Le récepteur des phospholipases A2 sécrétées (PLA2R1, 180 kDa) est une protéine membranaire de la famille des lectines de type C. PLA2R1 contrôle l'action de différentes phospholipases A2 impliquées dans des conditions physiologiques et physiopathologiques variées comme le métabolisme des lipides et l’inflammation. PLA2R1 est aussi l’auto-antigène majeur de la glomérulonéphrite extra-membraneuse (GEM), une maladie auto-immune rénale rare mais grave qui conduit à une forte protéinurie et à la perte des reins dans 30% des cas. Le titre des auto-anticorps PLA2R1 est corrélé à la sévérité de la maladie, mais leur valeur pronostique reste à démontrer. Le rôle pathogénique des anticorps n’est pas démontré. Enfin, 25% des patients sont négatifs pour PLA2R1, suggérant l’existence d'autres antigènes.Nous avons d’abord démontré la production d'une forme soluble sécrétée de PLA2R1, puis étudié les déterminants moléculaires du mécanisme de protéolyse. Concernant la GEM, nous avons développé plusieurs tests ELISA anti-PLA2R1 utilisant comme antigènes PLA2R1 humain, de lapin et de souris. Leur comparaison a montré des apports différents en diagnostic et pronostic. Nous avons aussi identifié 3 types d'auto-anticorps présents dans le sérum des patients. Ces anticorps ciblent 3 domaines épitopiques distincts de PLA2R1, sont liés par un mécanisme d’étalement épitopique, et la présence de plusieurs anticorps dans le serum est associé à un mauvais pronostic. Enfin, nous avons identifié la protéine membranaire THSD7A, distincte de PLA2R1, comme un second auto-antigène de la GEM, avec des auto-anticorps présents chez 2 à 5% des patients négatifs pour PLA2R1 / The phospholipase A2 receptor (PLA2R1, 180kDa) is a C-type lectin membrane protein. PLA2R1 binds secreted phospholipases A2 (sPLA2s) from snake venoms and mammalian tissues. Venom sPLA2s have multiple toxic and pharmacological properties, whereas mammalian sPLA2s are implicated in various physiological and pathophysiological conditions, including lipid metabolism and inflammation.PLA2R1 is also the major autoantigen in membranous nephropathy (MN), a rare but severe autoimmune kidney disease leading to high proteinuria and kidney failure in 30% of cases. Titers of PLA2R1 autoantibodies correlate with disease severity, but the prognosis value of the antibodies is not demonstrated. Their pathogenic role is also not proven. 25% of patients are negative for PLA2R1, suggesting other antigens involved in MN. We have first studied some molecular properties of PLA2R1 in the context of MN. We demonstrate the presence of a secreted soluble form of PLA2R1 produced by proteolytic shedding of the membrane protein and we have studied the molecular determinants of this mechanism. Regarding MN, we have developed several anti-PLA2R1 ELISA using human, rabbit and mouse PLA2R1 as antigens. Their comparison revealed differential contributions in diagnosis and prognosis. We have also identified in patients' sera 3 types of autoantibodies, which target three distinct epitope domains of PLA2R1, which are linked by a mechanism of epitope spreading and which are associated with disease worsening and poor prognosis. Finally, we identified THSD7A, a membrane protein distinct from PLA2R1, as a second autoantigen in MN, with autoantibodies present in 2-5% of PLA2R1-negative patients

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O anticorpo anti-proteína P ribossomal é um marcador sorológico do lúpus eritematoso sistêmico. Nós avaliamos a relevância do mesmo em discriminar os padrões histopatológicos de nefrite lúpica. O anti-P foi detectado em 18/81(22%) dos pacientes com envolvimento renal confirmado por biópsia. Foi observada uma freqüência aumentada deste anticorpo em pacientes com classe V (72%) comparado com outras classes de nefrite (28%), p=0.005. Dentro do esperado, pacientes anti-P positivos tiveram um nível médio de proteinúria mais elevado que pacientes anti-P negativos (6,4 + 4,8 vs. 4,7 + 3,9 g/dl, p= 0,046). É ainda interessante que a maioria dos pacientes com anti-P isolado tinha classe V, e 71%apresentaram o padrão membranoso puro. O anti-P parece ser um novo marcador sorológico para a nefrite lúpica membranosa. / Anti-ribosomal P antibody is a serological marker for systemic lupus erythematosus. We have evaluated its relevance in discriminating histopathologic patterns of lupus nephritis. Anti-P was detected in 18/81 (22%) patients with biopsy proven renal involvement. A higher frequency of this antibody was observed in patients with class V (72%) compared to other classes of renal disease (28%), p=0.005. Accordingly, anti-P positive patients had higher mean proteinuria level than anti-P antibody negative patients (6.4 + 4.8 vs. 4.7 + 3.9 g/dl, p= 0.046). Interestingly, the majority of patients with isolated anti-P had class V, and 71% displayed a pure membranous pattern. Anti-P seems to be a novel serological marker for membranous lupus nephritis.

O anticorpo anti-proteína P ribossomal é um marcador sorológico do lúpus eritematoso sistêmico. Nós avaliamos a relevância do mesmo em discriminar os padrões histopatológicos de nefrite lúpica. O anti-P foi detectado em 18/81(22%) dos pacientes com envolvimento renal confirmado por biópsia. Foi observada uma freqüência aumentada deste anticorpo em pacientes com classe V (72%) comparado com outras classes de nefrite (28%), p=0.005. Dentro do esperado, pacientes anti-P positivos tiveram um nível médio de proteinúria mais elevado que pacientes anti-P negativos (6,4 + 4,8 vs. 4,7 + 3,9 g/dl, p= 0,046). É ainda interessante que a maioria dos pacientes com anti-P isolado tinha classe V, e 71%apresentaram o padrão membranoso puro. O anti-P parece ser um novo marcador sorológico para a nefrite lúpica membranosa. / Anti-ribosomal P antibody is a serological marker for systemic lupus erythematosus. We have evaluated its relevance in discriminating histopathologic patterns of lupus nephritis. Anti-P was detected in 18/81 (22%) patients with biopsy proven renal involvement. A higher frequency of this antibody was observed in patients with class V (72%) compared to other classes of renal disease (28%), p=0.005. Accordingly, anti-P positive patients had higher mean proteinuria level than anti-P antibody negative patients (6.4 + 4.8 vs. 4.7 + 3.9 g/dl, p= 0.046). Interestingly, the majority of patients with isolated anti-P had class V, and 71% displayed a pure membranous pattern. Anti-P seems to be a novel serological marker for membranous lupus nephritis.

The present thesis entitled, "Molecular expression through fluorescence: Studies in probe design and aggregation" describes very simple bi-functional donor-acceptor poly-aromatic fluorophores that have been shown to possess distinctive properties depending on the context in which they are studied. In a sense, this work is an effort in exemplifying the inherent diversity and power of "molecular expression", with the central theme here being the phenomenon of fluorescence. The work has been divided into four chapters, each having a self-contained introduction. Chapter 1: First instance of metal ion (Zn2+) sensing exclusively at amphiphilic interfaces. (1 -pyrenyl)rnethyl-bis- [(2-pyridyl)methyl]amine (Pybpa), a simple, bi-functional fluorophore was synthesized. Pybpa has the modular design of a photoinduced electron transfer (PET) based analyte sensor. In Pybpa, a photoinduced electron transfer (PET) operates from the pyrenyl nitrogen (PyCH2-iV) to the excited pyrenyl (Py*) chromophore leading to fluorescence quenching. Zn2+ ion binding to the bis-picolyl (bpa) unit of Pybpa stops the PET process and leads to fluorescence enhancement. Thus Pybpa was able to sense Zn2+" in organic solvents. In water, however, Pybpa showed pronounced aggregation and the probe did not sense any metal ion. Surfactant micelles provide hydrophobic regions in water and the dynamic rnicellar assemblies could disrupt Pybpa aggregates. Pybpa monomers solubilized in micelles were responsive to Zn2+ in the low micro molar concentration range. The metal ion sensing on micelles was reflective of the charge of the interface. The sensing is negligible on cationic surface (CTAB), moderate on negatively charged surface (SDS micelles) and is the most efficient on neutral interface provided by TWEEN-20 micelles. With the Pybpa 'sensor*, no sensing is possible in water and hence the sensing is exclusive to the interface. Pybpa doped in membranous aggregates like phosphatidylcholine (PC) lipid bilayers, exists in monomeric form, and was able to sense Zn . The sensing on phosphatidylcholine (PC) bilayer vesicles was found to depend on the fluidity of the membrane. Zn2^ sensing with interfacially bound probe "was extended to a globular protein bovine serum albumin (BSA). BSA, a carrier protein, can bind hydrophobic molecules as well as metal ions like Zn2f. BSA was shown to disrupt Pybpa aggregation and bind Pybpa in a facile manner. BSA bound Pybpa was able to sense externally added Zn2+. Biological sensing of trace amounts of Zn2+ has been considered important since Zn2+ is crucial for eukaryotic systems. This is the first example of such 'exclusive' interfacial sensing of a metal ion. Chapter 2: Towards understanding and modulating self-assembly of pyrenyl bis-picolyl a mine: Organic nanoparticles that show tunable emission. Pybpa was found to aggregate in water in the size range of 80-250 nm. Evidence of aggregation was seen at concentrations as low as 1 |iM. The nanoscopic particles formed were characterized through transmission electron microscopy (TEM) and dynamic light scattering (DLS). Pybpa in water showed dual emission bands, with one band resernhling the emission from 'monomeric' Pybpa (as seen in solutions in organic solvents) and a broad red-shifted emission band (A,max ~ 480 ran) designated as "aggregate/nanoparticle" emission. Distinct excitation spectra for the two emission bands indicate that the bands (the '390 nm' band and the '480 nm' band) originate through distinct excitation/emission channels. The time resolved emission decay for the 'monomer' emission (397 nm) showed a substantial contribution from a long-lived pyrene-like excited state (x = 103.9 ns, 40% relative amplitude). On the other hand, the decay at 475 nm (for the nanoparticle/aggregate emission band) was considerably faster, with no evidence of any pyrene-like long-lived state. The short lifetimes indicated an exciplex nature of the red-shifted emission band, X-,nax~480 nm. The effect of temperature and urea on these aggregates was examined. The nanoparticles formed even in a concentrated urea solution (7.8 M). The aggregates formed in urea were found to be more emissive, indicating a 'looser' aggregate with reduced fluorescence quenching. Similar results were obtained on heating the aggregate. Increasing the concentration of Pybpa in water causes a change in the nature of the colloids formed as exemplified by increase in aggregate size and a decrease in the polydispersity index. Also seen was a substantial red shift in the 'aggregate emission'. At higher concentrations, the presence of three independent excitation/emission channels was observed. It is likely that a new type of aggregated Pybpa species formed at higher concentration, which emits at longer wavelength (A,rnax~540 nm), In such a scenario, it is possible to tune the emission wavelength by the choice of appropriate wavelength of excitation. Further, there is an opportunity to tailor the emission properties by controlling the aggregation behavior. The modulation of emission is one of the primary goals of research on fluorescent organic nanoparticles. Chapter 3: Photophysical properties of aryl-terpyridines in solution, solid and aggregated state: Unique CT emission from nanoparticles in water. Two aryl terpyridines, 4T-(l-pyrenyl)-2,2l:6'52fl-terpyridine (Pytpy) and 4'-(9-anthryl)-2,2':6',2n-terpyridine (Antpy), where the fluorophoric pyrene or anthracene unit is directly coupled to the terpyridine unit, were synthesized. The aryl terpyridines conjugates can be viewed as donor-acceptor molecules that are conformationally labile, with the possibility of rotation around three single bonds. It was of interest to see as to how conformational effects express themselves in different environments, especially in relation to the possibility of charge separation. Crystal structure data and Serni-empirical AMI calculations revealed a twisted molecular conformation for each of the molecules. Absorption and emission (steady state as well as tirne-resolved) behavior of Pytpy and Antpy in various organic solvents have been presented. The molecules showed only limited conjugation between the two units in the absorption behavior with the degree of conjugation being greater for Pytpy. In the emission behavior, only a single emission band (with a single lifetime) was observed in all organic solvent. Steady state and time resolved fluorescence data suggest the existence of a mixed or coupled, largely 7t—7i* state, with only marginal charge separation. The various photophysical parameters have been determined for the two systems. It appears that in the excited state, the inesomeric interactions show an increase for each of the two aryl-terpyridines, indicating at least a partially planar geometry in the excited state. Some specific solvent effects were observed for the molecules in alcoholic solvents and there was evidence of excited state H-bonding occurs for the aryl terpyridines in polar protic organic solvents, especially methanol. Pytpy and Antpy self-assembled in water over a large concentration range (1-100 |xM) to form spherical nanoparticles in the size range of 150-200 nm, as characterized by TEM and DLS. The absorption spectra for both conjugates showed red shift of the absorption bands in water (-10 nrn) along with significant tailing of the long-wavelength bands. The change in emission behavior in going from solution to the aggregates in water was very dramatic. Multiple, broadened, highly red-shifted emission bands for both Antpy and Pytpy were observed. Quite significantly, a long lifetime component in the emission decay was shown by the conjugates in water as compared to the lifetimes observed in solution. The data points towards a unique CT emission for Antpy and Pytpy aggregates in water. The excitation spectra for the multiple emission bands seen for Pytpy (or Antpy) were observed to be identical. Thus a single ground state population is responsible for emission over the entire range (approximately 420 nin - 600 nm). The existence of multiple emission bands and the large bathochromic shifts are exclusively due to excited state effects in the aggregated state in water. It appears that excited state H-bonding of the tpy N with water helps facilitate the excited state CT. The solid-state behavior of Pytpy and Antpy lias been examined and the emission from the two crystalline solids is very distinct. Antpy emission showed a X,,nax at -430 nm while Pytpy emission peaked at ~ 560 nm. The difference in the solid-state emission behavior exhibited by Pytpy and Antpy is explained through a consideration of the crystal packing for the two molecules. The degree of n-facial stacking was observed to be much greater for Pytpy. The observation of the distinct packing and emission shown by solid Pytpy and Antpy is highly significant if one considers the identical emission shown by the aqueous nanoparticles of the two molecules and brings to fore the 'nanoparticle effect' in water as compared to a simple concentration effect. It was also demonstrated that it was possible to modulate the aggregation of the terpyridines through additives, like metal ions Chapter 4: Pyrenyl terpyridine as a ratiometric fluorescence probe for sensing order and polarity of membranous aggregates. Pytpy was examined for its utility in probing surfactant aggregates, particularly membranous assemblies. la lipid bilayer vesicles made of phosphatidylcholine (PC) lipids (like dimyristoyl phosphatidylcholine, DMPC or egg-yolk PC) Pytpy showed an emission profile with marked similarity to that shown by the probe in water. Specifically, a broad red-shifted emission with A,maxin. the 500 nm region was observed. In addition, a peak in the -420 nm region was also seen. Fluorescence anisotropy was used to confirm the presence of vesicle-bound probe. Excitation spectra confirmed the presence of two distinct probe populations, om responsible for the '420 nm9 emission and another population responsible for the multiple, red-shifted emission bands. The emission behavior was indicative of aggregation of Pytpy on the vesicle surface and CT effects operating in conjunction with H-bonding. Fluorescence lifetime measurements, carried out at different Is suggest the CT nature of the red-shifted emission. The aggregation of the probe on the bilayer interface was confirmed by concentration and temperature dependence of the emission profile. The role of water in stabilizing this CT emission on bilayer surfaces was shown with use of a surface dehydrating agent polyethylene glycol (PEG). All these results helped build a model for the behavior of Pytpy in water. Pytpy aggregates on bilayer surface and shows a red-shifted CT emission with stabilization by interfacial water. Thus, the Pytpy 'aggregate' has a shallow, water accessible location in the bilayer. In addition to this, there is another Pytpy population responsible for the emission in the 420 nm region, and this second population might have a comparatively deeper location. The wavelength of the CT emission was sensitive to the polarity of the interface as evidenced "by the results obtained with bilayers made of a number of PC lipids. In general, the X™ax of the CTband showed a red shift with increasing polarity. The increase in polarity also caused an increase in the average lifetime of the probe. Pytpy could distinguish between vesicles made of lipids of different head groups. Aggregates made of phosphatidylethanolamine (PE) head group are in general less hydrated than PC lipid assemblies and Pytpy emission reflected this when examined in vesicles made of related lipids (dioleoyl lipids, DOPC and DOPE; dirnyristoyl lipids, DMPC and DMPE). Pytpy emission from PE vesicles was quenched and showed a pronounced blue shift in the emission Xmax vis-a-vis PC bilayers. Thus, dehydration of the interface consistently led to the destabilization of the CT state. Further, Pytpy emission was also responsive to hydration in more complex mixed PC-PE assemblies. Pytpy emission "behavior was also used to probe fluidity in complex "mixed" lipid assemblies- The effect of cholesterol on DMPC bilayers in terms of its known ability to dehydrate the bilayer was reported through a blue-shift Xmax of CT emission band. Further, cholesterol also causes drastic change in the bilayer at concentrations greater than ~ 30 mol%. This change in the bilayer was sensed through a sudden reduction in fluorescence intensity. Also from a careful analysis of Pytpy in various PC and PE vesicles, it emerged that the more fluid aggregates showed larger quantum yields. Thus, Pytpy could simultaneously report on both the polarity and fluidity of lipidic aggregates. Pytpy could also provide information about the order of an assembly. While the probe aggregated in bilayers and other membranous assemblies and showed water assisted CT emission, in more dynamic assemblies like micelles, Pytpy aggregates were not sustained, Pytpy in micelles showed emission spectra very similar to that seen in solutions in aprotic organic solvents. Thus, Pytpy proved to be a very useful ratiometric sensor for vesicle-to-rnicelle transition. Also, it has been possible to study some surfactant-lipid mixed assemblies that show phase separation. Pytpy reported the formation of a 'rigid', bilayer-like phases in mixed assemblies that are called bicelles.

Submitted by Irene Nascimento (irene.kessia@ufpe.br) on 2016-10-06T17:20:04Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertação Denise Maria do Nascimento Costa.pdf: 2568349 bytes, checksum: 97687424c47175731885cd254c815ad4 (MD5) / Made available in DSpace on 2016-10-06T17:20:04Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertação Denise Maria do Nascimento Costa.pdf: 2568349 bytes, checksum: 97687424c47175731885cd254c815ad4 (MD5) Previous issue date: 2016-07-21 / Introdução: Glomerulopatia membranosa (GM) é uma causa de síndrome nefrótica cuja etiologia pode ser primária (GMP) ou secundária, dentre estas é frequente o Lúpus eritematoso sistêmico (LES). Trata-se de uma doença imunologicamente mediada, caracterizada pela deposição de imunocomplexos no espaço subepitelial glomerular. A maioria dos antígenos envolvidos identificados são alvos da imunoglobulina G4 (IgG4), subclasse predominante em imunofluorescências renais na GMP, em contraste com a GM secundária a LES (GMS) na qual IgG1, IgG2 e IgG3 prevalecem. Apesar da IgG4 ser um subtipo de imunoglobulina com baixa capacidade de ativação do complemento, há várias evidências deste envolvimento na GMP. Esses dados, em conjunto com achados de depósitos glomerulares de lectina ligadora de manose (MBL), um dos principais componentes da via das lectinas do complemento, podem sugerir que tanto a via da lectina como a IgG4 estão envolvidas nesta patologia. Sabe-se ainda que o desenvolvimento de GMP também está associado a alterações genéticas. Entretanto, a etiopatogenia da GMP ainda não é totalmente conhecida e estudos para avaliação gênica do MBL2 e dosagem sérica de IgG em GM são escassos. Assim, foi realizado este estudo com o objetivo de avaliar a frequência de polimorfismos do gene MBL2 em portadores de GM, comparados a indivíduos saudáveis. Um segundo objetivo foi comparar pacientes com GMP e GMS quanto a diferenças do percentual de IgG4 sérico em relação a IgG (%IgG4) e da frequência de polimorfismos do MBL2. Métodos: Estudo realizado entre 2014 e 2015, em Pernambuco - Brasil. A amostra incluiu 60 pacientes adultos com diagnóstico histopatológico de GMP ou GMS. Outras causas de GM secundárias foram excluídas. Foram avaliados 35 pacientes com GMP e 24 com GMS, e um grupo controle (GC), formado por 101 indivíduos saudáveis. Resultados: O alelo mutante O do gene MBL2 foi mais frequente no grupo com GM comparados aos GC (42% x 22%; p < 0,001). A heterozigose A/O, em relação ao genótipo A/A, predominou entre os pacientes comparados ao GC, associando-se a GM com OR = 11,16 (95% IC = 4,77 - 28,41). À análise comparativa entre os pacientes com GMP e GMS, não houve diferença das frequências dos polimorfismos genéticos entre os grupos. O grupo GMP apresentou menor mediana de IgG sérica total (p = 0,008) e maior %IgG4 (p = 0,016), comparado ao grupo GMS. Nível sérico de IgG4 não diferiu significativamente entre os grupos GMP e GMS (p = 0,289). Conclusão: O polimorfismo do éxon 1 do gene MBL2 associou-se à GM, comparado a indivíduos saudáveis, porém sem diferença entre as etiologias avaliadas. Já o %IgG4 sérico foi maior na GMP em relação a GMS. Estes resultados sugerem que esta mutação genética possa conferir maior vulnerabilidade a GMP e que o %IgG4 sérico possa ser utilizado como marcador adicional para diagnóstico diferencial entre as duas etiologias da GM. / Introduction: Membranous glomerulopathy (MG) is a cause of nephrotic syndrome whose etiology may be primary (PMG) or secondary, wich is frequent systemic lupus erythematosus (SLE). It is an immune-mediated disease characterized by the deposition of immune complexes in the glomerular subepithelial space. Most of the identified antigens are targets to immunoglobulin IgG4, most common subclass in renal immunofluorescence in GMP, in contrast to the SLE secondary MG (SMG) in which IgG1, IgG2 and IgG3 prevail. Although IgG4 is a immunoglobulin subtype with low complement activation capacity, there is abundant evidence of this involvement in PMG. These data, together with glomerular deposits of mannose-binding lectin (MBL), a major component of the lectin pathway of complement, may suggest that both the lectin pathway and IgG4 are involved in this pathology. It is also known that the development of PMG is associated with genetic alterations. As the pathogenesis of PMG is not yet fully known, and studies for genetic evaluation of MBL2 and serum IgG in MG are scarce, this study was conducted to evaluate the frequency of MBL2 gene polymorphisms in patients with MG, compared to healthy subjects. A second objective was to compare patients with PMG and SMG with respect to the percentage of serum IgG4 (IgG4%) and frequency MBL2 polymorphisms. Methods: This study was conducted between 2014 and 2015 in Pernambuco - Brazil. The sample included 60 adult patients with histopathologic diagnosis of PMG or SMG. Other causes of secondary MG were excluded. Thity five patients with PMG and 24 with SMG were evaluated, compared to a control group (CG) of 101 healthy subjects. Results: The mutant allele O was more frequent in the MG population compared to CG (42% vs. 22%; p <0.001). The heterozygous A/O, compared to genotype A/A, predominated among patients compared to the control group, and was associated with MG (OR = 11.16; 95% CI = 4.77 to 28.41). In the comparative analysis between patients with PMG and SMG, there was no difference in the frequency of genetic polymorphisms between groups. The PMG group had lower median total serum IgG (p = 0.008) and higher IgG4% (p = 0.016) compared to the SMG group. Serum IgG4 did not differ significantly between the groups PMG and SMG (p = 0,289). Conclusion: The polymorphism of exon 1 MBL2 gene was associated with MG, compared to healthy subjects, but no difference between the assessed etiologies. Serum IgG4% was higher in PMG relative to SMG. These results suggest that this gene mutation can confer increased vulnerability to PMG and the serum IgG4% may be used as an additional marker for the differential diagnosis between the two etiologies MG.

Les ATPases F1F0 sont présentes chez la majorité des bactéries, notamment les mycoplasmes qui sont caractérisés par un génome réduit et un mode de vie parasitaire. En plus de l’opéron codant l’ATPase F1F0, des clusters apparentés de sept gènes ont été identifiés dans le génome de nombreux mycoplasmes. Au cours de cette thèse, nous avons cherché à caractériser l’évolution et la fonction de ces clusters supplémentaires. Quatre des protéines codées par ces clusters présentent des similarités structurales avec les sous-unités α, β,  et ε de l’ATPase F1F0, résultant en une potentielle structure F1-like. Les trois autres protéines ne présentent aucune similarité avec des protéines connues. Une localisation transmembranaire est prédite pour deux d’entre elles. Deux types d’ATPase F1-like, Type 2 et Type 3, ont été identifiés. Les clusters de Type 2 et de Type 3 pourraient être originaires du groupe phylogénétique Hominis, les clusters de Type 3 ayant vraisemblablement été disséminés par des transferts horizontaux de gènes entre mycoplasmes colonisant le même hôte. Les gènes du cluster de Type 3 de Mycoplasma mycoides subsp. mycoides sont organisés en opéron et exprimés en milieu axénique. Des études de mutagénèse et de complémentation démontrent que le cluster de Type 3 est associé à une activité ATPase majeure des fractions membranaires. Des analyses biochimiques suggèrent que l’activité ATPase du cluster est sensible au ∆pH mais pas au ∆Ψ. Ces analyses suggèrent que le sodium et le potassium ne sont pas impliqués dans le fonctionnement de l’ATPase F1-likeX0. Les sous-unités des ATPases F1-likeX0 et F1F0 présentent un comportement différent en présence de détergents. L’ensemble de ces expériences suggèrent que l’ATPase F1-likeX0 est un complexe plus fragile que l’ATPase F1F0. Nos résultats montrent qu’en dépit d’une tendance à la réduction de génome, les mycoplasmes ont développé et échangé des ATPases sans équivalent chez d’autres bactéries. Nous proposons un modèle dans lequel une structure F1-like est associée avec un domaine hypothétique X0, enchâssé dans la membrane des mycoplasmes. / F1F0 ATPases have been found in most bacteria, including mycoplasmas that are characterized by drastically reduced genomes and a parasitic lifestyle. In addition to the typical operon of eight genes encoding genuine F1F0 ATPase, related clusters of seven genes were identified in many mycoplasmas. In this work, we investigated the evolution and the function of these supplementary clusters. Four proteins encoded by these clusters present structural similarities with subunits α, β,  and ε of F1F0 ATPases, resulting in potential F1-like structures. The three other encoded proteins did not show any similarity to known proteins. Transmembrane helices were predicted for two of them, suggesting a membrane localisation. Two types of F1-like ATPases, Type 2 and Type 3, were identified. Clusters encoding Type 2 and Type 3 ATPases were assumed to originate from the Hominis group of mycoplasmas. Further spreading of Type 3 ATPases towards other phylogenetic groups by horizontal gene transfers in between mycoplasmas sharing a same host was proposed on the basis of phylogenetic trees and genomic context. Functional analyses indicated that genes of Type 3 cluster in the ruminant pathogen Mycoplasma mycoides subsp. mycoides were organized as an operon. Proteomic analyses indicated that the seven encoded proteins were produced during growth in axenic media. Mutagenesis and complementation assays demonstrated that Type 3 cluster was associated with a major ATPase activity of membrane fractions. Biochemical analyses indicated that this ATPase activity was sensitive to ΔpH but not to ΔΨ. These analyses suggested that Na+ and K+ were not involved in the F1-likeX0 functioning. Our results indicated a behaviour of F1-likeX0 ATPase subunits that is different to that of F1F0 ATPase subunits in presence of detergents. Altogether, these analyses suggest that the F1-likeX0 complex could be more fragile than the F1F0 complex. Our results showed that despite their tendency to genome reduction, mycoplasmas have evolved and exchanged specific F1-like ATPases with no known equivalent in other bacteria. We propose a model in which the F1-like structure is associated with a hypothetical X0 sector embedded in the membrane of mycoplasmal cells.

Le syndrome néphrotique idiopathique (SNI) est une néphropathie glomérulaire définie par une protéinurie massive associée à une hypoalbuminémie, sans lésions inflammatoires rénales, ni dépôts de complexes immuns circulants. Les travaux réalisés au cours de ma thèse concernent l’étude du rôle potentiel du gène c-mip dans la physiopathologie du SNI.Dans un premier temps, nous avons étudié la physiopathologie moléculaire de l’association maladie de Hodgkin et SNI. Nous avons démontré que cette association était liée à une forte induction de c-mip à la fois dans les cellules de Reed Sternberg (dont la présence signe le lymphome hodgkinien) et les podocytes qui sont des cellules spécialisées du glomérule rénal (Audard, et al. 2010). Nous avons montré que l’induction de c-mip résultait d’un défaut quantitatif et/ou qualitatif du gène Fyn, à la fois chez les patients et dans un modèle de souris déficiente en Fyn. Nous avons trouvé que c-mip était fortement induit dans les podocytes au cours du SNI ainsi que dans la glomérulopathie extramemenbraneuse (GEM). La surexpression de c-mip par transgénèse chez la souris déclenche une protéinurie néphrotique dont le mécanisme implique une rupture, médiée par c-mip, de la voie de signalisation de la néphrine (Science Signaling, 2010 co-auteur). L’étude de la néphrite de Heyman, le modèle expérimental de la GEM humaine, a permis de montrer que l’induction de c-mip coincidait avec l’apparition de la protéinurie et était associée à l’inhibition de l’activité RhoA, à une perte de la synaptopodine, à une diminution du VEGF tandis que l’expression de la DAPK (death-associated protein kinase) est fortement augmentée (Audard et al, manuscrit soumis 1). Nous avons recherché si l’hypogammaglobulinémie au cours du SNI était associée à des anomalies fonctionnelles des lymphocytes B (LB). Nous avons trouvé que c-mip interagit avec la sous unité régulatrice de la PI3 kinase et empêche la dissociation de la sous unité catalytique, p110, nécessaire à l’activation de la PI3 kinase. Enfin, l’expression de l’IL 21, une cytokine–clé secrétée par les lymphocytes T et intervenant dans la commutation isotypique, était fortement réduite dans le SNI (Audard et al, manuscrit en préparation 2). Ces résultats donnent un éclairage nouveau sur la physiopathologie moléculaire du SNI et suggèrent un rôle crucial de c-mip dans les anomalies lymphocytaires et podocytaires observées chez les patients / Idiopathic nephrotic syndrome comprises several podocyte diseases of unknown origin, affecting the glomerular podocyte, which plays a key role in controlling the permeability of the kidney filter to proteins. It is characterized by massive proteinuria and hypoalbuminemia, with no inflammatory lesions or cell infiltration. This works focused on the potential role of c-mip in the pathogenesis of INS. We showed that occurrence of minimal change nephrotic syndrome in the course of Hodgkin lymphoma (cHL-MCNS) is closely related to the induction of c-mip in both Hodgkin-Reed Sternberg cells and podocytes (Audard, et al. 2010), which is caused by a qualitative and/or quantitative defect in Fyn in both HRS and podocytes cells. We found that c-mip is upregulated in podocytes of patients with membranous nephropathy (MN). Transgenic mice overproducing c-mip in the podocytes developed heavy proteinuria without morphological alterations, inflammatory lesions or cell infiltrations. We showed that c-mip turned off podocyte proximal signaling by preventing the interaction between Fyn and nephrin, resulting in the inhibition of nephrin signaling pathway (Science signaling, 2010 coauthor). Moreover, the induction of c-mip in passive type Heymann nephritis (the experimental model of MN) was concomitant to proteinuria occurrence and is associated with reduction of RhoA activity, downregulation of synaptopodin and VEGF expression whereas DAPK expression is significantly increased (Audard et al manuscript submitted 1).We demonstrated that hypogammaglobulinemia, a common feature in INS patients, may result from a defect in B lymphocytes. We found that c-mip interacts with p85 regulatory subunit and prevent its dissociation from p110 catalytic subunit, resulting in inactivation of PI3 kinase. Finally, the expression of IL21, a key cytokine involved in class switching recombination, is repressed in active phases of INS, which may contribute for immunoglobulin disorders commonly observed in these patients (Audard et al manuscript in progress 2).Altogether, these results suggest that c-mip is a major player of lymphocyte and podocytes dysfunction observed in patients with INS

Η Ιδιοπαθής Μεμβρανώδης Σπειραματονεφρίτιδα (ΙΜΣ) ή νεφροπάθεια, η πιο συχνή αιτία νεφρωσικού συνδρόμου στους ενήλικες, συνήθως αντιμετωπίζεται με τη χορήγηση κορτικοειδών και κυτταροτοξικών φαρμάκων ή κυκλοσπορίνης (cyclosporine-A, CsA). Σκοπός της μελέτης ήταν η εκτίμηση της αποτελεσματικότητας της μακροχρόνιας χορήγησης CsA στην πρόκληση ύφεσης του νεφρωσικού συνδρόμου και των ιστολογικών αλλοιώσεων σε επαναληπτικές βιοψίες νεφρού μετά τη χορήγηση του δυνητικά νεφροτοξικού αυτού φαρμάκου. Επιπλέον, εκτιμήθηκε η αποτελεσματικότητα του Mycophenolate Mofetil (MMF) ως σχήματος θεραπείας της ΙΜΣ σε μικρό αριθμό ασθενών και η προγνωστική αξία των επιπέδων του αυξητικού παράγοντα TGF-β1 στα ούρα και στο πλάσμα ως δεικτών εξέλιξης της νόσου. Μελετήθηκαν 32 ασθενείς με ΙΜΣ οι οποίοι εμφάνιζαν νεφρωσικό σύνδρομο και είχαν ικανοποιητική νεφρική λειτουργία κατά τη διάγνωση της νόσου και στους οποίους χορηγήθηκε συνδυασμός πρεδνιζολόνης και CsA. Παρατηρήθηκε πλήρης ύφεση του νεφρωσικού συνδρόμου σε 18 (56%) και μερική ύφεση σε 10 ασθενείς (31%) μετά από 12 μήνες θεραπείας (συνολικά στο 87% των ασθενών). Επεισόδια υποτροπών παρατηρήθηκαν στο 39% και 60% των ασθενών με πλήρη ή μερική ύφεση αντίστοιχα, και πολλαπλές υποτροπές στο 25% των ασθενών, οι οποίοι παρουσίασαν βαθμιαία μείωση της απαντητικότητας στη CsA και επιδείνωση της νεφρικής λειτουργίας. Επαναληπτική βιοψία νεφρού έγινε σε 18 ασθενείς με ύφεση του νεφρωσικού συνδρόμου μετά από 24 μήνες θεραπείας για να εκτιμηθεί η δραστηριότητα της νόσου και οι πιθανές ιστολογικές αλλοιώσεις σε πλαίσια τοξικότητας από κυκλοσπορίνη. Στις επαναληπτικές βιοψίες παρατηρήθηκαν: εξέλιξη του σταδίου της νόσου, επιδείνωση της σπειραματοσκλήρυνσης και της διαμεσοσωληναριακής βλάβης στο 60% των ασθενών. Δεν παρατηρήθηκαν χαρακτηριστικές αλλοιώσεις νεφροτοξικότητας από την κυκλοσπορίνη. Η βαρύτητα των ιστολογικών αλλαγών συσχετίστηκε με το χρονικό διάστημα που είχε παρέλθει από την πρώτη βιοψία νεφρού (r = 0.452, p < 0.05) και θεωρήθηκε ως φυσική εξέλιξη της νόσου. Ικανοποιητικά αποτελέσματα διαπιστώθηκαν από τη χορήγηση Mycophenolate Mofetil σε 6 ασθενείς με ΙΜΣ, στους οποίους το MMF χρησιμοποιήθηκε σε συνδυασμό με μικρή δόση πρεδνιζολόνης, είτε λόγω ανθεκτικότητας του νεφρωσικού συνδρόμου στην CsA, είτε ως αρχική θεραπεία σε περιπτώσεις αντένδειξης στην χορήγηση CsA. Ύφεση του νεφρωσικού συνδρόμου παρατηρήθηκε σε 4 από τους 6 ασθενείς. Τα επίπεδα του TGF-β1 στα ούρα ασθενών με ΙΜΣ και λευκωματουρία ήταν σημαντικά υψηλότερα συγκριτικά με αυτά υγιών εθελοντών και ασθενών με άλλες σπειραματοπάθειες που δεν παρουσίαζαν λευκωματουρία και μειώθηκαν σημαντικά μετά από χορήγηση κορτικοειδών και κυκλοσπορίνης. Η συγκέντρωση του TGF-β1 στο πλάσμα δε διέφερε σημαντικά μεταξύ υγιών εθελοντών και ασθενών με ΙΜΣ και νεφρωσικό σύνδρομο, καθώς και μεταξύ ασθενών με ή χωρίς ύφεση της λευκωματουρίας μετά από τη θεραπευτική αγωγή. / Idiopathic membranous nephropathy (IMN), the most common cause of nephrotic syndrome in adults, is usually treated with a combination of corticosteroids with cytotoxic drugs or cyclosporin A (CsA). The aim of this study was the estimation of the effectiveness of long-term use of CsA in the remission and relapse rate of nephrotic syndrome along with histological changes in repeat renal biopsies after treatment with this potentially nephrotoxic drug, and the evaluation of Mycophenolate Mofetil (MMF) as a treatment regimen for IMN. In addition, urinary and plasma TGF-β1 levels were evaluated as markers of progression of kidney disease. Thirty-two nephrotic patients with well-preserved renal function treated by prednisolone and CsA were studied. Complete remission of nephrotic syndrome was observed in 18 (56%) and partial remission in 10 patients (31%) after 12 months of treatment (total 87%). Relapses were observed in 39% and 60% of patients with complete and partial remission, respectively, and multiple relapses in 25% of patients, who showed gradual unresponsiveness to CsA and decline of renal function. A repeat biopsy was performed in 18 patients with remission of nephrotic syndrome, after 24 months of treatment, to estimate the activity of the disease and features of CsA toxicity. Progression of the stage of the disease, more severe glomerulosclerosis and tubulointerstitial injury were recognized in 60% of patients in repeat renal biopsies. Features of CsA nephrotoxicity were not observed. The severity of histological changes was related to the time elapsed from the first biopsy (r = 0.452, P < 0.05). MMF was proved effective in a small number of nephrotic patients with IMN and well-preserved renal function. MMF in combination with small dose of prednisolone was given in 6 patients with either persistent nephrotic syndrome to CsA or as initial therapy because of contraindication to CsA administration. Remission of nephrotic syndrome was observed in 4 out of 6 MMF treated patients. Urinary and plasma TGF-β1 levels were examined as markers of progression of the disease. TGF-β1 levels in the urine of patients with proteinuria were significantly higher compared with those of healthy individuals and patients with other types of nephropathy without proteinuria. Furthermore, urinary TGF-β1 of nephrotic patients with membranous nephropathy significantly reduced after treatment with CsA and corticosteroids. Plasma TGF-β1 levels showed no difference between patients and healthy subjects as well as between patients with and without remission of proteinuria after treatment.

La morphologie des cellules et leurs interactions avec l’environnement découlent de divers procédés mécaniques qui contribuent à la richesse et à la diversité de la vie qui nous entoure. À titre d’exemple, les cellules mammifères se conforment à différentes géométries en fonction de l’architecture de leur cytosquelette tandis que les bactéries et les levures adoptent une forme circulaire par turgescence. Je présente, dans cette thèse, la découverte d’un mécanisme de morphogénèse supplémentaire, soit la déformation de surface cellulaire via l’assemblage de protéines par démixtion de phases aqueuses non miscibles et l’adhésion entre les matériaux biologiques. J’expose de façon spécifique comment ce mécanisme régule le recrutement et le mouvement dynamique des protéines qui induisent l’invagination de la membrane plasmique lors de l’endocytose clathrine-dépendante (CME). Le phénomène de démixtion des protéines dans le cytoplasme est analogue à la séparation de phase de l’huile en solution aqueuse. Il constitue un mécanisme cellulaire important et conservé, où les protéines s’agglomèrent grâce aux interactions intermoléculaires qui supplantent la tendance du système à former un mélange homogène. Plusieurs exemples de compartiments cellulaires dépourvus de membrane se forment par démixtion de phase, tels que le nucléole et les granules de traitement de l’ARN [1-6]. Ces organes ou compartiments dénommés NMO, du terme anglais « non-membranous organelles », occupent des fonctions de stockage, de traitement et de modification chimique des molécules dans la cellule. J’explore ici les questions suivantes : est-ce que les NMO occupent d’autres fonctions à caractère morphologique ? Quels signaux cellulaires régulent la démixtion de phase des protéines dans la formation des NMO ? Fondée sur la physique mécanique du contact entre les matériaux, j’émets l’hypothèse que des compartiments cellulaires nanoscopiques, formés par démixtion de phase, génèrent des forces mécaniques par adhésion interfaciale. Le travail mécanique ainsi obtenu déforme le milieu cellulaire et les surfaces membranaires adjacents au NMO nouvellement créé. Le but de mon doctorat est de comprendre comment les cellules orchestrent, dans le temps et l’espace, la formation des NMO associés au CME et comment ceux-ci génèrent des forces mécaniques. Mes travaux se concentrent sur les mécanismes de démixtion de phase et d’adhésion de contact dans le processus d’endocytose chez la levure Saccharomyces cerevisiae. Pour enquêter sur le rôle des modifications post-traductionnelles dans ces mécanismes, nous avons premièrement analysé la cinétique de phosphorylation des protéines en conditions de stress. Mes résultats démontrent que le recrutement et la fonction de certaines protéines impliquées dans le CME se régulent via des mécanismes de phosphorylation. Outre les processus de contrôle post-traductionnel, nous avons élucidé le rôle des domaines de faible complexité dans l’assemblage de plusieurs protéines associées avec le CME. De concert avec les modifications de phosphorylation, des domaines d’interaction protéine-protéine de type PrD (du terme « prion-like domains ») modulent directement le recrutement des protéines au sein des NMO associés au CME. La nature intrinsèquement désordonnée de ces PrD favorise un mécanisme d’assemblage des protéines par démixtion de phase tel que postulé. Finalement, mes travaux confirment que la formation de ces NMO spécifiques génère des forces mécaniques qui déforment la membrane plasmique et assurent le processus de CME. D’un point de vue fondamental, mes recherches permettent de mieux comprendre l’évolution d’une stratégie cellulaire pour assembler des compartiments cellulaires sans membrane et pour fixer les dimensions biologiques associées au CME. De manière plus appliquée, cette étude a le potentiel de générer des retombées importantes dans la compréhension et le traitement de maladies neurodégénératives souvent associées à une séparation de phase aberrante et à la formation d’agrégats protéiques liés à la pathologie. / Evolution has resulted in distinct mechanical processes that determine the shapes of living cells and their interactions with each other and with the environment. These molecular mechanisms have contributed to the wide variety of life we observe today. For example, mammalian cells rely on a complex cytoskeleton to adapt specific shapes whereas bacteria, yeast and plants use a combination of turgor pressure and cell walls to have their characteristic bloated form. In this dissertation, I describe my discovery of an unforeseen additional mechanism of morphogenesis: protein aqueous-aqueous phase separation and adhesive contact between biomaterials as a simple and efficient ways for cells to organize internal matter and accomplish work to shape internal structures and surfaces. I specifically describe how a fundamental process of phospholipid membrane and membrane-embedded protein recycling, clathrin-mediated endocytosis (CME), is driven by this mechanism. Analogous to water and oil emulsions, proteins, and biopolymers in general, can phase separate from single to a binary aqueous phase. For proteins that de-mix from the bulk environment, the intermolecular interactions (or cohesive energy) that favors protein condensation only needs to overcome the low mixing entropy of the system and represents a conserved and energy efficient cellular strategy [2, 3, 7, 8]. So far, various examples of phase separated cellular compartments, termed non-membranous organelles (NMOs), have been discovered. These include the nucleoli, germ line P granules and P bodies, to name a few [1-6]. NMOs are involved in many conserved biological processes and can function as storage, bioreactor or signaling bodies. Cells use phase separation as a scheme to organize internal matter, but do NMOs occupy other complex functions, such as morphogenesis? What specific signals trigger protein phase separation? Based on mechanical contact theory, I proposed that hundreds of nanometer- to micron-scale phase separated bodies can deform the cellular environment, both cytoplasm and membranes, through interfacial adhesion. I studied how mechanical contact between a phase-separated protein fluid droplet and CME nucleation sites on membranes drive endocytosis in the model organism budding yeast, Saccharomyces cerevisiae. Specifically, this dissertation describes first, my investigations of post-translational modifications (phosphorylation) of several CME-mediating proteins and the implications of these modifications in regulating CME. I then describe how my efforts to understand what was distinct about the proteins that are phosphorylated led me to propose their phase separation into droplets capable of driving invagination and vesicle formation from plasma membrane. I used fluorescence microscopy, mass spectrometry and micro rheology techniques to respectively determine the spatiotemporal dynamics, phosphorylation modifications and material properties of coalesced CME-mediating proteins. I further investigated how phase separation of these proteins might generate mechanical force. I demonstrate that changes in the phosphorylation of some endocytic proteins regulates their recruitment to CME nucleation sites. We achieved reliable predictions of functional phosphosites by combining information on the conservation of the post-translational modifications with analysis of the proportion of a protein that is dynamically phosphorylated with time. The same dynamically phosphorylated proteins were enriched for low amino acid compositional complexity “prion-like domains”, which we demonstrated were essential to these proteins undergoing aqueous-aqueous phase separation on CME nucleation sites. I then demonstrate how phase separated droplet can produce mechanical work to invaginate membranes and drive CME to completion. In summary, I have discovered a fundamental molecular mechanism by which phase separated biopolymers and membranes could apply work to shape each other. This mechanism determines the natural selection of spatial scale and material properties of CME. Finally, I discuss broader implications of this dissertation to mechanistic understandings of the origins of neurodegenerative diseases, which likely involve pathological forms of protein phase separation and/or aggregation.