Dissertations / Theses on the topic 'Cystinuria'

Cystinuria is a genetic disease leading a defective dibasic amino acid transporter in the renal proximal tubules resulting in an accumulation of urinary cystine. Urinary cystine precipitates into crystals which is believed to be a necessary step to stone formation. There is a wide variation in disease presentation that is not well understood and cannot be explained by either compliance with medical and dietary interventions, or differences in patient management. Predicting disease severity and managing patients expectantly is confounded by a paucity of validated methods to monitor disease activity and heterogeneity in how urinary dibasic amino acid levels are measured and reported in literature. Management of this disease is largely preventative and based on historical data. At Guy’s and St Thomas’, a clinic was set up to allow for a multidisciplinary approach to the management of these patients. It also provided a premise for research into patients with this disease. The objective of my research was threefold; to understand the factors that lead to severe disease, to investigate clinical markers of disease activity, and to understand the genetic mutations that cause the disease. This thesis is divided into seven chapters incorporating five supporting publications. Chapter one details what is already known on the subject. The evidence for dietary recommendations, current medical therapy and treatments are discussed. Chapter two describes the challenges to the clinician in the management of this disease. The basis of the specialist multidisciplinary clinic is outlined, the roles of each team member and the geographic distribution of our patient cohort. Chapter three investigates the utility of dibasic amino acids in the management of cystinuria in particular, the association between urinary dibasic amino acids levels and stone formation. Dr Caroline Pardy and Dr Erin Mozley contributed significantly to this chapter. Spot urine samples were collected at the time of each clinic visit. The levels of the urinary dibasic amino acids and the association with stone formation as evaluated by ultrasound were analysed. There was a statistically significant association between the levels of urinary ornithine and the presence of stones seen on ultrasound scan. However, as current cystinuria medications aim at reducing cystine levels only with no known effects on levels of urinary ornithine, the prognostic value of this remains uncertain. Chapter four describes original research on the diagnostic value of crystalluria. Separate early morning and clinic urine were collected at each clinic visit and the association between presence of crystals and presence of stones and new stone growth were analysed. Malassez counting chamber and conventional cytospin methods were also compared. The results demonstrated that the presence of crystals in patients with cystinuria is associated with stone formation and new stone growth when based on clinic urine using cytospin method. This may serve as a useful adjunct rather than as a single diagnostic tool. For the first time, the genetic mutations found in a UK population are characterized and described in chapter five. Dr Rachael Mein contributed significantly to this work. We found 23 new mutations in our UK population. We have found that in patients with mutations in SLC3A1, the presence of a missense mutation leads to lower levels of urinary lysine, ornithine and arginine. This is the first time such a genotype-phenotype association has been found and has the potential to improve our risk stratification of patients at the time of diagnosis and tailor their subsequent follow up. This association was not seen for cystine levels however there are limitations in current cystine assays that may account for this and is further discussed in the chapter. In chapter six, the use of protein modelling to model the two proteins encoded by SLC3A1 and SLC7A9 and the mutations that lead to protein dysfunction is described. The severity of the mutations in SLC7A9 as determined by the proximity of the mutation to the ligand binding sites and size of conformational change is shown to lead to a more severe biochemical phenotype as evidenced by raised levels of urinary dibasic amino acids. Further work in this area has the potential to lead to a personalized approach to the management of this disease. Finally, chapter seven summarises all the research findings, discusses the implications to current practice and future challenges in the management of these patients.

Cystinuria is an autosomal recessive disorder of the kidneys and intestine with defective luminal transport of cystine and other dibasic amino acids (ornithine, arginine, and lysine). Three phenotypes have been described, based on urinary excretion of these amino acids in obligate heterozygotes: Type I (silent carriers); Type II (moderate elevation); and Type III (mild elevation). The SLC3AI (D2H) protein has been shown to enhance cystine reabsorption and mutations in D2H have been reported in cystinuria. The aims of this study were to characterize D2H gene structure and to identify mutations in Quebec patients.<br>The D2H cDNA was used to isolate five genomic clones and to characterize the entire gene. The gene spans over 40 kb and contains 10 exons. SSCP and Southern blotting techniques were successful in identifying six novel mutations (2 large deletions, 3 missense mutations, and one 2bp deletion) in twenty cystinuric patients (8 Type I/I, 9 Type I/III, and 3 Type II/N).<br>Our group has identified mutations in the SLC3A1 gene on 15 of 25 cystinuria chromosomes. All but one of these mutations have been found on patients with Type I/I phenotype (the remaining mutation was identified on a Type I/III patient). These studies have revealed eight mutations unique to Quebec and indicate population-specificity and genetic heterogeneity. Furthermore, SLC3A1 mutations only account for Type I cystinuria. However, since only 1 SLC3A1 mutation was identified in 9 Type I/III patients, the data suggest that another gene(s) is (are) responsible for the Type I/N phenotype in some patients.

The aim of this work is to gain insight in the understanding of the biogenesis of membrane proteins, specially their folding, assembly and ER-exit. Our model is the human cystinuria transporter rBAT- b0,+AT. Disulfides and N-glycans are crucial for the correct folding, assembly and traffic of proteins. So we identified the disulfides and the N-glycans of the transporter and analysed their role in biogenesis of the transporter. In order to analyse the disulfide connectivity of rBAT, cysteine residues were mutated to serine, and we used mass-tagging of sulfhydryl (-SH) groups with mPEG5000-maleimide (mPEG) under denaturing conditions. A molecule of mPEG attaches to a –SH group, shifting the apparent molecular weight of the protein of interest by aprox. 5 kDa, which is easily detectable in SDS-PAGE. These experiments show that, in the presence of b0,+AT, the rBAT ectodomain is completely oxidised and contains 3 disulfide bonds. The pegylation pattern of C242S and C273A suggest that they form a disulfide bond. Pulse-chase analysis show that mutants C242S to C666S are retained in the ER and degraded, while C673S and C685S displayed slower stability and maturation rate. Interestingly, the double mutant C673S-C685S had a wild-type behaviour. These results strongly suggested that the other 2 disulfides present in rBAT ectodomain are C571-C666 and C673-C685. Further analysis showed that the only disulfide bond that is stably formed in the absence of the others is C242-C273, suggesting that it is the first to form in rBAT. It was also observed that when rBAT is expressed in the absence of b0,+AT, its oxidative folding is impaired, indicating that b0,+AT is necessary for the folding of rBAT. Its main role may be the stabilization of the oxidation of C571 to form the C571-C666 disulfide. This step seems to occur post-translationally and possibly defining the end of the oxidative folding process. In order to analyse the role of the N-glycans of rBAT the N-glycan consensus sites were mutated to serine or alanine. Pulse assays showed that rBAT contains 5 N-glycans. Pulse-chase and Endo H assays showed that all mutants display a wild-type like stability and maturation, except for the S577A mutant. Further studies showed that the N332 probably sustains degradation of unassembled rBAT and that the N-glycan N575 is necessary and sufficient for a wild-type-like maturation rate. The C-terminal loop of rBAT plays a key role in biogenesis as its deletion causes retention in the ER and subsequent degradation of rBAT. Analysis of this C-terminal loop showed that, when mutated, residues S675A, L678A and N679A have a similar maturation defect than the N-glycan mutant S577A and that mutants Y674A, L681A and Y682A showed little maturation and stability, explaining, at least in part, the results obtained when the whole loop is deleted. When expressed in the C673S-C685S background, some single loop mutants show an important decrease in rBAT stability and maturation. This synergistic effect suggests that the disulfide bond masks part of the maturation and stability effects of these loop residues, and that the absence of the disuflide bond potentiate the misfolding and maturation defects caused by these mutations. Finally, the biogenesis effects of some loop residues in the S577A background suggest that residues S675, L678 and N679 interact functionally and/or structurally with the N-glycan N575 and that they may form part of an ER-exit conformational signal in the luminal domain of the transporter.<br>El objetivo de este trabajo es el de estudiar la biogénesis de las proteínas de membrana. El modelo utilizado para ello es el transportador humano rBAT- b0,+AT, cuya ausencia causa cistinuria. Para estudiar el ensamblaje, el plegamiento y el tráfico del heterodímero se han analizado los puentes disulfuro, los N-glicanos y la cola C-terminal de rBAT. Los resultados muestran que el ectodominio de rBAT se encuentra completamente oxidado formando 3 puentes disulfuro: C242-C273, C571-C666 y C673-C685. Probablemente el primero en formarse es C242-C273 pues es el único capaz de formarse establemente en ausencia de los demás. Cuando una de estas cisteínas se encuentra desapareada, ésta interacciona con los demás residuos de cisteína evitando la correcta formación de los demás puentes disulfuro. La subunidad ligera b0,+AT es necesaria para el plegamiento oxidativo de rBAT. Parece que su presencia estabiliza la oxidación de C571 para formar el puente disulfuro C571-C666. Este puente disulfuro y el C242-C273 son esenciales para la biogénesis del transportador, no así el puente disulfuro C673-C685. rBAT contiene 5 N-glicanos. Ninguno de ellos es esencial para el transportador, aunque son necesarios para una degradación eficiente, para la salida del RE y para que el transportador sea plenamente funcional. De hecho, el N-glicano N575 es necesario y suficiente para conferir una eficiencia máxima de maduración al transportador. El mutante que elimina la cola C-terminal de rBAT, Δ673-685, es retenido en el RE y posteriormente degradado, lo que muestra la importancia de este element en la biogénesis del transportador. El estudio de los mutantes simples y dobles de esta región muestra que estos residuos son importantes para la estabilización y maduración del heterodímero, lo que explica, al menos en parte, el fenotipo observado en Δ673-685. El estudio de los mutantes dobles de la cola C-terminal de rBAT y del N-glicano N575 o del puente disulfuro C673-C685 sugiere que estos elementos podrían interaccionar física y/o funcionalmente con residuos de la cola C-terminal de rBAT para promover la maduración eficiente del transportador, y que podrían constituir parte de una señal conformacional de salida del RE en el dominio luminal de rBAT.

La lithiase rénale touche environ 10% de la population dans les pays industrialisés. 75% des calculs sont composés majoritairement d'oxalate de calcium; 10% sont composés de phosphate de calcium, 9% d'acide urique, 5% de struvite et moins de 1% de cystine. La composition des calculs dépend des espèces sursaturées dans les urines. Dans la première partie de ma thèse, je décris un modèle murin de cystinurie de type A lié à une mutation spontanée apparue dans la souche de souris 129S2/SvPasCrl. La cystinurie est une maladie autosomique récessive responsable de 7% des lithiases de l'enfant. Les calculs de cystine récidivent fréquemment et la cystinurie est caractérisée par un risque élevé de développer une insuffisance rénale chronique. Le modèle que nous proposons permet de tester de nouvelles thérapeutiques. Il met aussi en évidence une atteinte parenchymateuse avec un infiltrat inflammatoire associée aux calculs de cystine. Dans la deuxième partie de ma thèse, j'évalue le rôle des Escherichia coli dans la genèse des calculs phospho-calciques. J'ai étudié en microscopie électronique à balayage des calculs phosphocalciques issus de patients et analysé les propriétés calcifiantes de différentes souches bactériennes sauvages et mutées dans des milieux spécifiques et dans de l'urine. En milieu synthétique le rôle des phosphatases est déterminant mais le type de source de carbone influence l'activité des phosphatases. Dans les urines, certains E. coli induisent la précipitation de phosphate de calcium aussi rapidement que les Klebsiella sans moduler le pH. Le type de source de carbone dans les urines semble déterminant pour moduler la biominéralisation<br>Urolithiasis is a disease that corresponds to the presence of kidney stones in the urinary tract. It affects about 10% of the population in industrialized countries. About 75% of the stones are made of calcium oxalate. Less than 10% are made of calcium phosphate, 9% are made of uric acid, 5% are made of struvite and less than 1% are made of cystine. The composition depends on the species that are supersaturated in urine. In the first part of my thesis I will present a mouse model of cystinuria type A. Cystinuria is an autosomal recessive disease caused by the mutation of either SLC3A1 gene encoding for rBAT (type A cystinuria) or SLC7A9 gene encoding for b0,+AT (type B cystinuria). In 129S2/SvPasCrl strain, we evidenced cystine crystals, as well as cystine stones. We observed an heterogenous inflammatory infiltrate and cystine tubular casts in the parenchyma. We identified a single mutation and a defect of the heavy subunit rBAT. This mouse model could allow for further pathophysiological studies and may be useful to analyse the crystal/tissue interaction in cystinuria. In the second part of my thesis I will test the pathogenesis of E. coli in calcium phosphate stones. In this part, I observed calcium phosphate stones by scanning electron microscopy. I also analysed calcifying properties of wild type bacteria and mutant bacteria in urine or in specific calcifying medium. In synthetic medium phosphatases play a role in calcification but carbohydrate source seems to play a major part in the phosphatase activity. In urine some E. coli induce phosphate calcium precipitation as quickly as Klebsiella does

Thesis (M.S.) -- University of Maryland, College Park, 2005.<br>Thesis research directed by: Dept. of Animal and Avian Sciences. Title from t.p. of PDF. Includes bibliographical references. Published by UMI Dissertation Services, Ann Arbor, Mich. Also available in paper.

La lithiase rénale touche environ 10% de la population dans les pays industrialisés. 75% des calculs sont composés majoritairement d'oxalate de calcium; 10% sont composés de phosphate de calcium, 9% d'acide urique, 5% de struvite et moins de 1% de cystine. La composition des calculs dépend des espèces sursaturées dans les urines. Dans la première partie de ma thèse, je décris un modèle murin de cystinurie de type A lié à une mutation spontanée apparue dans la souche de souris 129S2/SvPasCrl. La cystinurie est une maladie autosomique récessive responsable de 7% des lithiases de l'enfant. Les calculs de cystine récidivent fréquemment et la cystinurie est caractérisée par un risque élevé de développer une insuffisance rénale chronique. Le modèle que nous proposons permet de tester de nouvelles thérapeutiques. Il met aussi en évidence une atteinte parenchymateuse avec un infiltrat inflammatoire associée aux calculs de cystine. Dans la deuxième partie de ma thèse, j'évalue le rôle des Escherichia coli dans la genèse des calculs phospho-calciques. J'ai étudié en microscopie électronique à balayage des calculs phosphocalciques issus de patients et analysé les propriétés calcifiantes de différentes souches bactériennes sauvages et mutées dans des milieux spécifiques et dans de l'urine. En milieu synthétique le rôle des phosphatases est déterminant mais le type de source de carbone influence l'activité des phosphatases. Dans les urines, certains E. coli induisent la précipitation de phosphate de calcium aussi rapidement que les Klebsiella sans moduler le pH. Le type de source de carbone dans les urines semble déterminant pour moduler la biominéralisation<br>Urolithiasis is a disease that corresponds to the presence of kidney stones in the urinary tract. It affects about 10% of the population in industrialized countries. About 75% of the stones are made of calcium oxalate. Less than 10% are made of calcium phosphate, 9% are made of uric acid, 5% are made of struvite and less than 1% are made of cystine. The composition depends on the species that are supersaturated in urine. In the first part of my thesis I will present a mouse model of cystinuria type A. Cystinuria is an autosomal recessive disease caused by the mutation of either SLC3A1 gene encoding for rBAT (type A cystinuria) or SLC7A9 gene encoding for b0,+AT (type B cystinuria). In 129S2/SvPasCrl strain, we evidenced cystine crystals, as well as cystine stones. We observed an heterogenous inflammatory infiltrate and cystine tubular casts in the parenchyma. We identified a single mutation and a defect of the heavy subunit rBAT. This mouse model could allow for further pathophysiological studies and may be useful to analyse the crystal/tissue interaction in cystinuria. In the second part of my thesis I will test the pathogenesis of E. coli in calcium phosphate stones. In this part, I observed calcium phosphate stones by scanning electron microscopy. I also analysed calcifying properties of wild type bacteria and mutant bacteria in urine or in specific calcifying medium. In synthetic medium phosphatases play a role in calcification but carbohydrate source seems to play a major part in the phosphatase activity. In urine some E. coli induce phosphate calcium precipitation as quickly as Klebsiella does

In the kidney, unbound amino acids are freely filtered into the lumen of the nephron. For reabsorption to occur, they must be transported across the phospholipid bilayers of the tubular epithelium by selective transport systems. Mutations in these transport systems can lead to disease though a conferred lack of amino acid re-absorption. One such disease is cystinuria, caused by mutations in SLC3A1 and SLC7A9, which encode the two protein subunits of System b0,+, rBAT and b0,+AT, respectively. In healthy individuals System b0,+ mediates Na+- independent reabsorption of dibasic amino acids, and the cysteine dimer, cystine, in exchange for neutral amino acids. In cystinuric patients, these amino acids are not sufficiently reabsorbed causing a dibasic aminoaciduria and the precipitation of cystine crystals, leading to the formation of renal calculi. A cohort of cystinuric patients was recruited to the study, and both genes were screened for causal variants. A range of techniques was employed to enable the detection of small point mutations and large genomic rearrangements. Four novel missense variants were detected in SLC3A1. These were M465K, N254T, L416P and Y579D. In silico homology modeling of rBAT against the crystal structure of B. cereus oligo-1,6-glucosidase (PDB code 1UOK), predicted the location of these mutations in the extracellular domain of the protein. When rBAT cRNA was injected into Xenopus oocytes, uptake of the prototypical System b0,+ substrate [3H]arginine was observed, following the association of human rBAT with an endogenous oocyte light chain. A series of techniques was optimised to allow the characterisation of FLAG-tagged rBAT function and expression in oocytes, 1-6 days postinjection of cRNA. Mutations in rBAT lead to a mis-folding of the protein and its early degradation in the ER, preventing successful trafficking of the System b0,+ heterodimer to the renal epithelial membrane. This aberrant trafficking leads to reduced rBAT expression and System b0,+ activity in oocytes. Functional characterisation of the novel mutant proteins led to a decrease in the Vmax of [3H]arginine transport. Over-expression of rBAT in oocytes apparently overcomes the defect and leads to a recovery of function over time. However, [3H]arginine uptake in M465Kexpressing oocytes was still lower than that observed with wild-type rBAT even at 6 days postinjection. These data were supported by immunofluorescent detection of rBAT and the mutant proteins at the plasma membrane of oocytes. Western blotting of total membrane proteins from oocytes expressing mutated rBAT showed decreased total protein, suggestive of an increased rate of degradation associated with the pathogenic variants. An increased understanding of the effect of these mutations on the biogenesis of rBAT will contribute to the identification of novel therapeutic targets in the treatment of cystinuria.

Un biomarqueur est une molécule (ou un ensemble de molécules) présente dans l’organisme qui témoigne de l’apparition d’un processus pathologique. Il permet ainsi de dépister une maladie, d’en prédire sa gravité ou encore d’évaluer l’efficacité d’un traitement. Les liquides biologiques représentent des milieux de choix pour la recherche de biomarqueurs en pathologie humaine car leur collection est habituelle dans la prise en charge des patients et moins invasive comparée aux biopsies d’organes ou de tissus. Dans cette thèse, nous nous sommes intéressés plus particulièrement aux exosomes présents dans ces liquides biologiques. Les exosomes sont des nanovésicules dont le diamètre est compris entre 30 et 100 nanomètres. Ils sont sécrétés par tous les types cellulaires et contiennent des protéines cytoplasmiques et membranaires spécifiques de leur cellule d’origine. L’intérêt majeur des exosomes isolés à partir des liquides biologiques, est qu’ils constituent une source de biomarqueurs. Ils peuvent donc être assimilés à une « biopsie liquide ». L’analyse des exosomes pourrait compléter utilement des examens classiques de dépistage, de diagnostic et de suivi d’une pathologie. Dans le cadre de projet de cette thèse, nous avons appliqué des techniques de protéomique à haut débit pour l’analyse des exosomes. Nous nous sommes tout d’abord intéressés à l’analyse du profil protéique des exosomes urinaires dans le contexte de deux pathologies du tractus urinaire : la cystinurie et le cancer du rein. La cystinurie est une néphropathie lithiasique d’origine génétique pour laquelle il y a peu de marqueurs biologiques pouvant prédire son évolution vers l’insuffisance rénale terminale. Nous avons développé une méthode de préparation des exosomes urinaire permettant d’analyser de façon reproductible leurs profils protéiques. Nous avons appliqué cette méthode à huit patients cystinuriques et comparé les résultats aux profils obtenus chez dix sujets sains. Un panel de 38 protéines différentiellement exprimé dans les exosomes des patients a été identifié et en partie validé par Western blot. Concernant le cancer du rein à cellules claires pour lequel le diagnostic nécessite des prélèvements invasifs par biopsie, nous avons analysé les exosomes urinaires de huit patients avant et après néphrectomie. Nous avons ainsi pu mettre en évidence un panel de 25 protéines surexprimées dans les exosomes des patients. Enfin, le dernier volet de cette thèse a été consacré à l’analyse des exosomes du lavage broncho-alvéolaire provenant de patients MV, maladie d’origine génétique qui atteint principalement les poumons. L’analyse des exosomes de lavage broncho-alvéolaire pourrait permettre de donner un éclairage nouveau sur la physiopathologie de la maladie. Nous avons réalisé la comparaison des profils protéiques des exosomes de quatre patients MV, et six patients asthmatiques. L’ensemble des résultats obtenus au cours de cette thèse montre que l’analyse protéomique des exosomes issus de fluides biologiques peut aider la recherche de biomarqueurs diagnostics ou pronostics de maladies<br>A biomarker is a molecule (or a cluster of molecule) which will reflect the occurrence of a pathological state, giving us the ability to detect a disease, to predict its severity or to assess drug efficiency. Biological fluids are the golden standards for biomarker research in human as they are routinely collected for patients’ follow-up and are less invasive than biopsies. During my PhD, I focused on exosomes that can be found in these biological fluids. Exosomes are nanovesicles with a diameter ranging between 30 and 100 nanometers. Exosomes are secreted by all cell types and harbor cytoplasmic and membranous proteins specific of their cells of origins. One of the major interest of exosomes enriched from biological fluids is that they represent a valuable source of biomarkers. They can be considered as a « liquid biopsy ». Their analysis could complete classical diagnosis and follow-up tools. In this project, we applied high resolution, high throughput proteomic techniques for exosomes analysis. We firstly focused on protein profiles in urinary exosomes in the context of two urinary tract diseases: cystinuria and kidney cancer. Cystinuria is an inherited autosomal recessive disease that is characterized by the formation of cystine stones in the kidneys. To date, there are no markers to predict the evolution toward end stage renal disease. We developed a method to prepare exosomes in order to reproducibly analyze their protein profiles. We applied this method to eight cystinuria patients and compared their profiles to those of ten healthy subjects. A panel of 38 differentially expressed proteins in patients were found and validated by western blots. We also applied this method to patients with clear cell renal cell carcinoma, for which invasive biopsies are necessary for clear diagnosis. We analyzed urinary exosomes form eight patients before and after nephrectomy. We were able to highlight 25 overexpressed proteins in patients’ exosomes. Eventually, the last part of my thesis was dedicated to the analysis of exosomes enriched from bronchoalveolar lavage fluid collected in cystic fibrosis patients, a disease that affects mostly the lungs. Bronchoalveolar lavage fluid exosomes analysis could give a new insight on the mechanisms of this disease. We compared protein profiles in exosomes from four cystic fibrosis patients and six asthmatic patients. The whole point of this work is to show that proteomic analysis of exosomes isolated from biological fluids could become a golden standard for the discovery of diagnosis or prognosis biomarkers

Peptidases regulate important physiological processes by controlling levels of bioactive peptides and occasionally through noncatalytic processes. This thesis presents a study of prolyl endopeptidase-like (PREPL), which is a peptidase involved in several human deletion syndromes, including hypotonia-cystinuria syndrome (HCS). Phenotypes tentatively attributed to PREPL deletion include hypotonia and decreased growth hormone (GH) levels. However, little is known about the mechanisms by which PREPL deletion causes these phenotypes. To better understand PREPL catalytic activity, we used an activity-based protein profiling fluorescence polarization screen to identify the first specific PREPL inhibitors. We proceeded to demonstrate the activity of these inhibitors in cells and discovered several classes of cell-active PREPL inhibitors. Further, one of these inhibitors, 1-isobutyl-3-oxo-3,5,6,7-tetrahydro-2H-cyclopenta[c]pyridine-4-carbonitrile, was able to enter mouse brains. To characterize PREPL substrate specificity, we performed several substrate profiling experiments, but no substrates could be identified, in line with reports from other groups who used related approaches to attempt to identify PREPL substrates. To characterize any noncatalytic functions of PREPL, we used an affinity purification-mass spectrometry approach (AP-MS) to search for any protein-protein interactions of PREPL. We identified brain-expressed X-linked 2 (BEX2) as a novel interactor of PREPL, and confirmed this interaction by immunoblot. Several other proteins identified in the AP-MS experiment, including several members of the STRIPAK complex are being further investigated for possible PREPL interaction. To determine whether HCS phenotypes are in fact due to PREPL deletion and to delineate the molecular pathways involved, we generated a conditional PREPL knockout mouse. These mice were visibly smaller than wildtypes and growth curve analysis verified that from week three of life, there was a significant difference in weight between wildtype and knockout mice. Initial surface righting task experiments also indicate that PREPL knockout pups may have a hypotonia phenotype. In summary, we have developed several new tools for studying PREPL catalytic and noncatalytic function, demonstrated that PREPL deletion causes a GH-related growth deficiency and possible hypotonia and thus moved several steps closer to understanding the molecular mechanisms underlying PREPL deletion phenotypes.<br>Chemistry and Chemical Biology

Trabalho Final do Mestrado Integrado em Medicina apresentado à Faculdade de Medicina<br>Introdução: A cistinúria é uma doença hereditária autossómica recessiva rara. Os principais objetivos deste estudo prenderam-se com a caracterização epidemiológica, bem como a avaliação da terapêutica tanto médica como cirúrgica a que os doentes com cistinúria foram sujeitos. Materiais e métodos: Analisámos retrospetivamente os processos clínicos de 15 doentes com cistinúria, seguidos no Centro Hospitalar e Universitário de Coimbra, que foram submetidos a procedimentos de fragmentação e extração de cálculos entre janeiro de 2005 e dezembro de 2019. Recolhemos os dados epidemiológicos dos doentes e registámos o número de intervenções a que estes foram sujeitos durante o período do estudo. Resultados: Dos 15 doentes em estudo, 9 eram do sexo masculino e 6 do sexo feminino, com idades médias de 47.2 anos. A média de idades na altura do diagnóstico destes doentes foi de 32.9 anos (compreendidas entre 12 e 60), com acometimento bilateral em 11 doentes (73.33%). Todos os doentes se encontravam sob terapêutica não farmacológica e a maior parte deles sob terapêutica farmacológica, com citrato de potássio e captopril. Os doentes foram submetidos a um total de 407 procedimentos, com uma média de 27.13 intervenções/pessoa e 1.81 intervenções/pessoa/ano. Destes 407 procedimentos, 330 corresponderam à Litotrícia Extracorpórea por Ondas de Choque (81.08%), com uma média de 22 sessões/pessoa e 1.47 sessões/pessoa/ano. Dos procedimentos invasivos, destacou-se a ureterorrenoscopia (11.06% dos procedimentos), com um total de 45 intervenções, numa média de 3/pessoa. Comparando os dois sexos, a Litotrícia Extracorpórea, como tratamento não invasivo, apresentou uma média de 21.33 tratamentos/pessoa no sexo masculino em relação aos 23.00 tratamentos/pessoa no sexo feminino. Quanto aos tratamentos invasivos, os doentes do sexo masculino apresentaram uma média de número de tratamentos mais alta, com exceção da pielolitotomia. Conclusão: Este estudo demonstrou a dificuldade no tratamento da cistinúria, com cada doente a ser submetido a um elevado número de procedimentos de fragmentação e extração de cálculos, com um seguimento de vários anos pela mesma. A maioria dos doentes encontrava-se sob terapêutica farmacológica, contudo não foi possível perceber o grau de adesão a esta nem à mudança de hábitos, ambas essenciais para minimizar a gravidade dos episódios sintomáticos da doença. A associação a sessões de Litotrícia Extracorpórea periódicas pode reduzir a necessidade de recurso a procedimentos invasivos. Palavras-chave: Cistinúria; Litíase; Litotrícia Extracorpórea por Ondas de Choque; Nefrolitotomia Percutânea; Ureterorrenoscopia; Pielolitotomia; Estudo retrospetivo.<br>Introduction: Cystinuria is a rare autosomal recessive hereditary disease. The main goals of this study were the epidemiological characterization and the evaluation of the therapy, both medical and surgical to which patients with cystinuria were submitted to. Material and methods: We analysed retrospectively the clinical records of 15 patients with cystinuria, followed at CHUC, who underwent fragmentation and stone extraction procedures between January 2005 and December 2019. We collected the epidemiological data from the patients and recorded the number of interventions they underwent during the study period. Results: Of the 15 patients in the study, 9 were male and 6 female, with an average age of 47.2 years. At the time of diagnosis of these patients, the average age was 32.9 years (range 12-60), with bilateral involvement in 11 patients (73.33%). All patients were on non-pharmacological therapy and most of them were on pharmacological therapy, with potassium citrate and captopril. Patients underwent a total of 407 procedures, with an average of 27.13 interventions/person and 1,81 interventions/person/year. Of these 407 procedures, 330 corresponded to Extracorporeal Shockwave Lithotripsy (81.08%), with an average of 22 sessions/person and 1.47 sessions/person/year. Of the invasive procedures, ureterorenoscopy (11.06% of the procedures) stand out, with a total of 45 interventions, an average of 3/person. Comparatively between the two genres, the Extracorporeal Shockwave Lithotripsy, as a non-invasive treatment, presented an average of 21.33 treatments/person in males compared to 23.00 treatments/person in females. As for invasive treatments, male patients had a higher average number of interventions, with the exception of pyelolithotomy. Conclusion: This study demonstrated the difficulty in the treatment of cystinuria, with each patient being subjected to a high number of fragmentation and stone extraction procedures, with a follow-up of several years. Most patients were on pharmacological therapy, however it was not possible to understand the compliance with it or with the dietary changes, both essential to minimize the severity of symptomatic episodes of the disease. The association with periodic Extracorporeal Shockwave Lithotripsy sessions can reduce the need for invasive procedures. Keywords: Cystinuria; Lithiasis; Extracorporeal Shockwave Lithotripsy; Percutaneous Nephrolithotomy; Ureterorenoscopy; Pyelolithotomy; Retrospective study.