The role of lipids in Wnt secretion and function

Wnt signaling pathways are a set of signal transduction cascades which are activated through the interaction of Wnt proteins with so-called Frizzled receptors [1-3]. These pathways are critically involved in many biological processes such as embryonic development, regeneration, organogenesis, cell division, cellular and tissue homeostasis, among many others [3, 4]. In addition, alterations of these signaling pathways have been linked to various types of diseases such as cancer [5-7], familial tooth agenesis [8], bipolar disease [9], Alzheimer's disease [10], and cardiac valve formation [11]. Wnt signaling components are accordingly promising drug targets to treat these diseases. Wnt pathways are probably among the best characterized receptor-ligand signaling pathways. Wnt proteins are therefore key players in biological signaling and promising drug targets to treat a plethora of diseases. Although several proteins involved in Wnt trafficking and secretion have been identified over the past years, little is known about the contribution of different lipid species into these processes. The trafficking and secretion of Wnts could be modulated by the type and number of acyl species covalently linked to Wnt proteins. Currently, the best described acyl modification is the palmiteoylation of a serine residue located around amino acids 205-215 mediated by the ER-resident O-acyltransferase Porcupine is responsible for this process. This lipid modification has been described for Wnt3a, Wnt5, xWnt8, and Wnt1, and it has been assumed to also take place in other members of the family of Wnt proteins. Despite the extensive data available, the debate around the lipid-modified amino acids in Wnt proteins has not yet reached a consensus. Recent results from O. Voloshanenko (M. Boutros group, DKFZ, Heidelberg, Germany) suggested that there may be other amino acid residues in Wnts that are lipidated, apart from this canonical serine residue. Furthermore, the specific saturation of the acylated chain that binds to Wnt remains inconclusive. In this thesis, I aimed to define other putative acylation types and lipid-modified sites in Wnt proteins and to determine the role of these alternative lipidations in Wnt secretion and signaling. Furthermore, I evaluated the impact of Wnt signaling and Wnt secretion on the lipidome of HEK293T and HCT116 cells. To achieve this, I employed a combination of chemical biology tools, mutagenesis experiments, and mass spectrometric measurements. In particular, I focused on Wnt11 as a working model. I studied its acylation using clickable lipids such as palmitic acid alkyne (cC16:0) and palmitoleic acid alkyne (cC16:1n-7). One of the early observations was that palmitoylation and secretion of Wnt11 were not wholly abolished in Porcupine knockout cells or some mutant variants of Wnt11. However, these observations seem to depend on the type of clickable fatty acid used. Our results suggest a lipid modification of Wnt11 at serine 215 via the monounsaturated fatty acid cC16:1n-7, consistent with the previously predicted models. However, lipid modification with the saturated fatty acid cC16:0 showed variations in the experimental replicates, which did not fully resolve whether Wnt11 contains another modification site. Importantly, our experiments stress that unsaturation is a key feature for Wnt acylation. The relevance of covalent lipid binding for the secretion and signaling activity of Wnts has also been assessed. It was demonstrated that lipidation is essential for the signaling activity of Wnt11 but is not strictly necessary for its secretion. In addition, an impact of Wnt protein expression on the overall cellular lipidome of HEK293T and HCT116 cells has been tested, yielding preliminary observations on the crosstalk between the Wnt signaling and the overall cellular lipid homeostasis. This study is expected to contribute to our understanding of how post-translational lipid modifications influence Wnt cellular secretion, signaling and, conversely, how proteins of the Wnt signaling pathway affect the lipid composition of cells

Efeito da pré-molhagem do agregado nas propriedades das argamassas com agregado reciclado deconcreto e agregado leve

This paper examines the suitability of partially replacing natural aggregate, sand, (NA) with recycled concrete aggregate (RCA) or lightweight aggregate (LWA) in mortars, under the hypothesis that pre-wetting aggregates would produce improvement in mortar properties. Fresh mortar properties such as density, entrained air content, consistency and heat of hydration, as well as hardened mortar properties such as dry density, compressive and flexural strength, and dimensional instability at 0% and 100% saturation were determined. The results show that mortars made with natural aggregate (75%) and recycled concrete aggregate (25%) have similar properties to mortars made with only natural aggregate (100%) and that pre-wetting the aggregates does not influence the properties of mortars significantly. Therefore, partial replacement with recycled concrete aggregate is a viable alternative for producing mortar.Peer ReviewedPostprint (published version

Mechanical and environmental behavior of cement mortars containing ladle furnace slag

At present, steel production is mainly done in basic oxygen furnaces (BOF) and electric arc furnaces (EAF) (Pauliuk, S. et al, 2013, Morfeldt, J. et al, 2015). According to the statistics, it is expected that the use of electric arc furnaces to supply the demand for steel will prevail. In 2017, in Europe, 41% of steel was produced in electric arc furnaces, while in Spain, according to the Union of Steel Companies (UNESID, 2016), the percentage was almost 66% in 2016. In 2016 the European Union generated approximately 18.4 Mt of slag (black and white), of which 1.5 Mt was produced in Spain. In other words, the amount of white slag produced was about 286 thousand tons, approximately between 20 and 30 kg of slag per ton of steel (UNESID, 2016, Euroslag, 2016). Refining slag (white slag) is the second most abundant waste in steel production, and, to date, its final destination is still the landfill. This study has used ladle slag from refined steelworks, as a replacement for cement in different proportions (0, 25, 50 and 75%), for the manufacture of mortars. A broad characterization of the ladle slag has been carried out, as well as determining the mechanical, durable, and environmental properties of the manufactured mortars. The results show that using the ladle slag as a replacement for cement produces a decrease in compressive strength, but the expansion behavior remains below the stability limit. It also has been seen that the slag incorporated into mortars, evaluated by leaching test at 28 days, shows inert material behavior.Postprint (published version

Long-term comparison between waste paper fly ash and traditional binder as hydraulic road binder exposed to sulfate concentrations

Sulfate attack is one of the drawbacks of cementitious materials for stabilized soils. In the current study, a durability comparison of stabilized soil with cement (Type IV) and waste paper fly ash (WPFA) was conducted. First, the treated soil’s unconfined compressive strength (UCS) was tested. Next, the treated soil was subjected to various wetting/drying cycles with various sulfate concentrations and temperatures for a year. In the meantime, samples were taken for DRX, FTIR, and TGA microstructural analyses. Additionally, samples were manufactured to track swelling over an 800 day period. The outcomes show that WPFA’s UCS remained constant. Furthermore, ettringite development can be seen in the microstructural studies, however testing on linear displacement over 800 days revealed no significant changes in swelling. Finally, SEM was used to verify the ettringite formation at 360 days in order to confirm the previous findings. All the results indicated that stabilizing soil with 5% of WPFA and 3% of cement IV is possible even in presence of high sulfate concentrations, while maintaining the durability of the structure.This research was funded by European Union’s Horizon 2020, grant number 730305.Peer ReviewedPostprint (published version

Mechanical and Environmental Behavior of Cement Mortars Containing Ladle Furnace Slag

At present, steel production is mainly done in basic oxygen furnaces (BOF) and electric arc furnaces (EAF) (Pauliuk, S. et al, 2013, Morfeldt, J. et al, 2015). According to the statistics, it is expected that the use of electric arc furnaces to supply the demand for steel will prevail. In 2017, in Europe, 41% of steel was produced in electric arc furnaces, while in Spain, according to the Union of Steel Companies (UNESID, 2016), the percentage was almost 66% in 2016. In 2016 the European Union generated approximately 18.4 Mt of slag (black and white), of which 1.5 Mt was produced in Spain. In other words, the amount of white slag produced was about 286 thousand tons, approximately between 20 and 30 kg of slag per ton of steel (UNESID, 2016, Euroslag, 2016). Refining slag (white slag) is the second most abundant waste in steel production, and, to date, its final destination is still the landfill. This study has used ladle slag from refined steelworks, as a replacement for cement in different proportions (0, 25, 50 and 75%), for the manufacture of mortars. A broad characterization of the ladle slag has been carried out, as well as determining the mechanical, durable, and environmental properties of the manufactured mortars. The results show that using the ladle slag as a replacement for cement produces a decrease in compressive strength, but the expansion behavior remains below the stability limit. It also has been seen that the slag incorporated into mortars, evaluated by leaching test at 28 days, shows inert material behavior

Implementación de políticas de gobierno abierto, su impacto en la gobernanza y democracia digital: el caso de San Isidro en el período 2015 2018

Busca ahondar en el proceso de implementación de políticas de gobierno abierto en la Municipalidad de San Isidro, de forma tal que se busca entender de que forma el uso de medios digitales ha generado un cambio en la forma de relacionamiento entre la ciudadanía con la administración municipal, en ese sentido se está ahondando en los tópicos de participación ciudadana, colaboración y transparencia como pilares de las políticas de gestión.Perú. Universidad Nacional Mayor de San Marcos. Vicerrectorado de Investigación y Posgrado. Programa de Promoción de Tesis de Pregrado. E18150634-PTPGRAD

Escala de expectativas de futuro en la adolescencia (EEFA): adaptación, validación y datos normativos en estudiantes de secundaria, Lima Metropolitana, 2021

La presente investigación de tipo aplicada y diseño instrumental tuvo como objetivo adaptar y evaluar las evidencias psicométricas de la Escala de expectativas de futuro en la adolescencia EEFA en estudiantes de secundaria de Lima Metropolitana. Participaron 707 adolescentes, entre 12 a 17 años. Se confirmó la validez basada en el contenido por medio de 5 jueces expertos, obteniéndose valores superiores a .93 en todos los ítems. Asimismo, se analizó la validez basada en la estructura interna por medio de un AFC, encontrándose adecuados índices de ajuste: x2/gl= 3.87, RMSEA= .057, SRMR= .038, CFI= .959 y TLI= .949, bajo la estructura multidimensional de segundo orden. Luego, se analizó la validez en relación con otras variables, hallándose correlaciones directas fuertes con autoestima (r=.549) y resiliencia (r=.562), y un tamaño del efecto grande (r2>.25). Además, se evaluó la confiabilidad mediante el coeficiente omega ordinal, obteniéndose =.94 para la escala general, además de valores superiores a =.70 para los factores. Seguidamente, se analizó la equidad y los resultados confirmaron que la escala es invariante a nivel de sexo y gestión educativa. Finalmente, se elaboraron los datos normativos para la interpretación de las puntuaciones, recomendando el uso del instrumento en ámbitos específicos de la psicología

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

A low-cost secure iot mechanism for monitoring and controlling polygeneration microgrids

The use of Internet-connected devices at homes has increased to monitor energy consumption. Furthermore, renewable energy sources have also increased, reducing electricity bills. However, the high cost of the equipment limits the use of these technologies. This paper presents a low-cost secured-distributed Internet of Things (IoT) system to monitor and control devices connected in a polygeneration microgrid, as a combined power system for local loads with renewable sources. The proposed mechanism includes a Wireless Sensor Actuator Networked Control System that links network nodes using the IEEE 802.15.4 standard. The Internet communication enables the monitor and control of devices using a mobile application to increase the efficiency. In addition, security mechanisms are implemented at several levels including the authentication, encryption, and decryption of the transmitted data. Furthermore, a firewall and a network intrusion detection-and-prevention program are implemented to increase the system protection against cyber-attack. The feasibility of the proposed solution was demonstrated using a DC microgrid test bench consisting of a diverse range of renewable energy sources and loads