Caracterización de co-cultivos entre células de cáncer de colon y células mononucleares de sangre periférica para el estudio del metabolismo glucolítico y el estrés oxidativo

En el microambiente tumoral, algunas condiciones como la heterogeneidad celular, las concentraciones de oxígeno y la inmunoedición ejercida por las células del sistema inmune, inducen un proceso de selección natural que favorece la adaptación de las células de cáncer al ambiente cambiante. Estas condiciones pueden potenciar el inicio, la supervivencia y proliferación de las células de cáncer. Aunque existen algunos trabajos que muestran los cambios que presentan las células del sistema inmune en el microambiente tumoral, se desconocen los posibles efectos que genera la presencia de este tipo de células sobre la regulación del metabolismo glucolítico y el estrés oxidativo en células de cáncer de colon y cómo esto puede fomentar la supervivencia de este tipo de células durante la inmunoedición. En este estudio se propone un sistema de co-cultivo in vitro entre Células Mononucleares de Sangre Periférica (CMSPs) y la línea celular de cáncer de colon humano HT29 para estudiar los posibles cambios metabólicos y de estrés oxidativo de las células cancerosas. Las células fueron co-cultivadas por 24 horas en relaciones HT29: CMSP como sigue, 1:0 (HT-29 en monocultivo), 1:1⁄2, 1:3, 1:5 y 1:10, en normoxia (20% O 2 ) o hipoxia (1% O 2 ). Se determinó la viabilidad celular, apoptosis, captación de glucosa, la concentración de lactato extracelular, los niveles de proteínas involucradas en el metabolismo glucolítico tales como Hexoquinasa 2 (HK2), la Isoenzima Piruvato Quinasa M2 (PKM2) y los niveles de especies reactivas de oxígeno (ROS). Los resultados mostraron una disminución en la viabilidad de las células HT-29 entre un 15 a 25% y un incremento en la apoptosis temprana entre un 10 a 15%, en la apoptosis tardía entre un 7 a 10% y en la necrosis del 5% al incrementar el número de CMSP en el co-cultivo. El consumo de glucosa en las células HT-29 y la concentración de lactato en el medio extracelular se vieron incrementados para las relaciones bajas del co-cultivo, pero disminuyó en la relación más alta. Los niveles de PKM2 no se vieron alterados en las diferentes relaciones evaluadas. Sin embargo, los niveles de HK2 disminuyeron ligeramente en la relación 1:10 en normoxia. Los niveles de ROS incrementaron gradualmente a medida que se incrementó el número de CMSPs en el co-cultivo en condiciones de normoxia, pero no en hipoxia. Los cambios metabólicos en las relaciones bajas del co-cultivo podrían estar asociadas a la glucólisis anaerobia o al efecto Warburg, mientras que las relaciones altas podrían estar asociadas a un efecto Warburg reverso. Este modelo podría ser aplicado para identificar cambios metabólicos en células tumorales que hayan sido co-cultivadas por contacto directo con células del sistema inmune.Abstract. In the tumoral microenvironment, some conditions such as the cellular heterogeneity, concentration of oxygen and immunoediting exerted by immune cells, could induce a natural selection process which favors the adaptation of cancer cells to the changing environment. These conditions could enhance the initiation, survival and proliferation of cancer cells. Although there are some works focused on the possible changes that suffer the immune system cells in the tumoral microenvironment, it is unknown the possible effect that the presence of immune cells has on the regulation of glycolytic metabolism and oxidative stress in colon cancer cells and how it could influence the survival of these type of cancer cells during immunoediting. This study proposed an in vitro co-culture system between peripheral blood mononuclear cells (PBMCs) and HT29 human colon cancer cells as an in vitro model to study possible changes in glycolytic metabolism an oxidative stress of cancer cells. The cells were co-cultured for 24h at ratios between HT29: PBMCs as follow, 1: 0 (HT-29 monoculture), 1: ½, 1: 3, 1: 5 and 1: 10, in normoxia (20% O2) or hypoxia (1% O2). Cell viability, apoptosis, glucose uptake, lactate concentration in the extracellular medium, the levels of proteins involved in the glycolytic metabolism, such as Hexokinase 2 (HK2) and Pyruvate kinase isozymes M2 (PKM2) and levels of reactive oxygen species (ROS) were determined. The results showed a decrease in the viability of the HT-29 cells between 15 and 25% and an increase in stages of early apoptosis between 10 and 15%, late apoptosis 7 to 10% and necrosis 5% when the number of PBMCs was increased in the co-culture. The glucose uptake and lactate concentration in extracellular medium increase for the low ratios of co-culture, but decrease for the highest ratio of co-culture. Levels of PKM2 were not altered at any of ratios tested. However, the levels of HK2 slightly decreased at 1:10 ratio in normoxia. The ROS levels increased gradually when the number of PBMCs was increased in normoxia but not in hypoxia. The metabolic changes at low ratios of co-culture could be linked with the anaerobic glycolysis or the Warburg effect, meanwhile the high ratios of co-culture could be linked with the reverse Warburg effect. This model could be applied to identify metabolic changes in the tumor cells following direct contact with immune cells.Maestrí

Propuesta para el cambio del Customer Experience En PDV Pronaca

This present thesis realizes an investigation and proposal for changes in Customer experience in Pronaca point of sales. Said evaluation is realized using the Net Promoter Score NPS tool, which helps us determine loyalty and experience lived by the customer with the company. It also gives us a clearer vision of customer’s expectations and feelings. If the experience is positive, the customer will increase their loyalty and become a promoter of the brand...En la presente tesis se realiza una investigación y propuesta para el cambio del Customer Experience en los puntos de venta Pronaca. Dicha evaluación se realiza mediante la herramienta Net Promoter Score NPS que nos ayuda a determinar la lealtad y experiencia que vive el cliente con la empresa y nos da una visión más clara de lo que un cliente espera y siente; si la experiencia es positiva este cliente aumentará su fidelidad y se convertirá en un recomendador de la marca..

Mutual A domain interactions in the force sensing protein von Willebrand factor

The von Willebrand factor (VWF) is a glycoprotein in the blood that plays a central role in hemostasis. Among other functions, VWF is responsible for platelet adhesion at sites of injury via its A1 domain. Its adjacent VWF domain A2 exposes a cleavage site under shear to degrade long VWF fibers in order to prevent thrombosis. Recently, it has been shown that VWF A1/A2 interactions inhibit the binding of platelets to VWF domain A1 in a force-dependent manner prior to A2 cleavage. However, whether and how this interaction also takes place in longer VWF fragments as well as the strength of this interaction in the light of typical elongation forces imposed by the shear flow of blood remained elusive. Here, we addressed these questions by using single molecule force spectroscopy (SMFS), Brownian dynamics (BD), and molecular dynamics (MD) simulations. Our SMFS measurements demonstrate that the A2 domain has the ability to bind not only to single A1 domains but also to VWF A1A2 fragments. SMFS experiments of a mutant [A2] domain, containing a disulfide bond which stabilizes the domain against unfolding, enhanced A1 binding. This observation suggests that the mutant adopts a more stable conformation for binding to A1. We found intermolecular A1/A2 interactions to be preferred over intramolecular A1/A2 interactions. Our data are also consistent with the existence of two cooperatively acting binding sites for A2 in the A1 domain. Our SMFS measurements revealed a slip-bond behavior for the A1/A2 interaction and their lifetimes were estimated for forces acting on VWF multimers at physiological shear rates using BD simulations. Complementary fitting of AFM rupture forces in the MD simulation range adequately reproduced the force response of the A1/A2 complex spanning a wide range of loading rates. In conclusion, we here characterized the auto-inhibitory mechanism of the intramolecular A1/A2 bond as a shear dependent safeguard of VWF, which prevents the interaction of VWF with platelets

Force-Sensitive Autoinhibition of the von Willebrand Factor ls Mediated by Interdomain Interactions

Von Willebrand factor (VWF) plays a central role in hemostasis. Triggered by shear-stress, it adheres to platelets at sites of vascular injury. Inactivation of VWF has been associated to the shielding of its adhesion sites and proteolytic cleavage. However, the molecular nature of this shielding and its coupling to cleavage under shear-forces in flowing blood remain unknown. In this study, we describe, to our knowledge, a new force-sensory mechanism for VWF-platelet binding, which addresses these questions, based on a combination of molecular dynamics (MD) simulations, atomic force microscopy (AFM), and microfluidic experiments. Our MD simulations demonstrate that the VWF A2 domain targets a specific region at the VWF A1 domain, corresponding to the binding site of the platelet glycoprotein Ibα (GPIbα) receptor, thereby causing its blockage. This implies autoinhibition of the VWF for the binding of platelets mediated by the A1-A2 protein-protein interaction. During force-probe MD simulations, a stretching force dissociated the A1A2 complex, thereby unblocking the GPIbα binding site. Dissociation was found to be coupled to the unfolding of the A2 domain, with dissociation predominantly occurring before exposure of the cleavage site in A2, an observation that is supported by our AFM experiments. This suggests that the A2 domain prevents platelet binding in a force-dependent manner, ensuring that VWF initiates hemostasis before inactivation by proteolytic cleavage. Microfluidic experiments with an A2-deletion VWF mutant resulted in increased platelet binding, corroborating the key autoinhibitory role of the A2 domain within VWF multimers. Overall, autoinhibition of VWF mediated by force-dependent interdomain interactions offers the molecular basis for the shear-sensitive growth of VWF-platelet aggregates, and might be similarly involved in shear-induced VWF self-aggregation and other force-sensing functions in hemostasis

Disulfide bond reduction and exchange in C4 domain of von Willebrand factor undermines platelet binding

Background The von Willebrand factor (VWF) is a key player in regulating hemostasis through adhesion of platelets to sites of vascular injury. It is a large, multi-domain, mechano-sensitive protein that is stabilized by a net of disulfide bridges. Binding to platelet integrin is achieved by the VWF-C4 domain, which exhibits a fixed fold, even under conditions of severe mechanical stress, but only if critical internal disulfide bonds are closed. Objective To determine the oxidation state of disulfide bridges in the C4 domain of VWF and implications for VWF’s platelet binding function. Methods We combined classical molecular dynamics and quantum mechanical simulations, mass spectrometry, site-directed mutagenesis, and platelet binding assays. Results We show that 2 disulfide bonds in the VWF-C4 domain, namely the 2 major force-bearing ones, are partially reduced in human blood. Reduction leads to pronounced conformational changes within C4 that considerably affect the accessibility of the integrin-binding motif, and thereby impair integrin-mediated platelet binding. We also reveal that reduced species in the C4 domain undergo specific thiol/disulfide exchanges with the remaining disulfide bridges, in a process in which mechanical force may increase the proximity of specific reactant cysteines, further trapping C4 in a state of low integrin-binding propensity. We identify a multitude of redox states in all 6 VWF-C domains, suggesting disulfide bond reduction and swapping to be a general theme. Conclusions Our data suggests a mechanism in which disulfide bonds dynamically swap cysteine partners and control the interaction of VWF with integrin and potentially other partners, thereby critically influencing its hemostatic function

DNA binds to a specific site of the adhesive blood-protein von Willebrand factor guided by electrostatic interactions.

Neutrophils release their intracellular content, DNA included, into the bloodstream to form neutrophil extracellular traps (NETs) that confine and kill circulating pathogens. The mechanosensitive adhesive blood protein, von Willebrand Factor (vWF), interacts with the extracellular DNA of NETs to potentially immobilize them during inflammatory and coagulatory conditions. Here, we elucidate the previously unknown molecular mechanism governing the DNA-vWF interaction by integrating atomistic, coarse-grained, and Brownian dynamics simulations, with thermophoresis, gel electrophoresis, fluorescence correlation spectroscopy (FCS), and microfluidic experiments. We demonstrate that, independently of its nucleotide sequence, double-stranded DNA binds to a specific helix of the vWF A1 domain, via three arginines. This interaction is attenuated by increasing the ionic strength. Our FCS and microfluidic measurements also highlight the key role shear-stress has in enabling this interaction. Our simulations attribute the previously-observed platelet-recruitment reduction and heparin-size modulation, upon establishment of DNA-vWF interactions, to indirect steric hindrance and partial overlap of the binding sites, respectively. Overall, we suggest electrostatics-guiding DNA to a specific protein binding site-as the main driving force defining DNA-vWF recognition. The molecular picture of a key shear-mediated DNA-protein interaction is provided here and it constitutes the basis for understanding NETs-mediated immune and hemostatic responses

Gain-of-Function Variant pPro2555Arg of von Willebrand Factor Increases Aggregate Size through Altering Stem Dynamics

The multimeric plasma glycoprotein (GP) von Willebrand factor (VWF) is best known for recruiting platelets to sites of injury during primary hemostasis. Generally, mutations in the VWF gene lead to loss of hemostatic activity and thus the bleeding disorder von Willebrand disease. By employing cone and platelet aggregometry and microfluidic assays, we uncovered a platelet GPIIb/IIIa-dependent prothrombotic gain of function (GOF) for variant p.Pro2555Arg, located in the C4 domain, leading to an increase in platelet aggregate size. We performed complementary biophysical and structural investigations using circular dichroism spectra, small-angle X-ray scattering, nuclear magnetic resonance spectroscopy, molecular dynamics simulations on the single C4 domain, and dimeric wild-type and p.Pro2555Arg constructs. C4-p.Pro2555Arg retained the overall structural conformation with minor populations of alternative conformations exhibiting increased hinge flexibility and slow conformational exchange. The dimeric protein becomes disordered and more flexible. Our data suggest that the GOF does not affect the binding affinity of the C4 domain for GPIIb/IIIa. Instead, the increased VWF dimer flexibility enhances temporal accessibility of platelet-binding sites. Using an interdisciplinary approach, we revealed that p.Pro2555Arg is the first VWF variant, which increases platelet aggregate size and shows a shear-dependent function of the VWF stem region, which can become hyperactive through mutations. Prothrombotic GOF variants of VWF are a novel concept of a VWF-associated pathomechanism of thromboembolic events, which is of general interest to vascular health but not yet considered in diagnostics. Thus, awareness should be raised for the risk they pose. Furthermore, our data implicate the C4 domain as a novel antithrombotic drug target

Autoregulation of von Willebrand factor function by a disulfide bond switch

Force-dependent binding of platelet glycoprotein Ib (GPIb) receptors to plasma von Willebrand factor (VWF) plays a key role in hemostasis and thrombosis. Previous studies have suggested that VWF activation requires force-induced exposure of the GPIb binding site in the A1 domain that is autoinhibited by the neighboring A2 domain. However, the biochemical basis of this “mechanopresentation” remains elusive. From a combination of protein chemical, biophysical, and functional studies, we find that the autoinhibition is controlled by the redox state of an unusual disulfide bond near the carboxyl terminus of the A2 domain that links adjacent cysteine residues to form an eight-membered ring. Only when the bond is cleaved does the A2 domain bind to the A1 domain and block platelet GPIb binding. Molecular dynamics simulations indicate that cleavage of the disulfide bond modifies the structure and molecular stresses of the A2 domain in a long-range allosteric manner, which provides a structural explanation for redox control of the autoinhibition. Significantly, the A2 disulfide bond is cleaved in ~75% of VWF subunits in healthy human donor plasma but in just ~25% of plasma VWF subunits from heart failure patients who have received extracorporeal membrane oxygenation support. This suggests that the majority of plasma VWF binding sites for platelet GPIb are autoinhibited in healthy donors but are mostly available in heart failure patients. These findings demonstrate that a disulfide bond switch regulates mechanopresentation of VWF.: This study was supported by grants from the National Health and Medical Research Council of Australia (P.J.H.), Royal College of Pathologists Foundation Kanematsu/Novo Nordisk Research Award (F.P. and L.J.), Diabetes Australia Research Trust grant G179720 and Sydney Medical School Early-Career Researcher Kickstart Grant (L.J.), National Heart Foundation of Australia Postdoctoral Fellowship (101285) (L.J.) and British Heart Foundation Intermediate Basic Science Research Fellowship (FS/11/51/28920) (B.M.L.), Deutsche Forschungsgemeinschaft (research unit FOR 1543 to C.A.-S., C.B., and F.G.), the Center for Modelling and Simulation in the Biosciences postdoctoral program of the Heidelberg University (A.B.), and the Klaus Tschira Foundation (F.G.). B.L. was supported by the Dutch Thrombosis Foundation through grant number 2016-03.

A conformational transition of the D9D3 domain primes von Willebrand factor for multimerization

Von Willebrand factor (VWF) is a multimeric plasma glycoprotein that is critically involved in hemostasis. Biosynthesis of long VWF concatemers in the endoplasmic reticulum and the trans-Golgi is still not fully understood. We use the single-molecule force spectroscopy technique magnetic tweezers to analyze a previously hypothesized conformational change in the D9D3 domain crucial for VWF multimerization. We find that the interface formed by submodules C8-3, TIL3, and E3 wrapping around VWD3 can open and expose 2 buried cysteines, Cys1099 and Cys1142, that are vital for multimerization. By characterizing the conformational change at varying levels of force, we can quantify the kinetics of the transition and stability of the interface. We find a pronounced destabilization of the interface on lowering the pH from 7.4 to 6.2 and 5.5. This is consistent with initiation of the conformational change that enables VWF multimerization at the D9D3 domain by a decrease in pH in the trans-Golgi network and Weibel-Palade bodies. Furthermore, we find a stabilization of the interface in the presence of coagulation factor VIII, providing evidence for a previously hypothesized binding site in submodule C8-3. Our findings highlight the critical role of the D9D3 domain in VWF biosynthesis and function, and we anticipate our methodology to be applicable to study other, similar conformational changes in VWF and beyond