Cholera is a water-borne disease that continues to ravage resource poor communities around the world especially those in developing countries. The disease is caused by Vibrio cholerae microorganisms whose natural habitat is the aquatic ecosystem. It is believed that this microorganism prior to becoming the primary cause of cholera acquired virulence factors expressed by two separate genetic elements. These genetic elements are known as VPIФ and CTXФ were acquired in that order for known physiological reasons. However only V. cholerae in possession of the CTX genetic element are capable of causing cholera disease. At present only two serotypes are known to have the ability to cause cholera and these are V. cholerae serotypes O1 and O139. SODIS (Solar disinfection) is an extremely low cost refined technology that can be used for the disinfection of water especially in areas where there is a considerable amount of sunshine. Although this technology is a composite of various factors the underlying principle is the use of solar ultraviolet radiation (SUVR). The preliminary target of SUVR is the cytoplasmic membrane and this was confirmed by flow cytometric analysis. The consequences of leaky cytoplasmic membrane include cellular death to the microorganism as well as an increase in cholera toxin secretion. The main objective of this study was to investigate the effect of solar ultraviolet radiation on the growth of V. cholerae and on the secretion of cholera toxin and to provide supporting information for the use of SODIS in South Africa while observing the possible role that climate may play in the onset of cholera disease. The initial part of the study evaluated the culturability, biomass increase and cholera toxin secretion in both a nutrient poor and a nutrient rich media by two toxigenic and one non toxigenic strain of V. cholerae. A series of pH and temperature combinations were used to achieve this objective. The result revealed that the microorganisms survived in both media. An increase in biomass was observed for all the bacteria grown in the nutrient rich media whereas in the poor nutrient media the bacteria remained culturable but no increase in biomass was observed. Interestingly lower temperatures seemed to provide more optimal growth conditions while high temperature on most occasions favoured cholera toxin secretion, in both media.The second part of the study required the exposure of the microorganisms to SUVR. A SODIS approach was used with a few modifications. The V. cholerae strains were exposed to solar radiation during all the seasons of the year. Evaluation of the viability, the increase in biomass and the detection of cholera toxin secretion was determined after each exposure to solar radiation. The results seem to suggest that the effect of SUVR depended on the season of the year, the nature of the media, strain, solar conditions and in the duration of solar exposure, in no particular order. The secretion of cholera toxin was mainly dependent on the media used, the season of the year and on the serotype of the strain. This study represents the first report on the evaluation of SUVR for the disinfection of water under South African conditions (Pretoria area) during all seasons of the year with variations in solar radiation levels and temperature. Furthermore what actually happened to V. cholerae during solar exposure in terms of cell morphology, cell viability and secretion of cholera toxin is also reported and this can give an insight of the possible role that SUVR may play in the onset of cholera. The main recommendation emanating from this study is the sensitisation of communities worldwide about the capacity that, SUVR carries to lighten the burden of communicable water borne diseases especially, in resource limited areas through the implementation of SODIS. / Life and Consumer Sciences / M. Sc. (Life Science)

The gonococcal transferrin binding proteins (Tbps) are two surface-exposed outer membrane proteins, TbpA and TbpB, which together function to remove and internalized iron from human transferrin. Iron is an essential nutrient to the gonococcus, without which it cannot survive. The Tbps have been established as virulence factors, demonstrating their importance in establishing infection. Both TbpA and TbpB are well conserved among gonococcal isolates, and have been considered potential vaccine targets. Vaccine studies with the closely related species Neisseria meningitidis, have demonstrated these proteins to be protective in murine challenge studies. Though the meningococcal Tbps have demonstrated promise, no similar gonococcal vaccine experiments have been conducted prior to the current studies. Here we demonstrate purification of recombinant TbpA and TbpB. These recombinant proteins were utilized to evaluate the human immune response to these proteins during natural infections, and their immunogenicity in murine vaccine studies. Our results demonstrate a paucity of antibodies elicited to these proteins during natural infections in serum and mucosal secretions from infected individuals. From this study we hypothesized the induction of both serum and genital antibodies to these proteins could serve to protect an individual from infection. To begin testing this hypothesis, we immunized mice both intranasally (IN) and subcutaneously (s.c.) with full-length Tbps in conjunction with the B subunit of cholera toxin (Ctb) as an adjuvant. We also performed another vaccine study using domains from both proteins in genetic fusions with Ctb and E. coli heat labile toxin IIb (LtbIIb). Both studies demonstrated that these antigens were immunogenic, as Tbp-specific antibodies were elicited in the serum and vaginal washes of female Balb/C mice. Intranasal immunization however was the only route with which we were able to elicit vaginal Tbp-specific IgA, and IgG, whereas subcutaneous immunization only elicited vaginal IgG. Furthermore, we found the full-length Tbps and the Ctb/LtbIIb chimeras were able to elicit bactericidal antibodies, which were also effective in killing heterologous gonococcal strains. This body of work comprises the first published study using the gonococcal transferrin binding proteins as vaccine antigens, and highlights their potential as vaccine antigens in the development of an efficacious gonococcal vaccine.

Cholera is a water-borne disease that continues to ravage resource poor communities around the world especially those in developing countries. The disease is caused by Vibrio cholerae microorganisms whose natural habitat is the aquatic ecosystem. It is believed that this microorganism prior to becoming the primary cause of cholera acquired virulence factors expressed by two separate genetic elements. These genetic elements are known as VPIФ and CTXФ were acquired in that order for known physiological reasons. However only V. cholerae in possession of the CTX genetic element are capable of causing cholera disease. At present only two serotypes are known to have the ability to cause cholera and these are V. cholerae serotypes O1 and O139. SODIS (Solar disinfection) is an extremely low cost refined technology that can be used for the disinfection of water especially in areas where there is a considerable amount of sunshine. Although this technology is a composite of various factors the underlying principle is the use of solar ultraviolet radiation (SUVR). The preliminary target of SUVR is the cytoplasmic membrane and this was confirmed by flow cytometric analysis. The consequences of leaky cytoplasmic membrane include cellular death to the microorganism as well as an increase in cholera toxin secretion. The main objective of this study was to investigate the effect of solar ultraviolet radiation on the growth of V. cholerae and on the secretion of cholera toxin and to provide supporting information for the use of SODIS in South Africa while observing the possible role that climate may play in the onset of cholera disease. The initial part of the study evaluated the culturability, biomass increase and cholera toxin secretion in both a nutrient poor and a nutrient rich media by two toxigenic and one non toxigenic strain of V. cholerae. A series of pH and temperature combinations were used to achieve this objective. The result revealed that the microorganisms survived in both media. An increase in biomass was observed for all the bacteria grown in the nutrient rich media whereas in the poor nutrient media the bacteria remained culturable but no increase in biomass was observed. Interestingly lower temperatures seemed to provide more optimal growth conditions while high temperature on most occasions favoured cholera toxin secretion, in both media.The second part of the study required the exposure of the microorganisms to SUVR. A SODIS approach was used with a few modifications. The V. cholerae strains were exposed to solar radiation during all the seasons of the year. Evaluation of the viability, the increase in biomass and the detection of cholera toxin secretion was determined after each exposure to solar radiation. The results seem to suggest that the effect of SUVR depended on the season of the year, the nature of the media, strain, solar conditions and in the duration of solar exposure, in no particular order. The secretion of cholera toxin was mainly dependent on the media used, the season of the year and on the serotype of the strain. This study represents the first report on the evaluation of SUVR for the disinfection of water under South African conditions (Pretoria area) during all seasons of the year with variations in solar radiation levels and temperature. Furthermore what actually happened to V. cholerae during solar exposure in terms of cell morphology, cell viability and secretion of cholera toxin is also reported and this can give an insight of the possible role that SUVR may play in the onset of cholera. The main recommendation emanating from this study is the sensitisation of communities worldwide about the capacity that, SUVR carries to lighten the burden of communicable water borne diseases especially, in resource limited areas through the implementation of SODIS. / Life and Consumer Sciences / M. Sc. (Life Science)

Diabetes mellitus is presently recognized as the sixth leading cause of death in the United States, affecting over 20 million people. Diabetes is a condition characterized by high blood glucose due to an insulin deficiency or resistance. Type I, which comprises 5-10% of all cases, results from the destruction of pancreatic beta cells in the islets of Langerhans. The current treatment for type I diabetes is insulin administration through injection or pump. Purification, production, and storage of this insulin proves to be quite costly. By producing biologically functional insulin with oral delivery capabilities through chloroplast genetic engineering, many of these costs could be cut back. In addition, the possibility of providing the C-peptide currently lacking in commercially available insulin becomes available. The Daniell lab inserted cholera toxin B-subunit (CTB) fused proinsulin, containing three furin cleavage sites, (CTB-pins Fx3) into the tobacco chloroplasts of Petit Havana via particle gun bombardment. The insertion of three furin cleavage sites along with fusion to CTB will enable the insulin to sustain function when orally administered. Transgenic plants from the second generation were then analyzed and tested for quantification of the CTB-pins Fx3 gene. Tobacco leaves of varying ages were compared to determine expression levels. The resulting data is pertinent for future production of both orally deliverable insulin and more cost effective injectable insulin.

Cholera toxin (CT) moves from the cell surface to the endoplasmic reticulum (ER) where the catalytic CTA1 subunit separates from the holotoxin and unfolds due to its intrinsic thermal instability. Unfolded CTA1 then moves through an ER translocon pore to reach its cytosolic target. Due to the instability of CTA1, it must be actively refolded in the cytosol to achieve the proper conformation for modification of its G protein target. The cytosolic heat shock protein Hsp90 is involved with the ER-to-cytosol translocation of CTA1, yet the mechanistic role of Hsp90 in CTA1 translocation remains unknown. Potential post-translocation roles for Hsp90 in modulating the activity of cytosolic CTA1 are also unknown. Here, we show by isotope-edited Fourier transform infrared (FTIR) spectroscopy that Hsp90 induces a gain-of-structure in disordered CTA1 at physiological temperature. Only the ATP-bound form of Hsp90 interacts with disordered CTA1, and its refolding of CTA1 is dependent upon ATP hydrolysis. In vitro reconstitution of the CTA1 translocation event likewise required ATP hydrolysis by Hsp90. Surface plasmon resonance (SPR) experiments found that Hsp90 does not release CTA1, even after ATP hydrolysis and the return of CTA1 to a folded conformation. The interaction with Hsp90 allowed disordered CTA1 to attain an active state and did not prevent further stimulation of toxin activity by ADP-ribosylation factor 6, a host cofactor for CTA1. This activity is consistent with its role as a chaperone that refolds endogenous cytosolic proteins as part of a foldosome complex consisting of Hsp90, Hop, Hsp40, p23, and Hsc70. A role for Hsc70 in CT intoxication has not yet been established. Here, biophysical, biochemical, and cell-based assays demonstrate Hsp90 and Hsc70 play overlapping roles in the processing of CTA1. Using SPR we determined that Hsp90 and Hsc70 could bind independently to CTA1 at distinct locations with high affinity, even in the absence of the Hop linker. Studies using isotope-edited FTIR spectroscopy found that, like Hsp90, Hsc70 induces a gain-of-structure in unfolded CTA1. The interaction between CTA1 and Hsc70 is essential for intoxication, as an RNAi-induced loss of the Hsc70 protein generates a toxin-resistant phenotype. Further analysis using isotope-edited FTIR spectroscopy demonstrated that the addition of both Hsc70 and Hsp90 to unfolded CTA1 produced a gain-of-structure above that of the individual chaperones. Our data suggest that CTA1 translocation involves a ratchet mechanism which couples the Hsp90-mediated refolding of CTA1 with extraction from the ER. The subsequent binding of Hsc70 further refolds CTA1 in a manner not previously observed in foldosome complex formation. The interaction of CTA1 with these chaperones is essential to intoxication and this work elucidates details of the intoxication process not previously known.

Este projeto teve como objetivo geral continuar a avaliar a adequação de anfifílicos que se agregam em solução aquosa formando bicamadas, para recobrir superfícies de sílica. Em paralelo, foi objetivada a reconstituição do reconhecimento receptor-ligante, tendo como modelo o monosialogangliosídio GM1 e a enterotoxina do vibrião da cólera. Os resultados obtidos poderão se mostrar de grande valor em aplicações práticas que envolvem a construção de sistemas de detecção e quantificação de substâncias ou moléculas específicas. A adsorção e estabilidade de bicamadas contendo fosfatidilcolina (PC) ou misturas de brometo de dioctadecildimetilamônio (DODAB) e dipalmitoilfosfatidilcolna (DPPC) sobre a superfície de nanopartículas de sílica foi estudada através de isotermas de adsorção por dosagem do fosfato inorgânico, análises de sedimentação por imagens fotográficas e medidas dos diâmetros médios (Dz) e potenciais-zeta (ζ) de partículas por espectroscopia de correlação de fótons. Foram avaliadas as afinidades entre as bicamadas e a superfície das nanopartículas e a estabilidade do sistema em função da força iônica, do pH e da concentração adicionada de lipídio. A afinidade das bicamadas pela superfície da sílica apresentou uma correlação com as forças de van der Waals e as pontes de hidrogênio que se formam entre os grupos químicos do anfifílico e aqueles de superfície da sílica. A formação de uma única bicamada lipídica de PC sobre a sílica foi detectada, o que levou à estabilidade coloidal do sistema particulado. As bicamadas mistas de DPPC/DODAB por sua vez apresentaram uma afinidade decrescente pela sílica com a elevação da porcentagem de DODAB na bicamada. Os dados de isoterma e de ζ das partículas sugerem uma separação física entre o DPPC e o DODAB. O conjunto otimizado partícula de sílica/bicamada de PC (partícula biomimética) foi assim utilizado para promover a incorporação do GM1 micelar nas bicamadas, medida por fluorescência com a utilização da sonda GM1 pireno, seguida do reconhecimento e ligação da toxina da cólera (CT) ao complexo formado. A transferência do GM1 das micelas para as partículas se mostrou dependente da disponibilidade de bicamadas adsorvidas nestas e da ausência de bicamadas não-adsorvidas, livres em dispersão. Para avaliar a ligação da toxina da cólera às partículas biomiméticas foram calculadas isotermas de adsorção a partir da dosagem de proteína não ligada que resta no sobrenadante. A ligação específica da CT na presença de bicamadas de PC e GM1 foi de 67% em massa do total da proteína adicionada, revelando uma ligação positiva entre receptor e ligante. A estequiometria revela que a proporção molar PC: GM1: CT é de 300: 5: 1 respectivamente. A proporção de 1 CT: 5 GM1 está de acordo com a literatura onde experimentos de difração de raios-X mostram a estrutura tridimensional do pentâmero CTB5 ligado a cinco unidades de pentasacarídeo do GM1. / The general objective of this project was to continue evaluating the suitability of amphiphiles that aggregate in aqueous solution forming bilayers, to cover surfaces of silica. A secondary objective pursued was to promote the reconstitution of the receptor-ligand function, having as a model the monosialoganglioside GM1 and the enterotoxin of the choleric vibrio. The results may prove to be valuable in pure research or practical applications for sensing and detection of biomolecules. The adsorption and stability of phosphatidylcholine (PC) bilayers or mixtures of dipalmitoylphosphatidylcholine (DPPC) and dioctadecyldimethylammonium bromide (DODAB) over surfaces of silica nanoparticles were evaluated through adsorption isotherms by inorganic phosphate dosage, sedimentation analysis by means of photographic images and measurements of hydrodynamic diameter (Dz) and zeta-potentials (ζ) of particles by photon correlation spectroscopy. The affinity between the bilayers and the nanoparticles surfaces and the general system stability were evaluated as a function of ionic strength, pH and added lipid concentration. The affinity showed a correlation with the van der Waals and the hydrogen bridges forces that form between the chemical groups of the amphiphile and the silica surface. The formation of a single lipid PC bilayer over the silica was detected, leading to the colloidal stability of the particulate system. Mixed bilayers of DPPC/DODAB on the other hand showed a decreasing affinity for silica with an increasing DODAB percentage in the bilayer. Data from isotherms and ζ of particles suggests a physical separation of the DPPC and DODAB. The optimized array of silica particle/PC bilayer (biomimetic particles) was thus used to promote the incorporation of micellar GM1 into the bilayers, measured by fluorescence with a GM1 pirene probe, followed by the cholera toxin (CT) binding to the resulting array. GM1 transfer from micelles to particles showed dependence on the adsorbed bilayers availability and on the absence of non-adsorbed bilayers, free in the bulk solution. To evaluate CT binding to biomimetic particles adsorption isotherms were calculated from the dosage of unbound protein remaining in the supernatant. Specific binding of CT in the presence of PC and GM1 bilayer was 67% mass of total protein added, indicating a positive binding between receptor and ligand. Stoichiometry revealed that the molar proportion PC: GM1: CT is 300: 5: 1 respectively. The proportion of 1 CT: 5 GM1 is in good agreement with data in the literature where X-ray diffraction tests show the 3-dimensional structure of the CTB5 pentamer bound to five units of the pentasaccharide GM1.

O estudo do sistema visual de macacos-prego representa importante questão devido ao aspecto evolutivo que a espécie apresenta. Foram utilizados cinco macacos-prego, 2 kg. Foi efetuda injeção intra-ocular de 100 <font face=\"symbol\">ml de solução aquosa de toxina colérica subunidade B (CTb) a 1%, sendo a perfusão realizada 15 dias após a injeção intra-ocular. As retinas intactas e os encéfalos foram submetidos à procedimento de imunoistoquímica para análise. A caracterização da retina evidenciou dois tipos distintos de células bipolares, além disto, subunidades de receptores gabaérgicos co-localizam em retinas de macacos-prego, diferente dos resultados apresentados em outras espécies. As projeções retinianas foram observadas em todas as estruturas do sistema visual primário, óptico acéssório e de temporização circadiana, além de projeções para áreas adicionais. Os resultados evidenciam diferenças interespecíficas sugerindo que a extrapolação dos resultados adquiridos em diferentes espécies devam ser extrapolados com cautela. / The diurnal habits and its complex SNC, make the tufted capuchin monkey an important subject for the study of the visual system. In the present study, five tufted capuchins received a single intraocular neuronal tracer subunit B of cholera toxin (CTb) injection and perfused 15 days later. The retina and brain were removed from the animals and processed with immunohistochemical techniques. The CTb analysis showed that the retina send projections to several structures, such as primary visual, optical accessory and circadian control systems. The immunohistochemical characterization also showed two different types of bipolar cells in the retina. These cells, differently from other species, were co-localized with gabaergic receptors. Overall our results showed several interspecies differences suggesting that comparison of the visual system between species must be undertaken with great caution.

Este projeto teve como objetivo geral continuar a avaliar a adequação de anfifílicos que se agregam em solução aquosa formando bicamadas, para recobrir superfícies de sílica. Em paralelo, foi objetivada a reconstituição do reconhecimento receptor-ligante, tendo como modelo o monosialogangliosídio GM1 e a enterotoxina do vibrião da cólera. Os resultados obtidos poderão se mostrar de grande valor em aplicações práticas que envolvem a construção de sistemas de detecção e quantificação de substâncias ou moléculas específicas. A adsorção e estabilidade de bicamadas contendo fosfatidilcolina (PC) ou misturas de brometo de dioctadecildimetilamônio (DODAB) e dipalmitoilfosfatidilcolna (DPPC) sobre a superfície de nanopartículas de sílica foi estudada através de isotermas de adsorção por dosagem do fosfato inorgânico, análises de sedimentação por imagens fotográficas e medidas dos diâmetros médios (Dz) e potenciais-zeta (&#950;) de partículas por espectroscopia de correlação de fótons. Foram avaliadas as afinidades entre as bicamadas e a superfície das nanopartículas e a estabilidade do sistema em função da força iônica, do pH e da concentração adicionada de lipídio. A afinidade das bicamadas pela superfície da sílica apresentou uma correlação com as forças de van der Waals e as pontes de hidrogênio que se formam entre os grupos químicos do anfifílico e aqueles de superfície da sílica. A formação de uma única bicamada lipídica de PC sobre a sílica foi detectada, o que levou à estabilidade coloidal do sistema particulado. As bicamadas mistas de DPPC/DODAB por sua vez apresentaram uma afinidade decrescente pela sílica com a elevação da porcentagem de DODAB na bicamada. Os dados de isoterma e de &#950; das partículas sugerem uma separação física entre o DPPC e o DODAB. O conjunto otimizado partícula de sílica/bicamada de PC (partícula biomimética) foi assim utilizado para promover a incorporação do GM1 micelar nas bicamadas, medida por fluorescência com a utilização da sonda GM1 pireno, seguida do reconhecimento e ligação da toxina da cólera (CT) ao complexo formado. A transferência do GM1 das micelas para as partículas se mostrou dependente da disponibilidade de bicamadas adsorvidas nestas e da ausência de bicamadas não-adsorvidas, livres em dispersão. Para avaliar a ligação da toxina da cólera às partículas biomiméticas foram calculadas isotermas de adsorção a partir da dosagem de proteína não ligada que resta no sobrenadante. A ligação específica da CT na presença de bicamadas de PC e GM1 foi de 67% em massa do total da proteína adicionada, revelando uma ligação positiva entre receptor e ligante. A estequiometria revela que a proporção molar PC: GM1: CT é de 300: 5: 1 respectivamente. A proporção de 1 CT: 5 GM1 está de acordo com a literatura onde experimentos de difração de raios-X mostram a estrutura tridimensional do pentâmero CTB5 ligado a cinco unidades de pentasacarídeo do GM1. / The general objective of this project was to continue evaluating the suitability of amphiphiles that aggregate in aqueous solution forming bilayers, to cover surfaces of silica. A secondary objective pursued was to promote the reconstitution of the receptor-ligand function, having as a model the monosialoganglioside GM1 and the enterotoxin of the choleric vibrio. The results may prove to be valuable in pure research or practical applications for sensing and detection of biomolecules. The adsorption and stability of phosphatidylcholine (PC) bilayers or mixtures of dipalmitoylphosphatidylcholine (DPPC) and dioctadecyldimethylammonium bromide (DODAB) over surfaces of silica nanoparticles were evaluated through adsorption isotherms by inorganic phosphate dosage, sedimentation analysis by means of photographic images and measurements of hydrodynamic diameter (Dz) and zeta-potentials (&#950;) of particles by photon correlation spectroscopy. The affinity between the bilayers and the nanoparticles surfaces and the general system stability were evaluated as a function of ionic strength, pH and added lipid concentration. The affinity showed a correlation with the van der Waals and the hydrogen bridges forces that form between the chemical groups of the amphiphile and the silica surface. The formation of a single lipid PC bilayer over the silica was detected, leading to the colloidal stability of the particulate system. Mixed bilayers of DPPC/DODAB on the other hand showed a decreasing affinity for silica with an increasing DODAB percentage in the bilayer. Data from isotherms and &#950; of particles suggests a physical separation of the DPPC and DODAB. The optimized array of silica particle/PC bilayer (biomimetic particles) was thus used to promote the incorporation of micellar GM1 into the bilayers, measured by fluorescence with a GM1 pirene probe, followed by the cholera toxin (CT) binding to the resulting array. GM1 transfer from micelles to particles showed dependence on the adsorbed bilayers availability and on the absence of non-adsorbed bilayers, free in the bulk solution. To evaluate CT binding to biomimetic particles adsorption isotherms were calculated from the dosage of unbound protein remaining in the supernatant. Specific binding of CT in the presence of PC and GM1 bilayer was 67% mass of total protein added, indicating a positive binding between receptor and ligand. Stoichiometry revealed that the molar proportion PC: GM1: CT is 300: 5: 1 respectively. The proportion of 1 CT: 5 GM1 is in good agreement with data in the literature where X-ray diffraction tests show the 3-dimensional structure of the CTB5 pentamer bound to five units of the pentasaccharide GM1.

O estudo do sistema visual de macacos-prego representa importante questão devido ao aspecto evolutivo que a espécie apresenta. Foram utilizados cinco macacos-prego, 2 kg. Foi efetuda injeção intra-ocular de 100 <font face=\"symbol\">ml de solução aquosa de toxina colérica subunidade B (CTb) a 1%, sendo a perfusão realizada 15 dias após a injeção intra-ocular. As retinas intactas e os encéfalos foram submetidos à procedimento de imunoistoquímica para análise. A caracterização da retina evidenciou dois tipos distintos de células bipolares, além disto, subunidades de receptores gabaérgicos co-localizam em retinas de macacos-prego, diferente dos resultados apresentados em outras espécies. As projeções retinianas foram observadas em todas as estruturas do sistema visual primário, óptico acéssório e de temporização circadiana, além de projeções para áreas adicionais. Os resultados evidenciam diferenças interespecíficas sugerindo que a extrapolação dos resultados adquiridos em diferentes espécies devam ser extrapolados com cautela. / The diurnal habits and its complex SNC, make the tufted capuchin monkey an important subject for the study of the visual system. In the present study, five tufted capuchins received a single intraocular neuronal tracer subunit B of cholera toxin (CTb) injection and perfused 15 days later. The retina and brain were removed from the animals and processed with immunohistochemical techniques. The CTb analysis showed that the retina send projections to several structures, such as primary visual, optical accessory and circadian control systems. The immunohistochemical characterization also showed two different types of bipolar cells in the retina. These cells, differently from other species, were co-localized with gabaergic receptors. Overall our results showed several interspecies differences suggesting that comparison of the visual system between species must be undertaken with great caution.

Les voies d’endocytose qui régissent l’internalisation d’éléments extracellulaires peuvent être classées selon que la protéine de manteau, la clathrine, est impliquée ou non dans le processus. Les voies indépendantes de la clathrine sont utilisées par de nombreuses toxines, des virus et des protéines endogènes. Les mécanismes permettant d’induire le recrutement des protéines cargoes et la déformation de la membrane plasmique dans le contexte de l'endocytose clathrine-indépendant restent encore mal compris. Cette étude montre que la galectine 3, une protéine humaine qui se lie aux glucides, induit la formation d’invaginations de la membrane plasmique de manière indépendante de la clathrine. Les glycosphingolipides (molécules jouant un rôle majeur dans la physiologie de la cellule) sont essentielles pour permettre à la galectine 3 d’induire ces invaginations et d’être internalisée dans la cellule. Les structures tubulaires induites par la galectine 3 présentent une morphologie étonnamment similaire à celle de compartiments intermédiaires de transport décrits dans la littérature pour l’endocytose indépendante de la clathrine. Des cargos utilisant la voie indépendante de la clathrine, tels que CD44 et les intégrines α5 et β1, sont retrouvés dans les tubules induits par la galectine 3. De plus, cette dernière est nécessaire à l’internalisation de CD44. Cela indique donc que la galectine 3 pourrait relier des protéines cargos glycosylés à des glycosphyngolipides de la membrane plasmique et ainsi induire une déformation de la membrane et leur internalisation dans les cellules. Ce mécanisme diffère de celui utilisé par la toxine pentamérique de Shiga et par la toxine cholérique, qui sont leur protéines cargos propores et interagissant directement avec le glycosphyngolipide leur servant de récepteur. Les tubules induits par la galectine 3 sont distincts de ceux induit par les autres lectines. Celles-ci présentent des spécificités différentes de liaison aux glucides, montrant ainsi la l'importance des interactions entre les lectines et les sucres dans ce processus. De plus, nous avons constaté que la galectine 3 module l’équilibre à l’état basal de l’intégrine β1 à la surface de la cellule. Cette protéine étant capitale pour les phénomènes d’adhésion et de migration cellulaires, nous avons donc exploré le rôle conjoint de la galectine 3 et des glycosphingolipides dans la migration cellulaire. La galectine 3 inhibe la migration des cellules humaines de carcinomes mammaires alors qu’elle stimule au contraire celle de cellules de tumeurs mammaires murines. Or, nous avons montré que la régulation par la galactine 3 de la migration de différentes lignées cellulaires est dépendante des glycosphingolipides. Il ressort donc de cette étude que la galectine 3 et les glycosphingolipides contribuent de manière synergique au processus d’induction de déformation de la membrane, à l’endocytose de protéines cargos et à la migration cellulaire. / Endocytic processes which govern the uptake of extracellular material into the cell can be classified based on their dependence on the coat protein, clathrin. Clathrin-independent mechanisms are used by many toxins, viruses and endogenous proteins. How cargo is recruited and membranes are bent is not well understood in these cases. Here, we discovered that galectin 3, a human carbohydrate binding protein induced the clathrin-independent formation of endocytic plasma membrane invaginations. Glycosphingolipids, which have established functions in key physiological processes, were found to be essential for the formation of galectin 3-induced invaginations and for the efficient uptake of the protein into the cell. Galectin 3-induced tubular structures were found to have a strikingly similar morphology to that of the clathrin-independent carriers described in literature. Clathrin-independent endocytic cargoes such as CD44, α5 and β1 integrin were present in galectin 3-induced tubules, and galectin activity and glycosphingolipids were required for the uptake of CD44. This indicated that galectin 3 could link glycosylated cargoes with glycosphingolipids for cargo recruitment and membrane bending. In contrast, the pentameric Shiga and cholera toxins are their own cargoes and drive membrane deformations by directly binding to their respective glycosphingolipid receptors. Galectin 3-induced tubules were distinct from those induced by lectins with different carbohydrate binding specificities, which revealed the importance of lectin-glycan interaction in this process. Further, we observed that galectin 3 modulated the steady state surface dynamics of β1 integrin, a protein which like CD44 is critical for cell adhesion and migration. Subsequently, we explored the interplay of galectins and glycosphingolipids in cell migration. Galectin 3 inhibited cell migration in human breast carcinoma cells, and stimulated migration in a mouse mammary tumor cell line. However, the regulation of migration by galectin 3 was in both cases found to be dependent on glycosphingolipids. In conclusion, galectin 3 and glycosphingolipids synergistically contribute to the clathrin-independent curvature generation process, cargo endocytosis and cell migration.