Jurkat/A4 cells with multidrug resistance exhibit reduced sensitivity to quercetin

Background: While multidrug resistance of cancer cells is a well-known phenomenon, little is known on the cross resistance between cytotoxic chemotherapeutical agents and unrelated substances such as natural flavonoids. Aim: To compare the effects of cytotoxic drug, vepeside and natural flavonoid, quercetin in Jurkat cells and their multidrug-resistant subline Jurkat/A4, in particular to analyze the effector mechanisms of apoptosis and the profiles of several pro- and antiapoptotic proteins in these cells upon exposure to vepeside or quercetin. Methods: Apoptosis and poly (ADP-ribose) polymerase cleavage were assessed by flow cytometry. Expression of apoptosisrelated proteins was analyzed by Western blotting. Results: Jurkat/A4 cells are less sensitive to antiproliferative effects of quercetin as compared with the parental Jurkat cell line. While vepeside as well as quercetin initially induces apoptosis in both cell lines, the following survival of the exposed cells is essentially different. In resistant Jurkat/A4 cells, vepeside or quercetin treatment activates significantly less caspase-9 and -3 as compared with that in the parental cells. The expression of Bad and BNip1 proteins in Jurkat/A4 cells is lower than in the parental cell line. At the same time, XIAP and CAS levels in Jurkat/A4 cells increase. Upon apoptosis induction, XIAP and CAS levels in Jurkat cells decrease, this effect being negligible in resistant cells. Conclusion: Multidrug-resistant Jurkat/A4 cells exhibit reduced sensitivity to cytotoxic effects of quercetin. The expression profile of Jurkat/A4 cells is characterized by the increased levels of XIAP and CAS representing the endogenous inhibitors of apoptosis

Development of monoclonal antibodies specific to ribosomal protein S6 kinase 2

Ribosomal protein S6 kinase 2 (S6K2) is a serine/threonine kinase that belongs to the family of AGC kinases, which includes PKB/Akt, PKC, PDK1, and SGK1. Mammalian cells express two isoforms of S6K, termed S6K1 and S6K2. Each of these has nuclear and cytoplasmic spicing variants, which originate from different initiation start codons. Nuclear isoforms of S6K1 and S6K2 are slightly longer, as they possess additional sequences at the N-terminus with nuclear localization signals. Biochemical and genetic studies implicated S6Ks in the regulation of cell size, growth, and energy metabolism. Deregulation of S6K signaling has been linked to various human pathologies, making them excellent targets for drug discovery. The aim of this study was to produce monoclonal antibodies directed at the N-terminal regulatory region of S6K2, which shows very low homology to S6K1 or other members of the AGC family. To achieve this goal, two S6K2 fragments covering 1-64aa and 14-64aa N-terminal sequences were expressed in bacteria as GST/6His fusion proteins. Affinity purified recombinant proteins were used as antigens for immunization, hybridoma screening, and analysis of generated clones. We produced a panel of S6K2-specific antibodies, which recognized recombinant S6K2 proteins in ELISA and Western blot analysis. Further analysis of selected clones revealed that three clones, termed B1, B2, and B4, specifically recognized not only recombinant, but also endogenous S6K2 in Western blot analysis of HEK293 cell lysates. Specificity of B2 clone has been confirmed in additional commonly used immunoassays, including immunoprecipitation and immunocytochemistry. These properties make B2 MAb particularly valuable for elucidating signal transduction pathways involving S6K2 signaling under physiological conditions and in human pathologies

Generation and characterization of polyclonal antibodies specific to N-terminal extension of p85 isoform of ribosomal protein S6 kinase 1 (p85 S6K1)

Aim. Generation of polyclonal antibodies specific to the ribosomal protein S6 kinase isoform – p85S6K1 and directed to the N-terminal (1–23 aa) extension of p85S6K1. Methods. Animal immunization with synthetic (1–23 aa) peptide, ELISA, Western blot, Immunoprecipitation, immunofluorescent analysis. Results. Polyclonal antibodies have been generated, which specifically recognize only p85 but not p70 isoform of S6K1 in western blot, immunoprecipitation and immunofluorescence analysis. Conclusions. The obtained antibodies can be recommended for studies on the p85S6K1 and other S6K1 isoforms possessing the N-terminal extension – the identification of binding protein partners, analysis of subcellular localization under different physiological conditions, elucidation of the signal transduction pathways involving different S6K1 isoforms.Мета. Отримати поліклональні антитіла специфічні до p85 ізоформи S6K1 и спрямовані до N-кінцевого (1–23 a.к.) подовження p85S6K1. Методи. Іммунизація тварин синтетичним (1–23 а.к.) пептидом, ІФА, вестерн блот, іммунопреципітація, іммуноцитохимічний аналіз. Результати. Отримано поліклональні антитіла, що розпізнають p85, але не p70 ізоформу S6K1 в вестерн блот анализі, іммунопреципітації і іммуноцитохімичному аналізі. Висновки. Отримані антитіла можуть бути рекомендованими для дослідження p85S6K1 та інших ізоформ S6K1, що мають N- кінцеве подовження – ідентифікації білків-партнерів, аналізу субклітинної локалізації за різних фізіологічних умов, аналізі ролі S6K1 ізоформ в сигнальній трансдукції.Цель. Получить полииклональные антитела специфичные к p85 изоформе S6K1 и направленные против N-концевого (1–23 a.к.) удлинения p85S6K1. Методы. Иммунизация животных синтетическим (1–23 а.к.) пептидом, ИФА, вестерн блот, иммунопреципитация, иммуноцитохимический анализ. Результаты. Получены поликлональные антитела распознающие p85, но не p70 изоформу S6K1 в вестерн блот анализе, иммунопреципитации и иммуноцитохимическом анализе. Выводы. Полученные антитела могут быть рекомендованы для изучения p85S6K1 и других изоформ S6K1 обладающих N-концевым удлинением – идентификации белков партнеров, анализе субклеточной локализации при разных физиологических условиях, анализе роли S6K1 изоформ в сигнальной трансдукции

Generation of monoclonal antibodies specific to ribosomal protein S6 kinase 1

The aim of this study was to produce monoclonal antibodies directed to the N-terminal regulatory region of S6K1, which shows very low homology to S6K2. Methods. Hybridoma technology, ELISA, Western blot, Immunoprecipitation. Results. Three hybridoma clones (A1, A2 and A3) producing mAbs specific to ribosomal protein S6 kinase 1 have been generated. Specificity of mAbs has been confirmed by Western blot and immunoprecipitation technique. Conclusions. The obtained antibodies are suitable for elucidating signal transduction pathways involving S6K1. Keywords: ribosomal protein S6 kinase, mAbs.Мета. Отримати моноклональні антитіла проти N-кінцевої регуляторної ділянки кінази рибосомного білка S6-S6K1, який має низький рівень гомології з ізоформою S6K2. Методи. Гібридомна технологія, ІФА, Вестерн-блот, імунопреципітація. Результати. Створено тригібридомних клони (A1, A2 і A3), які продукують моноклональні антитіла, специфічні до кінази S6K1. Висновки. Одержані моноклональні антитіла за своїми характеристикам можна використовувати для дослідження S6K1-залежних сигнальних шляхів. Ключові слова: кіназа рибосомного білка S6, МкАТ.Цель. Получить моноклональные антитела против N-концевого регуляторного участка киназы рибосомного белка S6 – S6K1, имеющего низкий уровень гомологии с S6K2 изоформой. Методы. Гибридомная технология, ИФА, Вестерн-блот, иммунопреципитация. Результаты. Созданы три гибридомных клона (A1, A2 и A3), продуцирующих моноклональные антитела, специфичные к киназе S6K1. Специфичность антител подтверждена методами Вестерн блота и иммунопреципитации. Выводы. Полученные моноклональные антитела по своим характеристикам можно использовать для изучения S6K1-зависимых сигнальных путей. Ключевые слова: киназа рибосомного белка S6, МкАТ

Molecular cloning of CoA synthase - The missing link in CoA biosynthesis

Coenzyme A functions as a carrier of acetyl and acyl groups in living cells and is essential for numerous biosynthetic, energy-yielding, and degradative metabolic pathways. There are five enzymatic steps in CoA biosynthesis. To date, molecular cloning of enzymes involved in the CoA biosynthetic pathway in mammals has been only reported for pantothenate kinase. In this study, we present cDNA cloning and functional characterization of CoA synthase. It has an open reading frame of 563 aa and encodes a protein of similar to60 kDa. Sequence alignments suggested that the protein possesses both phosphopantetheine adenylyltransferase and dephospho-CoA kinase domains. Biochemical assays using wild type recombinant protein confirmed the gene product indeed contained both these enzymatic activities. The presence of intrinsic phosphopantetheine adenylyltransferase activity was further confirmed by site-directed mutagenesis. Therefore, this study describes the first cloning and characterization of a mammalian CoA synthase and confirms this is a bifunctional enzyme containing the last two components of CoA biosynthesis

Application of Studies of Demographical Processes of the Iecava Municipal Area in Classes of Geography

Diplomdarbs ir veltīts projektu metodes izmantošanas iespēju novērtējumam ģeogrāfijas mācību vielas apguvei skolā. Diplomdarbā „Iecavas novada demogrāfisko procesu pētījumu izmantošana ģeogrāfijas stundās” veicam ar skolēniem projekta nedēļā pētījumu par Iecavas novada iedzīvotāju svarīgākajiem demogrāfiskajiem indikatoriem. Pēdējo gadu laikā mūsu valstī ir novērojama iedzīvotāju depopulācija. Kopš Latvija ir neatkarīga valsts, ir parādījusies jauna sociāla iedzīvotāju grupa – bezdarbnieki. Zemes īpašums vairs nav tikai valsts rīcībā, bet pieder gan privātpersonām, gan pašvaldībai un juridiskām personām. Iedzīvotāji vēlas sasniegt zināmu labklājības līmeni, tāpēc ir svarīgi iegūt labu, profesionālu izglītību un darba tirgū pieprasītu specialitāti. Iecavas novads, kā viens no Latvijas Republikas novadiem, īpaši neatšķiras ar iedzīvotājiem saistītajos procesos. Pēc veiktā pētījuma un raksturojuma diplomdarbā, secināju, ka Iecavas novadā ir novērojama iedzīvotāju depopulācija gan dabiskās, gan mehāniskās kustības rezultātā. Pieaug veco ļaužu skaits un samazinās bērnu īpatsvars. Par saprotamu parādību Iecavas novadā ir kļuvis bezdarbs, jo trūkst darba piedāvājumu, tajā pašā laikā ir problēmas atrast kvalificētu darbaspēku. Iecavas novadā ir iespēja iegūt labu vispārējo izglītību, bet nav motivācijas atgriezties pēc diploma saņemšanas, tāpēc no novada aizplūst jauni cilvēki. Darba atslēgas vārdi: projektu darbs, Iecavas novads, demogrāfiskie procesi.Diploma paper is meant for opportunities of projects methods usage in geography studies acquirement at school. In diploma paper "Application of Studies of Demographical Processes of the Iecava Municipal Area in Classes of Geography" is used research in project week about the most important demographic indicators of Iecava municipality inhabitants. Recent years in our country has been observed depopulation of inhabitants. Since Latvia is independent country, has been appeared a new social group of inhabitants - unemployed. Land belongs not only to country, but also to private owners, to municipalities and legal persons. Inhabitants gain to achieve the level of welfare, because is really important to get a good education and trade what is enquired in labour market. Iecava municipality in Latvia does not differ from other parts of Latvia in processes inhabitants involved. After research and characteristics in diploma paper, was concluded that in Iecava municipality is observed depopulation of inhabitants as natural as mechanical result of movement. The amount of old people increases but the amount of young people decreases. Unemployment has become common thing, because of lack of job offers, but in the same time has been observed problems to find qualified labour. There is opportunity to get a good secondary education, but lack of motivation to stay and work in home place. Young people flow away to other work places. The key words of diploma paper: project work, Iecava municipality, demographic processes

Feasibility study: Implementation of Industry 4.0. principles in the creation of a new business model

RESUMEN: El siguiente Trabajo Fin de Grado pivota sobre la elaboración de un plan de empresa y el consiguiente estudio de viabilidad de un modelo de negocio basado en el alquiler de movilidad, siendo la base del mismo un dispositivo de control capaz de monitorizar el buen uso del servicio. Este dispositivo forma parte de una red más grande y autónoma capaz de recoger datos, procesarlos y tomar decisiones en consecuencia, abordando uno de los pilares de la Industria 4.0 aplicada a un servicio (el internet de las cosas). Dicho trabajo comienza con una descripción detallada del modelo de negocio, justificado por los correspondientes análisis del entorno, el sector, la competencia y los clientes potenciales. A continuación, se ahonda en el desarrollo de las herramientas necesarias para desarrollar la actividad tal y como se plantea. Posteriormente se establecen las bases acerca de los recursos necesarios para emprender la actividad, ya sean humanos, financieros, de localización o de equipamiento. Por último, se realiza un estudio y previsión a 5 años vista de las ventas, teniendo en cuenta las etapas de desarrollo de la empresa y la política de expansión del plan de negocio, finalizando con las conclusiones de la viabilidad del proyecto y bajo qué circunstancias.ABSTRACT: This final degree project is about the development of a business plan and the subsequent feasibility study of a business model based on carsharing. The technical part is based on a control device capable of monitoring the good use of borrowed car. This device is part of a larger and autonomous network capable of collecting data, processing it and making decisions, consistent with one of the pillars of Industry 4.0 applied to a service (Internet of Things). This essay begins with a detailed description of the business model, justified by the corresponding analyzes of the environment, the sector, the competition and potential customers. Next, development of the necessary tools is studied. Subsequently, the bases about the necessary resources to undertake the activity, whether human, financial, location or equipment are established. Finally, a 5-year study and forecast of sales is carried out, considering the stages of development of the company and the expansion policy of the business plan, ending with the conclusions of the viability of the project and necessary circumstances to achieve it.Grado en Ingeniería en Tecnologías Industriale

Study of facilities, habits, and maintenance of a hotel establishment. Proposal of actions focused on energy efficiency

Planteamiento del problema: Los suministros energéticos suponen una fuente importante de gasto y, al mismo tiempo, de incertidumbre. Para la electricidad, durante el 2023, su precio medio ha sufrido una variación de más del 200 % y para el suministro de gasóleo para calefacción, esta variación en su precio ha supuesto más del 30 % entre su valor más alto y más bajo. Con esta elevada dependencia del mercado, los grandes consumidores se plantean si el estado de sus instalaciones es el óptimo, si es posible realizar acciones que favorezcan la eficiencia energética y si existen alternativas que mitiguen el coste y la incertidumbre. Es el caso del Hotel el Infantado, de más de 40 habitaciones, sito en Cantabria, objeto del presente estudio. Descripción del proyecto: Durante el desarrollo del estudio se mantendrán conversaciones con la gerencia para contextualizar el establecimiento, se realizará un estudio del estado y consumo de los principales demandantes energéticos, se valorará el mantenimiento realizado a las instalaciones y se hará seguimiento de los hábitos que influyen en el funcionamiento y consumo de los equipos. Ante lo estudiado, se planteará la resolución de las incidencias detectadas, se generarán propuestas de planes de mantenimiento y se harán llegar sugerencias en cuanto a los hábitos de uso de las instalaciones. También se propondrán iniciativas de nueva instalación o sustitución de tecnologías actuales en los diferentes sistemas para mejorar el desempeño energético, obtener una mayor estabilidad presupuestaria y una mayor independencia del mercado. Siempre cumpliendo e incluso mejorando los altos estándares de servicio con los que ya cuenta el establecimiento. Conclusiones / Presupuesto Tras el análisis, se han planteado iniciativas diferenciadas en tres horizontes temporales que persiguen reducir el coste en los suministros energéticos, velar por la seguridad y asegurar el mejor servicio. También se ha aportado información de utilidad para poder tomar decisiones estratégicas a largo plazo, basadas en el estado actual y las tecnologías disponibles, fomentando la rentabilidad, el servicio y la sostenibilidad.Problem statement: Energy supplies represent an important source of expense and, simultaneously, uncertainty. For electricity, during 2023, its average price[1] has undergone a variation of over 200%, and for heating diesel supply[2], this price variation has exceeded 30% between its highest and lowest values. With this high market dependency, major consumers question whether the state of their facilities is optimal, if actions favoring energy efficiency are possible, and if there are alternatives to mitigate costs and uncertainty. This study focuses on Hotel el Infantado, with over 40 rooms, located in Cantabria. Project description: Conversations with management will be held to contextualize the establishment, a study of the state and consumption of the main energy demanders will be conducted, maintenance of the facilities will be assessed, and habits influencing the operation and consumption of equipment will be monitored. Based on the findings, resolutions to detected issues will be proposed, maintenance plans will be suggested, and recommendations regarding facility usage habits will be provided. Initiatives for new installations or replacement of current technologies in different systems will also be proposed to improve energy performance, achieve greater budget stability, and increase independence from the market. All while maintaining or enhancing the establishment's existing high service standards. Conclusions: Following the analysis, differentiated initiatives have been proposed across three temporal horizons, aiming to reduce costs in energy supplies, ensure safety, and guarantee the best service. Additionally, valuable information has been provided for making long-term strategic decisions based on the current state and available technologies, promoting profitability, service quality, and sustainability.Máster en Ingeniería Industria

Generation and characterization of polyclonal antibodies specific to N-terminal extension of p85 isoform of ribosomal protein S6 kinase 1 (p85 S6K1)

Aim. Generation of polyclonal antibodies specific to the ribosomal protein S6 kinase isoform – p85S6K1 and directed to the N-terminal (1–23 aa) extension of p85S6K1. Methods. Animal immunization with synthetic (1–23 aa) peptide, ELISA, Western blot, Immunoprecipitation, immunofluorescent analysis. Results. Polyclonal antibodies have been generated, which specifically recognize only p85 but not p70 isoform of S6K1 in western blot, immunoprecipitation and immunofluorescence analysis. Conclusions. The obtained antibodies can be recommended for studies on the p85S6K1 and other S6K1 isoforms possessing the N-terminal extension – the identification of binding protein partners, analysis of subcellular localization under different physiological conditions, elucidation of the signal transduction pathways involving different S6K1 isoforms.Мета. Отримати поліклональні антитіла специфічні до p85 ізоформи S6K1 и спрямовані до N-кінцевого (1–23 a.к.) подовження p85S6K1. Методи. Іммунизація тварин синтетичним (1–23 а.к.) пептидом, ІФА, вестерн блот, іммунопреципітація, іммуноцитохимічний аналіз. Результати. Отримано поліклональні антитіла, що розпізнають p85, але не p70 ізоформу S6K1 в вестерн блот анализі, іммунопреципітації і іммуноцитохімичному аналізі. Висновки. Отримані антитіла можуть бути рекомендованими для дослідження p85S6K1 та інших ізоформ S6K1, що мають N- кінцеве подовження – ідентифікації білків-партнерів, аналізу субклітинної локалізації за різних фізіологічних умов, аналізі ролі S6K1 ізоформ в сигнальній трансдукції.Цель. Получить полииклональные антитела специфичные к p85 изоформе S6K1 и направленные против N-концевого (1–23 a.к.) удлинения p85S6K1. Методы. Иммунизация животных синтетическим (1–23 а.к.) пептидом, ИФА, вестерн блот, иммунопреципитация, иммуноцитохимический анализ. Результаты. Получены поликлональные антитела распознающие p85, но не p70 изоформу S6K1 в вестерн блот анализе, иммунопреципитации и иммуноцитохимическом анализе. Выводы. Полученные антитела могут быть рекомендованы для изучения p85S6K1 и других изоформ S6K1 обладающих N-концевым удлинением – идентификации белков партнеров, анализе субклеточной локализации при разных физиологических условиях, анализе роли S6K1 изоформ в сигнальной трансдукции