Parmi les voies de signalisation, la voie hedgehog (HH) intervient dans la formation de la polarité segmentaire. Si elle est défectueuse, elle entraine plusieurs malformations. De nombreux cancers présentent une suractivation de cette voie. La voie HH activée par la fixation du ligand HH sur le récepteur Patched (hPtc) et fait intervenir plusieurs partenaires cytoplasmiques dont Supressor of Fused (SUFU).Peu de données moléculaires et structurales sont disponibles pour cette voie et pourtant, ces données sont nécessaires pour comprendre sans ambiguïté son fonctionnement. De plus, la voie HH a été proposée comme pouvant être la cible de traitements chimio thérapeutiques mais, la protéine hPtc est impliquée dans l’efflux des drogues anticancéreuses. Une inhibition de hPtc par la fixation de son ligand entraine l’inhibition de l’efflux de drogues. Néanmoins, le site de fixation de HH sur son récepteur n’a pas encore été déterminé.Durant cette thèse, les travaux effectués ont permis l’étude structurale de la protéine hPtc notamment la détermination du site de fixation de HH. Dans un deuxième volet de cette thèse, j’ai effectué des études structurales de certaines protéines SUFU.Dans un premier temps, je me suis concentrée sur les domaines extracellulaires de hPtc qui ont été décrits comme nécessaires pour la fixation du ligand HH. J’ai cloné une protéine chimère constituée de ces deux domaines liés par le lysozyme du phage T4 (hPtcD1D2). Cette construction a été exprimée dans la bactérie E.coli. Les conditions d’expression testées permettent d’obtenir la protéine sous forme de corps d’inclusion dans le cytoplasme de la bactérie. Dans un deuxième temps, j’ai cloné la protéine dans un vecteur d’expression en levure. De manière concomitante, j’ai exprimé la protéine hPtc tronquée de ses régions N et C terminales (hPtcΛNΛC). Ce sont des régions intrinsèquement désordonnées qui ne permettraient pas une bonne cristallisation de la protéine. L’expression a été effectuée dans la levure. La solubilisation de cette protéine membranaire est en cours d’expérimentation.Ce travail a permis de poser les bases de l’expression de hPtcD1D2 et de hPtcΛNΛC. Ceci va notamment permettre la surexpression de la protéine et sa cristallisation afin de déterminer sa structure 3D et de caractériser le site de fixation de son ligand.Enfin, j’ai entrepris des études structurales des protéines SUFU. Un nouveau site de fixation du Zn a été caractérisé. En effet, après purification de la protéine, j’ai effectué des mesures d’affinité à l’aide d’un composé colorimétrique, le PAR et des expériences de spectroscopie d’émission atomique dans lesquelles j’ai fait varier le pH et la concentration en Zn. Ainsi, j’ai pu déterminer que SUFU a une affinité nanomolaire pour le Zn meilleure à pH 8 qu’à pH 6,5. La fixation du Zn se ferait donc sur un site basique. La structure de SUFU a été publiée en 2013 par deux équipes, je me suis inspirée des conditions de cristallisation de ces deux articles, pour cristalliser SUFU en présence de Zn. Les expériences de dichroïsme circulaire ont permis d’affirmer que ces protéines sont organisées en hélices α et en feuillets β. De plus, grâce à la diffusion des rayons X aux petits angles, j’ai pu déterminer que dSUFU, hSUFU et zSUFU n’ont pas la même conformation en solution. Alors que SUFU de drosophile est un monomère globulaire, les protéines humaine et de poisson zèbre seraient plutôt allongées et dimériques. La région N-terminale potentiellement impliquée dans la dimérisation de hSUFU a été tronquée et hSUFUΛ30 présente des différences d’état d’oligomérisation. / The hedgehog (HH) signalling pathway is involved in the segmentary polarity formation. A dysfunction of this pathway is involved in several malformations. Many cancers are caused by an overactivation of this pathway. The HH signalling pathway is activated by the binding of HH on the receptor Patched (hPtc) and included many cytoplasmic partners such as Suppressor of Fused (SUFU). Few molecular and structural data are available on this pathway even if these data are important to fully understand the pathway functioning. Furthermore, the HH signalling pathway maybe be the target of chemotherapy treatments. However, hPtc is involved in drugs efflux. Inhibition of hPtc by the binding of its ligand HH may lead to this efflux inhibition. Yet, the binding site of HH on its receptor hPtc is not yet determined.During this thesis, the structural study of hPtc have been engaged especially the study of the binding site of HH. On the second hand, I have structurally studied some SUFU proteins.First of all, I have expressed the extracellular domains of hPtc. These domains have been described as necessary for HH binding. I have cloned a chimeric protein made by the extracellular domains of hPtc associated with the lysozyme T4 (hPtcD1D2). This protein have been expressed in the E.Coli bacteria. The protein expressed in inclusion bodies in the cytoplasm of the bacteria. In the other hand, I have cloned the protein in a yeast expression vector. Part of this, I have also expressed the protein hPtc without its N and C terminus regions (hPtcNC). These regions are intrinsically disrupted. They may lead to crystallization problems. The protein has been expressed in yeast.This work permits to expressed hPtcD1D2 and hPtcΛNΛC. This will lead to the expression of the protein and its crystallisation in order to determine its 3D structure and to characterize its ligand binding site.Finally, I structurally studied the protein SUFU. A novel Zn binding site has been characterized. In fact, after the protein purification, I have made affinity measures using a colorimetric compound, PAR. I also performed spectroscopic experiments in which I varied the pH and the Zn concentration. I determined the SUFU has a nanomolar affinity for the Zn best at pH 8 than pH 6.5. Indeed, the Zn binding site may be basic.The SUFU 3D structure has been published in 2013 by two teams. Inspired by their crystallization conditions, I crystallized SUFU with Zn. Circular dichroism experiments permitted to know that the proteins are organized in  helices and  sheets. Moreover, small angles X ray spectroscopy experiments show that dSUFU, hSUFU and zSUFU did not have the same conformation in solution. Drosophila SUFU is globular and human and zebrafish SUFU are long and dimeric. The N-terminal region involved in hSUFU has been removed and hSUFUΛ30 is present in different oligomerization forms.

Exosomer är extracellulära vesiklar i nanostorlek som frigörs från cellerna till den extracellulära matrisen. Exosomer är laddade med nukleinsyror, proteiner, och lipider, och fungerar som kommunikatorer mellan celler. Det har gjort dem mycket attraktiva for forskning inom terapi, diagnostik och transport av läkemedel. För att använda exosomer i kliniska tillämpningar behöver standardiserade metoder för isolering, rening och analys av exosomer att utvecklas.  Detta projekt syftar till att sätta upp en snabb metod som använder "multiangle light scattering" (MALS) i kombination med kromatografi for att bestämma storleken på exosomer. Olika storlekskromatografi (SEC)/jonbyte (IEX)-kolonner kommer att undersökas och användas for analys av storlekar på exosomer. Partikelstorleken som erhålls från MALS kommer sedan att verifieras med nanoparticle tracking analysis (NTA).  För SEC-MALS-analyserna eluerades exosomerna i dödvolymen. IEX-MALS-metoden separerade exosomerna enligt resultatet. Exosomer i storlek mellan 60-110 nm eluerades ut med 800 mM NaCl. Större exosomer och eventuella aggregat i storlek 120-200 nm eluerades ut med 1200 mN NaCl.  Resultatet visar att signalen från MALS indikerar var exosomer i kromatogrammen elueras ut och kan ge värdefull information om storleksfördelningen i en topp. SEC-MALS-resultatet är dock inte reproducerbart, eftersom det ibland sker en förändring i storleksfördelningen över toppen. Resultatet har också visat att arean for toppen i dödvolymen varierar mellan körningarna, vilket förmodligen orsakats av att exosomer interagerar med den stationära fasen eller filtret i kolonnen. Förskjutningen i storleksfördelning observerades också i IEX­MALS-metoden. Medelvärdet av partikelstorlekarna som beräknades från SEC-MALS överensstämmer med storlekarna beräknade med NTA. SEC-MALS-metoden behöver förbättras för att fa reproducerbara resultat. Den beräknade storleken från IEX-MALS stämde inte överens med storleken från NT A. Jonbytesanalysen skulle kunna upprepas och fraktioner skulle kunna analyseras med andra tekniker för att verifiera resultatet i framtida arbete. Effekten av den höga saltkoncentrationen på exosomerna behöver också undersökas ytterligare. / Exosomes are nanosized extracellular vesicles released from the cells into the extracellular space. Exosomes are loaded with nucleic acids, proteins, and lipids, and work as communicators among cells. This has made them very attractive for research in therapeutics, diagnostics and drug delivery applications. Standardised exosome isolation, purification, and analysis methods need to be developed to use exosomes in clinical applications.  This project aimed to set up a quick method using multiangle light scattering (MALS) combined with chromatography techniques to determine exosome sizes. Different size exclusion (SEC)/ion exchange (IEX) columns will be investigated and used to analyse exosome sizes. The particle size obtained from MALS will then be verified with nanoparticle tracking analysis (NTA).  For the SEC-MALS analysis, the exosomes were eluted in the void volume. The IEX-MALS method separated the exosome sample. The exosome eluted with 800 mM NaCl ranged between 60-110 nm in diameter. The exosomes eluted with 1200 mM NaCl ranged between 120-200 nm in diameter. The result shows that the light scattering intensity from MALS indicates where the exosomes elute in the chromatograms and gives valuable information about the size distribution in a peak. However, the SEC-MALS result is not reproducible, as sometimes, a shift in the size distribution over the peak occurs.  The result has also shown that the void peak area varies between the runs, caused mainly by the exosomes interacting with the resin or the column's filter. The shift in size distribution was also observed in the IEX-MALS method.  The average sizes calculated from SEC-MALS agree with the sizes calculated with NTA. The SEC-MALS method needs to be improved to obtain reproducible results. The calculated size from IEX-MALS did not agree with the size from NTA. The ion exchange analysis could be repeated and further analysed with other techniques to verify the result in future work. The effect of the high salt concentration on the exosomes also needs to be further investigated.

The objective of this research is to further define the relationship between the charge state of insulin, and the self assembly properties of insulin and PEGylated insulin in solution. Polyethylene glycol (PEG) chains were covalently attached to insulin in order to evaluate their impact on insulin's systemic duration of action after pulmonary dosing. This thesis will focus on the assembly properties of the PEG-insulin and insulin, and also demonstrate how the charge state, which was modified by the covalent attachment of PEG, relates to different modes of behavior by anion and cation exchange chromatography. In addition, explain how modifying the assembly state extends to improving formulation properties of spray-dried insulin powders. This thesis is an investigation into the relationship of insulin's charge state controlled by pH and how the charge state affects the self assembly of insulin, especially when the zinc ion is removed. Ionic interaction is one of the major forces affecting insulin assembly. The theory that a change in the charge state of insulin could modulate the ionic interaction and reduce hexamer formation at alkaline conditions was investigated. Experiments were designed to measure the level of hexamer with light scattering, and the amount of hexamer was then correlated with the pH and zinc content of the solutions. The importance of the charge state of the monomer and its behavior extends to chromatography and purification modes as well. Specifically, the purification of various species of PEGylated insulin presents a challenge. By varying mobile phase pH which induces the charge to insulin, an ion exchange method demonstrated very high resolution and controllable interaction between the ion exchange media and the insulin derivatives. A highly accurate method for determining molecular weight and thus the average associated state of insulin in solution has been developed using the MALS (Multi-Angle Light Scattering). Insulin concentration, pH, and metal ion concentrations, were in pharmaceutically relevant ranges. The MALS method was developed to evaluate how the parameters above affect the self-assembly properties of insulin, and use the assembly properties to improve formulations of insulin or PEGylated insulin. To use the light scattering technique the dn/dc (change in refractive index with change in concentration) is required. During the method development, the dn/dc of insulin was measured at 690 nm, and a value of 0.185 mL/g based on theory was confirmed. A novel approach for preparing insulin powders with improved chemical stability, based on maintaining the dissociation of hexamers in solution during the spray drying process was developed. The mode presented here is to remove the zinc ions from solution, increase the pH from 6.6 to 7.8, and maintain a low concentration of insulin approximately 2 to 15 mg/mL. Each of these factors alone decreases the hexamer population in solution, but by combining all three factors, hexamers are driven to very low levels of equilibrium. The increased stability of the powders is predominately related to the decrease in covalent insulin dimer (CID). The data presented correlates a reduced hexamer population in the solution with lower levels of CID's in the dry powder compared to controls. The CID formation rate was reduced by 40% compared to a control.

The diploma thesis is focused on the study of interactions between hyaluronan of various molecular weights with CAE surfactant. This surfactant does not have the exact composition and there is not known their molecular weight. One of the main parameters needed to describe the interactions between surfactant and hyaluronan, there is the determination of critical micellar concentration. The value of critical micellar concentration of CAE is not known yet. All measurements were made for solutions in aqueous solution and 0.15 M NaCl. The determination of the molecular weight of this surfactant was performed by the technique SEC-MALS-dRI. High resolution ultrasonic spectroscopy was chosen to determine the critical micellar concentration and tensiometry was chosen as a complementary method. The interactions of CAE surfactant with polysaccharide of hyaluronan were showed by high resolution ultrasonic spectroscopy and densitometry. Compressibility was calculated from ultrasonic velocity and density.

The objective of this research is to further define the relationship between the charge state of insulin, and the self assembly properties of insulin and PEGylated insulin in solution. Polyethylene glycol (PEG) chains were covalently attached to insulin in order to evaluate their impact on insulin¿s systemic duration of action after pulmonary dosing. This thesis will focus on the assembly properties of the PEG-insulin and insulin, and also demonstrate how the charge state, which was modified by the covalent attachment of PEG, relates to different modes of behavior by anion and cation exchange chromatography. In addition, explain how modifying the assembly state extends to improving formulation properties of spray-dried insulin powders. This thesis is an investigation into the relationship of insulin¿s charge state controlled by pH and how the charge state affects the self assembly of insulin, especially when the zinc ion is removed. Ionic interaction is one of the major forces affecting insulin assembly. The theory that a change in the charge state of insulin could modulate the ionic interaction and reduce hexamer formation at alkaline conditions was investigated. Experiments were designed to measure the level of hexamer with light scattering, and the amount of hexamer was then correlated with the pH and zinc content of the solutions. The importance of the charge state of the monomer and its behavior extends to chromatography and purification modes as well. Specifically, the purification of various species of PEGylated insulin presents a challenge. By varying mobile phase pH which induces the charge to insulin, an ion exchange method demonstrated very high resolution and controllable interaction between the ion exchange media and the insulin derivatives. A highly accurate method for determining molecular weight and thus the average associated state of insulin in solution has been developed using the MALS (Multi-Angle Light Scattering). Insulin concentration, pH, and metal ion concentrations, were in pharmaceutically relevant ranges. The MALS method was developed to evaluate how the parameters above affect the self-assembly properties of insulin, and use the assembly properties to improve formulations of insulin or PEGylated insulin. To use the light scattering technique the dn/dc (change in refractive index with change in concentration) is required. During the method development, the dn/dc of insulin was measured at 690 nm, and a value of 0.185 mL/g based on theory was confirmed. A novel approach for preparing insulin powders with improved chemical stability, based on maintaining the dissociation of hexamers in solution during the spray drying process was developed. The mode presented here is to remove the zinc ions from solution, increase the pH from 6.6 to 7.8, and maintain a low concentration of insulin approximately 2 to 15 mg/mL. Each of these factors alone decreases the hexamer population in solution, but by combining all three factors, hexamers are driven to very low levels of equilibrium. The increased stability of the powders is predominately related to the decrease in covalent insulin dimer (CID). The data presented correlates a reduced hexamer population in the solution with lower levels of CID¿s in the dry powder compared to controls. The CID formation rate was reduced by 40% compared to a control.

This master thesis is dealing with the use of light scattering techniques in the study on formation of polyelectrolyte complexes in the system biopolymer-surfactant. Sodium hyaluronate was chosen as biopolymer and cetyltrimethylammonium bromide and karbethopendecinium bromide were selected as surfactants. The first precipitation experiments were performed to determine the optimal concentration range of surfactants for subsequent titration measurements performed on Zetasizer Nano ZS and for SEC-MALS analysis. The key parameters obtained from these measurements were the values of critical aggregation concentrations in hyaluronate-surfactant systems. It was found out that the increasing molecular weight of hyaluronan in these systems is causing reduction of obtained values of critical aggregation concentrations. Changes of Z-average particle size, zeta potential, polydispersity index, conformation plot, radius of gyration and molecular weight in the hyaluronan-surfactant system were monitored during experiments.

Owing to their key role in mucosal functioning as surface barriers with biospecific interaction potentials, the mucins are interesting candidates for use as surface modifiers in biomaterials applications. In this work, “mild” fractionation procedures were used to prepare mucins of bovine (BSM), porcine (PGM), and human (MG1) origin. Biophysicochemical analysis showed the prepared mucins to differ in size, charge, conformation, and composition. In turn, these factors were shown to govern mucin adsorption on hydrophilic and hydrophobic model surfaces. To enable for detailed coating analysis, methods for the qualitative and quantitative analysis of mucin-based coatings were developed. Of particular interest, a method for the determination of the fraction of surface-exposed, presumed bioactive proteins in a complex mucin coating was described. It was shown, using microscopy and activation assays, that mucin precoating effectively suppresses the neutrophil response towards a polymeric model biomaterial. Under optimal coating conditions, all mucins performed equally well, thus indicating them to be functionally similar. Coating analysis suggested that efficient mucin surface-shielding is critical for good mucin coating performance. Following a study on the complexation of albumin with preadsorbed mucin, we investigated the effect of mucin precoating on the conformation and neutrophil-activating properties of adsorbed host proteins. We found that mucin precoating greatly reduces the strong immune-response normally caused by adsorbed proinflammatory proteins (IgG and sIgA). Detailed coating analysis revealed that the fraction of surface-exposed protein in the mucin-protein composite influences the neutrophil response. Unexpectedly low neutrophil activation for composites containing near-monolayer concentrations of exposed IgG, suggested IgG to act synergistically with mucin on the surface. Conformational analysis supported this by showing that a preadsorbed mucin layer could stabilize adsorbed IgG through complexation. Our findings link well to the complex in vivo situation and suggest that functional mucosal mimics can be created in situ for improved biomaterials performance.

Biopolymer surfactant systems have been studied by using different physico chemical methods. As the biopolymer, it was chosen high (1400–1600 kDa), medium (250–450 kDa) and low (8–15 kDa) molecular weight sodium hyaluronate. Two cationic surfactants – cetyltrimethylammonium bromide (CTAB) and carbethopendecinium bromide (Septonex) were selected to provide polyelectrolyte complexes with oppositely charged hyaluronan. The critical aggregation concentration of the surfactant in the system was monitored by dynamic light scattering (Zetasizer Nano ZS). SEC-MALS method was used for the description of the conformation of sodium hyaluronate of different molecular weights and polyelectrolytes complexes hyaluronan surfactant after reaching the aggregation point. Isothermal titration calorimetry (ITC) was used to determine the critical aggregation concentration of the surfactant by monitoring the thermal changes accompanying the aggregation of the system. It was found out that the increasing molecular weight of hyaluronan in these systems decreases the critical aggregation concentrations and conversely.

L'objectiu d'aquesta tesi és estudiar el funcionament real de la servitud catalana medieval. Per això s'estudien els homenatges i els cobraments dels mals usos rebuts i aplicats per l'Almoina del Pa de la Seu de Girona sobre els seus remences al llarg dels segles XIV i XV i, més concretament, entre 1331 i 1458. Aquestes dates han estat determinades per la documentació generada per l'esmentada institució benèfica. El primer llibre de comptes conservat és de l'any 1331. En aquests llibres de comptes els pabordes encarregats de gestionar l'Almoina hi consignaven tots els seus ingressos i totes les despeses. La data final també ha estat fixada per la documentació, perquè a partir d'aquest moment deixem de trobar constància escrita del pagament dels mals usos i de la prestació d'homenatges.La importància dels mals usos, és a dir, aquells pagaments que gravaven als serfs pel fet de ser-ho, a la Catalunya de la baixa edat mitjana és una qüestió fora de discussió. Bona part dels historiadors -Hinojosa, Vicens Vives, Freedman, etc.- atribueixen als mals usos, als homenatges i a la seva continuada exigència els dos alçaments remences contra les senyories feudals a partir de l'any 1462. Segons aquestes hipòtesis, la lluita per suprimir els mals usos i aconseguir la llibertat individual és la raó de les guerres remences de finals del segle XV. Com és sabut van quedar resoltes amb la Sentència Arbitral de Guadalupe, dictada pel rei Ferran II, que va suprimir definitivament la servitud de les terres catalanes.Malgrat la importància que els historiadors han concedit a l'existència dels mals usos i, sobretot, a la manca de llibertat dels remences, no hi ha estudis sistemàtics sobre la seva aplicació a la pràctica. Per això, l'objectiu d'aquesta tesi és estudiar tots els mals usos i tots els homenatges aplicats i rebuts per una sola senyoria -l'Almoina del Pa de la Seu de Girona-, els remences de la qual van participar activament en ambdues guerres i que és representativa, sense cap mena de dubte, del que succeïa en la diòcesi gironina. A més a més, cal assenyalar que l'estudi comprèn un període de temps que inclou circumstàncies tan cabdals com la pesta negra i la resta de catàstrofes del segle XIV i el segle XV fins a la primera guerra remença.Com és sabut, els remences catalans estaven sotmesos a sis mals usos: la redempció de persones (mitjançant la qual aconseguien la seva llibertat), les firmes d'espoli forçades (que havien de pagar quan es casaven en determinades circumstàncies), la intèstia i l'eixòrquia (que gravaven la mort intestada i sense descendents), l'àrsia (que penalitzava la crema accidental del mas o la masada) i la cugúcia (exigida a les dones considerades adúlteres). Els remences confirmaven la seva dependència d'una senyoria en els corresponents homenatges o reconeixements de domini que havien de prestar quan n'eren requerits.Aquesta tesi consta de deu capítols a més d'una introducció (o primer capítol) i d'unes conclusions. El segon capítol és dedicat a la descripció de les fonts utilitzades, entre les que destaquen els llibres de comptes dels pabordes, i on queda prou palesa la importància del fet d'haver pogut disposar d'una excepcional font seriada, a més a més de pergamins. El tercer correspon a l'estudi de la institució tractada, que tenia terres a les actuals comarques del Gironès, La Selva, l'Alt i el Baix Empordà i el Pla de l'Estany. En el quart capítol hi analitzo els problemes generats a l'hora d' intentar conèixer el nombre de persones que eren pròpies de l'Almoina i saber de quins masos provenien.A continuació segueixen els quatre capítols que constitueixen el cos central de la tesi. Al seu torn, la seva anàlisi va ocasionar l'elaboració dels tres darrers. En el capítol cinquè s'estudien tots els homenatges rebuts per l'Almoina entre els anys 1300 i 1457, tant els que figuren en els manuals de comptes com els conservats en pergamí. En total, tenim documentats 1258 dels homenatges o reconeixements de domini rebuts per la institució. El capítol següent és dedicat a l'estudi de les firmes d'espoli forçades i als 424 cobraments fets per l'Almoina per aquest concepte, entre els anys 1331 i 1452. En els capítols setè i vuitè, s'analitzen els mals usos que gravaven les sortides del domini, tant les voluntàries com les involuntàries. El resultat obtingut és que entre 1331 i 1458, la institució va concedir la llibertat a 557 persones bona part de les quals van tornar a adscriure's de nou a una altra senyoria. El nombre d'aquests sortides contrasta amb el cobrament per part de l'Almoina, entre aquestes mateixes dates, de només 105 intèsties i eixòrquies; dit d'una altra manera, fins l'any 1445 va ingressar diners en concepte de 23 intèsties, fins el 1458 per 68 eixòrquies i fins el 1406 per 14 intèsties i/o eixòrquies. Els capítols 9 i 10 tracten del significat i les limitacions que comportaven els mals usos, com a trets definitoris de la pertinença a la servitud, sobre les persones que hi estaven sotmeses. Finalment, el darrer capítol analitza el compliment de la sentència dictada pel rei Alfons el Magnànim l'any 1457 en la que suspenia la servitud al Principat de Catalunya. Queda fora de dubte que pocs anys abans de la primera guerra remença els homenatges i els mals usos havien deixat d'aplicar-se. / The aim of ths thesis is to study the real functioning of the mediaeval Catalan servitude. To accomplish this purpose, the servile homages and the bad customs' payments received and applied by the Almoina of the Cathedral of Girona from his remences (servile persons) are studied in depth. Theses homages had place along XIV and XV centuries and, more precisely, among 1331 and 1458, dates fixed by the documentation generated by that charitable institution, as the first account book preserved is from 1331. In these accounts books the Almoina's provosts consigned all its incomes and all its expenses. The final date has also been fixed by the documentation, because from that year on we don't have any more written evidences of bad customs' payments and of homages. The importance of the bad customs -that is to say, those payments that the remences had to pay just because they were serfs- in the mediaeval Catalonia is a question out of argument. Most historians - such as Hinojosa, Vicens Vives, Freedman, etc.- attribute to the bad customs, to the servile homage and to its continued demand both remença uprisings against the feudal lordships from 1462 on. According to these hypotheses, the fight to suppress the bad customs and to achieve the individual freedom is the reason of the remença wars at the end of the 15th century. How it is well known, the Sentència Arbitral de Guadalupe, pronounced by the king Ferran II, suppressed definitively the servitude in Catalonia. In spite of the importance that historians have granted to the bad customs and, especially, to the lack of freedom of the remences, there are not studies about its real application. That's why it was so important to study -not theoretically but with real data- all the bad customs and all the servile homages applied and received by one only lordship: the Almoina of the Cathedral of Girona, whose remences participated actively in both wars and that is representative, with no doubt, of what happened in the diocese of Gerona. Besides, it is necessary to point out that the study includes a period of time that contains circumstances such important as the Black Death and the rest of 14th and 15th centuries' catastrophes until the first remença war.How it is known, the Catalan remences were subjected to six bad customs: the redemption of persons (through which they achieved their personal freedom), the firma d'espoli forçada (payment in return for the lord's guarantee of nuptial agreements over dowry and marriage portion), the intèstia (the lord's right to a substantial part of the movables left by a peasant householder who died intestate), the eixòrquia (a levy in the event of death without direct legitimate heirs), the àrsia (a fine resulting from the deliberate or accidental burning of a peasant's house or other property) and the cugúcia (the confiscation of a portion of a peasant's property by reason of his wife's adultery). The remences confirmed their lordship' dependence in a servile homage or dominium recognitionis that they had to do when were required.This thesis is organized in ten chapters, an introduction (or first chapter) and the conclusions. The second chapter is devoted to the description of the sources used, among those highlight the annual accounts books of the provosts besides the parchments. The third chapter studies the Almoina institution, which had properties in the current regions of Gironès, Selva, Alt and Baix Empordà and Pla de l'Estany. In the fourth chapter, data problems are analyzed; problems that came when trying to know the exact amount of people, or the exact number of persons bounded to the Almoina and from which manses they came.The next four chapters constitute the main part of the thesis. In turn, its analysis brought the need of writing the three last ones. In the fifth chapter I study all the servile homages received by the Almoina between 1300 and 1457, those that appear in the account books as well as the ones preserved in parchment. In total, we have data of 1258 homages or dominium recognitionis received by the institution. The following chapter is devoted to the study of the firmes d'espoli forçades and to the 424 payments collected by the Almoina by this concept, between 1331 and 1452. In the seventh and eighth chapters , I analyze the bad customs required to the exits of the lordship, the volunteers as well as the involuntary ones. The obtained result is that among 1331 and 1458, the institution granted the freedom to 557 persons; surprisingly, most of them bounded again to another lordship. The amount of these exits contrasts with the payment to the same lordship during these same dates, for only 105 intèsties and eixòrquies. In other words, until 1445 the provost charged by 23 intèsties, until 1458 by 68 eixòrquies and until 1406 by 14 intèsties and/or eixòrquies Chapters 9 and 10 are about the meaning and the limitations that entailed the bad customs, as features of the belonging to the servitude, over the persons who were bounded to a lordship. Finally, the last chapter analyzes the fulfillment of the sentence dictated by the king Alfons el Magnànim in 1457, where suspended the servitude in Catalonia. There is no doubt that few years before the first remença war the servile homages and the bad customs were not required any more.

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