Modulación del TLR4 : estudios de reconocimiento molecular y diseño de fármacos por modelado molecular

Tesis de la Universidad Complutense de Madrid, Facultad de Farmacia, Departamento de Química Orgánica y Farmacéutica, leída el 04/05/2018The heterodimeric complex, formed by Toll-Like Receptor 4 (TLR4) and its accessory protein Myeloid Differentiation factor 2 (MD-2) is responsible of activating the innate immune system when sensing the presence of particular pathogen-associated molecular patterns (PAMPs) from bacteria. The outer membrane of Gram-negative bacteria is primarily populated by lipopolysaccharides (LPS) which are essential for their growth and survival. These LPSs are specifically recognized by the TLR4/MD-2 complex as follows: an LPS binds to MD-2 inside a deep molecular hydrophobic pocket causing molecular rearrangements of the receptorial complex resulting in the dimerization of another TLR4/MD-2 unit. TLR4 ectodomains dimerization event brings together the TLR4 intercellular domains initiating the activation of innate immune system signaling pathways. Interestingly, this activation is not only modulated by naturally occurring LPSs from many different Gram-negative bacteria but also by non-naturally occurring glycolipids and other non-LPS like molecules...El complejo heterodimérico, formado por el receptor Toll-like 4 (Toll-like receptor 4, TLR4) y su proteína accesoria, el Myeloid Differentiation factor 2 (MD-2), es responsable de activar la respuesta del sistema inmune innato cuando detecta la presencia de patrones moleculares asociados a patógenos (pathogen associated molecular patterns, PAMPs), que provienen de bacterias y virus. En concreto, la membrana externa de bacterias Gram-negativas está poblada principalmente por lipopolisacáridos (lipopolisaccharides, LPS), compuestos que son esenciales para su crecimiento y supervivencia. Estos LPS son reconocidos de forma específica por el complejo TLR4/MD-2 de la siguiente manera: una molécula de LPS se une a la proteína MD-2 dentro de un profundo bolsillo hidrofóbico dando lugar al reordenamiento molecular del complejo resultando en la dimerización de otra unidad de TLR4/MD-2. El evento de dimerización de los ectodominios del TLR4 hace que se acercan los dominios intracelulares que inician la activación de las vías de señalización del sistema inmune innato. Curiosamente, esta activación no sólo está modulada por LPS naturales de muchas bacterias Gram-negativas distintas, sino también por glicolípidos no naturales y otras moléculas de estructura química diferente a los LPS...Fac. de FarmaciaTRUEunpu

Gigantic vulvar masses: a case series of benign large vulvar tumors

Benign vulvar masses are uncommon condition of the lower genital tract. To date, there is no recognized classification for benign tumors. Most of the vulvar tumors show no symptoms, unless large enough to be noted on self-examination. Presented are 3 cases of large vulvar masses. The first case is a Bartholin’s gland cyst in 56 years old, G5P4 (4014) with a 5-year history of gradually enlarging mass characterized as fleshy, approximately 16x14x12 cm, soft, non-tender located in the medial portion of the left labia majora. Grossly, the specimen consists of a unilocular cyst filled with serous fluid and had a smooth glistening inner capsule wall. The second case is a cellular angiofibroma in 61 years old, G0, who developed a fleshy mass, approximately 13x18x15 cm, soft, non-tender, attached to the left labia majora via a pedicle, which developed for 10 years. The specimen is described as an irregular mass with well circumscribed nodules with soft to rubbery, flesh cut surface. The third case is lipoblastoma-like tumor of the vulva in 31 years old, G2P2 (2002), who had a fleshy hypopigmented mass, approximately 15x12x10 cm, pendulous, soft, smooth, non-tender in the right inferolateral labia majora, which grew for 8 years. The specimen is characterized as an irregular mass partially overlain by skin with cut sections showing cream fatty lobulated surfaces. Benign vulvar masses may present similarly to one another hence careful and detailed assessment should be done. Histologic evaluation is critical in establishing an accurate diagnosis due to the fact that multiple diagnoses may have similar gross characteristics. Management is based on type and size of mass and symptomatology of patient

Novel carboxylate-based glycolipids: TLR4 antagonism, MD-2 binding and self-assembly properties

New monosaccharide-based lipid A analogues were rationally designed through MD-2 docking studies. A panel of compounds with two carboxylate groups as phosphates bioisosteres, was synthesized with the same glucosamine-bis-succinyl core linked to different unsaturated and saturated fatty acid chains. The binding of the synthetic compounds to purified, functional recombinant human MD-2 was studied by four independent methods. All compounds bound to MD-2 with similar affinities and inhibited in a concentration-dependent manner the LPS-stimulated TLR4 signaling in human and murine cells, while being inactive as TLR4 agonists when provided alone. A compound of the panel was tested in vivo and was not able to inhibit the production of proinflammatory cytokines in animals. This lack of activity is probably due to strong binding to serum albumin, as suggested by cell experiments in the presence of the serum. The interesting self-assembly property in solution of this type of compounds was investigated by computational methods and microscopy, and formation of large vesicles was observed by cryo-TEM microscopy.TOLLerant project (H2020-MSC-ETN-642157), the Italian Ministry for Foreign Affairs and International Cooperation (MAECI) and Spanish MINECO (CTQ2014-57141-R and CTQ2017-88353-R grants) are acknowledged. Vesna Hodnik of University of Ljubljana for the help with SPR, Sandra Delgado of CIC BioGUNE for the Cryo-TEM images. RJ was partially funded by the research program P4-0176 by the Slovenian Research Agency

Protein kinase A controls yeast growth in visible light

Background: A wide variety of photosynthetic and non-photosynthetic species sense and respond to light, having developed protective mechanisms to adapt to damaging effects on DNA and proteins. While the biology of UV light-induced damage has been well studied, cellular responses to stress from visible light (400–700 nm) remain poorly understood despite being a regular part of the life cycle of many organisms. Here, we developed a high-throughput method for measuring growth under visible light stress and used it to screen for light sensitivity in the yeast gene deletion collection. Results: We found genes involved in HOG pathway signaling, RNA polymerase II transcription, translation, diphthamide modifications of the translational elongation factor eEF2, and the oxidative stress response to be required for light resistance. Reduced nuclear localization of the transcription factor Msn2 and lower glycogen accumulation indicated higher protein kinase A (cAMP-dependent protein kinase, PKA) activity in many light-sensitive gene deletion strains. We therefore used an ectopic fluorescent PKA reporter and mutants with constitutively altered PKA activity to show that repression of PKA is essential for resistance to visible light. Conclusion: We conclude that yeast photobiology is multifaceted and that protein kinase A plays a key role in the ability of cells to grow upon visible light exposure. We propose that visible light impacts on the biology and evolution of many non-photosynthetic organisms and have practical implications for how organisms are studied in the laboratory, with or without illumination

Rectangular to Large Diameter Conical Corrugated Waveguide Converter Based on Stacked Rings

This paper considers the design and manufacture, using stacked rings, of a standard corrugated antenna for the WM-380 band for use as a converter from a WM-380 rectangular aperture to a large diameter conical corrugated waveguide. In-house mode matching software is utilised for the design and three prototypes manufactured using stacked rings. The level of agreement of the stacked ring prototypes with the design predictions for the return loss and HE11 modal coupling is measured and found to demonstrate high levels of agreement

Rectangular to Large Diameter Conical Corrugated Waveguide Converter Based on Stacked Rings

This paper considers the design and manufacture, using stacked rings, of a standard corrugated antenna for the WM-380 band for use as a converter from a WM-380 rectangular aperture to a large diameter conical corrugated waveguide. In-house mode matching software is utilised for the design and three prototypes manufactured using stacked rings. The level of agreement of the stacked ring prototypes with the design predictions for the return loss and HE11 modal coupling is measured and found to demonstrate high levels of agreement

3-D printed band-pass combline filter

This article describes a fourth-order 3-D printed combline filter with a Chebyshev response, operating at central frequency 3 GHz and having a 3% fractional bandwidth. The filter is designed using the coupling matrix theory, fabricated, and experimental results are presented. Comparison between simulations and measurements shows good agreement

Effect of ultrapure lipopolysaccharides derived from diverse bacterial species on the modulation of platelet activation

Platelets are small circulating blood cells that play essential roles in the maintenance of haemostasis via blood clotting. However, they also play critical roles in the regulation of innate immune responses. Inflammatory receptors, specifically Toll-like receptor (TLR)-4, have been reported to modify platelet reactivity. A plethora of studies have reported controversial functions of TLR4 in the modulation of platelet function using various chemotypes and preparations of its ligand, lipopolysaccharide (LPS). The method of preparation of LPS may explain these discrepancies however this is not fully understood. Hence, to determine the impact of LPS on platelet activation, we used ultrapure preparations of LPS from Escherichia coli (LPSEC), Salmonella minnesota (LPSSM), and Rhodobacter sphaeroides (LPSRS) and examined their actions under diverse experimental conditions in human platelets. LPSEC did not affect platelet activation markers such as inside-out signalling to integrin IIb3 or P-selectin exposure upon agonist-induced activation in platelet-rich plasma or whole blood whereas LPSSM and LPSRS inhibited platelet activation under specific conditions at supraphysiological concentrations. Overall, our data demonstrate that platelet activation is not largely influenced by any of the ultrapure LPS chemotypes used in this study on their own except under certain conditions