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Carbon nanotubes (CNTs) are of special interest to industry and they have been increasingly utilised as advanced nanovectors in drug/gene delivery systems. They possess significant advantages including high surface area, welldefined morphologies, unique optical property, superior mechanical strength and thermal conductivity. However, despite their unique and advanced physicochemical properties, the low compatibility of some of those materials [e.g. multiwalled CNTs (MWCNTs)] in most biological and chemical environments has also generated some serious health and environment concerns. Chemical functionalization broadens CNT applications, conferring new functions, and at the same time was found potentially altering toxicity. Although considerable experimental data related to functionalised CNT toxicity, at the molecular and cellular levels, have been reported, there is very limited information available for the corresponding mechanism involved (e.g. cell apoptosis, genotoxicity. The toxicity of carbon nanotubes has been confirmed on many cell lines including A549 (lung cancer cell line) and MRC-5 (lung fibroblasts). However, the sensitivity of each cell line in terms of cellular morphology, apoptosis and DNA damage are still unknown. In this report the different levels of cellular response to oxidative stress and phagocytosis have been investigated in A549, MCF-7 and MRC-5 cell lines to better understand the mechanisms of the toxicity pathway. siRNA as an ideal personalized therapeutics can specifically regulate gene expression, but efficient delivery of siRNA is difficult while it has been shown that MWCNTs protect siRNA, facilitate entry into cells. In this study, we comprehensively evaluated the in vitro cytotoxicity of pristine and functionalized (-OH, -COOH) multi-wall carbon nanotubes (MWCNTs), via cell viability test, reactive oxygen species (ROS) generation test, cell apoptosis and DNA mutation detection, to investigate the non-toxic dose and influence of functional group in A549, MCF-7 and MRC-5 cells exposed to 1-1000 μg/mL MWCNTs from 6 to 72 hours. In addition, 84 toxicity related genes have been detected to investigate the change of RNA regulation after treatment with MWCNTs. The research findings suggest that functionalized MWCNTs are more genotoxic compared to their pristine form, and the level of both dose and dispersion in the matrix used should be taken into consideration before applying further clinical applications of MWCNTs. Among all three cell lines, MCF-7 was the most sensitive to cell death and DNA damage induced by pristine carbon nanotubes. The majority of MCF-7 cell death was in necrotic. In A549 cells, apoptosis played a notable role in cytotoxicity. MRC-5 didn’t show significant cell loss or membrane damage, which might be explained by its low cell growth rate, notably however, a great reduction of the F-actin and attachment points was observed after treatment which indicates that MRC-5 cells are under very unhealthy condition and less attached to the bottom of flasks. Despite their toxicity, which is still being researched, carbon nanotubes have a great potential in clinical medicine. Thus, understanding the sensitivity of different cell lines could offer a more individualized approach for future treatment regimes. In regards to gene delivery, MWCNTs were found to be less toxic than chemical agents (positive control) without weakening the delivery efficiency, which proves that MWCNTs have a good potential in medicine area.

Les cellules NK sont capables d’ADCC (Antibody Dependent Cytotoxicity) suite à l’engagement durécepteur Fc!RIIIa/CD16a, et de fonctions effectrices directes antivirales et anti-tumorales: c’est la«cytotoxicité naturelle ». Ainsi activées elles peuvent également répondre en produisant des cytokines, commel’IFN-!. La dégranulation et la synthèse d’IFN-! par les cellules NK observées après engagement du récepteurCD16a, dont l’expression est indépendante du polymorphisme V158F, ont été largement supérieures à cellesobtenues avec les autres récepteurs activateurs. Son engagement par les AcMor thérapeutiques a produit desréponses fonctionnelles variables selon l’AcMor, et selon les donneurs de cellules. La perte d’expression duCD16a membranaire s’est révélé être un marqueur sensible de l’activation des cellules NK, même quand cedernier n’était pas engagé. Enfin, l’emploi de d’inhibiteur d’ADAM17 (TMI-2 et TIMP3) a permis d’observerle maintien de l’expression du CD16a après activation cellulaire sans augmenter les réponses fonctionnelles.Ce travail souligne la place centrale de l’engagement du CD16a dans l’activation NK. / NK cell can trigger ADCC (Antibody Dependent Cytotoxicity) through the engagement of theFc!RIIIa/CD16a receptor, and « Natural Cytotoxicity » after integration of cellular signals coming from theiractivating and inhibitory receptors. Moreover, activated NK cells produce cytokines such as IFN-!.Engagement by monoclonal antibodies (mAb) of CD16a was strongly more efficient than that of any otheractivating receptor to induce degranulation and IFN-! synthesis. Functional responses depend on thetherapeutic mAb used to engage CD16a and on the donor of NK cells. CD16a down-modulation was a verysensitive marker of NK cell activation, whatever the mean of activation. It was inhibited in the presence ofTMI-2 and TIMP3 (ADAM17 inhibitors), whereas CD16-dependent functional responses were not increased.This work highlighted the major role of the CD16a receptor in the activation of NK cells.

De nombreuses données expérimentales et cliniques ont montré l'importance des cellules Natural Killer (NK) dans l'immunosurveillance antitumorale. Les stratégies thérapeutiques basées sur les cellules NK pourraient donc être une alternative de choix dans le traitement de certains types de cancers. Nous avons focalisé notre étude sur deux types de cancer incurables malgré les récents progrès thérapeutiques : la leucémie lymphoïde chronique B (LLC-B) et le cancer de la prostate. Le but de notre étude est une meilleure compréhension des mécanismes mis en place par les cellules B leucémiques et les cellules tumorales prostatiques pour échapper à la réponse antitumorale des cellules NK. La connaissance de ces mécanismes d'échappement est un pré-requis indispensable à l'utilisation des cellules NK dans les thérapies antitumorales. Concernant la LLC-B, nos résultats suggèrent que les cellules NK de patients, fonctionnellement compétentes, ne peuvent pas initier une réaction immunitaire appropriée envers les cellules B leucémiques due au manque de reconnaissance de ces dernières. Concernant le cancer de la prostate, nos données préliminaires montrent que les cellules NK circulantes de patients sont également fonctionnellement compétentes, quelque soit le stade de la maladie, malgré la diminution significative de l'expression du récepteur NKp30. Ainsi, le degré d’immunogénicité des cellules B leucémiques et celui des cellules tumorales prostatiques devra être autant pris en compte que la fonctionnalité des cellules NK dans les stratégies visant à optimiser l'activité antitumorale de ces dernières. / Many experimental and clinical data have enlightened the importance of Natural Killer (NK) cells in tumor immunosurveillance. Therapeutic strategies based on NK cells could be an alternative in the treatment of certain cancers. We focused our study on two types of incurable cancers despite recent advances in treatment: B chronic lymphocytic leukemia (B-CLL) and prostate cancer. The aim of our study is a better understanding of the mechanisms set up by leukemic B cells and prostate cancer cells to escape from NK antitumor response. The knowledge of these escape mechanisms is an essential prerequisite to the use of NK cells in antitumor therapies. Regarding B-CLL, our results suggest that NK cells, although functionally competent, can not initiate an appropriate immune response against leukemic B cells due to a lack of recognition of the latter. Concerning the prostate cancer, our preliminary data show that circulating NK cells are functionally competent, whatever the stage of disease, despite the significant decrease in expression of the receptor NKp30. Thus, the degree of immunogenicity of leukemic B cells and of the prostate cancer cells must be taken into account as well as the functionality of NK cells in strategies aiming at improving NK antitumor activity.

The gene for methylthioadenosine phosphorylase (MTAP) is absent in almost 30% of cancers, opening a door for selective chemotherapy. One strategy to target the absence of MTAP involves the design of a cytotoxic methylthioadenosine (MTA) analogue. Non-cancerous cells would break down the cytotoxic analogue, since they contain MTAP, but cancerous cells would not, since they do not have MTAP. However, before such a compound can be made, we need to better understand the types of substrates accommodated by MTAP. The purpose of this thesis was therefore to explore a series of MTA analogues, probing the structure-function relationships between MTAP and specific structural modifications of MTA. Nine phenylthioadenosine (PTA) derivatives bearing ortho-, meta-, or para- methyl carboxylate, carboxylate, and hydroxymethyl substituents were synthesized and tested for cytotoxicity and as substrates for MTAP. The biological results of these nine compounds suggested that addition of substituents to the ortho-position was not tolerated by MTAP, and substituents similar to the hydroxymethyl might be accommodated by MTAP. None of the compounds were cytotoxic. This informed the design of ten more PTA derivatives, most of which were synthesized and tested for cytotoxicity and as substrates for MTAP. The range of functionalities included an amine, an acetamide, a urea, an isovaleramide, and an N-nitrosourea group inspired by the known anticancer agent lomustine. The amine derivatives of PTA were the best substrates of all MTA analogues tested (including PTA). The meta-amine derivative and the meta-isovaleramide showed minor cytotoxicity. Finally, the urea derivatives were moderate substrates of MTAP, and this pointed towards the future creation of other nitrosoureas as potential cytotoxic MTAP substrates. / Graduate / 2017-08-25

Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Jesika Štorkánová Supervisor: RNDr. Eva Novotná, Ph.D. Title of diploma thesis: Study of the cytotoxicity of selected chemotherapeutics for the treatment of leukemia in human tumor cell lines Leukemia represents a diverse group of malignant diseases with a hematopoietic disorder with different prognoses. As the incidence of patients with leukemia is increasing, is an effort to establish the treatment that will lead to successful therapy. One of the basic approaches to the treatment of leukemias is chemotherapy. Today it is known that the effectiveness of chemotherapy is influenced by a number of factors which can significantly affect the treatment strategy and thus decide on the outcome of the treatment itself. An important approach in chemotherapy is the selection of cytostatics with maximum efficacy for oncological disease and elimination cytostatics to which the cells are resistant based on the findings in in vitro conditions. The aim of this diploma thesis was to determine the inhibitory effects of in vitro selected chemotherapeutics in cell tumor lines. For determine the inhibitory effect, HCT116, HepG2 and HL-60 cell lines were selected using a colorimetric method based on the...

O desenvolvimento de adesivos bioativos mostra-se como uma alternativa interessante para agregar benefícios aos novos materiais. O objetivo do estudo foi sintetizar nanopartículas de prata (NAg) e de hidroxiapatita (NHA), avaliar sua influencia no efeito antibacteriano, citotoxicidade de um adesivo com NAg e sua resistência de união à dentina associado ao uso de NHA. As nanopartículas foram caracterizadas por fluorescência de raio x por reflexão total (TRXF), difratometria de raio x (DRX), microscopia eletrônica de transmissão (TEM) e espalhamento de luz visível (DLS). O sistema Scotchbond Multi-Purpose (SBMP) foi modificado com adição de 0,05% e 0,1% de NAg no primer e no bond. Molares humanos foram restaurados com o sistema adesivo modificado após o pre-tratamento da dentina com suspensão aquosa de NHA a 0,5% e 1% para realização do ensaio de resistência de união imediata (24h) e após 1 ano de envelhecimento. A interface adesiva foi caracterizada por Microscopia Confocal Raman (MCR). Para mensurar o efeito antibacteriano (S.mutans) do adesivo com NAg foi realizado o ensaio de difusão em ágar com template e contagem de Unidades Formadoras de Colônias (UFC). A citotoxicidade foi avaliada com uso de células tronco da polpa dentária (DPSCs) em contato com adesivos não polimerizados e polimerizados em diferentes concentrações de meio condicionado (0% 25%, 75% e 100%). Foram produzidas NAg esféricas, com estrutura cúbica de face centrada, e com 16 nm de diâmetro médio. As NHA exibiram estrutura prismática com tamanho médio de 79,4 nm. O adesivo com NAg e o tratamento da dentina com NHA não afetaram a resistência de união imediata ou após 1 ano de envelhecimento, e as interfaces adesivas mostraram-se íntegras. Os adesivos modificados por NAg exibiram citotoxicidade similar ao adesivo controle e maior efeito antibacteriano. Os adesivos com adição de NAg são promissores quanto à obtenção de um material bioativo antimicrobiano que não altera a resistência de união ou a biocompatibilidade. / The development of bioactive adhesives is shown an interesting alternative to add benefits to the new materials. The aim of the study was to synthesize silver nanoparticles (NAg) and hydroxyapatite (NHA) and to evaluate their influence on the bond strength, antimicrobial effect, and biomaterials cytotoxicity. The nanoparticles were characterized by x-ray fluorescence by total reflection (TRXF), x-ray diffractometry (XRD), transmission electron microscopy (TEM) and visible light scattering (DLS). The Scotchbond Multi-Purpose system (SBMP) was modified with 0.05% and 0.1% silver in the primer and bond. Human molars were restored with the modified adhesive after pre-treatment of dentin with 0.5% and 1% NHA for the immediate bond strength test (24h), and after 1 year of aging. The adhesive interface was characterized by Confocal Raman Microscopy (MCR). To measure the antibacterial effect (S.mutans) of the adhesive with NAg, the agar diffusion assay with template was performed and counting of Colony Forming Units (UFC). In order to evaluate the cytotoxicity, dental pulp stem cells (DPSCs) were used in contact with unpolymerized and polymerized adhesives in different concentrations of conditioned medium (0% 25%, 75% and 100%). Spherical NAg, with a cubic face centered structure, and 16 nm in diameter were produced. The NHA exhibited a prismatic structure with approximately 79.4 nm. The adhesive with NAg and the treatment of dentin with NHA did not affect the bond strength immediately or after 1 year of aging, and the adhesive interfaces were shown to be intact. NAg-modified adhesives exhibited cytotoxicity similar to the control adhesive and higher antibacterial effect. The adhesives with NAg addition are promising in obtaining a bioactive antimicrobial material that does not alter the bond strength or the biocompatibility.

Antraciclinas estão entre os mais eficazes quimioterápicos contra o cancer. São fármacos glycosídicos compostos pelo carboidrato daunosamina ligado a uma aglicona hidróxi antraquinona, e atuam por intercalação ao DNA, geração de estresse oxidative e envenenamento de topoisomerase II. Apesar de sua utilidade terapêutica, multirresistência e cardiotoxicidade grave são importantes limitações decorrentes do tratamento com antraciclinas, estimulando a descoberta de novos análogos, por exemplo através de glicodiversificação. Este trabalho objetivou explorar azido glicosídeos, a serem combinados com agliconas de antraciclinas para gerar novos glicosídeos. Em uma estratégia semi-sintética, daunorrubicinona e doxorrubicinona protegida foram glicosiladas com doadores 2-azido glucosídicos e -galactosídicos, além de glicais. Uma varredura de metodologias de glicosilação envolveu cloretos, imidatos e tioglicosídeos, sendo os promotores com melhores rendimentos HgO/HgBr2 (4-52%) e TMSOTf (38-41%); para glucais e galactais, catalisadores de Au(I) and Cu(I) forneceram moderados rendimentos (15-46%), mas o sistema mais eficiente foi o organocatalisador de tiouréia e ácido fosfórico (18-95%). A clivagem dos grupos de proteção foi desafiadora, dificultando e atrasando a obtenção dos glicosídeos livres. Mediante desproteção, os glicosídeos obtidos incluíram glucosídeo 49 (13%), 2-azido glucosídeo 51 (34%), 2-desóxi glucosídeo 58 (11%) e 2-desóxi galactosídeo 61 (85%), todos com o esqueleto de daunorrubicina. Em ensaios de proliferação celular, os glicosídeos 61? e 61? foram testados em linhagens de células tumorais humanas HeLa, MDA-MB-231 e MCF-7 e um modelo de células sadias (HDF), com IC50 na faixa de 27.1 a 74.6 ?M para o anômero ?, e superior a 250 ?M para o anômero ?. Estudos preliminares com cardiomiócitos humanos derivados de células-tronco induzidas foram inconclusivos para estabelecer um modelo experimental de toxicidade cardíaca. / Anthracyclines are ranked among the most effective chemotherapeutics against cancer. They are glycoside drugs comprised by the aminosugar daunosamine linked to a hydroxyanthraquinone aglycone, and act by DNA-intercalation, oxidative stress generation and topoisomerase II poisoning. Regardless of their therapeutic value, multidrug resistance and severe cardiotoxicity are important limitations arising from anthracycline treatment, prompting the discovery of novel analogues, for instance through glycodiversification. This work aimed to exploit azido glycosides, to be combined with anthracycline aglycone and generate novel glycosides. In a semi-synthesis approach, both daunorubicinone and protected doxorubicinone were glycosylated with conveniently functionalised 2-azido glucosyl and galactosyl donors, as well as glycals. A screening of glycosylation protocols involved glycosyl chlorides, imidates and thioglycosides with the most successful promoters being HgO/HgBr2 (4-52% yield) and TMSOTf (38-41%); for glucals and galactals, Au(I) and Cu(I) catalysts gave moderate yields (15-46%), but thiourea-phosphoric acid was the most efficient catalyst system (18-95%). Cleavage of protecting groups proved challenging, hampering and delaying the obtention of free glycosides. Upon deprotection, the glycosides obtained included glucoside 49 (13%), 2-azido glucoside 51 (34%), 2-deoxyglucoside 58 (11%), and 2-deoxygalactoside 61 (85%), all with the daunorubicin scaffold. In cell proliferation assays, glycosides 61? and 61? were tested against human cancer cell lines HeLa, MDA-MB-231 and MCF-7 and a model of healthy cells (HDF), with IC50 in the range of 27.1 to 74.6 ?M for the ? anomer, and higher than 250 ?M for the ? anomer. Preliminary studies with human cardiomyocytes derived from induced pluripotent stem cells were inconclusive to establish a cardiac toxicity experimental model.

Les sarcomes sont des cancers rares touchant toutes les zones du corps humain, caractérisés par une grande diversité de nature, de comportement clinique et de réponse aux thérapies existantes, certains étant de bon pronostic, d’autres très difficilement curables.Leur traitement de référence est la chirurgie ; la radiothérapie et les protocoles de chimiothérapie n’ayant que peu évolué lors des 30 dernières années.Récemment des caractéristiques génétiques leur étant propres ont été découvertes, prédictives de leur agressivité et contre lesquelles il est possible de diriger des drogues spécifiques pouvant améliorer le pronostic et diminuer les effets secondaires des thérapies conventionnelles.C’est sur l’inhibition d’Aurora Kinase A et B et CHK1 que s’est focalisé ce travail avec le test des effets de deux nouvelles drogues sur 9 types de cellules cancéreuses sarcomateuses avec des résultats très prometteurs, qu’il conviendra de conforter par d’autres expériences, notamment sur l’animal. / Sarcomas are rare cancers, which may arise in all parts of human body. They are characterized by great diversity in their nature, clinical behavior and response to existing therapeutics. Some are of good prognosis and others hard to cure.Their treatment essentially relies on surgery and radiotherapy or chemotherapy haven’t know major breakthrough over the last 3 decades.Recently new genetics abnormalities linked to sarcomas have been discovered. Their analysis can predict their aggressiveness and it is now possible to develop targeted therapies against them. This could help improving cancer prognosis and/or limiting conventional drugs adverse effects.Our work focused on Aurora Kinase A and B and CHK1 inhibition, testing the effects of 2 new drugs on 9 types of sarcoma cells with promising results, which we will confort by other experiments, including on the animal.