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341 p., Esta tesis pretende explorar la función de los esfingolípidos en el crecimiento celular y en la composición y las propiedades físicas de las membranas. Para ello se han utilizado células que poseen una síntesis limitada de esfingolípidos. Tanto células modificadas genéticamente como tratadas químicamente.A lo largo del estudio se han utilizado diferentes técnicas biofísicas para la caracterización de estas propiedades: sobre todo la espectroscopía y microscopía fluorescente, la microscopia de fuerza atómica y la espectrometría de masas.Esta investigación demuestra que existe una clara correlación entre la disminución del contenido de SL de las membranas y los cambios biofísicos en las propiedades biofisicas de las mismas: Las membranas celulares se vuelven más fluidas al medirlas mediante la polarización general de laurdan y son más penetrables al inducir su ruptura mediante la espectrometría de fuerza (AFM).Cuando las cantidades de SL se reducen en las membranas celulares, las células CHO y HAP1 intentan mantener su orden de membrana nativo mediante una respuesta homeostática en su síntesis lipídico: Sintetizan más glicerofosfolípidos totalmente saturados y menos insaturado. Las LY-B (SPT deficiente) mantienen el orden molecular de sus membranas solo parcialmente, ya que su membrana plasmática es más penetrable en comparación a las de la línea celular CHO (wild type). Por otro lado, la línea celular humana HAP1-SPT (SPT deficiente) mantiene el orden de sus membranas, ya que no existen diferencias en comparación a la penetrabilidad de la membrana plasmática de las células HAP1 (wild type).

N-maleimide-derivatized phospholipids are often used to facilitate protein anchoring to membranes. In autophagy studies, this is applied to the covalent binding of Atg8, an autophagy protein, to a phosphatidylethanolamine (PE) in the nascent autophagosome. However, the question remains on how closely the N-maleimide PE derivative (PE-mal) mimicks the native PE in the bilayer. In the present paper, spectroscopic and calorimetric techniques have been applied to vesicles containing either PE or PE-mal (together with other phospholipids) to compare the properties of the native and derivatized forms of PE. According to differential scanning calorimetry, and to infrared spectroscopy, the presence of PE-mal did not perturb the fatty acyl chains in the bilayer. Fluorescence spectroscopy and microscopy showed that PE-mal did not alter the bilayer permeability either. However, fluorescence emission polarization of the Laurdan and DPH probes indicated an increased order, or decreased fluidity, in the bilayers containing PE-mal. In addition, the infrared spectral data from the phospholipid phosphate region revealed a PE-mal-induced conformational change in the polar heads, accompanied by increased hydration. Globally considered, the results suggest that PE-mal would be a reasonable substitute for PE in model membranes containing reconstituted proteins.

Cardiolipin (CL) is a key lipid for damaged mitochondrial recognition by the LC3/GABARAP human autophagy proteins. The role of ceramide (Cer) in this process is unclear, but CL and Cer have been proposed to coexist in mitochondria under certain conditions. Varela et al. showed that in model membranes composed of egg sphingomyelin (eSM), dioleoyl phosphatidylethanolamine (DOPE), and CL, the addition of Cer enhanced the binding of LC3/GABARAP proteins to bilayers. Cer gave rise to lateral phase separation of Cer-rich rigid domains but protein binding took place mainly in the fluid continuous phase. In the present study, a biophysical analysis of bilayers composed of eSM, DOPE, CL, and/or Cer was attempted to understand the relevance of this lipid coexistence. Bilayers were studied by differential scanning calorimetry, confocal fluorescence microscopy, and atomic force microscopy. Upon the addition of CL and Cer, one continuous phase and two segregated ones were formed. In bilayers with egg phosphatidylcholine instead of eSM, in which the binding of LC3/GABARAP proteins hardly increased with Cer in the former study, a single segregated phase was formed. Assuming that phase separation at the nanoscale is ruled by the same principles acting at the micrometer scale, it is proposed that Cer-enriched rigid nanodomains, stabilized by eSM:Cer interactions formed within the DOPE- and CL-enriched fluid phase, result in structural defects at the rigid/fluid nanointerfaces, thus hypothetically facilitatingLC3/GABARAP protein interaction.

In macroautophagy, the autophagosome (AP) engulfs portions of cytoplasm to allow their lysosomal degradation. AP formation in humans requires the concerted action of the ATG12 and LC3/GABARAP conjugation systems. The ATG12–ATG5-ATG16L1 or E3-like complex (E3 for short) acts as a ubiquitin-like E3 enzyme, promoting LC3/GABARAP proteins anchoring to the AP membrane. Their role in the AP expansion process is still unclear, in part because there are no studies comparing six LC3/GABARAP family member roles under the same conditions, and also because the full human E3 was only recently available. In the present study, the lipidation of six members of the LC3/GABARAP family has been reconstituted in the presence and absence of E3, and the mechanisms by which E3 and LC3/GABARAP proteins participate in vesicle tethering and fusion have been investigated. In the absence of E3, GABARAP and GABARAPL1 showed the highest activities. Differences found within LC3/GABARAP proteins suggest the existence of a lipidation threshold, lower for the GABARAP subfamily, as a requisite for tethering and inter-vesicular lipid mixing. E3 increases and speeds up lipidation and LC3/GABARAP-promoted tethering. However, E3 hampers LC3/GABARAP capacity to induce inter-vesicular lipid mixing or subsequent fusion, presumably through the formation of a rigid scaffold on the vesicle surface. Our results suggest a model of AP expansion in which the growing regions would be areas where the LC3/GABARAP proteins involved should be susceptible to lipidation in the absence of E3, or else a regulatory mechanism would allow vesicle incorporation and phagophore growth when E3 is present.

[EN] Suppression of a specific gene effect can be achieved by genetic as well as chemical methods. Each approach may hide unexpected drawbacks, usually in the form of side effects. In the present study, the specific inhibitor myriocin was used to block serine palmitoyltransferase (SPT), the first enzyme in the sphingolipid synthetic pathway, in CHO cells. The subsequent biophysical changes in plasma membranes were measured and compared with results obtained with a genetically modified CHO cell line containing a defective SPT (the LY-B cell line). Similar effects were observed with both approaches: sphingomyelin values were markedly decreased in myriocin-treated CHO cells and, in consequence, their membrane molecular order (measured as laurdan general polarization) and mechanical resistance (AFM-measured breakthrough force values) became lower than in the native, non-treated cells. Cells treated with myriocin reacted homeostatically to maintain membrane order, synthesizing more fully saturated and less polyunsaturated GPL than the non-treated ones, although they achieved it only partially, their plasma membranes remaining slightly more fluid and more penetrable than those from the control cells. The good agreement between results obtained with very different tools, such as genetically modified and chemically treated cells, reinforces the use of both methods and demonstrates that both are adequate for their intended use, i.e. the complete and specific inhibition of sphingolipid synthesis in CHO cells, without apparent unexpected effects.

Externalization of the phospholipid cardiolipin (CL) to the outer mitochondrial membrane has been proposed to act as a mitophagy trigger. CL would act as a signal for binding the LC3 macroauto-phagy/autophagy proteins. As yet, the behavior of the LC3-subfamily members has not been directly compared in a detailed way. In the present contribution, an analysis of LC3A, LC3B and LC3C interaction with CL-containing model membranes, and of their ability to translocate to mitochondria, is described. Binding of LC3A to CL was stronger than that of LC3B; both proteins showed a similar ability to colocalize with mitochondria upon induction of CL externalization in SH-SY5Y cells. Besides, the double silencing of LC3A and LC3B proteins was seen to decrease CCCP-induced mitophagy. Residues 14 and 18 located in the N-terminal region of LC3A were shown to be important for its recognition of damaged mitochondria during rotenone- or CCCP-induced mitophagy. Moreover, the in vitro results suggested a possible role of LC3A, but not of LC3B, in oxidized-CL recognition as a counterweight to excessive apoptosis activation. In the case of LC3C, even if this protein showed a stronger CL binding than LC3B or LC3A, the interaction was less specific, and colocalization of LC3C with mitochondria was not rotenone dependent. These results suggest that, at variance with LC3A, LC3C does not participate in cargo recognition during CL-mediated-mitophagy. The data support the notion that the various LC3-subfamily members might play different roles during autophagy initia-tion, identifying LC3A as a novel stakeholder in CL-mediated mitophagy.

In the early 1970s, the existence of a “lipid annulus” stably surrounding the individual intrinsic protein molecules was proposed by several authors. They referred to a number of lipid molecules in slow exchange with the bulk lipid in the bilayer, i.e., more or less protein-bound, and more ordered than the bulk lipid. The annular lipids would control enzyme activity. This idea was uncritically accepted by most scientists working with intrinsic membrane proteins at the time, so that the idea operated like a myth in the field. However, in the following decade, hard spectroscopic and biochemical evidence showed that the proposed annular lipids were not immobilized for a sufficiently long time to influence enzyme or transporter activity, nor were they ordered by the protein. Surprisingly, forty years later, the myth survives, and the term ‘annular lipid’ is still in use, in a different, but even more illogical sense.

In the 1960s, Biophysics was an unheard of scientific field in Spain, and even outside Spain, the distinction between Biophysics and Molecular Biology was not clear at the time. This paper describes briefly the developments that led to the foundation of the Spanish National Committee for Biophysics (1981) and of the Spanish Biophysical Society (1987), the incorporation of Spain into IUPAB and EBSA, and the normalized presence of Biophysics as a compulsory subject in undergraduate curricula in Spain.

Lipidomic analysis of the N-acyl components of sphingolipids in different mammalian tissues had revealed that brain tissue differed from all the other samples in that SM contained mainly C18:0 and C24:1N-acyl chains, and that the most abundant Cer species was C18:0. Only in the nervous system was C18:0 found in sizable proportions. The high levels of C18:0 and C16:0, respectively in brain and non-brain SM, were important because SM is by far the most abundant sphingolipid in the plasma membrane. In view of these observations, the present paper is devoted to a comparative study of the properties of C16:0 and C18:0 sphingolipids (SM and Cer) pure and in mixtures of increasing complexities, using differential scanning calorimetry, confocal microscopy of giant unilamellar vesicles, and correlative fluorescence microscopy and atomic force microscopy of supported lipid bilayers. Membrane rigidity was measured by force spectroscopy. It was found that in mixtures containing dioleoyl phosphatidylcholine, sphingomyelin and cholesterol, i.e. representing the lipids predominant in the outer monolayer of cell membranes, lateral inhomogeneities occurred, with the formation of rigid domains within a continuous fluid phase. Inclusion of saturated Cer in the system was always found to increase the rigidity of the segregated domains. C18:0-based sphingolipids exhibit hydrocarbon chain-length asymmetry, and some singularities observed with this N-acyl chain, e.g. complex calorimetric endotherms, could be attributed to this property. Moreover, C18:0-based sphingolipids, that are typical of the excitable cells, were less miscible with the fluid phase than their C16:0 counterparts. The results could be interpreted as suggesting that the predominance of C18:0 Cer in the nervous system would contribute to the tightness of its plasma membranes, thus facilitating maintenance of the ion gradients.

The basic matrix of cellular membranes consists of a double layer (bilayer) of phospholipids. Semisynthetic lipid bilayers are commonly used in biophysical studies of membranes. According to tem-perature and composition, lipid bilayers can exist in liquid-crystalline (or liquid-disordered), liquid -ordered, rippled, and gel phases. In the present study, the hydrophobic, solvatochromic molecule Nile red has been used as a fluorescent probe to examine the physical state of bilayers of different composi-tions in the 15-60 degrees C range. Phospholipids with saturated or unsaturated acyl chains, in the presence or absence of cholesterol have been studied. Nile red shows absorption maxima at 520-550 nm and emis-sion maxima at 580-640 nm, single photon excitation not being damaging to the system. A red/orange intensity ratio (ROIR) index has been used to normalize the results. ROIR varies clearly and reproducibly with the lipid phase, increasing in the order: liquid-ordered < gel < rippled < liquid-crystalline. It increases with temperature and decreases with cholesterol contents in the bilayers. Nile red allows an unusually clear observation of the rippled-to-liquid crystalline phase transition in saturated phospho-lipids. FLIM studies with Nile red also show differences between lamellar phases. Rotational relaxation times have been determined for Nile red in liquid-disordered (0.72 +/- 0.010 ns), gel (1.16 +/- 0.070 ns), and liquid-ordered (1.79 +/- 0.14 ns) phases, the large value of the liquid-ordered phase being an indication of the sterol hindering probe tumbling in the hydrophobic matrix.

Autophagy is a process in which parts of the eukaryotic cell are selectively degraded in the lysosome. The materials to be catabolized are first surrounded by a double-membrane structure, the autophagosome. Autophagosome generation is a complex event, in which many proteins are involved. Among the latter, yeast Atg8 or its mammalian orthologues are essential in autophagosome membrane elongation, shaping and closure. A subfamily of the human Atg8 orthologues is formed by the proteins LC3A, LC3B, and LC3C. Previous studies suggest that, at variance with the other two, LC3C does not participate in cardiolipin-mediated mitophagy. The present study was devoted to exploring the binding of LC3C to lipid vesicles, bilayers and monolayers, and the ensuing protein-dependent perturbing effects, in the absence of the mitochondrial lipid cardiolipin. All Atg8 orthologues are covalently bound to a phospholipid prior to their involvement in autophagosome elongation. In our case, a mutant in the C-terminal amino acid, LC3C G126C, together with the use of a maleimide-derivatized phosphatidyl ethanolamine, ensured LC3C lipidation, up to 100% under certain conditions. Ultracentrifugation, surface pressure measurements, spectroscopic and cryo-electron microscopic techniques revealed that lipidated LC3C induced vesicle aggregation (5-fold faster in sonicated than in large unilamellar vesicles) and inter-vesicular lipid mixing (up to 82%), including inner-monolayer lipid mixing (up to 32%), consistent with in vitro partial vesicle fusion. LC3C was also able to cause the release of 80–90% vesicular aqueous contents. The data support the idea that LC3C would be able to help in autophagosome elongation/fusion in autophagy phenomena

Lipid model membranes are important tools in the study of biophysical processes such as lipid self-assembly and lipid–lipid interactions in cell membranes. The use of model systems to adequate and modulate complexity helps in the understanding of many events that occur in cellular membranes, that exhibit a wide variety of components, including lipids of different subfamilies (e.g., phospholipids, sphingolipids, sterols…), in addition to proteins and sugars. The capacity of lipids to segregate by themselves into different phases at the nanoscale (nanodomains) is an intriguing feature that is yet to be fully characterized in vivo due to the proposed transient nature of these domains in living systems. Model lipid membranes, instead, have the advantage of (usually) greater phase stability, together with the possibility of fully controlling the system lipid composition. Atomic force microscopy (AFM) is a powerful tool to detect the presence of meso- and nanodomains in a lipid membrane. It also allows the direct quantification of nanomechanical resistance in each phase present. In this review, we explore the main kinds of lipid assemblies used as model membranes and describe AFM experiments on model membranes. In addition, we discuss how these assemblies have extended our knowledge of membrane biophysics over the last two decades, particularly in issues related to the variability of different model membranes and the impact of supports/cytoskeleton on lipid behavior, such as segregated domain size or bilayer leaflet uncoupling.

Sphingolipids (SL) are ubiquitous in mammalian cell membranes, yet there is little data on the behavior of cells under SL- restriction conditions. LY- B cells derive from a CHO linein whichserine palmitoyl transferase (SPT), thus de novo SL synthesis, is suppressed, while maintaining the capacity of taking up and metabolizing exogenous sphingoid bases from the culture medium. In this study, LY- B cells were adapted to grow in a fetal bovine serum (FBS)- deficient medium to avoid external uptake of li-pids. The lowest FBS concentration that allowed LY- B cell growth, though at a slow rate, under our conditions was 0.04%, that is, 250- fold less than the standard (10%) concentration. Cells grown under limiting SL concentrations remained viable for at least 72hours. Enriching with sphingomyelin the SL- deficient medium allowed the recovery of growth rates analogous to those of control LY- B cells. Studies in-cluding whole cells, plasma membrane preparations, and derived lipid vesicles were carried out. Laurdan fluorescence was recorded to measure membrane molecular order, showing a significant decrease in the rigidity of LY- B cells, not only in plasma membrane but also in whole cell lipid extract, as a result of SL limitation in the growth medium. Plasma membrane preparations and whole cell lipid extracts were also studied using atomic force microscopy in the force spectroscopy mode. Force measurements demonstrated that lower breakthrough forces were required to pen-etrate samples obtained from SL- poor LY- B cells than those obtained from control cells. Mass- spectroscopic analysis was also a helpful tool to understand the rear-rangement undergone by the LY- B cell lipid metabolism. The most abundant SL in LY- B cells, sphingomyelin, decreased by about 85% as a result of SL limitation in the medium, the bioactive lipid ceramide and the ganglioside precursor hexosylcera-mide decreased similarly, together with cholesterol. Quantitative SL analysis showed that a 250- f

341 p., Esta tesis pretende explorar la función de los esfingolípidos en el crecimiento celular y en la composición y las propiedades físicas de las membranas. Para ello se han utilizado células que poseen una síntesis limitada de esfingolípidos. Tanto células modificadas genéticamente como tratadas químicamente.A lo largo del estudio se han utilizado diferentes técnicas biofísicas para la caracterización de estas propiedades: sobre todo la espectroscopía y microscopía fluorescente, la microscopia de fuerza atómica y la espectrometría de masas.Esta investigación demuestra que existe una clara correlación entre la disminución del contenido de SL de las membranas y los cambios biofísicos en las propiedades biofisicas de las mismas: Las membranas celulares se vuelven más fluidas al medirlas mediante la polarización general de laurdan y son más penetrables al inducir su ruptura mediante la espectrometría de fuerza (AFM).Cuando las cantidades de SL se reducen en las membranas celulares, las células CHO y HAP1 intentan mantener su orden de membrana nativo mediante una respuesta homeostática en su síntesis lipídico: Sintetizan más glicerofosfolípidos totalmente saturados y menos insaturado. Las LY-B (SPT deficiente) mantienen el orden molecular de sus membranas solo parcialmente, ya que su membrana plasmática es más penetrable en comparación a las de la línea celular CHO (wild type). Por otro lado, la línea celular humana HAP1-SPT (SPT deficiente) mantiene el orden de sus membranas, ya que no existen diferencias en comparación a la penetrabilidad de la membrana plasmática de las células HAP1 (wild type).

BACKGROUND Arachidyl amido cholanoic acid (Aramchol) is a potent downregulator of hepatic stearoyl-CoA desaturase 1 (SCD1) protein expression that reduces liver triglycerides and fibrosis in animal models of steatohepatitis. In a phase IIb clinical trial in patients with nonalcoholic steatohepatitis (NASH), 52 wk of treatment with Aramchol reduced blood levels of glycated hemoglobin A1c, an indicator of glycemic control. AIM To assess lipid and glucose metabolism in mouse hepatocytes and in a NASH mouse model [induced with a 0.1% methionine and choline deficient diet (0.1MCD)] after treatment with Aramchol. METHODS Isolated primary mouse hepatocytes were incubated with 20 mu mol/L Aramchol or vehicle for 48 h. Subsequently, analyses were performed including Western blot, proteomics by mass spectrometry, and fluxomic analysis with(13)C-uniformly labeled glucose. For thein vivopart of the study, male C57BL/6J mice were randomly fed a control or 0.1MCD for 4 wk and received 1 or 5 mg/kg/d Aramchol or vehicle by intragastric gavage for the last 2 wk. Liver metabolomics were assessed using ultra-high-performance liquid chromatography-time of flight-MS for the determination of glucose metabolism-related metabolites. RESULTS Combination of proteomics and Western blot analyses showed increased AMPK activity while the activity of nutrient sensor mTORC1 was decreased by Aramchol in hepatocytes. This translated into changes in the content of their downstream targets including proteins involved in fatty acid (FA) synthesis and oxidation [P-ACC alpha/beta(S79), SCD1, CPT1A/B, HADHA, and HADHB], oxidative phosphorylation (NDUFA9, NDUFB11, NDUFS1, NDUFV1, ETFDH, and UQCRC2), tricarboxylic acid (TCA) cycle (MDH2, SUCLA2, and SUCLG2), and ribosome (P-p70S6K[T389] and P-S6[S235/S236]). Flux experiments with(13)C-uniformely labeled glucose showed that TCA cycle cataplerosis was reduced by Aramchol in hepatocytes, as indicated by the increase in the number of rounds that malate remaine

292 p., El objetivo general de esta tesis es ampliar el conocimiento sobre la fisiopatología de la EA e investigar la posible interrelación existente entre tres hechos biológicos cerebrales que podrían estar involucrados en los mecanismos patológicos de la EA. Estos hechos fisiopatológicos se refieren a cambios en la homeostasis del colesterol, la microestructura de la SB y la amiloidogénesis.En el Capítulo 2 el objetivo es analizar si el metabolismo lipídico sistémico está involucrado con cambios en la microestructura de la SB. Se realiza un estudio transversal para analizar si la variabilidad interindividual de los niveles plasmáticos de colesterol total (CTOT), lipoproteínas de colesterol de alta densidad (HDL), lipoproteínas de colesterol de baja densidad colesterol (LDL) y triglicéridos (TG) se asocia a la variabilidad en la microestructura de la SB de las regiones relacionadas con la cognición, en sujetos sanos. La hipótesis es que los valores altos de HDL se relacionan con los valores altos de FA y AxD e inferiores de MD y RD y que por el contrario los valores altos de los TG, LDL y CTOT se asocian con niveles inferiores de FA y AxD y superiores de MD y RD. También se plantea la hipótesis de que estasasociaciones estarían influidas por el genotipo APOE, la presión arterial o el uso de las estatinas. Además, se examina también si la posible asociación entre los lípidos plasmáticos y la difusividad axial está relacionada con la pérdida axonal, medida a través de los niveles de NfL en LCR.En el Capítulo 3 el objetivo es entender mejor la relación entre el metabolismo del colesterol cerebral, concretamente sus oxiesteroles, la microestructura de la SB y los biomarcadores de EA en LCR, especialmente en regiones relacionadas con la cognición. Este estudio se centra en los dos principales oxiesteroles sintetizados enzimáticamente, 24-hidroxicolesterol (24-OHC) y 27-hidroxicolesterol (27-OHC); y en otros dos sintetizados no-enzimáticamente en condiciones de estrés oxidativo

The biophysical properties of sphingolipids containing lignoceric (C24:0) or nervonic (C24:1) fatty acyl residues have been studied in multicomponent lipid bilayers containing cholesterol (Chol), by means of confocal microscopy, differential scanning calorimetry and atomic force microscopy. Lipid membranes composed of dioleoyl phosphatidylcholine and cholesterol were prepared, with the addition of different combinations of ceramides (C24:0 and/or C24:1) and sphingomyelins (C24:0 and/or C24:1). Results point to C24:0 sphingolipids, namely lignoceroyl sphingomyelin (lSM) and lignoceroyl ceramide (lCer), having higher membrane rigidifying properties than their C24:1 homologues (nervonoyl SM, nSM, or nervonoyl Cer, nCer), although with a similar strong capacity to induce segregated gel phases. In the case of the lSM-lCer multicomponent system, the segregated phases have a peculiar fibrillar or fern-like morphology. Moreover, the combination of C24:0 and C24:1 sphingolipids generates interesting events, such as a generalized bilayer dynamism/instability of supported planar bilayers. In some cases, these sphingolipids give rise to exothermic curves in thermograms. These peculiar features were not present in previous studies of C24:1 combined with C16:0 sphingolipids. Conclusions of our study point to nSM as a key factor governing the relative distribution of ceramides when both lCer and nCer are present. The data indicate that lCer could be easier to accommodate in multicomponent bilayers than its C16:0 counterpart. These results are relevant for events of membrane platform formation, in the context of sphingolipid-based signaling cascades.

151 p., El desarrollo de esta tesis doctoral ha aportado luz sobre los mecanismos que subyacen a la enfermedad del hígado graso no alcohólico (NAFLD por sus siglas en inglés), ya que los resultados han revelado la existencia de al menos 2 subtipos diferentes de pacientes al comparar (mediante cromatografía de líquidos acoplada a espectrometría de masas) el perfil metabolómico sérico de estos con el perfil metabolómico de dos modelos animales de esta enfermedad: ratones MAT1A-KO y 0.1MCD.Por otra parte, experimentos in vitro, in cellulo e in vivo han revelado parte del mecanismo de acción del Aramchol®, una molécula compuesta por un ácido biliar y un ácido graso saturado. El tratamiento con esta molécula en ratones 0.1MCD ha mostrado una clara reducción en la acumulación de grasa en el hígado, así como una disminución de la fibrosis y de un aumento en la capacidad antioxidante, todo ello disminuyendo los síntomas de esta enfermedad. Así mismo, el estudio clínico en humanos muestra el efecto del Aramchol en el metabolismo hepático de la glucosa, resultados que hemos complementado y validado en esta tesis con el modelo 0.1MCD.Por último, hemos caracterizado una cohorte de personas pertenecientes a población general, para determinar mediante Resonancia Magnética Nuclear (RMN) una huella metabólica en orina que nos permita distinguir pacientes con síndrome metabólico de personas sanas

Ceramide is a major actor in the sphingolipid signaling pathway elicited by various kinds of cell stress. Under those conditions ceramide (Cer) is produced in the plasma membrane as a product of sphingomyelin (SM) hydrolysis, and this may lead to apoptosis. Thus, SM and Cer coexist in the membrane for some time, and they are known to separate laterally from the (more abundant) glycerolipids, giving rise to highly rigid domains or platforms. The properties of these domains/platforms are rather well understood, but the underlying SM:Cer molecular interactions have not been explored in detail. Infrared (IR) spectroscopy is a powerful analytical technique that provides information on all the chemical groupings in a molecule, and that can be applied to membranes and lipid bilayers in aqueous media. IR spectra can be conveniently retrieved as a function of temperature, thus revealing the thermotropic transitions of SM and its mixtures with Cer. Four regions of the IR spectrum of these sphingolipids have been examined, two of them dominated by the hydrophobic regions in the molecules, namely the C-H stretching vibrations (2800-3000 cm(-1)), and the CH2 scissoring vibrations (1455-1485 cm(-1)), and two others arising from chemical groups at the lipid-water interface, the sphingolipid amide I band (1600-1680 cm(-1)), and the phosphate vibrations in the 1000-1110 cm(-1) region. The latter two regions have been rarely studied in the past. The IR data from the hydrophobic components show a gel (or ripple)-fluid transition of SM at 40 degrees C, that is shifted up to about 70 degrees C when Cer is added to the bilayers, in agreement with previous studies using a variety of techniques. IR information concerning the polar parts is more interesting. The amide I (carbonyl) band of pure SM exhibits a maximum at 1638 cm(-1) at room temperature, and its position is shifted by about 10 cm(-1) in the presence of Cer. Cer causes also a change in the overall band shape, but no signs of ban

Programmed cell death or apoptosis is a central biological process that is dysregulated in many diseases, including inflammatory conditions and cancer. The detection and quantification of apoptotic cells in vivo is hampered by the need for fixatives or washing steps for non-fluorogenic reagents, and by the low levels of free calcium in diseased tissues that restrict the use of annexins. In this manuscript, we report the rational design of a highly stable fluorogenic peptide (termed Apo-15) that selectively stains apoptotic cells in vitro and in vivo in a calcium-independent manner and under wash-free conditions. Furthermore, using a combination of chemical and biophysical methods, we identify phosphatidylserine as a molecular target of Apo-15. We demonstrate that Apo-15 can be used for the quantification and imaging of drug-induced apoptosis in preclinical mouse models, thus creating opportunities for assessing the in vivo efficacy of anti-inflammatory and anti-cancer therapeutics. Programmed cell death or apoptosis is an essential biological process that is impaired in some diseases and can be used to assess the effectiveness of drugs. Here the authors design Apo-15 as a fluorogenic peptide for the detection and real-time imaging of apoptotic cells.

This study was aimed at preparing and characterizing plasma membranes (PM) from Chinese Hamster Ovary (CHO) cells. Two methods of PM preparation were applied, one based on adhering cells to a poly-lysine-coated surface, followed by hypotonic lysis and removal of intracellular components, so that PM patches remain adhered to each other, and a second one consisting of bleb induction in cells, followed by separation of giant plasma membrane vesicles (GPMV). Both methods gave rise to PM in sufficient amounts to allow biophysical and biochemical characterization. Laurdan generalized polarization was used to measure molecular order in membranes, PM preparations were clearly more ordered than the average cell membranes (GP ≈0.450 vs. ≈0.20 respectively). Atomic force microscopy was used in the force spectroscopy mode to measure breakthrough forces of PM, both PM preparations provided values in the 4–6 nN range, while the corresponding value for whole cell lipid extracts was ≈2 nN. Lipidomic analysis of the PM preparations revealed that, as compared to the average cell membranes, PM were enriched in phospholipids containing 30–32 C atoms in their acyl chains but were relatively poor in those containing 34–40 C atoms. PM contained more saturated and less polyunsaturated fatty acids than the average cell membranes. Blebs (GPMV) and patches were very similar in their lipid composition, except that blebs contained four-fold the amount of cholesterol of patches (≈23 vs. ≈6 mol% total membrane lipids) while the average cell lipids contained 3 mol%. The differences in lipid composition are in agreement with the observed variations in physical properties between PM and whole cell membranes.

The photoacoustic effect is generated when a variable light interacts with a strongly light-absorbing material. In water, it may produce hot bubbles and shock waves that could affect the integrity of nearby cellular membranes, opening transient pores (photoporation). In this study, we have evaluated the effect of pulsed laser-irradiated carbon nanoparticles (cNP) on model membranes and on Chinese hamster ovary (CHO) cells. Fluorescence lifetime measurements of calcein-loaded liposomes support the notion that the photoacoustic effect causes transient openings in membranes, allowing diffusion fluxes driven by gradient concentrations. With CHO cells, we have shown that this effect can induce either intracellular delivery of calcein, or release of cellular compounds. The latter process has been recorded live with multiphoton excitation microscopy during pulsed infrared laser irradiation. Calcein loading and cell viability were assayed by flow cytometry, measuring necrotic cells as well as those in early apoptosis. To further assess long-term cell recovery after the rather harsh treatment, cells were reseeded and their behaviour recorded for 48h. These extended studies on cell viability show that pulsed laser cNP photoporation may be considered an adequate intracellular delivery technique only if employed with soft irradiation conditions (below 50mJ/cm2).

152 p., Viruses are parasitic biological agents that in the virion state contain differentmorphologies and assembly organisations. In some virions, a lipid membrane of cellularorigin is a structural component of the infectious particle. This membrane can belocated as the outermost layer or internally depending on the species. Our work aims atunderstanding the mechanism of assembly and the viral proteins involved in cellularinfection, using for that Cryo-electron microscopy (Cryo-EM) and Cryo-electrontomography (Cryo-ET).In the first case, we present the 3D structures of the archaeal, halophilic, internalmembrane-containing Haloarcula californiae icosahedral virus 1 (HCIV-1) andHaloarcula hispanica icosahedral virus 2 (HHIV-2) at 3.7 and 3.8 Å resolution,respectively, using single-particle cryo-electron microscopy. These structures reveal theviral assembly mechanisms of the capsid shell over the internal membrane and themorphology of the viral receptors.In the second case (ongoing study), using Cryo-ET technique, we have morphologicallycharacterised pleomorphic Bovine Viral Diarrhea Virus 1 (BVDV), as very little isknown about the assembly, virus entry and antibody recognition. Thus, combining cryo-ET with sub-tomogram averaging, we present the preliminary cryo-ET average map ofthe BVDV type 1 bound to mAbs against the glycoproteins Erns.

Mintz ereduak, bai zelularen, baita zelularen organuluen arkitektura, imita dezaketenez, proteina-lipido elkarrekintzak, geruza bikoitzean txertatutako proteinen egiturak edota mintzen dinamika ikertzeko erabilgarriak izan daitezke. Gainera, mintz ereduen diseinu kontrolatu bat egiteko aukera existitzen denez, beraien konposizio fisiko-kimiko eta tamaina jakineko egitura lipidikoak sortuz, hauek izan ditzaketen erabilerak gero eta ugariagoak izaten ari dira, horien artean aurkitzen dira hain zuzen ere fusio prozesua ikertzeko entseguak. Jakina da, zeluletan fusioa oso prozesu garrantzitsua dela; espermatozoideek burutzen duten obuluaren ernalketa, exozitosi konstitutibo eta arautua edota organuluen biogenesia esaterako, fusio prozesuetan oinarritzen dira. Ikerketa honetan, izozte-urtze ziklo bidezko mintz ereduen fusio kontrolatu bat burutzeko teknikaren efikazia neurtu da. Tamaina (SUV, LUV eta MLV) eta konposizio molekular ezberdineko (fosfatidilkolina edota fosfatidilkolina, esfingomielina, fosfatidiletanolamina eta kolesterol kantitate ezberdineko) mintz ereduak erabili dira. Fusio maila erresonantzia bidezko energia transferentzia (FRET) izeneko teknikaz neurtu da, honetarako NBD-PE eta Rho-PE markatzaile fluoreszenteak mintz ereduetan txertatuak izan direlarik. Horrela ba, ikerketa honetan, saiakuntza mota hauetan erabili beharko litezkeen mintz eredu eta lipido mota egokienak, markatzaile fluoreszente kantitate eta izozte-urtze ziklo kopuru optimoa eta fusio osteko egitura lipidikoen morfologia ikertu dira. Azkenik, teknika hau GUV izenez ezagutzen diren mintz ereduak sortzeko baliogarria izan daitekeela frogatu da; Model membranes are models that somehow mimic natural biomembranes. They have being widely used for investigating many cell events as protein-lipids interaction, the architecture of proteins embedded in the membranes or the membrane dynamic. In addition, the design of model membranes can be controlled in order to form specific physicochemical comp

Sphingolipids contain in their polar heads chemical groups allowing them to establish a complex network of H-bonds through different –OH and –NH–groups with other lipids in the bilayer. In the recent years the specific interaction of sphingomyelin with cholesterol has been examined, largely in the context of the “lipid raft” hypothesis. Formation of sphingomyelin - Ceramide complexes, proposed to exist in cell membranes in response to stress, has also been described. In addition, data demonstrating direct Cholesterol-Ceramide interaction are becoming available. Finally, a more recent emphasis on the variability of sphingolipids is advancing our knowledge in the field, as more studies are performed on different variants of sphingolipids, such as deoxysphingolipids, and reports on the effects of the N-acyl chain length or unsaturation are becoming available. This could be of importance in the currently growing field of developing ceramide-related therapeutic drugs or treatments for different illnesses, being the most relevant the cancer-related therapies.; Esfingolipidoek, beren buru polarrean dauzkaten talde polarren bidez, geruza bikoitzean existitzen diren beste lipido batzuekin sare konplexu bat eratzeko ahalmena daukate. Sare konplexu honen eraketa, -OH eta -NH- taldeekin hidrogeno-zubiak eratuz lortzen dute hain zuzen ere. Azken urteotan, lipido-urmaelaren hipotesiaren barruan, esfingomielinaren eta kolesterolaren arteko elkarrekintza espezifikoa asko ikertzen ari da, eta ikerketa honen emaitza da, estres-egoeren aurrean, zelulen mintzetan, esfingomielina-zeramida konplexuak sortzen direla proposatu izana. Gainera, kolesterol-zeramida elkarrekintzak ere gertatzen direla erakusten duten artikuluak gero eta ugariagoak dira. Existitzen diren esfingolipido moten inguruan jartzen ari den arretak gaiaren inguruko jakinduria handitzera garamatza. Izan ere, azkenaldian, deoxiesfingolipidoak bezalako esfingolipido aldakorren eta N-azil katearen luzerak edota lipidoen ase

The properties of bilayers composed of pure brain cerebroside (bCrb) or of binary mixtures of bCrb with brain ceramide, cholesterol, egg phosphatidylcholine or brain sphingomyelin have been studied using a combination of physical techniques. Pure bCrb exhibits a rather narrow gel-fluid transition centred at approximate to 65 degrees C with a half-width at half-height T-1/2 approximate to 3 degrees C. bCrb mixes well with both fluid and gel phospholipids and ceramide, and it rigidifies bilayers of egg phosphatidylcholine or brain sphingomyelin when the latter are in the fluid state. Cholesterol markedly widens the bCrb gel-fluid transition, while decreasing the associated transition enthalpy, in the manner of cholesterol mixtures with saturated phosphatidylcholines, or sphingomyelins. Laurdan and DPH fluorescence indicate the formation of fluid ordered phases in the bCrb:cholesterol mixtures. Macroscopic phase separation of more and less fluid domains is observed in giant unilamellar vesicles consisting of bCrb:egg phosphatidylcholine or bCrb:sphingomyelin. Crb capacity to induce bilayer permeabilization or transbilayer (flip-flop) lipid motion is much lower than those of ceramides. The mixtures explored here contained mostly bCrb concentrations >50 mol%, mimicking the situation of cell membranes in Gaucher's disease, or of the Crb-enriched microdomains proposed to exist in healthy cell plasma membranes.

202 p., Malformación de la membrana de núcleos esta observada en casos de distrofia muscular, así como en ciertos tipos de cáncer, como el de mama o el de riñones. Los mecanismos que intervienen en la integridad de eta membrana están controlados por las interacciones proteíns con lípidos. Hace poco, se ha demostrado que las propiedades físicas de lípidos específicos tienen una función esencial en el proceso de formación de la membrana de núcleos en el modelo del erizo de mar. Para comprender como la arquitectura de la membrana está regulada, utilizamos técnicas cuantitativas como la espectrometría de mesa y la RMN para determinar la composición en lípidos de la membrana y sus propiedades dinámicas. La extracción del núcleo se realizó a partir de células renales humanas, HEK 293T. Se ha optimizado una extracción física basada en un tratamiento a presión y un gradiente de sucrosa para obtener una gran cantidad de núcleos intactos (lípidos de la membrana nuclear) necesarios para nuestros experimentos. Estos núcleos purificados incluyen un mínimo de residuos celulares, retículo endoplásmico y apartato golgi. Después, lípidos de núcleos han sido extraídos con un método de Folch modificado. Los experimentos de RMN en líquidos mostraron que la membrana nuclear estaba compuesta de muchos tipos de fosfolípidos y de los cuales la fosfatidilcolina era el lípido principal. Además, la composición muestra una gran proporción de fosfatidilcolina en comparación con otros tipos de membranas. Experimentos de espectrometríaa de masa han demostrado que la membrana nuclear estaba compuesta principalmente de fosfolípidos altamente insaturados y con cadenas que contenían un promedio de 34 átomos de carbono. Los análisis por RMN del sólido realizados en membranas reconstituidas a partir de lípidos de núcleos han revelado propiedades físicas atípicas. La temperatura de transición de fase fluido-gel está muy baja y amplia (-10Cº-15ºC)lo que puede venir de la presencia de muchas especias de fosfolípi

265 p., La presente tesis se ha centrado el estudio de las interacciones lípido-lípido asociadas a la segregación lateral de esfingolipidos y colesterol. Estos estudios se han llevado a cabo utilizando sistemas de membranas modelo compuestos por tres o cuatro componentes, y utilizando diferentes técnicas biofísicas, principalmente calorimetría diferencial de barrido, espectroscopia y microscopía de fluorescencia y microscopía de fuerza atómica, con los siguientes objetivos:- Caracterizar la estructura de las fases de la eSM y la pSM por debajo de su temperatura de transición principal de fase.- Analizar el comportamiento de la ceramida y el colesterol con un fosfolípido saturado en un sistema modelo de tres componentes.- Estudiar el efecto de la incorporación de esfingolipidos insaturados en membranas fosfolipídicas modelo.¿Describir la interacción de glicoesfingolipidos naturales (cerebrósidos) en sistemas binarios con esfingolípidos, glicerolípidos o colesterol.

The mechanisms of Pb(II) toxicity have been studied in human red blood cells using confocal microscopy, immunolabeling, fluorescence-activated cell sorting and atomic force microscopy. The process follows a sequence of events, starting with calcium entry, followed by potassium release, morphological change, generation of ceramide, lipid flip-flop and finally cell lysis. Clotrimazole blocks potassium channels and the whole process is inhibited. Immunolabeling reveals the generation of ceramide-enriched domains linked to a cell morphological change, while the use of a neutral sphingomyelinase inhibitor greatly delays the process after the morphological change, and lipid flip-flop is significantly reduced. These facts point to three major checkpoints in the process: first the upstream exchange of calcium and potassium, then ceramide domain formation, and finally the downstream scramblase activation necessary for cell lysis. In addition, partial non-cytotoxic cholesterol depletion of red blood cells accelerates the process as the morphological change occurs faster. Cholesterol could have a role in modulating the properties of the ceramide-enriched domains. This work is relevant in the context of cell death, heavy metal toxicity and sphingolipid signaling.

138 p., La porfiria eritropoyética congénita es una enfermedad rara autosómica recesiva producida por una actividad deficiente en la uroporfirinógeno III sintasa, la cuarta enzima de la ruta biosintética del grupo hemo. La enfermedad afecta a diversos órganos, llegando a ser potencialmente peligroso para la vida, careciendo actualmente de tratamientos curativos. Bioquímicamente, las mutaciones hereditarias de mayor frecuencia reducen la estabilidad del enzima, alterando su homeostasis, que, en última instancia, reducen la producción de grupo hemo intracelular. Esto da como resultado la acumulación de subproductos de uroporfirina que se distribuyen y depositan por todos los tejidos, agravando la patología con síntomas tales como fotosensibilidad de la piel y lesiones cutáneas fototóxicas desfigurantes. En el presente trabajo, demostramos como el sintético antifúngico y microbiano fármaco comercial denominado ciclopirox, se asocia al enzima estabilizándolo. Ciclopirox asiste al enzima mediante unión alostética, distante del centro activo, sin afectar, por tanto, a su función catalítica. El fármaco es capaz de reestablecer la actividad in vitro, in cellula e in vivo, llegando incluso a aliviar la mayoría de los síntomas clínicos en un modelo de ratón bona fide de la enfermedad, actuando a concentraciones sub-tóxicas, estableciendo una nueva línea de intervención terapéutica contra la porfiria eritropoyética congénita. Aplicable a la mayoría de las mutaciones sin sentido perjudiciales que causan esta devastadora enfermedad.

170 p, El retrómero es un complejo multiproteíco que participa en múltiples etapas del tráfico vesicular, controlando el reciclaje de cargos presentes en los endosomas a la membrana plasmática, la red Trans-Golgi, así como a diferentes orgánulos distales. Debido a su papel esencial, el retrómero está implicado en numerosos procesos fisiológicos y patológicos de gran relevancia. En este trabajo, hemos caracterizado estructural (cristalografía de rayos X) y funcionalmente la unión del retrómero con el efector de Legionella pneumophila, RidL, así como con diferentes proteínas accesorias y los cargos implicados en la enfermedad de Alzheimer APP y BACE. Todo ello, con el fin de contribuir al establecimiento de un modelo organizativo del retrómero que ayude a entender las bases moleculares que rigen el reconocimiento y selección de receptores transmembrana de manera específica, evitando su degradación en los lisosomas.

Los capítulos 3 y 4 están sujetos a confidencialidad por el autor. 69 p., La protección del genoma viral durante el viaje extracelular es un requisito absoluto para la supervivencia y replicación del virus. Una vez que la partícula viral se une a la célula huésped, puede comenzar la eyección del material genómico. Además de las cápsides proteicas casi universales, ciertos virus poseen una capa de membrana que encierra su genoma de ADN bicatenario (ds) dentro de la cubierta proteica. Aunque las propiedades mecánicas de otros virus se han estudiado anteriormente, esta tesis investiga un virus con una membrana bajo su cápside proteica. Usando el virus enterobacterial PRD1 como prototipo de virus sin cola que contienen membrana, y una combinación de ensayos de nanoindentación mediante la microscopía de fuerza atómica y modelado de elementos finitos, mostramos que la arquitectura jerárquica de PRD1 (cubierta de proteína, vesícula proteolipidica, ds ADN) proporciona una mayor estabilidad contra el estrés mecánico que la lograda por otros virus icosaédricos ds ADN que carecen de una membrana. La combinación de un caparazón proteínico rígido y quebradizo junto con una vesícula de membrana blanda y dócil produce un nanomaterial compuesto resistente adecuado para proteger el ADN viral durante el transporte extracelular. Además, observamos la estructura y las propiedades mecánicas que implican el ensamblaje del tubo de eyección de ADN de dicho virus. Si bien los estudios han resuelto la estructura de varios virus en tubos, aquí investigamos un fago que forma el tubo de eyección de ADN al unirse. Empleamos la reconstrucción con crio-microscopía electrónica (cryo-EM) para mostrar que esta partícula viral está produciendo un tubo estructurado, no helicoidal, con una simetría axial de siete veces, que se forma utilizando la membrana proteolípica. En combinación con ensayos de nanoindentación de microscopía de fuerza atómica (AFM), visualizamos partículas individuales que producen tubos y mostramos que el tubo de PRD1 tiene una rigidez comparable a estru

252 p., La homeostasis de tejidos es considerado como el equilibrio entre la muerte y proliferación celular, y es por ello que su desregulación a menudo está asociada a diversas situaciones patológicas, tales como enfermedades neurodegenerativas o el cáncer. Este equilibrio, se encuentra regulado principalmente por un mecanismo denominado apoptosis o suicidio celular. La familia de proteínas BCL2 se erige como pieza clave en la regulación de este mecanismo, principalmente, a nivel de la permeabilización de la membrana externa mitocondrial (MEM). En las últimas décadas, se han realizado avances de notoria importancia en el conocimiento sobre los mecanismos de acción e interacción de esta familia de proteínas, Sin embargo, importantes aspectos del proceso de activación de las proteinas proapoptóticas tipo BAX (BAX, BAK y quizás BOK) se encuentran vagamente descritos, desconociendo entre otros aspectos la disposición estructural exacta que la proteína BAX adopta en membrana o la cantidad mínima de subunidades de BAX necesarias para la formación del poro. Por otra parte, puesto que la membrana conforma el locus biológico de estas proteinas, el estudio del componente lipídico y de la propia membrana se encuentra actualmente en boga. Concretamente, el lípido especifico de la mitocondria, cardiolipina (CL), se ha erigido como uno de los moduladores claves de la apoptosis. Sin embargo, a pesar de su importancia, la función exacta de la CL en particular y de los lípidos en general, en las actividades de membrana de las BCL2, se encuentra vagamente descrita. Además, dilucidar el modo de acción de las proteinas antiapoptóticas o tipo BCL2, mediadoras del bloqueo de la actividad de las proapoptóticas, es considerado uno de los pilares fundamentales en el desarrollo de posibles dianas terapéuticas. El objetivo de esta Tesis Doctoral ha sido avanzar en la comprensión de los mecanismos de acción de la familia de proteínas BCL2, así como de los factores reguladores que modulan su actividad

213 p., La colicina A y en concreto el dominio formador de poros, pfColA es expresado por Escherichia coli y resulta tóxico para cepas relacionadas. Las bacterias productoras de la toxina expresan la proteína de inmunidad Cai que las protege de la misma toxina que producen. Cai inhibe la formación de poros y subsecuente muerte celular mediante una interacción directa hélice-hélice en la membrana interna de la bacteria. En el presente trabajo se analizó la interacción entre Cai y pfColA en diferentes entornos como micelas de detergente, vesículas lipídicas o en la membrana bacteriana. El estudio funcional en vesículas lipídicas demostró que pfColA es capaz de conducir protones y que esta actividad se ve inhibida por Cai. Esta actividad resultó ser dosis dependiente, saturable y de la que se deduce un complejo 1:1. El par Cai-pfColA fue co-expresado en la membrana interna de E. coli y se consiguió aislar el complejo integro en detergente de tipo alkilglucósido. Por último, la formación de complejo fue testada también en solución en micelas de detergente, donde se forma un complejo muy estable con una afinidad en el rango de micromolar.

Specific protein-lipid interactions lead to a gradual recruitment of AuTophaGy-related (ATG) proteins to the nascent membrane during autophagosome (AP) formation. ATG3, a key protein in the movement of LC3 towards the isolation membrane, has been proposed to facilitate LC3/GABARAP lipidation in highly curved membranes. In this work we have performed a biophysical study of human ATG3 interaction with membranes containing phosphatidylethanolamine, phosphatidylcholine and anionic phospholipids. We have found that ATG3 interacts more strongly with negatively-charged phospholipid vesicles or nanotubes than with electrically neutral model membranes, cone-shaped anionic phospholipids (cardiolipin and phosphatidic acid) being particularly active in promoting binding. Moreover, an increase in membrane curvature facilitates ATG3 recruitment to membranes although addition of anionic lipid molecules makes the curvature factor relatively less important. The predicted N-terminus amphipathic a-helix of ATG3 would be responsible for membrane curvature detection, the positive residues Lys 9 and 11 being essential in the recognition of phospholipid negative moieties. We have also observed membrane aggregation induced by ATG3 in vitro, which could point to a more complex function of this protein in AP biogenesis. Moreover, in vitro GABARAP lipidation assays suggest that ATG3-membrane interaction could facilitate the lipidation of ATG8 homologues.

159 p., Las membranas biológicas son complejas estructuras con una base lipídica y cruciales para lasupervivencia y función de las células. La ceramida es un lípido de membrana con unas característicasfísico-químicas singulares, capaz de alterar profundamente las propiedades biofísicas de las membranas.La ceramida es también un lípido bioactivo, con un papel importante en diversos procesos celulares como proliferación, inflamación y apoptosis.Esta tesis se ha centrado en el estudio de las propiedades biofísicas de la ceramida empleando vesículas lipídicas como membranas modelo y valiéndose de diversas técnicas biofísicas, principalmente espectroscopía y microscopía de fluorescencia. La tesis se ha centrado en tres áreas principales: laidoneidad de análogos fluorescentes de ceramida para su uso como sondas de membrana, el efecto de laincorporación de ceramida y colesterol en membranas de fosfolípidos saturados y el efecto de lasceramidas sobre la integridad de la membrana y el mecanismo de permeabilización de las membranasinducido por la ceramida. Los resultados obtenidos en esta tesis contribuyen a una mejor comprensión de las propiedades de la ceramida y de su papel en las membranas celulares en condiciones tanto normales como patológicas.

Se analizan los procesos bioquímicos que se pueden dar en la elaboración de platos en la cocina

La membrana biológica es una barrera hidrofóbica semipermeable de naturaleza compleja, cuya estructura consiste en una doble capa de moléculas de lípidos en la cual las proteínas están insertadas. Existen diferentes modelos para su estudio, uno de los cuales se basa en la utilización de membranas modelo. Por otra parte, la radioterapia es uno de los principales tratamientos para los distintos tipos de cáncer. En este trabajo se estudia el efecto de la radiación ionizante sobre la permeabilidad de membranas modelo, con objeto de comprender los mecanismos de su acción sobre dicho tratamiento.

Bordetella pertussis causes whooping cough, a respiratory infectious disease that is the fifth largest cause of vaccine-preventable death in infants. Though historically considered an extracellular pathogen, this bacterium has been detected both in vitro and in vivo inside phagocytic and non-phagocytic cells. However the precise mechanism used by B. pertussis for cell entry, or the putative bacterial factors involved, are not fully elucidated. Here we find that adenylate cyclase toxin (ACT), one of the important toxins of B. pertussis, is sufficient to promote bacterial internalisation into non-phagocytic cells. After characterization of the entry route we show that uptake of "toxin-coated bacteria" proceeds via a clathrin-independent, caveolae-dependent entry pathway, allowing the internalised bacteria to survive within the cells. Intracellular bacteria were found inside non-acidic endosomes with high sphingomyelin and cholesterol content, or "free" in the cytosol of the invaded cells, suggesting that the ACT-induced bacterial uptake may not proceed through formation of late endolysosomes. Activation of Tyr kinases and toxin-induced Ca2+-influx are essential for the entry process. We hypothesize that B. pertussis might use ACT to activate the endocytic machinery of non-phagocytic cells and gain entry into these cells, in this way evading the host immune system.

Los lípidos de las membranas biológicas no son perfectamente miscibles entre sí, con frecuencia dan origen a “dominios” bidimensionales separados lateralmente cuando se resuspenden en agua. Las mezclas esfingomielina/dioleilfosfatidilcolina/colesterol dan origen con frecuencia a dominios, pero esto no ocurre cuando la esfingomielina es insaturada (p. ej. N-nervonil esfingomielina). En este trabajo se han aplicado técnicas calorimétricas y estructurales para estudiar el comportamiento de mezclas N-nervonil esfingomielina/dioleilfosfatidilcolina/colesterol/ceramida. En presencia de ceramida y de N-nervonil esfingomielina se observa formación de dominios laterales, al contrario de lo que ocurría en ausencia de ceramida.

Recent research regarding 2-hydroxylated fatty acids (2OHFAs) showed clear evidence of their benefits in the treatment of cancer, inflammation, and neurodegenerative disorders such as Alzheimer’s disease. Monolayer compressibility isotherms and isothermal titration calorimetry of 2OHFA (C18−C22) in phosphatidylcholine/phosphatidylethanolamine/sphingomyelin/cholesterol (1:1:1:1 mole ratio), a mixture that mimics the composition of mammalian plasma membrane, were performed to assess the membrane binding capacity of 2OHFAs and their natural, nonhydroxylated counterparts. The results show that 2OHFAs are surfaceactive substances that bind membranes through exothermic, spontaneous processes. The main effects of 2OHFAs are a decrease in lipid order, with a looser packing of the acyl chains, and a decreased dipole potential, regardless of the 2OHFAs’ relative affinity for the lipid bilayer. The strongest effects are usually observed for 2-hydroxyarachidonic (C20:4) acid, and the weakest one, for 2-hydroxydocosahexaenoic acid (C22:6). In addition, 2OHFAs cause increased hydration, except in gel-phase membranes, which can be explained by the 2OHFA preference for membrane defects. Concerning the membrane dipole potential, the magnitude of the reduction induced by 2OHFAs was particularly marked in the liquid-ordered (lo) phase (cholesterol/sphingomyelin-rich) membranes, those where order reduction was the smallest, suggesting a disruption of cholesterol−sphingolipid interactions that are responsible for the large dipole potential in those membranes. Moreover, 2OHFA effects were larger than for both lo and ld phases separately in model membranes with liquid disordered (ld)/lo coexistence when both phases were present in significant amounts, possibly because of the facilitating effect of ld/lo domain interfaces. The specific and marked changes induced by 2OHFAs in several membrane properties suggest that the initial interaction with the membrane and subsequent reorganization mig

Proteolytic enzymes have evolved several mechanisms to cleave peptide bonds. These distinct types have been systematically categorized in the MEROPS database. While a BLAST search on these proteases identifies homologous proteins, sequence alignment methods often fail to identify relationships arising from convergent evolution, exon shuffling, and modular reuse of catalytic units. We have previously established a computational method to detect functions in proteins based on the spatial and electrostatic properties of the catalytic residues (CLASP). CLASP identified a promiscuous serine protease scaffold in alkaline phosphatases (AP) and a scaffold recognizing a beta-lactam (imipenem) in a cold-active Vibrio AP. Subsequently, we defined a methodology to quantify promiscuous activities in a wide range of proteins. Here, we assemble a module which encapsulates the multifarious motifs used by protease families listed in the MEROPS database. Since APs and proteases are an integral component of outer membrane vesicles (OMV), we sought to query other OMV proteins, like phospholipase C (PLC), using this search module. Our analysis indicated that phosphoinositide-specific PLC from Bacillus cereus is a serine protease. This was validated by protease assays, mass spectrometry and by inhibition of the native phospholipase activity of PI-PLC by the well-known serine protease inhibitor AEBSF (IC50 = 0.018 mM). Edman degradation analysis linked the specificity of the protease activity to a proline in the amino terminal, suggesting that the PI-PLC is a prolyl peptidase. Thus, we propose a computational method of extending protein families based on the spatial and electrostatic congruence of active site residues.

[EN] We propose that accumulated membrane bending energy elicits a neutral sphingomyelinase (SMase) activity in human erythrocytes. Membrane bending was achieved by osmotic or chemical processes, and SMase activity was assessed by quantitative thin-layer chromatography, high-performance liquid chromatography, and electrospray ionization-mass spectrometry. The activity induced by hypotonic stress in erythrocyte membranes had the pH dependence, ion dependence, and inhibitor sensitivity of mammalian neutral SMases. The activity caused a decrease in SM contents, with a minimum at 6 min after onset of the hypotonic conditions, and then the SM contents were recovered. We also elicited SMase activity by adding lysophosphatidylcholine externally or by generating it with phospholipase A2. The same effect was observed upon addition of chlorpromazine or sodium deoxycholate at concentrations below the critical micellar concentration, and even under hypertonic conditions. A unifying factor of the various agents that elicit this SMase activity is the accumulated membrane bending energy. Both hypoand hypertonic conditions impose an increased curvature, whereas the addition of surfactants or phospholipase A2 activation increases the outer monolayer area, thus leading to an increased bending energy. The fact that this latent SMase activity is tightly coupled to the membrane bending properties suggests that it may be related to the general phenomenon of stressinduced ceramide synthesis and apoptosis.

[EN] The binding and early stages of activity of a phospholipase C/sphingomyelinase from Pseudomonas aeruginosa on giant unilamellar vesicles (GUV) have been monitored using fl uorescence confocal microscopy. Both the lipids and the enzyme were labeled with specifi c fl uorescent markers. GUV consisted of a mixture of phosphatidylcholine, sphingomyelin,phosphatidylethanolamine, and cholesterol in equimolar ratios, to which 5–10 mol% of the enzyme endproduct ceramide and/or diacylglycerol were occasionally added. Morphological examination of the GUV in the presence of enzyme reveals that, although the enzyme diffuses rapidly throughout the observation chamber, detectable enzyme binding appears to be a slow, random process, with new bound-enzyme-containing vesicles appearing for several minutes. Enzyme binding to the vesicles appears to be a cooperative process. After the initial cluster of bound enzyme is detected, further binding and catalytic activity follow rapidly. After the activity has started, the enzyme is not released by repeated washing, suggesting a “scooting” mechanism for the hydrolytic activity. The enzyme preferentially binds the more disordered domains, and, in most cases, the catalytic activity causes the disordering of the other domains. Simultaneously, peanut- or fi gure-eight-shaped vesicles containing two separate lipid domains become spherical. At a further stage of lipid hydrolysis, lipid aggregates are formed and vesicles disintegrate.

Hot-cold hemolysis is the phenomenon whereby red blood cells, preincubated at 37 °C in the presence of certain agents, undergo rapid hemolysis when transferred to 4 °C. The mechanism of this phenomenon is not understood. PlcHR2, a phospholipase C/sphingomyelinase from Pseudomonas aeruginosa, that is the prototype of a new phosphatase superfamily, induces hot-cold hemolysis. We found that the sphingomyelinase, but not the phospholipase C activity, is essential for hot-cold hemolysis because the phenomenon occurs not only in human erythrocytes that contain both phosphatidylcholine (PC) and sphingomyelin (SM) but also in goat erythrocytes, which lack PC. However, in horse erythrocytes, with a large proportion of PC and almost no SM, hot-cold hemolysis induced by PlcHR2 is not observed. Fluorescence microscopy observations confirm the formation of ceramide-enriched domains as a result of PlcHR2 activity. After cooling down to 4 °C, the erythrocyte ghost membranes arising from hemolysis contain large, ceramide-rich domains. We suggest that formation of these rigid domains in the originally flexible cell makes it fragile, thus highly susceptible to hemolysis. We also interpret the slow hemolysis observed at 37 °C as a phenomenon of gradual release of aqueous contents, induced by the sphingomyelinase activity, as described by Ruiz-Argu¨ello et al. [(1996) J. Biol. Chem. 271, 26616]. These hypotheses are supported by the fact that ceramidase, which is known to facilitate slow hemolysis at 37 °C, actually hinders hot-cold hemolysis. Differential scanning calorimetry of erytrocyte membranes treated with PlcHR2 demonstrates the presence of ceramide-rich domains that are rigid at 4 °C but fluid at 37 °C. Ceramidase treatment causes the disapperance of the calorimetric signal assigned to ceramide-rich domains. Finally, in liposomes composed of SM, PC, and cholesterol, which exhibit slow release of aqueous contents at 37 °C, addition of 10 mol % ceramide and transfer to 4 °C caus