMX with colistin enhanced their antimicrobial efficacy against E. coli, P. aeruginosa and S. marcescens. Compounds 1a and 1b, like TMP, strongly inhibited the activity of the E. coli DHFR. Additionally, it was detected that the heterocyclic ring of the compound 1a fills the pocket occupied by the nicotinamide ring of NADPH. CONCLUSIONS: The present PhD thesis has led to some relevant conclusions. With respect to Microcin MccJ25, the chemical modification of the compound avoided its detection by the membrane receptor FhuA. Moreover, it did not affect the interaction with the target and, as the polymyxin facilitated the microcin entrance across the membrane, once inside the cell, the new compound retained its ability to inhibit the growth of bacteria. When referring to Trimethoprim (TMP), the derivatives showed interesting antimicrobial activities acting synergistically with SMX. The combination of TMP, TMP- like molecules and SMX with colistin enhances their antimicrobial efficacy by permeabilizing the cells. Compounds 1a and 1b, like TMP, strongly inhibited the activity of the E. coli DHFR and it was suggested that both molecules interact with the analogues during inhibition. The search of new antimicrobial compounds is one of the main pathways to overtake bacterial resistance to antibiotics. All putative compounds should be tested in conditions in which outer membrane role as permeability barrier is inactivated. Their assay together with sublethal concentrations of colistin is proposed as one of the methods of election.
Irrigation with Laser-Activated sodium hypochlorite: An antimicrobial alternative in endodontics
(Universitat de Barcelona, 2019-10-21) Betancourt Henríquez, Pablo Andrés; Viñas, Miquel; Arnabat Domínguez, Josep; Universitat de Barcelona. Departament de Patologia i Terapèutica Experimental
[eng] Bacteria and their sub-products are the main cause of the occurrence and perpetuation of endodontic infection. However, only a few bacterial species are seen as responsible of persistent endodontic infections, among them Enterococcus faecalis is the most frequently isolated species. It has several virulence factors, such as aggregation substances, enterococcal surface protein (Esp), “endocarditis and biofilm-associated pili” (ebp) and cytolysin. Moreover, its high resistance to antibacterial agents is emhanced by its ability to form biofilm. Due to the complex and unpredictable root canal morphology, the complete removal of smear layer and bacterial biofilm is difficult. Recently, laser-activated irrigation (LAI) has been introduced as an alternative to achieve a deeper cleaning and disinfection of the root canal system. Its mechanism of action is based on the generation of cavitation bubbles, through the absorption of laser energy by the irrigant. The most used lasers are from the Erbium family, Er, Cr: YSGG (2780nm) and Er: YAG (2980nm). Sodium hypochlorite (NaOCl) is considered the "gold standard" of endodontic irrigators. It has a broad antibacterial spectrum and is capable of dissolving organic tissue. It is used in a range between 0.5% and 6%, varying its degree of effectiveness. Nevertheless, at high concentrations it is toxic, causing damage to endothelial cells and the periodontal ligament cells, which generates an acute inflammatory reaction and pain. Hence, the aim of this thesis was to explore the bactericidal effect of low concentration of NaOCl activated by Er, Cr: YSGG laser-activated irrigation against E. faecalis biofilms in root canals in order to decipher if it may be similar to the one achieved by high concentrations of NaoCl. IN IN VITRO ROOT CANAL MODEL: The main objective of the first stage of this thesis was to build a laboratory model to simulate the conditions inside a single-tooth root canal. The Pasteur pipettes were inoculated with E. faecalis ATCC 29212 for 24 hours bacterial colonization and the subsequent formation of biofilm in the proposed in vitro model was demonstrated by atomic force microscope. The second essential point of this part was to determine antimicrobial capacity of Er,Cr:YSGG laser against E.faecalis. In addition, passive ultrasonic irrigation was also tested. Laser-activated irrigation demonstrated higher antimicrobial activity than passive ultrasonic irrigation. The final stage of this first part consisted in the analysis and measurement of the nano-roughness by atomic force microscopy of the cells treated and its comparison with that of untreated cells. EXTRACTED TEETH: This part was focused on the endodontic preparation of extracted human teeth. The root canals were instrumented by a crown-down / step-back technique using conventional sequence of 0.02 taper files up to the master # 55. The teeth were inoculated with E. faecalis ATCC 29212 for 10 days. The following stage was focused on the study of the antibacterial action of low concentration of NaOCl activated by Er,Cr: YSGG laser in extracted human teeth. The antimicrobial effectiveness of laser-activated irrigation was compared with passive ultrasonic irrigation activation and conventional manual irrigation. Er,Cr:YSGG laser and 0.5% NaOCl showed a considerable synergistic action. Finally, the last part was focused on the microscopic visualization of the samples, to complement the results of the microbiological count. The scanning electron microscope was used to determine the degree of effectiveness of bacterial biofilm and smear layer removal both on the dentin surface and inside the dentinal tubules. Additionally, the confocal scanning electron microscope was used to visualize the proportion of alive and dead bacteria after treatment. The results obtained in this thesis show that laser-activated irrigation is a therapeutic alternative for infections caused by E. faecalis inside of root canals.
Desarrollo de la podología en España
(Universitat de Barcelona, 2009-09-09) Novel Martí, Virginia; Corbella i Corbella, Jacint, 1937-; Universitat de Barcelona. Departament de Patologia i Terapèutica Experimental
[spa] La Podología es una especialidad sanitaria que a lo largo de su desarrollo ha estado sometida a dualidades empíricas y científica, lo que ha originado que su evolución le haya relegado a un nivel supeditado a otras disciplinas médicas hasta mediados del siglo XIX. Se abordan algunos aspectos de esta evolución , así como también aquellas disposiciones legales que han influido en la profesionalización de la Podología. El hecho de describir la evolución de una profesión, no deja de ser equivalente a la descripción de una evolución cultural de la sociedad, historia y legislación. Especial interés, el análisis de las interacciones que configuran la profesionalización y el papel del podólogo en relación a otras profesiones sanitarias. Esta tesis ha pretendido reflejar la existencia y evolución de una actividad antigua que ha originado una profesión sanitaria.
Experimental Approaches for Pulp Tissue Regeneration
(Universitat de Barcelona, 2019-10-22) Bucchi Morales, María Cristina; Manzanares Céspedes, María Cristina; Anta i Vinyals, Josep Maria de; Universitat de Barcelona. Departament de Patologia i Terapèutica Experimental
[eng] The aim of this PhD thesis was to study experimental approaches for revitalization of necrotic teeth. Revitalization, also known as regenerative endodontic procedures (REPs), is a relatively new treatment for necrotic teeth which tries to regenerate the dentine-pulp complex instead of obturating the root canal with biologically inert materials (root canal treatment). Until very recently, the most reliable option for the treatment of immature necrotic teeth was apexification followed by root canal treatment. However, endodontically treated teeth remain devitalized throughout the patient's lifetime and therefore defenceless to new caries lesions, as the absence of pulp implies the lack of tooth immune mechanisms. On the contrary, the regeneration of the dentine-pulp complex allows further root development and aims to recover the natural immune and secretory system of the pulp, making teeth more resistant to future lesions or traumatisms. The therapy was developed to treat necrotic immature teeth (i.e. those that have not completed their root development). Clinically, the outcomes can be considered successful since there is a resolution of the symptomatology, healing of the apical pathosis and further root development in most cases. However, histological analysis has demonstrated that the tissues formed after the therapy are reparative tissues – such as cementum-like tissue – instead of dentine, as well as an unorganized connective tissue, instead of pulp with its characteristic odontoblast layer. Currently, numerous efforts are being made to shed light on the clinical and biological aspects involved in the regeneration of pulp. Chapter 1: As previously said, evidence shows that no dentine but reparative tissues (cementum-like tissue) are responsible for the root development after regenerative endodontics. As cementum is less hard and less elastic than dentine, the question arises whether a root with apposition of cementum can endure mechanical stress similarly to roots completed by dentine. Thus, one of the objectives of this thesis was to compare the biomechanical performance of cementum- and dentine-reinforced teeth, and therefore to evaluate the biomechanical advantages of dentine regeneration after regenerative endodontics. We developted a finite element model of cementum- and dentinereinformed teeth and studied the stress distribution after the simulation of biting, trauma and orthodontic movement. The results showed that apposition of hard tissue (whether cementum or dentine) after REPs reduces mechanical stress on 17 immature teeth and, more important, that the formation of dentine is advantageous because it, unlike cementum, facilitates an even stress distribution throughout the root. As far as we know, ours was the first study showing the biomechanical advantages of dentine regeneration. Chapter 2: Odontoblasts are post-mitotic cells that secrete dentine. The isolation and culture of odontoblasts may open numerous possibilities to study this cell type under standardized conditions, shedding light on their roles in dentine formation, immune defence and transmission of external stimuli. We evaluated different protocols of enzymatic treatment to isolate primary odontoblasts from human molars. The results showed that, regardless of the enzymatic solution used, odontoblasts in culture did not remain viable after 24 h. Additionally, we identified increased expression of nestin (NE), bone sialoprotein (BSP) and dentine matrix acidic phosphoprotein 1 (DMP1) in the odontoblast layer compared to pulp fibroblasts. Though primary odontoblasts can still not be cultivated after isolation, characteristic genes were identified to differentiate odontoblasts from pulp fibroblasts. Chapter 3: We analysed the effects of autologous platelet concentrates (APCs) in the clinical and histological outcomes of the therapy and the different clinical protocols clinically used through systematic reviews. The results indicated that APCs improve the clinical and radiographic outcomes of regenerative endodontics since the teeth treated with APCs achieved significantly better thickening of the dentine walls and root lengthening. However, true regeneration of pulp was not achieved with the addition of platelet concentrates, which only stimulated tissue repair. Additionally, most of the studies did not follow a standard clinical protocol for regenerative endodontic therapy and used irritant and intracanal medicaments that are cytotoxic and affect the differentiation and adherence of the stem cells. Chapters 4 and 5: As will be mentioned in detail, a small apical foramen acts as a physical barrier that hinders tissue ingrowth into the root canal and therefore reduces the possibility of revitalization of mature teeth. We studied different methods for apical foramen enlargement of mature teeth as a basis to apply it in a further animal study. We analysed manual instrumentation at different working lengths and apicoectomy on extracted human teeth and in situ teeth. We concluded that apicoectomy is not an effective technique for apical foramen enlargement and therefore should not be used for that purpose. Instrumentation 18 0.5mm beyond the apex resulted in the most effective technique. Later, we performed an animal study and evaluated pulp tissue regeneration/repair in mature teeth and the differentiation of the stem cells from the periapical tissues into odontoblast-like cells by adding preameloblast-conditioned medium. Preameloblast-conditioned medium was applied in pulpectomized ferret canines, whose apical foramina were enlarged using the previously developed method. We observed vascularized connective tissue occupying the apical third of the canal space in 50% of the teeth, showing the potential of revascularization of mature teeth. However, no odontoblast-like cells were observed showing that in vivo odontoblast-like differentiation of stem cells is still not possible with the tested technique. Chapter 6: Finally, we present here the preliminary data of characterization and odontoblast-like differentiation of amnion epithelial cells. Human amnion epithelial cells (hAECs) express pluripotent stem cell markers and have been proven to differentiate in cells of the three embryologic layers. However, as far as we know, these are the first experiments that have proved the potential of odontoblast-like differentiation of these cells in vitro. To induce the odontoblast-like differentiation, we seeded hAECs over dentine disks treated with EDTA and evaluated the morphological characteristic of cells. We observed that hAECs present a characteristic odontoblast-like morphology, with cytoplasmic processes located in dentinal tubuli, after 48 h. Further studies will be carried out with known concentrations of dentine matrix proteins and qPCR.
Canales de calcio y dolor: Investigación del mecanismo de acción de la pregabalina sobre la neurotransmisión
(Universitat de Barcelona, 2019-04-26) Martínez San Segundo, Pablo; Llobet Berenguer, Artur, 1972-; Universitat de Barcelona. Departament de Patologia i Terapèutica Experimental
[spa] Los gabapentinoides son una familia de fármacos que se unen con gran afinidad a la subunidad α2δ-1 de los canales de calcio voltaje-dependientes (VGCCs). Fármacos de esta familia como la pregabalina o la gabapentina se utilizan como tratamientos de primera línea para aliviar el dolor neuropático en enfermedades como la fibromialgia, la neuropatía diabética o la neuralgia del trigémino. Sin embargo, su uso extensivo contrasta con el limitado conocimiento sobre las implicaciones funcionales que produce la interacción de los gabapentinoides con la subunidad α2δ-1 y cuáles son sus efectos sobre la neurotransmisión. Los resultados generados a lo largo de esta tesis ayudan a obtener una imagen más nítida sobre la capacidad de la pregabalina para modificar la neurotransmisión de forma aguda. Estudiando la población homogénea de sinapsis presentes en los cultivos autápticos de neuronas colinérgicas del ganglio cervical superior de rata (SCMs), demostramos que la interacción de la pregabalina y del ectodominio de la neurexina-1α con la subunidad α2δ-1 inhibe la liberación sincrónica de vesículas sinápticas controlada por los canales de calcio dependientes de voltaje de tipo N, de forma aguda. Visualizando de manera simultánea la entrada de calcio presináptico mientras registramos las respuestas postsinápticas, comprobamos que el efecto de la pregabalina no está relacionado con una reducción significativa en la entrada de calcio presináptico. Por el contrario, el efecto reside en una alteración de la organización física establecida entre los canales de calcio y las vesículas sinápticas de la zona activa, reduciéndose de manera notable el tamaño efectivo del depósito de vesículas de liberación rápida. Asimismo, comprobamos que la sobreexpresión de la subunidad α2δ-1 potencia los efectos observados con la pregabalina y la neurexina-1α en la neurotransmisión; efectos que son mutuamente excluyentes. En esta tesis desvelamos la implicación de la región extracelular de la neurexina-1α en la regulación de la liberación de neurotransmisores. Debido a su privilegiada localización extracelular en las zonas activas, las subunidades α2δ sinápticas emergen como una diana sináptica común desde donde fármacos como los gabapentinoides o moléculas endógenas como el ectodominio de la neurexina-1 pueden controlar de forma aguda el acoplamiento entre los canales de calcio voltaje-dependientes con la liberación de las vesículas sinápticas del RRP. La variabilidad en la composición molecular de las sinapsis, como por ejemplo los niveles de α2δ presentes en los pacientes que padecen dolor neuropático, así como un posible incremento de los niveles de la región extracelular de la neurexina-1α durante las condiciones inflamatorias relacionadas con el dolor neuropático, podrían ser factores claves para explicar la efectividad del tratamiento con los gabapentinoides.

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