Modification by glycosylation gives proteins a range of diverse functions reflecting their structural variability. N-glycans regulate many biological outcomes in mammalian cells under both normal and pathological conditions. They play a major role in various pathologies such as cancer and lysosomal storage diseases. Interplay between N-glycans and other regulators, such as membrane lipid domains, in the control of signaling pathways remains poorly understood. My thesis therefore focuses on how N-glycans and membrane lipid domains oppose and/or work together at different cellular levels to regulate various processes such as receptor signaling and diffusion, endocytosis and lysosomal organelle biogenesis. Mgat5 encodes for ß1,6-N-acetylglucosaminyltransferase V that produces N-glycans, the preferred ligand for galectins. In tumor cells, galectins bind glycosylated receptors at the cell surface forming a lattice, that restricts receptor endocytosis and enhances its residency at the plasma membrane. In the first part of my thesis, I report that Galectin/receptor crosslinking opposes receptor sequestration by oligomerized caveolin-1 (Cav1) domains overriding its negative regulation of epidermal growth factor receptor (EGFR) signaling, cell surface diffusion and tumor growth. These results identify Cav1 as a conditional tumor suppressor. I also demonstrate that Cav1 is a negative regulator of lipid raft-mediated endocytosis. Cav1 indirectly regulates the internalization of cholera toxin b subunit to the Golgi apparatus independently of caveolae formation. That identifies a new role for caveolin-1 outside caveolae in the regulation of raft-dependent endocytosis Finally, Mgat5 overexpression in pneumocytes is associated with the expression of a lysosomal organelle, the multilamellar body (MLB), via autophagy. MLB expression is also a characteristic of various lysosomal storage diseases. I demonstrate that cholesterol accumulation can override the need for Mgat5 overexpression in MLB formation indicating that they may form via multiple mechanisms. However, I also demonstrate that a contribution of the autophagic pathway is a common determinant of biogenesis of MLB of various lipid compositions. In conclusion, Mgat5-dependent protein glycosylation and Cav1/raft domains therefore both function as regulators of plasma membrane interactions, endocytosis and lysosomal organelle biogenesis. Understanding of this interplay is crucial for the understanding of the mechanisms involve in various pathologies such as cancer and lysosomal storage diseases. / Medicine, Faculty of / Graduate

In der vorliegenden Arbeit wurde der Einfluss mehrfach ungesättigter Fettsäuren (PUFA) auf das Phospho- und Sphingolipidmuster der Non-raft- und Lipid raft-Bereiche in Membranen von RAW264.7-Makrophagen massenspektrometrisch untersucht. Außerdem wurden mittels Fluoreszenzmikroskopie die Auswirkungen einer PUFA-Zugabe auf die Lokalisation des immunologisch bedeutsamen Mustererkennungs-Rezeptor TLR2 und seinen Ko-Rezeptoren TLR1 und TLR6 untersucht.

Vesicles are a type of biological or biomimetic particle consisting of one or more often spherical bilayers made up of amphipathic molecules, creating a closed system. They can function as an encapsulating device, holding hydrophilic molecules on the inside of the bilayer membrane(s) or hydrophobic molecules in the non-polar interstitial space in the middle of the bilayers. Because of this capacity to carry molecules, vesicles are a premier system for drug delivery and even theranostics in vivo. A peptide-based approach to release of encapsulated molecules has previously been developed but since drug delivery vesicles are in the size range of nanometers, the mechanisms have not been visualized. This project aims to produce giant unilamellar vesicles as a model system used to visualize membrane interactions vital to the understanding and further development of smaller vesicle-based systems for drug delivery. Giant unilamellar vesicles were produced successfully and a preparation protocol was established. Additionally, some membrane interactions were investigated using fluorescence microscopy.

No description available.

No description available.

Coastal karst aquifers are important water resources, often providing the only source of freshwater to coastal communities for agriculture, industrial usage and human consumption. In order to implement management strategies and preventative measures for future perseveration of this resource, it is imperative to understand how coastal groundwater conditions are controlled by the interaction of freshwater/seawater on local and regional scales, but also over recent and past time periods. However, there is a limited resource of published hydrological data on recent aquifer conditions. In the Yucatán Peninsula and other anchialine environments, this lack of information inhibits the understanding of the spatial and temporal interaction of the meteoric and marine water masses. Documenting how the aquifer is responding to forcing mechanisms such as large precipitation events, seasonal cycles and short-term sea level rise (e.g. storm surge) will assist in understanding modern aquifer condition but also the interpretation of paleo-records. Utilization of water level and salinity sensors in strategic positions in the aquifer demonstrate that meteoric water mass salinity varies over wet and dry seasons with the movement of the halocline, but also on a short-term basis though large rainfall events. Salinity in the meteoric water mass is influenced by mixing with the marine water mass during intense precipitation events associated with Hurricane Ingrid (2013), Tropical Storm Hanna (2014) and a series of unnamed events in 2015. During wet periods, induced flow from increase precipitation causes turbulent mixing with the marine water mass, increasing salinity in the upper meteoric lens. On the contrary, during dry periods, mixing is reduced, therefore making the meteoric lens less saline. This contemporary understanding of meteoric/marine water mass dynamics can be applied to developing and calibrating the geochemical record of calcite rafts, calcite precipitation at the air-water interface of cave pools, as a hydrological proxy for aquifer conditions. Our monitoring of calcite raft formation, deposition and geochemistry shows that raft accumulations (e.g., raft piles/cones) can offer a good paleoenvironmental archive of changing hydrological conditions. Based on a 2-year observational record, results indicate that calcite raft precipitation/formation occurs continuously but with only minor biases with intense rainfall events altering supersaturation conditions in the surface waters. Testing the use of calcite rafts in sediment cores from Hoyo Negro show that geochemical analyses (87Sr/86Sr, δ18O, δ13C, Sr/Ca and Cl/Ca) show that meteoric water mass salinity varied during the Holocene (~ 8.5 Ka – present) likely due to changing rainfall and or cave passage geomorphology, which is coherent with other independent climate records. Prior to this study, calcite rafts have never been considered a paleo-hydrological archive for aquifer conditions, however, the consistency and cross-validation with independent records demonstrates great potential for future paleohydrological reconstructions. / Thesis / Doctor of Philosophy (PhD)

Proteolytic processing of the amyloid precursor protein (APP) may lead to the formation of the Abeta peptide, the major constituent of amyloid plaques in Alzheimer`s disease. The full-length APP is a substrate for at least 2 different (alpha and beta) proteases ("secretases"). The beta-secretase, BACE, cleaves APP in the first step of processing leading to the formation of the neurotoxic Abeta. BACE competes for APP with alpha-secretase, which cleaves APP within its Abeta sequence, thus precluding Abeta formation. It is thus important to understand how is the access of the alpha- and beta-secretase to APP regulated and how are the individual activities of these secretases modulated. Both these regulatory mechanisms, access to substrate and direct activity modulation, can be determined by the lipid composition of the membrane. Integral membrane proteins (like APP and BACE), can be viewed as solutes in a two-dimensional liquid membrane, and as such their state, and biological activity, critically depend on the physico-chemical character (fluidity, curvature, surface charge distribution, lateral domain heterogeneity etc.) of the lipid bilayer. These collective membrane properties will influence the activity of embedded membrane proteins. In addition, activity regulation may involve a direct interaction with a specific lipid (cofactor or co-structure function). Interactions of membrane proteins are furthermore affected by lateral domain organization of the membrane. Previous results had suggested that the regulation of the activity of the alpha- and beta-secretases and of their access to APP is lipid dependent, and involves lipid rafts. Using the baculovirus expression system, we have purified recombinant human full-length APP and BACE to homogeneity, and reconstituted them in large (~100nm, LUVs) and giant (10-150microm, GUVs) unilamellar vesicles. Using a soluble peptide substrate mimicking the beta-cleavage site of APP, we have examined the involvement of individual lipid species in modulating BACE activity in LUVs of various lipid compositions. We have identified 3 groups of lipids that stimulate proteolytic activity of BACE: 1.cerebrosides, 2.anionic glycerophospholipids, 3. cholesterol. Furthermore, we have co-reconstituted APP and BACE together in LUVs and demonstrated that BACE cleaves APP at the correct site, generating the beta-cleaved ectodomain identical to that from cells. We have developed an assay to quantitatively follow the beta-cleavage in proteoliposomes, and we have shown that the rate of cleavage in total brain lipid proteoliposomes is higher than in phosphatidylcholine vesicles. We have also studied partitioning of APP and BACE in GUVs between liquid ordered (lo) and liquid disordered (ld) phases. In this system, significant part of the BACE pool (about 20%) partitions into the lo phase, and its partitioning into lo phase can be further enhanced by cross-linking of membrane components. Only negligible fraction of APP can be found in the lo phase. We continue to study the behavior of co-reconstituted APP and BACE in GUVs The work presented in this thesis has yielded some interesting results and raised further questions. One of the important assignments of this project will in the next stage be the characterization of the impact of membrane domain organization on the beta-cleavage. Different domain arrangements that can be hypothesized in cell membranes can be modeled by varying the degree of phase fragmentation in proteoliposomes comprising reconstituted APP and BACE.

Die Arbeit umfasst die Synthese von lipophilen Inositolen und Glycerollipiden, welche auf ihre Raftophilie getestet wurden. Des weiteren wurden eine Reihe neuer Ceramide synthetisiert und diese in Bioassays auf ihre Wirksamkeit gegenüber diversen Krankheiten wie Influenza getestet.

Aims: T1α/(podoplanin) is abundantly expressed in the alveolar epithelial type I cells (ATI) of rodent and human lungs. Caveolin-1 is a classical primary structural protein of plasmalemal invaginations, so-called caveolae, which represent specialized lipid rafts, and which are particularly abundant in ATI cells. The biological functions of T1α in the alveolar epithelium are unknown. Here we report on the characteristics of raft domains in the microplicae/microvillar protrusions of ATI cells, which contain T1α. Methods: Detergent resistant membranes (DRMs) from cell lysates of the mouse epithelial ATI-like cell line E10 were prepared using different detergents followed by flotation in a sucrose gradient and tested by Western and dot blots with raft markers (caveolin-1, GM1) and nonraft markers (transferrin receptor, PDI and β-Cop). Immunocytochemistry was employed for the localization of T1α in E10 cells and in situ in rat lungs. Results: Our biochemical results showed that the solubility or insolubility of T1α and caveolin-1 differs in Triton X-100 and Lubrol WX, two distinct non-ionic detergents. Caveolin-1 was unsoluble in both detergents, whereas T1α was Triton X-100 soluble but Lubrol WX insoluble. Immunofluorescence double stainings revealed that both proteins were colocalized with GM1, while caveolin-1 and T1α were not colocalized in the plasma membrane. Cholesterol depletion modified the segregation of T1α in Lubrol WX DRMs. Cellular processes in ultrathin sections of cultured mouse E10 cells were immunogold positive. Immunoelectron microscopy (postembedding) of rat lung tissue revealed the preferential localization of T1α on apical microvillar protrusions of ATI cells. Conclusion: We conclude that T1α and caveolin-1 are located in distinct plasma membrane microdomains, which differ in their protein-lipid interactions. The raft-associated distribution of T1α may have an impact on a specific, not yet clarified function of this protein in the alveolar epithelium. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Proteolytic processing of the amyloid precursor protein (APP) may lead to the formation of the Abeta peptide, the major constituent of amyloid plaques in Alzheimer`s disease. The full-length APP is a substrate for at least 2 different (alpha and beta) proteases ("secretases"). The beta-secretase, BACE, cleaves APP in the first step of processing leading to the formation of the neurotoxic Abeta. BACE competes for APP with alpha-secretase, which cleaves APP within its Abeta sequence, thus precluding Abeta formation. It is thus important to understand how is the access of the alpha- and beta-secretase to APP regulated and how are the individual activities of these secretases modulated. Both these regulatory mechanisms, access to substrate and direct activity modulation, can be determined by the lipid composition of the membrane. Integral membrane proteins (like APP and BACE), can be viewed as solutes in a two-dimensional liquid membrane, and as such their state, and biological activity, critically depend on the physico-chemical character (fluidity, curvature, surface charge distribution, lateral domain heterogeneity etc.) of the lipid bilayer. These collective membrane properties will influence the activity of embedded membrane proteins. In addition, activity regulation may involve a direct interaction with a specific lipid (cofactor or co-structure function). Interactions of membrane proteins are furthermore affected by lateral domain organization of the membrane. Previous results had suggested that the regulation of the activity of the alpha- and beta-secretases and of their access to APP is lipid dependent, and involves lipid rafts. Using the baculovirus expression system, we have purified recombinant human full-length APP and BACE to homogeneity, and reconstituted them in large (~100nm, LUVs) and giant (10-150microm, GUVs) unilamellar vesicles. Using a soluble peptide substrate mimicking the beta-cleavage site of APP, we have examined the involvement of individual lipid species in modulating BACE activity in LUVs of various lipid compositions. We have identified 3 groups of lipids that stimulate proteolytic activity of BACE: 1.cerebrosides, 2.anionic glycerophospholipids, 3. cholesterol. Furthermore, we have co-reconstituted APP and BACE together in LUVs and demonstrated that BACE cleaves APP at the correct site, generating the beta-cleaved ectodomain identical to that from cells. We have developed an assay to quantitatively follow the beta-cleavage in proteoliposomes, and we have shown that the rate of cleavage in total brain lipid proteoliposomes is higher than in phosphatidylcholine vesicles. We have also studied partitioning of APP and BACE in GUVs between liquid ordered (lo) and liquid disordered (ld) phases. In this system, significant part of the BACE pool (about 20%) partitions into the lo phase, and its partitioning into lo phase can be further enhanced by cross-linking of membrane components. Only negligible fraction of APP can be found in the lo phase. We continue to study the behavior of co-reconstituted APP and BACE in GUVs The work presented in this thesis has yielded some interesting results and raised further questions. One of the important assignments of this project will in the next stage be the characterization of the impact of membrane domain organization on the beta-cleavage. Different domain arrangements that can be hypothesized in cell membranes can be modeled by varying the degree of phase fragmentation in proteoliposomes comprising reconstituted APP and BACE.