No description available.

No description available.

M. Tech. (Department of Chemistry, Faculty of Applied and Computer Sciences), Vaal University of Technology. / The world is continuously losing the battle against superbugs (resistant bacteria towards commonly used antibiotics), hence there is an urgent need to develop novel antibacterial agents. In this study, green synthesized silver nanoparticles (AgNPs) and eucalyptus oil, were incorporated into the polymer blend fibres of polyvinyl alcohol (PVA) and cellulose nanocrystals (CNC’s). Various techniques were used to characterize the AgNPs, PVA/CNC polymer fibres, and PVA/CNC incorporated with AgNPs/eucalyptus oil. The morphology of AgNPs synthesized using an increasing concentration of the Citrus sinensis peels (CSP) extract was obtained from transmission electron microscopy (TEM). AgNPs synthesized using 1 and 2 % m/v (CSP) were agglomerated and whereas those synthesized using 3 % m/v of the extract were spherical with an average particle size 10 ± 1.2 nm. UV/Visible absorption spectra for all the synthesized AgNPs exhibited a surface plasmon resonance (SPR) peak at around 400 nm which is a characteristic peak of silver. Significant shifts in the absorption peaks or maxima were observed to signify changes in the shape and size of the nanoparticles. Scanning electron microscopy (SEM) was used to study the morphology of the fabricated polymer fibres. The Addition of CNC’s to PVA resulted in an increase in fibre diameter due to an increase in viscosity of the solution. An increase in the concentration of silver nanoparticles and the eucalyptus oil in the PVA/CNC resulted in a decrease in fibre diameter due to an increase in conductivity of the material. The fibres with AgNPs were smooth while the ones with the eucalyptus oil were beaded. X-ray diffraction (XRD) showed the presence of the AgNPs in the polymer fibres and Fourier transform infrared (FTIR) showed the presence of the functional groups that are available in the eucalyptus oil. The antibacterial efficiency of the PVA/CNC incorporated with AgNPs, eucalyptus oil, and the mixture of AgNPs and the eucalyptus oil was investigated using S. aureus and K. pneumoniae. All the materials showed significant inhibition of the growth of the selected bacterial strains. PVA/CNC polymer fibres incorporated with AgNPs showed higher antibacterial activity compared to PVA/CNC polymer fibres incorporated with eucalyptus oil.

Les extrémités des chromosomes eucaryotes ressemblent à une cassure double brin et, en tant que telles, peuvent conduire à l'activation indésirable de la réponse aux dommages de l'ADN. Les télomères sont une structure ribonucléoprotéique qui coiffe les extrémités des chromosomes et les protège contre l'activation indésirable de la réparation des dommages à l'ADN. Après chaque division cellulaire, on observe un raccourcissement progressif des télomères, ce qui limite leur potentiel prolifératif. Une enzyme spécialisée, la télomérase, reconstitue les télomères pour contrebalancer leur érosion. La télomérase est régulée à la baisse dans la plupart des cellules somatiques. Cependant, l'activité de la télomérase est détectée dans la plupart des cellules souches adultes, bien qu'à de faibles niveaux. Le déficit en télomérase a été associé à un groupe de "troubles de la biologie des télomères" (ou téloméropathies), englobant des maladies de vieillissement prématuré, des syndromes d'insuffisance de la moelle osseuse, des fibroses pulmonaires et des maladies du foie. À l'inverse, dans le cancer, environ 85 % des types de tumeurs sont positifs à la télomérase. Par conséquent, l'inhibition de la télomérase est depuis longtemps considérée comme une cible attrayante pour le traitement du cancer. Dans la présente étude, nous avons cherché à découvrir les facteurs qui affectent la fonction de la télomérase humaine et d'autres protéines associées aux télomères ou à la télomérase. Tout d'abord, nous nous sommes concentrés sur l'identification de nouveaux inhibiteurs de la télomérase à partir de composés naturels. Une nouvelle catéchine a été identifiée dans les extraits végétaux de Burkea africana. Les catéchines sont une classe de molécules que l'on trouve couramment dans le thé vert. La catéchine isolée a inhibé la télomérase humaine recombinante in vitro avec un IC50 de 16,19 μM. Dans un deuxième chapitre, nous avons utilisé un criblage d'édition de bases CRISPR dans une lignée cellulaire humaine pour étudier des mutations cliniquement pertinentes dans 22 gènes importants pour l'homéostasie des télomères. Nous avons identifié des variantes qui affectent négativement l'aptitude cellulaire, y compris certaines variantes précédemment annotées comme variantes de signification incertaine. Nous avons également détecté pour la première fois des variantes hTERT qui confèrent une résistance à la petite molécule BIBR1532, un inhibiteur de la télomérase. Nous avons montré que ces allèles résistants aux médicaments permettent l'immortalisation cellulaire et ont un potentiel tumorigène accru. L'ensemble de ces études souligne l'importance de la télomérase humaine pour le maintien des télomères et la santé cellulaire, contribuant ainsi à une meilleure compréhension du rôle de la télomérase dans le cancer et les troubles de la biologie des télomères. / The extremities of eukaryotic chromosomes resemble a double-stranded break and, as such, can lead to the unwanted activation of the DNA damage response. Telomeres are a ribonucleoprotein structure that caps the ends of the chromosomes and protects them from the unwanted activation of DNA damage recognition and repair processes. After each cellular division, progressive telomere shortening is observed, limiting cellular proliferative potential. A specialized enzyme called telomerase replenishes telomeres to counterbalance telomere erosion. Telomerase is downregulated in most somatic cells. However, telomerase activity is detected in most adult stem cells, although at low levels. Telomerase deficiency has been linked to a group of “Telomere Biology Disorders” (or telomeropathies), encompassing premature aging diseases, bone marrow failure syndromes, pulmonary fibrosis and liver diseases. Conversely, in cancer, around 85% of tumour types are telomerase-positive. Therefore, telomerase inhibition has long been considered an attractive target for cancer therapy. In the present study, we aimed to uncover factors that affect the function of human telomerase and other telomere or telomerase-associated proteins. Firstly, we focused on identifying new telomerase inhibitors from natural compounds. A new catechin was identified in the plant extracts from Burkea africana. Catechins are a class of molecules commonly found in green tea. The isolated catechin inhibited recombinant human telomerase in vitro with an IC50 of 16.19 μM. In the second chapter, we employed a CRISPR base editing screen in a human cell line to investigate clinically-relevant mutations in 22 genes important for telomere homeostasis. We identified variants that negatively affected cell fitness, including some variants previously annotated as variants of uncertain significance. Also, we uncovered hTERT variants that confer resistance to the small molecule BIBR1532, a telomerase inhibitor. We showed that these drug-resistant alleles permit cellular immortalization and exhibit tumorigenic potential at levels comparable to wild-type telomerase. Combined, these studies highlight the importance of human telomerase for telomere maintenance and cell fitness, thereby furthering our understanding of the role of telomerase in cancer and telomere biology disorders.

<p dir="ltr">Lung cancer is the leading cause of cancer-related deaths, and although important therapy advancements have been achieved, ~1.6 million people die from lung cancer annually. Non-small cell lung cancer (NSCLC), which makes up ~85% of lung cancer cases, is mainly treated with radiotherapy, chemotherapies, and targeted agents. Targeted agents are selected based on the mutation spectrum of the tumor. In NSCLC the epidermal growth factor receptor (EGFR) is commonly mutated and, leads to increased proliferation and cell survival. The standard-of-care treatment for patients with activating mutations in EGFR is treatment with tyrosine kinase inhibitors (TKI), such as erlotinib. While tumors initially respond to TKIs, after 1-2 years most patients develop resistance. In ~60% of TKI resistant tumors, resistance is the result of a secondary mutation in EGFR, whereas in the remaining 20%, tumors turn on bypass track-signals to overcome inhibition of the EGFR pathway. However, 15-20% of the cases the mechanisms underlying resistance are unknown. Most studies focus on the gain of function of oncogenes as mediators of resistance; however, little is known about the role that tumor suppressors play in TKI resistance. Hence, we performed a genome-wide CRISPR Cas9 knock-out screen to identify genes that when knocked-out would drive erlotinib resistance, and KMT5C was identified as the top candidate. KMT5C is a histone methyltransferase that trimethylates H4K20 (H4K20me3), enabling the establishment of constitutive and facultative heterochromatin. Data from human samples suggests that the <i>KMT5C</i> transcript is globally downregulated in NSCLC and in tumor samples resistant to the third generation TKI osimertinib. Additionally, loss of the modification H4K20me3, influences prognosis of NSCLC, indicating that loss of KMT5C function is a crucial mechanism in carcinogenesis. Here we describe how loss of KMT5C leads to increased transcription of the oncogene MET, due to a loss in H4K20me3-mediated repression of a long non-coding RNA transcription (LINC01510) upstream of MET. This mechanism was found to be partially responsible in driving TKI resistance in EGFR mutant cells. Historically, KMT5C has been associated with generation of constitutive heterochromatin (cHC); however, recent reports, including our own, indicate that KMT5C also regulates transcription in regions outside of cHC. Our preliminary evidence suggests that deposition of H42K0me3 via KMT5C in regions outside of cHC, is less stable than in cHC regions. This novel finding led us to hypothesize that regulation of KMT5C and H42K0me3 at different regions of heterochromatin is a dynamic process.</p>

Castration-resistant prostate cancer (CRPC) remains an aggressive incurable disease in men mainly due to treatment resistance. Current treatments do not effectively eradicate cancer stem cells (CSCs), which play a pivotal role in tumour maintenance, progression and drug resistance. Aldehyde dehydrogenases (ALDHs) have been used in some tumour types as CSC markers. Their high expression and high functional activity found in CSCs is also associated with drug resistance. Emerging evidence suggests deregulation of certain ALDH isoforms have implications in cancer. The role of ALDHs in prostate cancer as potential biomarkers and therapeutic targets has not been fully explored yet. Accordingly, this study investigated the expression, regulation and function of selected ALDH isoforms in prostate cancer. This study showed that ALDH1A3, ALDH1B1, ALDH2 and ALDH7A1 are highly expressed in primary prostate cancer cells (n=9) compared to benign (n=9) prostate cells. The expression of ALDH1A3 was high in the stem cells (SCs) (n=3) as well as the more differentiated counterparts (n=16). Treatment of both benign and malignant primary prostate cancer cells with all-trans retinoic acid (atRA) also resulted in increased expression of ALDH1A3 and ALDH3A1, supporting a feedback loop between atRA and ALDHs. Furthermore, SerBob, Bob and LNCaP cells were sensitive to treatment with epigenetic drugs and led to significantly higher expression of ALDH1A2, ALDH3A1 and ALDH7A1 respectively. Importantly, siRNA suppression of ALDH1A3 and ALDH7A1 led to reduced SC properties of primary prostate cultures including reduced cell viability, migration and colony formation, and increased differentiation of transit amplifying (TA) cells to committed basal (CB) cells. Novel ALDH-affinic probes showed reduced cell viability of primary prostate epithelial cultures as a single agent and also when used in combination with docetaxel. The results indicate the potential of using ALDH-affinic compounds as single agents for therapeutic intervention or in combination with docetaxel to sensitise resistant cells to this anticancer drug. The data in this thesis provides novel findings, which supports ALDH1A2, -1A3 and -7A1 as potential biomarkers and/or therapeutic targets for drug intervention. Although, a study analysing a larger number of samples is necessary to fully understand ALDH isoform expression in CSC, TA and CB cells it is envisaged that an ALDH-targeted therapy have potential in future treatment strategies for prostate cancer. / Prostate Cancer UK

Tuberculosis (TB) is a respiratory disease caused by Mycobacterium tuberculosis (Mtb) that kills around 1.3 million people annually. Multi-drug resistant TB (MDR-TB) strains are increasingly encountered, in part resulting from shortcomings of current TB drug regimens that last between six to nine months. Patients may stop taking the antibiotics during their allotted regimen, leading to drug resistant TB strains. Novel drug screening platforms are therefore necessary to find drugs effective against MDR-TB. In order to discover compounds that target under-exploited pathways that may be essential only in vivo, the proposed screening platform will use a novel approach to drug discovery by blocking essential protein-protein interactions (PPI). In Mtb, PPI can be monitored by mycobacterial protein fragment complementation (M-PFC). This project will re-engineer the M-PFC assay to include the red fluorescent mCherry reporter for increased efficiency and sensitivity in high-throughput screening applications. To optimize the mCherry assay, we have developed fluorescent M-PFC reporter strains to monitor distinct PPI required for Mtb virulence: homodimerization of the dormancy regulator DosR. A drug screen will then identify novel compounds that inhibit this essential PPI. The screen will involve positional-scanning combinatorial synthetic libraries, which are made up of chemical compounds with varying side chains. This work will develop novel tools for TB drug discovery that could identify new treatments for the emerging world threat of MDR-TB.

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[ES] La ingeniería tisular ha evolucionado hacia el modelado de la fisiología humana in vitro. El microambiente de la médula ósea (BM) es también hogar de procesos malignos. El mieloma múltiple (MM) es una neoplasia hematológica caracterizada por proliferación y acumulación en la BM de células plasmáticas monoclonales. Los tratamientos han mejorado, sin embargo, sigue siendo incurable. Moléculas de la matriz extracelular como fibronectina (FN) o ácido hialurónico (HA) tienen un papel reconocido en la resistencia a fármacos (DR). La inadecuación de los modelos preclínicos bidimensionales es una de las bases del problema de DR. Se han intentado diferentes enfoques in vitro, sin embargo, se basan en hidrogeles y andamios celulares diseñados para células adherentes, mientras que las células de MM presentan crecimiento en suspensión. El objetivo principal de esta Tesis es desarrollar, optimizar y validar una plataforma de cultivo 3D, denominada microgel, basada en microesferas en un medio líquido y que coexisten con células de MM creciendo dinámicamente en suspensión. Se desarrollaron y caracterizaron diferentes microesferas con diferentes funcionalizaciones. Optimizamos un protocolo de polimerización en suspensión para la obtención de microesferas a base de acrilatos con dos composiciones diferentes (presencia (10%) o ausencia (0%) de ácido acrílico (AA)) i dos distribuciones de tamaño diferentes (< 60 y > 70 ¿m). La FN se adsorbió en la superficie de la microesfera, mientras que el HA, colágeno I y diferentes secuencias peptídicas se injertaron covalentemente. Se modificaron las microesferas comerciales Cytodex 1 para adaptar sus características a la plataforma. Se utilizaron técnicas capa por capa (LbL) para introducir HA y sulfato de condroitina (CS) en su superficie. Por tanto, se ha generado un amplio repertorio de microesferas para desarrollar microgeles. Se optimizaron y validaron las condiciones de cultivo para la plataforma de microgel. Las condiciones óptimas se establecieron como 150 rpm de velocidad de agitación utilizando un agitador orbital y microesferas de < 60 ¿m. Los microgeles con diferentes composiciones y funcionalizaciones permitieron una buena proliferación de las líneas RPMI8226, U226 y MM1.S. Todos los sistemas respetaron el patrón de crecimiento en suspensión, factor que ha demostrado ser clave para su buen desempeño en cultivo 3D. En estudios iniciales de DR, la línea celular RPMI8226 cultivada en microgeles que contenían AA mostró una resistencia significativamente mayor a la dexametasona que sus cultivos en suspensión. Y las líneas RPMI8226, U226 y MM1.S cultivadas en microgeles que contenían AA mostraron una resistencia significativamente mayor a bortezomib que sus cultivos en suspensión. Por lo tanto, la presencia de AA en la matriz polimérica mostró un efecto positivo en la generación de DR in vitro y requerirá más estudios. Se ha validado la reducción de escala del sistema para trabajar con volúmenes más pequeños de microesferas y números reducidos de células, lo que es de gran relevancia para su traslación clínica. Finalmente, se han realizado cultivos preliminares con la línea celular RPMI8226 en los microgeles basados en Cytodex 1. Las microesferas de Cytodex 1 sin modificación tuvieron un efecto negativo sobre la viabilidad de las células de MM. La modificación mediante LbL con los pares quitosano/CS y quitosano/HA aumentó la viabilidad y proliferación. Sin embargo, estos sistemas no respetaron el carácter no adherente de las células MM. Hemos desarrollado y validado un novedoso sistema de cultivo basado en un medio 3D semisólido definido por microesferas y células de MM especialmente diseñado para células en suspensión. Este sistema constituye una herramienta versátil que debe explorarse más a fondo para el cultivo 3D de neoplasias hematológicas y para estudios de resistencia a fármacos in vitro. / [CAT] L'enginyeria tissular ha evolucionat cap al modelat de la fisiologia humana in vitro. El complex microambient de la medul·la òssia (BM) és també llar d'alguns processos malignes. El mieloma múltiple (MM) és una neoplàsia hematològica caracteritzada per una proliferació i acumulació a la BM de cèl·lules plasmàtiques monoclonals. Els tractaments han millorat, no obstant, el MM segueix sent incurable. Molècules de la matriu extracel·lular com fibronectina (FN) o àcid hialurònic (HA) tenen un paper reconegut en la generació de resistència a fàrmacs (DR) en MM. La inadequació dels models preclínics bidimensionals és una de les bases del problema de DR. Per això, s'han intentat diferents aproximacions in vitro, tanmateix es basen en hidrogels i andamis cel·lulars dissenyats per a cèl·lules adherents, mentre que les cèl·lules de MM presenten creixement en suspensió. L'objectiu principal d'aquesta Tesi és desenvolupar, optimitzar i validar una plataforma de cultiu 3D, denominada microgel, basada en microesferes en un medi líquid i que coexisteixen amb cèl·lules de MM que creixen dinàmicament en suspensió. S'han produït i caracteritzat diferents microesferes amb diferents funcionalitzacions. S'ha optimitzat un protocol de polimerització en suspensió per a l'obtenció de microesferes d'acrilats amb dues composicions diferents (presència (10%) o absència (0%) d'àcid acrílic (AA)) i amb dos distribucions de diàmetres diferents (< 60 y > 70 ¿m). La FN es va adsorbir, mentre que el HA, el col·lagen I i diferents seqüències peptídiques es van unir covalentment. S'han modificat microesferes comercials Cytodex 1 per tal d'adaptar les seves característiques a la plataforma del microgel. Mitjançant tècniques capa a capa (LbL) s'han introduït HA i sulfat de condroïtina (CS) a la seua superfície. Per tant, s'ha generat un ampli repertori de microesferes per desenvolupar microgels. Es van optimitzar i validar les condicions de cultiu per a la plataforma de microgel. Les condicions òptimes de cultiu es varen establir com a 150 rpm de velocitat d'agitació utilitzant un agitador orbital i microesferes de < 60 ¿m. Els microgels amb diferents composicions i funcionalitzacions van permetre una bona proliferació de les línies RPMI8226, U226 i MM1.S. Tots els sistemes van respectar el patró de creixement en suspensió, factor que ha demostrat ser clau per al seu bon rendiment en cultius 3D. En estudis inicials de DR línia cel·lular RPMI8226 cultivada en microgels que contenien AA va mostrar una resistència significativament major a la dexametasona que els seus cultius en suspensió convencionals. Línies RPMI8226, U226 y MM1.S cultivades en microgels que contenien AA mostraren una resistència significativament major a bortezomib que els seus cultius en suspensió convencionals. Per tant, la presencia d'AA a la matriu polimèrica de les microesferes va mostrar un efecte positiu en termes de generació de DR in vitro, cosa que requerirà estudis futurs. S'ha validat la reducció de l'escala del sistema per treballar amb volums més petits de microesferes i menys cèl·lules, el que és de gran rellevància per a la seva translació clínica. Finalment, s'han realitzat cultius preliminars amb la línia cel·lular RPMI8226 en els microgels basats en les Cytodex 1. Les microesferes de Cytodex 1 sense modificar van mostrar efecte negatiu sobre la viabilitat de les cèl·lules de MM. La modificació mitjançant LbL amb els parells quitosà/CS i quitosà/HA va augmentar la viabilitat i proliferació de cèl·lules MM. No obstant, aquests sistemes no respectaren el caràcter no adherent de les cèl·lules de MM. S'ha desenvolupat i validat un nou sistema de cultiu cel·lular basat en un medi 3D semisòlid definit per microesferes i cèl·lules de MM, especialment dissenyat per a cèl·lules no adherents. Aquest sistema constitueix una eina versàtil que ha de ser explorada per al cultiu 3D de neoplàsies hematològiques i per a estudis de resistència a fàrmacs in vitro. / [EN] Tissue engineering has evolved towards modeling of human physiology in vitro. The bone marrow (BM) microenvironment is likewise the home of some malignant processes. Multiple myeloma (MM) is a hematological neoplasia characterized by proliferation and BM accumulation of monoclonal plasma cells. Treatments have improved; however, MM remains incurable. Extracellular matrix molecules such as fibronectin (FN) or hyaluronic acid (HA) have a recognized role in drug resistance (DR). The inadequacy of two-dimensional preclinical models is one cause of the DR problem, different in vitro approaches have been developed, however, all these studies are based on hydrogels and scaffolds designed for adherent cells while MM cells are suspension growing cells. The main objective of this Thesis is to develop, optimize and validate a 3D culture platform, termed as microgel, based on microspheres suspended in a liquid media and coexisting with MM cells growing dynamically in suspension. Different microspheres with different functionalities were developed and characterized. We optimized a suspension polymerization protocol for the obtention of acrylates-based microspheres with two different compositions: with presence (10%) or absence (0%) of acrylic acid (AA). We obtained two different size distributions (< 60 and > 70 ¿m). FN was adsorbed on microsphere surface, while HA, collagen I and different peptide sequences were covalently grafted. Commercial Cytodex 1 microspheres were modified to adapt their characteristics to the microgel platform. Layer-by-layer (LbL) technics were used to introduce HA and chondroitin sulfate (CS) on Cytodex 1 surface. Therefore, a wide repertoire of microspheres has been generated to develop microgels. The culture conditions for the microgel platform were optimized and validated. Agitation is needed to keep microspheres and cells in suspension. Optimal culture conditions were 150 rpm of stirring speed using orbital shaker and < 60 ¿m diameter microspheres. Microgels with different compositions (0% AA, 10% AA) and functionalizations (none, HA, FN, collagen 1 and peptide sequences) allowed good proliferation of RPMI8226, U226 and MM1.S cells under 3D conditions. All the 3D systems respected the suspension growth pattern which appears as key factor for their good performance in 3D culture. In the initial DR studies, we found that MM cell line RPMI8226 cultured in microgels containing AA showed significantly higher resistance to dexamethasone than their conventional suspension cultures. And that MM cell lines RPMI8226, U226 and MM1.S cultured in microgels containing AA showed significantly higher resistance to bortezomib than their conventional suspension cultures. Thus, AA in the polymeric microsphere matrix showed a positive effect on the generation of DR in vitro and will require further studies. The scale-down of the system to work with smaller volumes of microspheres and reduced cell numbers has been validated, this is of great relevance for their clinical application. Finally, preliminary cultures with the cell line RPMI8226 have been performed with the Cytodex 1-based microgels. Cytodex 1 microspheres without modification had a negative effect on MM cells viability. LbL modification with the pairs chitosan/CS and chitosan/HA increased MM cells viability and proliferation. However, these systems did not respect the non-adherent character of MM cells. We have developed and validated a novel cell culture system based on a semi-solid 3D media defined by microspheres and MM cells which is specially designed for cells in suspension. It represents a versatile tool that should be further explored for the 3D culture of hematological malignancies and drug resistance studies in vitro. / Me gustaría agradecer al Servicio de Microscopía de la UPV y a sus técnicos por su valiosa ayuda con las técnicas de microscopía electrónica, a la Agencia Estatal de Investigación (proyecto PID2019-106099RB-C41 / AEI / 10.13039/501100011033) y al Ministerio de Ciencia, Innovación y Universidades (ayuda predoctoral FPU17/05810) que han financiado esta Tesis. / Clara Trujillo, S. (2022). Development of a 3D in Vitro Disease Model for Multiple Myeloma [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/186054