Minimal change disease (MCD) and primary (idiopathic) focal segmental glomerulosclerosis (1FSGS), referred to collectively as “primary podocytopathies”, are major causes of nephrotic syndrome in children and adults, and are thought to be due to direct podocyte damage visible only at the electron microscopic level. Lupus podocytopathy (LP) is a newly recognized entity that involves severe podocyte injury in the setting of systemic lupus erythematosus, in the complete absence of peripheral capillary wall immune deposits. All of these pathologic diagnoses hinge on the ultrastructural finding of severe podocyte injury and foot process effacement. In addition to these ultrastructural changes, we have observed the presence of fine granular anti-IgG antibody immunoreactivity on podocyte cell bodies in kidney biopsies of patients with MCD, LP, and some patients with the tip lesion variant and NOS variants of 1FSGS. To validate this finding, we compared antibody staining from primary podocytopathy biopsies with those in biopsies from patients with other disease states, including lesions associated with severe podocyte injury in the absence of immune deposits: secondary (adaptive) focal segmental glomerulosclerosis, thin basement membrane disease, diabetic nephropathy, and renal amyloidosis. We found that a fine granular pattern of anti-IgG immunoreactivity on podocyte cell bodies is a specific morphologic feature of the primary podocytopathies, including virtually all cases of MCD that we encountered, some instances of tip lesion variant and NOS variant of 1FSGS, and one cases of LP. The antigen targeted by the anti-IgG immunostaining in these biopsies exhibited one of several oligoclonal IgG heavy chain subtype plus light chain profiles. Ultra-high resolution microscopy revealed fine linear anti-IgG staining along filtration slit diaphragms, suggesting that IgG deposition may potentially be targeting a filtration slit-associated antigen such as podocin. Our findings suggest the possibility of a direct antibody-mediated mechanism of podocyte injury in the primary podocytopathies, one that potentially targets podocyte-specific protein structures, and which may provide a specific and more rapid diagnostic marker for this group of diseases. The findings also suggest an etiologic relationship between MCD and some instances of 1FSGS.

Nephrotic syndrome (NS) is a set of symptoms defined by heavy proteinuria and associated with a host of kidney diseases that cause injury to the glomerulus, the filtration apparatus of the kidney. Primary podocytopathies (Px), a group of diseases including minimal change disease (MCD), primary focal segmental sclerosis (pFSGS), and lupus podocytopathy (LP), are the principal cause of idiopathic NS in both children and adults. The hallmark feature of Px is the ultrastructural finding of podocyte foot process effacement (FPE), so current differential diagnosis of Px relies on technically exhaustive electron microscopy (EM) analysis. During routine immunofluorescence (IF) microscopy of many Px cases, we have observed punctate IgG (P-IgG) immunoreactivity in the glomerulus. P-IgG may represent a disease-specific reactivity that could not only provide clues for understanding Px etiology but could also serve as a diagnostic tool. However, we have found that P-IgG may be misinterpreted as protein reabsorption granules (PRGs), a morphological feature seen in proteinuric conditions. We sought to definitively characterize the key characteristics of PRGs and P-IgG in order to differentiate these features. To accomplish this, we reviewed prior IF immunostaining of MCD biopsies to evaluate anti-human IgG and anti-human albumin staining. We conducted additional IF staining on archived MCD biopsies using antibodies against IgG and against the markers of PRGs: albumin, megalin, and cubilin. We found that the P-IgG demonstrates a diffuse, global distribution pattern that is specific to glomerular epithelium and is fine and scattered. Conversely, the PRGs are coarse, clustered, and frequently demonstrate a focal, segmental pattern in the glomeruli and tubules. Co-staining with albumin and megalin revealed that the P-IgG and the PRGs do not colocalize in the tissue. While the cubilin antibody positively stained the tubular epithelium, it did not stain glomeruli. Our results showed that punctate IgG and protein reabsorption granules are morphologically and constitutionally distinct and do not colocalize with each other, indicating that P-IgG is highly likely to represent a distinct process from epithelial protein reabsorption.

Cette thèse traite du problème de l'intégration d'une nouvelle information dans un système d'argumentation abstrait. Un tel système est un graphe orienté dont les nœuds représentent les arguments, et les arcs représentent les attaques entre arguments. Il existe divers moyen de décider quels arguments sont acceptés par l'agent qui utilise un tel système pour représenter ses croyances.Il peut arriver dans la vie d'un agent qu'il soit confronté à une information du type "tel argument devrait être accepté", alors que c'est en contradiction avec ses croyances actuelles, représentées par son système d'argumentation.Nous avons étudié dans cette thèse diverses approches pour intégrer une information à un système d'argumentation.Notre première contribution est une adaptation du cadre AGM pour la révision de croyances, habituellement utilisé lorsque les croyances de l'agent sont représentées dans un formalisme logique. Nous avons notamment adapté les postulats de rationalité proposés dans le cadre AGM pour pouvoir caractériser des opérateurs de révision de systèmes d'argumentation, et nous avons proposé différents moyens de générer les systèmes d'argumentation résultant de la révision.Nous avons ensuite proposé d'utiliser la révision AGM comme un outil pour réviser les systèmes d'argumentation. Il s'agit cette fois-ci d'une approche par encodage en logique du système d'argumentation, qui permet d'utiliser les opérateurs de révision usuels pour obtenir le résultat souhaité.Enfin, nous avons étudié le problème du forçage d'un ensemble d'arguments (comment modifier le système pour qu'un ensemble donné soit une extension). Nous avons proposé une nouvelle famille d'opérateurs qui garantissent le succès de l'opération, contrairement aux opérateurs de forçage existants, et nous avons montré qu'une traduction de nos approches en problèmes de satisfaction ou d'optimisation booléenne permet de développer des outils efficaces pour calculer le résultat du forçage. / This thesis tackles the problem of integrating a new piece of information in an abstract argumentation framework. Such a framework is a directed graph such that its nodes represent the arguments, and the directed edges represent the attacks between arguments. There are different ways to decide which arguments are accepted by the agent who uses such a framework to represent her beliefs.An agent may be confronted with a piece of information such that "this argument should be accepted", which is in contradiction with her current beliefs, represented by her argumentation framework.In this thesis, we have studied several approaches to incorporate a piece of information in an argumentation framework.Our first contribution is an adaptation of the AGM framework for belief revision, which has been developed for characterizing the incorporation of a new piece of information when the agent's beliefs are represented in a logical setting. We have adapted the rationality postulates from the AGM framework to characterize the revision operators suited to argumentation frameworks, and we have identified several ways to generate the argumentation frameworks resulting from the revision.We have also shown how to use AGM revision as a tool for revising argumentation frameworks. Our approach uses a logical encoding of the argumentation framework to take advantage of the classical revision operators, for deriving the expected result.At last, we have studied the problem of enforcing a set of arguments (how to change an argumentation framework so that a given set of arguments becomes an extension). We have developed a new family of operators which guarantee the success of the enforcement process, contrary to the existing approaches, and we have shown that a translation of our approaches into satisfaction and optimization problems makes possible to develop efficient tools for computing the result of the enforcement.

Immunolocalization of proteins within the cell is a significant and powerful tool that improves understanding of cellular functions and processes, such as molecule secretion during immune responses. Immunogold electron microscopy (IEM) is an immunohistochemistry technique that uses gold-conjugated antibodies and electron microscopy (EM) to identify and localize antigens at the ultrastructural level. Here, we are trying to develop and optimize an IEM staining protocol that targets glomerular proteins of interest in Minimal Change Disease (MCD), and eliminates background staining, and preserves tissue morphology. Using this optimized protocol, we hope to learn more about the relationship between IgG and Nephrin in MCD. Kidney biopsies diagnosed with MCD, Membranous Nephropathy (MN), and Thin Basement Membrane Disease (TBMD) and previously embedded in paraffin blocks were retrieved from the tissue archive of the Renal Pathology Laboratory at Boston Medical Center. MN and TBMD were selected as positive controls for IgG and Nephrin staining protocols, respectively. Co-staining of IgG and Nephrin was performed after the protocols for each target were optimized. During protocol development, it was observed that section quality is significantly affected by the angle and sharpness of the knife, and the thickness of the section. Moreover, section quality highly impacted gold particle localization. Ultimately, co-staining of IgG and Nephrin was successful in MCD cases. However, further improvements are needed to optimize IgG and Nephrin staining, and in turn, our understanding of MCD.

La pathogénie du syndrome néphrotique idiopathique est inconnue, mais de nombreux arguments clinques et expérimentaux favorisent l’hypothèse d’une pathogénie dys-immunitaire à expression immunologique et rénale, au cours de laquelle on observerait une altération des lymphocytes T. Cependant, le mécanisme exact reste encore mal connu. Récemment, le Rituximab, un anticorps dirigé contre l’antigène CD20, a montré une efficacité à induire une rémission à moyen et long terme suggérant l’implication d’une dysfonction des lymphocytes B et/ou un défaut de coopération T-B. Notre laboratoire a isolé un nouveau gène C-MIP dont l’expression est induite dans certaines sous-populations lymphocytaires T et B, ainsi que dans les podocytes de patients atteints de SNI en phase de poussée mais quasiment indétectable chez les sujets sains.Dans ces travaux, ancillaires au PHRC NEPHRUTIX, nous avons étudié les perturbations lymphocytaires T, avant, au moment de la rechute et en période de rémission au cours de syndrome néphrotique à lésions glomérulaires minimes et l’effet du traitement par le Rituximab. Dans cette étude, nous avons mis en évidence que la rechute était associée à un effondrement des lymphocytes T régulateurs, une baisse profonde de l’interleukine-2 ainsi qu’à une surexpression significative de C-MIP, précédant la survenue de la rechute. Ces modifications se restaurent en rémission. Enfin, la rémission obtenue dans le bras Rituximab, entraîne une diminution des lymphocytes T folliculaires (Tfh), des iNKT et des cellules double-négatives DN-TCR Vα24, suggérant que le SNLGM implique un défaut des réponses immunitaires innées et adaptatives, qui peut être stabilisé par un traitement par Rituximab.Afin d’étudier le rôle de C-MIP, nous avons généré des souris transgéniques sur-exprimant ce gène dans les lymphocytes T matures périphériques. Cette surexpression est à l’origine d’un phénotype lymphocytaire altéré marqué par une accumulation de lymphocytes T naïfs, un effondrement des cytokines activatrices de type Th1 et Th2 et une accumulation des formes inactives des Src kinases. Ces résultats suggèrent que C-MIP, en inhibant les Src kinases, est un régulateur négatif de l’activation T impliqué dans la signalisation proximale et pourrait être impliqué dans l’hypo-réactivité lymphocytaire T observée chez les patients atteints de SNLGM actif. / The pathogenesis of minimal-change nephrotic syndrom (MCNS) is unknown, but, supported by many clinical and experimental arguments, it was suggested that MCNS is a dys-immune disorder with immunogical and renal expression, during which T-cell alteration would be observed. However, the exact mechanism remains unknown. Recently, Rituximab, a B-cell depleting agent, is effctive in inducing mid- and long-term remission suggesting involvement of B-cell dysfunction and/or lack of T-B cooperation. Our laboratory identified a new gene: C-MIP. We have shown that C-MIP abundance is increased in some T and B lymphocyte subpopulations, as well as in podocytes of MCNS patients during relapse phase but undetectable in healthy subjects.In this work, ancillary to the NEPHRUTIX PHRC, we studied T-cell disturbances before and during the relapse or during the remission time in MCNS and the effect of Rituximab therapy. In this study, we found that relapses were associated with significant decrease in regulatory T cell and interleukin-2 expression, while C-MIP abundance was significantly increased. These changes are restored during remission time. Finally, remission after Rituximab therapy leads to a decrease in follicular T cells (Tfh), iNKT and double-negative (CD4- CD8-) T cells expressing the invariant Vα24 chain, suggesting that MCNS involves a disorder of innate and adaptative immune response, which can be stabilized by Rituximab treatment.In order to study the C-MIP role, we generated transgenic mice overexpressing this gene in the peripheral mature T-cells. This overexpression leads to an altered lymphocyte phenotype with an accumulation of naive T lymphocytes, a significant decrease of Th1 and Th2 activating cytokines and accumulation of inactive Src kinases. These results suggest that, by inhibiting Src kinases, C-MIP is a negative regulator of activation T involved in proximal signalling and may be responsable of the lymphocyte T hypo-reactivity observed in patients with active MCNS.

Nephrotic syndrome is characterized by proteinuria, hypoproteinemia, edemas and hyperlipidemia. It occurs in primary (e.g. focal segmental glomerulosclerosis, FSGS or minimal change disease, MCD) and in secondary glomerulopathies (e.g. kidney amyloidosis). In primary forms, great attention is paid to the potential genetic background of the disease and due to new molecular genetic methods genes, whose mutations cause different nephropathies (e.g. ACTN4 or INF2) were identified. The aims of presented doctoral thesis were following. Firstly, to continue the mutational analysis of ACTN4 that was described in the author's diploma thesis in other glomerulopathies. Secondly, to implement the mutational analysis of INF2 and subsequently analyse this gene in patients with FSGS/MCD and in patients from special group characterized by positive family history for end stage renal disease (ESRD) in combination with advanced chronic kidney disease (CKD) or already developed ESRD at the time of diagnosis. Thirdly, mutational analysis of NPHS2 and TRPC6 (methods implemented in laboratory earlier) in selected patients from the special group. Finally, expression analyses of genes important for podocyte function or connected with human immune system. This part also verifies the applicability of NPHS2/SYNPO expression...

Combinatorial objects can be represented by strings, such as 21534 for the permutation (1 2) (3 5 4), or 110100 for the binary tree corresponding to the balanced parentheses (()()). Given a string s = s1 s2 sn, the right-shift operation shift(s, i, j) replaces the substring si si+1..sj by si+1..sj si. In other words, si is right-shifted into position j by applying the permutation (j j−1 .. i) to the indices of s. Right-shifts include prefix-shifts (i = 1) and adjacent-transpositions (j = i+1). A fixed-content language is a set of strings that contain the same multiset of symbols. Given a fixed-content language, a shift Gray code is a list of its strings where consecutive strings differ by a shift. This thesis asks if shift Gray codes exist for a variety of combinatorial objects. This abstract question leads to a number of practical answers. The first prefix-shift Gray code for multiset permutations is discovered, and it provides the first algorithm for generating multiset permutations in O(1)-time while using O(1) additional variables. Applications of these results include more efficient exhaustive solutions to stacker-crane problems, which are natural NP-complete traveling salesman variants. This thesis also produces the fastest algorithm for generating balanced parentheses in an array, and the first minimal-change order for fixed-content necklaces and Lyndon words. These results are consequences of the following theorem: Every bubble language has a right-shift Gray code. Bubble languages are fixed-content languages that are closed under certain adjacent-transpositions. These languages generalize classic combinatorial objects: k-ary trees, ordered trees with fixed branching sequences, unit interval graphs, restricted Schr oder and Motzkin paths, linear-extensions of B-posets, and their unions, intersections, and quotients. Each Gray code is circular and is obtained from a new variation of lexicographic order known as cool-lex order. Gray codes using only shift(s, 1, n) and shift(s, 1, n−1) are also found for multiset permutations. A universal cycle that omits the last (redundant) symbol from each permutation is obtained by recording the first symbol of each permutation in this Gray code. As a special case, these shorthand universal cycles provide a new fixed-density analogue to de Bruijn cycles, and the first universal cycle for the "middle levels" (binary strings of length 2k + 1 with sum k or k + 1).