In this work the involved mechanisms between Ro/SSA complex, composed also by the tripartite motif 21 (TRIM21alpha) and trove domain 2 (TROVE2) proteins, with respect to systemic lupus erythematosus (SLE) autoantibodies is studied. The work is divided in three chapters: I- In vitro and in silico analysis of the molecular recognition between lupus autoantibodies and TRIM21alpha Fc Receptor ; II- In vitro evidence of bipolar-bridged immune TROVE2 complexes in the pathogenesis of systemic lupus erythematosus and III- Label-free piezoelectric biosensor for prognosis and diagnosis of Systemic Lupus Erythematosus. Samples of lupic patients and health subjects were kindly provided by La Fe hospital, accordingly the required protocols. After its extraction and purification, the immunoglobulin samples were obtained to study in vitro protein interactions and the involved mechanism by using a quartz crystal microbalance with dissipation factor attributions, and dual polarization interferometry. Techniques such, polarization modulation infrared interferometry, x-ray photoelectron interferometry and contact angle measurement were used in order to characterize surfaces. Pre-steady-state analysis revealed an antibody bipolar bridging involved in both TRIM21alpha and TROVE2 proteins. Identification of the main immunodominant human linear epitope for TRIM21alpha was finely mapped using a series of overlapping synthetic polypeptides with a size of 21 amino acids. The epitopes recognised by autoantibodies for this protein spanned the linear sequence from the aminoacid 151 to 183, and a conformational epitope for SLE patients and healthy subjects, respectively. Autoantibodies from lupic patients targeted protein epitopes, allowing health subjects to be discriminated. Major Histocompatibility Complex Class-II binding peptide prediction results corroborated the sequence as the immunodominant linear epitope, mostly coded as the HLA DRB1*1304 allele for SLE patients, and HLA DRB1*0806 for controls. The subdominant epitope corresponded to the PRY-SPRY domain, recently known as mammalian Fc receptor. Finally, the TRIM21alpha protein structure was modeled by a new homology modeling, never before presented. From the TROVE2 protein, the major linear epitope recognized by autoantibodies correspond to the sequence from the aminoacid 160 to 210 for healthy subjects. However, the major epitope in SLE serum is undiscovered. We suggest that the difference between epitopes could correspond to a majority necrosis-induced specificity in SLE patients, and an apoptotic via in healthy subjects. TROVE2 showed the ability to bind to Fcs, depending on alkaline earth cations in solution. The results suggest that the TROVE2-TRIM21alpha binding is a calcium-dependent protein interaction linked through the MIDAS-like motif in the vWFA-like domain. Finally, a pratical consequence of all study was the development of label-free biosensing method, based in microbalance technology, for in vitro diagnostics of systemic lupus erythematosus patients, allowing the premature sensing of autoantibodies against TRIM21alpha and TROVE2 protein, in advance of the clinical illness symptoms appear. / En este trabajo se ha estudiado el mecanismo involucrado entre el complejo Ro/SSA, compuesto también por las proteínas tripartite motif 21 alpha(TRIM21alpha;) y trove domain 2 (TROVE2) con respecto a autoanticuerpos de pacientes que tienen lupus eritematoso sistémico, en comparación con autoanticuerpos de personas sanas. El estudio comprende tres capítulos: I- Análisis in vitro e in silico del reconocimiento molecular entre autoanticuerpos de lupus y receptor TRIM21alpha; Fc; II- Evidencias in vitro de complejos inmunes TROVE2 bipolares con puentes en la patogénesis del lupus eritematoso sistémico y III- Biosensor piezoeléctrico libre de marcaje para el pronóstico y el diagnóstico del lupus eritematoso sistémico. Las muestras de pacientes lúpicos y personas sanas fueron proporcionadas por el hospital La Fe de acuerdo con los protocolos establecidos. Tras una etapa de extracción y purificación de las inmunoglobulinas fueron estudiadas la interacción de proteínas in vitro utilizando una microbalanza de cristal de cuarzo con atribución de factor de disipación e interferometria de polarización dual. Técnicas de caracterización como espectroscopía infrarroja de reflexión-absorción por modulación de la polarización, espectroscopía fotoelectrónica de rayos-x y análisis de ángulo de contacto fueron utilizadas con la finalidad de caracterizar superficies. El análisis del estado pre estacionario ha revelado un mecanismo de puente bipolar para las dos proteínas, TRIM21alpha; y TROVE2. Tras su identificación, el epítopo linear inmunodominante fue mapeado para TRIM21alpha;, utilizando una serie de polipéptidos sintéticos superpuestos de 21 aminoácidos. Los epitopos reconocidos por autoanticuerpos para esta proteína abarca la secuencia lineal a partir del aminoácido 151 hasta el 183 para epitopos de pacientes lúpicos y sujetos sanos, respectivamente. Autoanticuerpos de pacientes lúpicos reconocieron epítopos de proteínas, permitiendo la discriminación de pacientes sanos. Los resultados de la unión del Complejo Mayor de Histocompatibilidad clase II con el péptido de unión corroboraron la secuencia cómo el epítopo lineal inmunodominante, codificado como el alelo HLA DRB1 * 1304 para pacientes con LES y HLA DRB1 * 0806 para los controles. El epitopo subdominante corresponde al dominio PRY-SPRY, recientemente conocido receptor Fc de mamífero. Finalmente, la estructura de la proteína TRIM21alpha; fue determinada utilizando un nuevo modelo de homología no presentado antes. De la proteína TROVE2, el epitopo lineal immunodominante reconocido por los autoanticuerpos corresponde a la secuencia que pudiera corresponder del aminoácido 160 hasta 210 para sujetos sanos. Sin embargo, el epitopo mayoritario en sueros lúpicos no fue determinado. Se sugiere que la diferencia entre los epitopos se corresponde mayoritariamente a una necrosis-inducida en pacientes lúpicos, y a una vía apoptótica en pacientes sanos. TROVE2 presentó la habilidad de unirse a Fcs dependiendo de los cationes alcalinos presentes en la disolución. Los resultados sugieren que la unión TROVE2-TRIM21alpha; es dependiente de la interacción con calcio vinculada a través del motivo MIDAS en el dominio vWFA. Finalmente, una consecuencia práctica de todo el estudio fue el desarrollo de un biosensor libre de marcaje para diagnóstico in vitro de lupus eritematoso sistémico, permitiendo la detección prematura de autoanticuerpos anti TRIM21alpha; y anti TROVE2, varios años antes de la aparición de los síntomas clínicos de la enfermedad. / En aquest treball s'ha estudiat el mecanisme involucrat en el complex Ro/SSA, compost per les proteïnes tripartite motif 21 (TRIM21alpha;) i trove domain 2 (TROVE2) respecte a autoanticossos de pacients que tenen lupus eritematós sistèmic, en comparació amb autoanticossos de persones sanes. L'estudi es divideix en tres capítols: : I- Anàlisi in vitro i in silico del reconeixement molecular entre autoanticossos de lupus i receptor TRIM21alpha; Fc; II- Evidències in vitro de complexos immunes TROVE2 bipolars amb ponts en la patogènesi del lupus eritematós sistèmic i III-Biosensor piezoelèctric lliure de marcatge per al pronòstic i el diagnòstic del lupus eritematós sistèmic. Les mostres de pacients lúpics y persones sanes van ser amablement proporcionades per l'hospital La Fe d'acord amb els protocols establerts. Després d'una etapa de purificació adequada, el pool de mostres de immunoglobulines va ser estudiat les interaccions in vitro de les proteïnes utilitzant una microbalança de cristall de quars amb atribució de factor de dissipació i interferometria de polarització dual. Tècniques de caracterització, como ara espectroscòpia de infraroja de reflexió-absorció per modulació de la polarització, espectroscòpia fotoelèctrica de rajos X i anàlisi d'angle de contacte van ser emprades amb per tal de caracteritzar les superfícies. L¿anàlisi de l`estat preestacionari ha revelat un mecanisme de pont bipolar que involucra les dos proteïnes, TRIM21alpha; i TROVE2. Una vegada identificat, va ser mapat l'epítop immunodominant lineal per a TRIM21alpha; emprant una sèrie de polipèptids sintètics superposats de 21 aminoàcids. Els epítops reconeguts per autoanticossos per a aquesta proteïna engloben la seqüència lineal a partir de l'aminoàcid 151 fins al 183 per a epítops de pacients lúpics y subjectes sans, respectivament. Autoanticossos de pacients lúpicos van reconèixer epítops de proteïnes, fet que va permetre la discriminació de pacients sans. Els resultats de la unió del Complexe Major de Histocompatibilitat classe II amb el pèptid de unió van corroborar la seqüència com l'epítop lineal immunodominant, codificat com l'al·lel HLA DRB1 * 1304 per a pacients amb LES i HLA DRB1 * 0806 per als controls. L'epítop subdominant correspon al domini PRY-SPRY, recentment conegut receptor Fc de mamífer. Finalment, l'estructura de la proteïna TRIM21alpha; va ser determinada utilitzant un nou model d'homologia no presentat abans. De la proteïna TROVE2, l'epítop lineal immunodominant reconegut pels autoanticossos correspon a la seqüència que pogués correspondre l'aminoàcid 160 fins al 210 per a subjectes sans. No obstant això, l'epítop majoritari en sèrums lúpics no va ser determinat. Es suggereix que la diferència entre els epítops es correspon majoritàriament a una necrosis induïda en pacients lúpics i a una via apoptòtica en pacients sans. TROVE2 va mostrar l'habilitat de unir-se a Fcs en funció dels cations alcalins presents en la dissolució. Els resultats suggereixen que la unió TROVE2-TRIM21alpha; depèn de la interacció amb calci vinculada a través del motiu MIDAS en el domini vWFA. Finalment, la conseqüència pràctica de tot l'estudi va ser el desenvolupament d'un biosensor sense marcatge per al diagnòstic in vitro de lupus eritematós sistèmic, el qual permet la detecció prematura d'autoanticossos cap a les proteïnes TRIM21alpha; i TROVE2 anys abans de l'aparició dels símptomes clínics de la malaltia. / Do Nascimento, NM. (2017). The Ro/SSA Complex in Systemic Lupus Erythematosus Patients [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/80534 / TESIS

This study investigates the characteristics of and the interrelationships between disease, disability, service use and social support in a random sample of 647 community dwellers aged 75 years and over. The two broad objectives of the study are: to examine the physical aspects and manifestations of health by investigating disease and disability and the interrelationships between these two factors, and; to examine the social aspects of health by investigating service use and social support and the interrelationships between these two factors. Given the dramatic population ageing in Australia, particularly in the very old age groups, the health, well-being and quality of life of older Australians are of paramount importance and will be well into the future. The proportion of the population with diseases and disabilities increases significantly with age. As the physical aspects of health are manifested with increasing age the social aspects of health also become increasingly important. Older adults, particularly those in advanced old age, are disproportionately high users of health and community services. Despite the high use of services in this age group, far more older adults living in the community rely on their families, friends and neighbours for social support and many older adults use a combination of formal services and informal social support. Little is known about people aged 75 years and over living in the community in Australia. In particular, significant knowledge gaps exist with regard to the relationship between disease and disability and that between service use and social support. The characteristics of social support in this group of older adults are also largely unknown. The papers presented in this thesis are based on data collected in The Sydney Older Persons Study (SOPS). This is a large longitudinal multidisciplinary project which began in 1991 in order to investigate the health and service use patterns of people aged 75 years and over living in the community in the Central Sydney Health Area. The initial sample consisted of two groups: first, the Australian Bureau of Statistics (ABS) selected census districts with probability proportional to size and 9271 households were door-knocked to obtain a random sub-sample of the general community (n=320, response rate 73%); second, community-living veterans and war widows residing in the Central Sydney Health Area were selected at random from a list provided by the Department of Veterans Affairs to obtain a veteran/war widow sub-sample (n=327, response rate 82%). Respondents participated in both an interview conducted by a social scientist and a medical assessment performed by a medical practitioner with experience in geriatric medicine. An informant was sought for each respondent and this informant participated in a phone interview conducted by a social scientist. The first paper in this thesis investigates the characteristics of diseases (neurodegenerative, systemic and psychiatric) including their prevalence and association with age. The second paper extends the first by examining the nature of the relationship between disease and disability and in particular which individual diseases and groups of diseases have the greatest impact on disability. The third paper expands the analysis in the second paper by focusing in greater detail on the relationship between disease and disability. The contribution of clinically-diagnosed individual diseases and groups of diseases to three different measures of disability (clinician-rated, informant-rated or proxy and self-report) is investigated here. The fourth paper examines the possibility of disease and disability being the major predictors of service use and social support. It focuses on the determinants of service use and social support using Andersen's behavioral model. The fifth paper investigates the characteristics of social support, in particular gender differences and the socio-demographic variables associated with social support. This is an important research area because lower levels of social support have been found to predict mortality, disease and lower levels of well-being. Finally, the sixth paper links the major themes of the fourth and fifth papers by investigating the relationship between service use and social support. This paper tests Cantor's 'hierarchical-compensatory' mechanism, which predicts a negative association between service use and social support, and the 'bridging' mechanism which predicts a positive association between these two factors. Thus it assesses the extent to which demands for service use and for social support are made together or in a compensatory fashion for respondents of equal disease and disability. The presented work demonstrates that neurodegenerative diseases [dementia, cognitive impairment, parkinsonism, instability (gait ataxia), immobility (gait slowing) and motivation loss/behaviour change] have the largest and most significant increases with age of all disease groups. Therefore the hypothesis made in paper one that neurodegenerative diseases will come to dominate the health care needs of older adults, particularly when combined with population ageing, is supported. Further, results of papers two and three indicate that neurodegenerative diseases result in greater levels of disability, lending credence to the finding that it is these neurodegenerative diseases that are of central importance to the future of the health care needs of older adults of advanced age. While systemic diseases play an important role in disability, the neurodegenerative diseases are under-recognised by self-report and yet are most strongly associated with severe disability. A major recommendation of this study is that assessments and diagnosis of neurodegenerative diseases be included in disability assessments. With regard to the social aspects of health, the fourth paper finds that disease and disability are the main predictors of service use and social support. The fifth paper highlights important gender differences in social support and also finds that lower levels of social support are associated with increased age, male gender, single marital status and lower socioeconomic status. Because it is widely accepted that social support is protective against adverse health outcomes and low levels of wellbeing, these groups of older adults are at risk of poorer health and wellbeing. Finally the sixth paper fills some knowledge gaps with regard to the relationship between service use and social support. It shows that with regard to IADL (instrumental activities of daily living) services and IADL social support, Cantor's 'hierarchical-compensatory' mechanism (negative correlation) applies but with regard to medical services and both ADL (activities of daily living) and IADL social support the 'bridging' mechanism (positive correlation) is supported. These complex interrelationships between disease, disability, service use and social support are summarised schematically in a model. In light of significant population ageing, substantial resources in the form of medical and community services and social support from carers, family, friends and neighbours will need to be devoted to older adults with diseases, in particular neurodegenerative diseases, and to those with disabilities. Given the increasing importance of disease, disability, service use and social support in very old age, it is crucial that knowledge and understanding of these factors and their interrelationships be advanced in order to better allocate and sustain resources and to ultimately improve the health, well-being and quality of life of very old adults.