Syftet med detta projekt var att undersöka hur vanligt använda cancerläkemedel påverkar mRNA-uttryck och kromatinorganisation i humana erytroleukemiceller. Som modell användes K562-celler från en patient i blastocystkris (2), för att utvärdera leukemicellernas svar på cancerläkemedel vinblastin och doxorubicin. Vinblastin och doxorubicin valdes på grund av deras distinkta mekanismer i cancercellen: medan doxorubicin interkaleras i DNA, hämmar topoisomeras II-aktivitet vilket orsakar celldöd, riktar vinblastin sig mot mikrotubuli för att stoppa mitotisk delning och proliferation. Uttryck av mRNA undersöktes i celler vid 0-timmar, 6-timmar och 24-timmar drogbehandling, samt efter en veckas återhämtning från 24-timmars drogbehandling. Kromatintillgänglighet med ATAC-seq undersöktes i K562-celler vid 0- timmar, 1-timmar, 6-timmar, 24-timmar och 24-timmar + en veckas återhämtning. Därefter utfördes DNA (ATAC-seq) och RNA (mRNA-seq) extraktion och biblioteksberedning på tre biologiska replikat, och öppna DNA-regioner samt mRNA expression undersöktes via sekvensering. Resultaten visade en stark korrelation mellan de biologiska replikaten, vilket indikerar att resultaten var upprepbara. Differentiellt uttryck av mRNA vid doxorubicin- och vinblastinbehandlingar utfördes genom att jämföra mRNA-nivåerna i läkemedelsbehandlade prover med obehandlade (0-timmar). Uppreglerade och nedreglerade gener identifierades och MA-grafer genererades för att visuellt analysera de differentiellt uttryckta generna vid olika tidpunkter efter läkemedelsbehandling och en veckas återhämtning. För att hitta anrikningar av funktionella genkategorier bland de läkemedelsinducerade eller -undertryckta generna, utfördes genontologianalyser. Slutligen användes verktyget Integrative Genomics Viewer (IGV) för att visuellt utforska mRNA-nivåerna och deras differentiella uttrycksmönster under läkemedelsbehandlingar. För ATAC-seq utfördes inte detaljerad dataanalys på grund av tidsbegränsning, men genomets öppenhet undersöktes visuellt genom IGV. Sammantaget inducerade doxorubicinbehandling en långsamt men långvarig förändring av genuttrycket, vilket involverade flera olika biologiska processer. Doxorubicinbehandlade K562-celler ändrade genuttryck att stöda kemoresistens snarare än att inducera apoptos eller celldöd. Behandlingen hade en långvarig inverkan på mRNA-nivåer som sträckte över återhämtningsveckan. Den totala uttrycksförändringen i återhämtningsproverna var förknippad med återhämtning av tumörigena egenskaper och återställning av mekanismener som stöder cellernas tillväxt. Vinblastine förorsakade snabb ökning av mRNA involverade i cytoskelettet. Vid 24-timmars vinblastinbehandling upplevde tumörcellerna stress på grund av grovt elongerad struktur, och de inducerade gener som stöder tumörbildning. En ökning av totala mRNA-nivåer detekterades i vinblastinbehandlade K562-leukemiceller, vilket var särskilt tydligt under återhämtningen. Resultaten visade att cellerna som överlevde vinblastinbehandling fokuserade på att återställa sin strukturella form. Sammantaget visade resultaten att monoterapi inte fungerar effektivt mot leukemiceller eftersom K562-leukemiceller inte bara överlevde läkemedelsbehandlingarna utan också inducerade mRNA som är involverade i resistens mot läkemedelsbehandlingar. / The primary objective of this project is to investigate how commonly used cancer drugs affect mRNA expression and chromatin organization in human erythroleukemia cells. As a model, K562 cells derived from a patient in blastocyst crisis (2) were utilized, evaluating the leukemia cells’ cellular responses to cancer medicines vinblastine and doxorubicin. Vinblastine and doxorubicin were chosen due to the distinct pathways they target in the cell: while doxorubicin intercalates into DNA and inhibits topoisomerase II activity, which eventually cause cell death, vinblastine targets microtubules to stops mitotic division and excessive proliferation. Expression of mRNA was investigated in cells harvested at 0h, 6h, 24h and 24h + one week recovery. Chromatin accessibility with ATAC-seq was investigated in K562 cells harvested at 0h, 1h, 6h, 24h and 24h + one week recovery. Then DNA (ATAC-seq) and RNA (mRNA-seq) extraction and library preparation were performed on three replicates, and the genome-wide results was investigated via sequencing. The results showed a strong correlation between the biological replicates, indicating that the experimental conditions were sustained in these biological variables. Differential Expression of mRNA upon doxorubicin and vinblastine treatments was performed by comparing the mRNA levels in drug-treated samples to non-treated (0h) upregulated and down regulated genes were identified and MA plots generated to visually analyze the differentially expressed genes at different time points after drug treatment and one week recovery. To find enrichments of functional gene categories among the drug-induced or -repressed genes, gene ontology analyses were performed. Finally, the Integrative genomics viewer (IGV) tool was used to visually explore the mRNA levels and their differential expression pattern during drug treatments. For ATAC-seq, detailed data analysis was not performed due to limitation of time, and data was only visually explored through IGV. Taken together, doxorubicin treatment showed slow initial response within 6h followed by an extensive change in gene expression in 24h, involving several different biological processes. The response was more inclined towards chemoresistance rather than inducing apoptosis or cell death. There was a sustained increase in mRNA levels of doxorubicin treated leukemia cells during recovery week. The overall expression change in the recovery samples was majorly linked with not only gaining back the tumourigenic properties and restoring the mechanism which were affected by doxorubicin action but, based on changes in mRNA expression, it looks like doxorubicin treatment made the tumour cells more aggressive. The initial, 6h, response to vinblastine increases mRNAs involved in cytoskeleton. Upon 24h vinblastine treatment the tumour cells experienced stress due to shear force and structural deformity, and they induced genes supporting tumourigenesis. An increase in total mRNA levels was detected in vinblastine-treated K562 leukemia cells, which was particularly evident during recovery. The results indicated that the cells that survived vinblastine treatment focused on recovering its structural form. Overall, the results indicated that monotherapy does not effectively work against leukemia cells as K562 leukemia cells not only survived the drug treatments but also induced mRNAs involved in resistance against drug treatment.

Σήμερα η μελέτη των βιοϋλικών προσανατολίζεται σε δύο κατευθύνσεις, την ανάπτυξη συστημάτων μεταφοράς φαρμάκων και συστημάτων κατάλληλων να διεγείρουν κυτταρικές λειτουργίες. Η έρευνά μας έχει σχέση με την συγκριτική μελέτη συστημάτων μεταφοράς φαρμάκων κατάλληλων για εφαρμογή σε οστικούς καρκίνους. Τέτοιου είδους συστήματα θα πρέπει, αρχικά να είναι ικανά να μεταφέρουν τα φαρμακευτικά μόρια και στη συνέχεια να μπορούν να επάγουν οστεογένεση. Η δεύτερη λειτουργικότητα είναι ιδιαιτέρως σημαντική καθώς έχει σαν αποτέλεσμα την πλήρωση του οστικού ελλείμματος που προκαλείται από την δράση των καρκινικών κυττάρων. Για τον σκοπό αυτό, διερευνήθηκε η ικανότητα του υαλώδους δικτύου να μεταφέρει φαρμακευτικά μόρια μέσω παραδοσιακών συστημάτων μεταφοράς. Συνεπώς, ακολούθησε η ex vitro μελέτη pH-ευαίσθητων τροποποιημένων πυριτικών ξηρών πηκτών στα οποία είχε συνδεθεί το αντικαρκινικό φάρμακο δοξορουπμυσίνη. Συγκεκριμένα, πυριτικά ξηρά πηκτώματα συντέθηκαν με την μέθοδο sol-gel και τροποποιήθηκαν περαιτέρω με χημεία καρβοδιϊμιδίου. Η τροποποίηση είχε σαν αποτέλεσμα την επιφανειακή σύνδεση υδροπηκτών δεξτράνης που παρουσιάζουν ευαισθησία στο pH. Στη συνέχεια, ακολούθησε η σύνθεση των ανόργανων βιοενεργών νανοσφαιρών. Για την σύνθεση των υαλωδών νανοσφαιρών με εσωτερική κοιλότητα ακολουθήθηκε η διαδικασία επικάλυψης sol-gel, κατά την οποία έγινε η ηλεκτροστατική επικάλυψη νανοσωματιδίων πολυστυρενίου με αποτέλεσμα την σύνθεση ανόργανων πυριτικών και φωσφοπυριτικών νανοσφαιρών. Επιπλέον, μελετήθηκε η εφαρμογή του συμπολυμερούς του πολυμεθακρυλικού μεθυλεστέρα – υδρομεθακρυλικού προπυλεστέρα ως υποστρώματος καθώς το PMMA αποτελεί βασικό συστατικό των οστικών τσιμέντων και παρουσιάζει βελτιωμένες μηχανικές ιδιότητες. Προκειμένου να ολοκληρωθεί η συγκριτική μελέτη μας, ακολούθησε η ex vitro ανάλυση των παραπάνω υβριδικών φωσφοπυριτικών νανοσφαιρών καθώς επίσης των πυριτικών νανοσφαιρών PS και PMMA-co-HPMA. Η επώαση σε διάλυμα SBF οδήγησε στον σχηματισμό ανθρακικού πυριτικού υδροξυαπατίτη με το μέγεθος των κρυσταλλιτών να μην υπερβαίνει τα 45 nm και έντονη παρουσία συσσωματωμάτων. Βέλτιστες ιδιότητες βιοενεργότητας παρουσιάζουν οι τροποποιημένες με αμίνες υβριδικές νανοσφαίρες PMMA-co-HPMA, οι οποίες έχουν επίσης την δυνατότητα να χρησιμοποιηθούν ως μεταφορείς φαρμακευτικών μορίων σε όξινο καθώς επίσης και σε φυσιολογικό pH με παρατεταμένη δυνατότητα αποδέσμευσης. / Recently the study of biomaterials has moved in two directions, the evolution of drug delivery systems and of systems that can stimulate specific cellular responses. Our investigation aims to the study of drug delivery systems for bone cancer therapy. These systems must fulfill two important functionalities. At first, they should be able to deliver drug molecules to bone cancer environment through loading or surface conjugation and subsequently to cause osteogenesis. Their second functionality is especially important since it leads to substitution of bone defects caused from the action of cancer cells. For this purpose, the ability of the glassy network to deliver drug molecules was studied. For this purpose, expanded ex vitro research was followed in DOX conjugated pH-sensitive functionalized silica xerogels. Specifically, silica xerogels were synthesized through a sol-gel process and further functionalized with carbodiimide chemistry. The functionalization process resulted in pH-sensitive dextran hydrogels. The study of the enhanced properties of glassy substrates was followed by the synthesis of amorphous bioactive nanospheres. Moreover, the change of the organic core and the use of PMMA-co-HPMA were advantageous due to the enhanced mechanical properties of PMMA-co-HPMA and its use in bone cements. In order to accomplish our comparative investigation, we followed the ex vitro study of the above hybrid binary silicate, ternary and quaternary phosphosilicate nanospheres as well as the silicate PS and PMMA-co-HPMA nanospheres. The incubation in SBF solution resulted in the formation of a silica-substituted carbonate hydroxyapatite (Si-HCA) a with crystallite size of around 45 nm and extended surface aggregates. The amino-modified PMMA-co-HPMA hybrid nanospheres present enhanced biocompatible properties, with prolonged release ability as drug delivery systems, in acidic as well as physiological pH.

Polymeric monodispersed nanocarriers with controlled shape and size have been fabricated in the literature primarily using top down processes such as imprint lithography. In this dissertation, the geometric and material property limits of imprint based techniques have been studied. The resulting insight has led to the creation of new processes that significantly extend the limits of imprint processes in several ways: (i) Ability to print nanocarriers with ultra-soft biomaterials (<1MPa modulus); (ii) Sub-50nm diameter cylindrical particles with >3:1 aspect ratio with >5x enhanced wafer yield; (iii) Creation of reentrant barrel shapes that have the potential to be valuable in cellular uptake, such shapes being significant as they lead to fundamental demolding challenges in prior imprint processes; and (iv) Multi-layer nanocarriers which can potentially provide sophisticated functionality such as tailored release kinetics of one or more drugs. By understanding the requirements of bio-functional nanocarriers and related manufacturing constraints, a previously explored Bio Jet and Flash Imprint Lithography (Bio J-FIL) process was refined to perform successful imprints and improve the nanocarrier fabrication scalability. Next, two new fabrication processes have been developed. The first process is called Decoupled Functional Imprint Lithography (D-FIL) which allows fabrication of ultra-soft bio-functional materials (modulus of <1 MPa), challenging sizes (sub-50nm diameter cylinders with aspect ratio > 3:1), and reentrant barrel shapes. The second decoupled process, Dual Removable Layer Lithography (DRLL), has been developed to specifically create multi-layered cylindrical nanocarriers. Nanocarriers fabricated with D-FIL and DRLL process have been shown to chemically bind with an imaging agent, and model anti-cancer drugs. Drug (siRNA) retention (>90% over 9 days) and stimuli triggered release studies were performed on sub-100nm cylindrical PEGDA nanocarriers. It was found that these nanocarriers show accelerated triggered drug release when exposed to a hydrolase, Cathepsin B. While the exact mechanisms causing the triggered release are not fully understood, a few possible explanations are provided based on the experiments reported. Finally, the D-FIL, the DRLL, and the refined Bio J-FIL processes have been successfully demonstrated at the prototype scale as well as at the pilot scale in collaboration with an industrial partner, Molecular Imprints Inc. / text

Des nanoparticules d’oxydes de fer superparamagnétiques (SPIONs) PEGylées ont servi de plateforme pour la formulation de nanovecteurs théragnostiques pour la délivrance d’un agent anticancéreux, la doxorubicine (DOX). Le chargement de la DOX sur les nanovecteurs à l’aide d’un complexe avec l’ion fer (II) a été optimisé. Ce complexe se dissocie en milieu acide, typique des compartiments intracellulaires. La spectroscopie Raman exaltée de surface (SERS) a confirmé que les nanovecteurs libèrent la DOX sous forme non complexée. La cytotoxicité in vitro induite par la libération de la DOX a été évaluée sur différentes lignées cellulaires de cancer du sein, et comparée à celle de la DOX en solution. Les voies d’internalisation des nanovecteurs ont été explorées en microscopie électronique en transmission (MET), et le devenir intracellulaire de la DOX a été suivi en imagerie confocale multispectrale (ICMS). Enfin, un protocole thérapeutique in vivo chez la souris tumorisée a permis d’évaluer la capacité de la nanoformulation à limiter la croissance tumorale, la possibilité d’un ciblage magnétique, et la réduction des effets secondaires induits par la DOX. / PEGylated superparamagnetic iron oxide nanoparticles (SPIONs) were used as a platform to build theranostic nanovectors for the delivery of an anticancer drug, doxorubicin (DOX). The DOX loading on nanocarriers via a DOX-iron (II) complex was optimized. The complex dissociates at low pH, typical of intracellular compartments. Surface enhanced Raman scattering (SERS) confirmed that the nanovectors released DOX under free form. In vitro cytotoxicity due to DOX loaded on nanocarriers was performed on different breast cancer cells, and compared to that of DOX in solution. Internalization pathways of nanovectors were explored with transmission electron microscopy (TEM), and intracellular fate of DOX was monitored by confocal spectral imaging (CSI). To finish, a therapeutical protocol was performed on tumorized mice, in order to evaluate the efficacy of the nanoformulation on tumor reduction, the possibility of magnetic targeting, and the decrease of side effects induced by DOX.

Tumor diseases are severe problem worldwide with increasing number of patients suffering from various types of malignancies. Many of approved therapeutics cause serious side toxicities. Therefore, there are intensive efforts to improve cancer treatment protocols. The aim of this study was to deplete regulatory T (Treg) cells without affecting other immunocompetent cells playing a positive role in tumor eradication. Treg cells were reported to hamper anti-tumor immunity and promote tumor growth and survival. Thus, their selective elimination could lead to induction of anti-tumor responses and tumor rejection if combined with chemotherapy with selected N-(2- hydroxypropyl)methacrylamide (HPMA) copolymer-bound drug conjugates. Original approach was to deplete of Treg cells without the use of anti-CD25 mAb that has been widely exploited for Treg cell elimination; however, its long-term persistence in circulation together with inhibitory effect on activated effector cells (CD25+ ) are its main disadvantages. Thus, Treg cells were sensitized to cell cycle-specific cytostatic drugs via application of IL-2/anti-IL-2 JES6.1 mAb immunocomplexes that induce vigorous selective proliferation of this cell population. Subsequent application of cell cycle-specific cytostatics showed steep decrease of Treg cell...

L’auto-assemblage de copolymères à blocs amphiphiles est un outil puissant de la chimie supramoléculaire pour la conception de nano-objets complexes et fonctionnels. Dans ces travaux de thèse, l’étude approfondie d’un copolymère à blocs « hybride » synthétique-b-peptidique poly(triméthylène carbonate)-b-poly(acide glutamique) pour des applications de vectorisation a été menée. Des morphologies vésiculaires, obtenues par auto-assemblage en voie « co-solvant » et présentant une grande stabilité ainsi qu’un caractère stimulable ont été mises en évidence. Une transition inédite en température, par des phénomènes de fusion et de fission, a pu être observée. L’encapsulation dans ces vésicules polymères d’un principe actif anti-tumoral et de nanoparticules magnétiques, à des taux très élevés, permet d’améliorer le contraste en IRM ainsi que de moduler la libération de la molécule par une variation des paramètres environnementaux (pH, T) ou par un effet d’hyperthermie magnétique. / Block copolymer self-assembly is a powerful tool within supramolecular chemistry to design smart and functional nano-objects. In this thesis work, comprehensive study of hybrid poly(trimethylene carbonate)-b-poly(glutamic acid) block copolymers for drug delivery applications has been conducted. Highly stable vesicular morphologies presenting stimuli-responsive behaviour were prepared using a solvent-injection method. In particular, original temperature responsiveness mediated by fusion and fission events has been evidenced. Dual loading of an anticancer drug and superparamagnetic nanoparticles in these vesicles, at very high loading contents, allows enhancing MRI contrast and controlling drug release kinetics by varying environmental conditions (pH, T) or by using a magnetic hyperthermia effect.

O objetivo geral deste trabalho foi explorar a versatilidade de filmes multicamadas de polieletrólitos (PEM) e suas aplicações em sistemas de entrega de drogas e como filmes funcionais para aplicações biomédicas. Filmes PEM montados pela técnica de camada por camada (layer­-by­-layer, LbL), foram explorados em três aplicações principais. Na primeira, foi explorado o desenvolvimento de um protocolo de funcionalização em filmes de poli(alilamina)/poli (estireno sulfonato), PAH/SPS. Os parâmetros de construção do filme para biotinilação dos grupamentos amina do PAH foram otimizados e aplicados na captura e detecção do antígeno específico da próstata (PSA), na concentração de 100 a 0,1 ng/mL, usando pontos quânticos (Qdots). Em comparação com outros trabalhos, este sistema apresentou uma boa sensibilidade na detecção de PSA, dentro do limite de detecção clínica de 0,4 a 0,1 ng/mL. A segunda aplicação envolveu o desenvolvimento de filmes de sacrifício baseados nas interações naturais da mucina submandibular bovina e da lectina, jacalina (BSM/JAC). Filmes de BSM/JAC apresentaram estabilidade quando submetidos a uma ampla faixa de pH (pH 3-­-9) e em solução de alta força iônica (5 M NaCl). A dissolução dos filmes BSM/JAC pôde ser seletivamente desencadeada mediante à incubação em solução de melibiose, 37 °C, pH 7,4, sem apresentar citotoxicidade às células. Na última parte deste trabalho, a incorporação de lipossomos ecogênicos (ELIP) em mochilas celulares foi investigada. Mochilas celulares são \"patches\" de 7­-10 &#181;m de diâmetro que podem ser fabricados por meio de deposição alternada de polímeros utilizando-­-se a técnica de LbL, sobre uma matriz pré­-moldada obtida por fotolitografia, a fim de criar um sistema composto por três multicamadas estratificadas: uma região de liberação, para promover o destacamento do substrato, uma região de carga de droga, e uma região adesiva às células. O uso de ELIP permitiu incorporação de até 9x mais doxorrubicina (DOX) se comparado com o fármaco livre em solução absorvido pelos dos filmes. A liberação de DOX pelos filmes foi monitorado por 25 dias. Mochilas contendo ELIP-­DOX foram então aderidos a monócitos, e sua viabilidade monitorados por 72h. Mochilas vazias mostraram diminuir a proliferação de monócitos ao longo das 72 horas, enquanto mochilas carregadas com ELIP-­DOX mostraram uma diminuição dramática na população celular, apontando uma potencialização dos efeitos da droga pela sua proximidade com as células. / The overall goal of this thesis was to exploit the versatility of polyelectrolite multilayers (PEM) to be applied in drug delivery systems and biofunctionalizable films for biomedical applications. PEM films assembled by the layer-by­-layer technique were explored in three main applications. In the first part of this work, the development of a functionalization protocol of poly(allylamine)/poly(styrene sulfonate), PAH/SPS was explored. The optimal film parameters to the use of biotinylated multilayers were applied for the capture and detection of prostate specific antigen (PSA) protein in the range of 100 to 0.1 ng/mL, by using quantum dots. Compared to previous work, this system presented a good sensitivity for PSA detection that is within the clinical limit range of 0.4 to 0.1 ng/mL. The second application involved the creation of a novel sacrificial multilayer film. Films based in natural interactions of bovine submaxillary mucin and the lectin jacalin, BSM/JAC were assembled. BSM/JAC films showed stability when underwent a wide rage of pH (pH 3 to 9) and high ionic strength (5 M NaCl) solutions. BSM/JAC dissolution could be triggered released by incubation in melibiose at 37 °C in pH 7.4 buffer, without cytotoxicity. In the last part of this work the incorporation of echogenic liposomes (ELIP) into cell backpacks was investigated. Cell backpacks are 7-10 &#181;m diameter patches that can be fabricated through LbL polymer deposition onto a photopatterned array to create a stacked composite of three stratified multilayer systems: a releasable region for easy detachment from the substrate, a drug payload region, and a cell adhesive region. The use of ELIP allowed up to 9x more doxorubicin (DOX) loading when compared to free drug in solution adsorbed through the films. DOX release from films was monitored for over 25 days. ELIP­-DOX backpacks were then attached to mouse monocytes and their viability monitored by 72h. Empty backpacks showed to decrease monocytes proliferation over the course of 72h, while ELIP­-DOX backpacks showed a dramatic decrease in cell population, showing that DOX effects were enhancement in drug potency by its proximity.

Pacientes com câncer de mama localmente avançado são submetidas à quimioterapia neoadjuvante na tentativa de reduzir a dimensão do tumor e aumentar a possibilidade da realização de uma cirurgia conservadora. Nosso grupo identificou previamente através da tecnologia de cDNA microarray, trios de genes, incluindo BZRP, CLPTM1, MTSS1, NOTCH1, NUP210, PRSS11, RPL37A, SMYD2 e XLHSRF-1, cuja expressão era capaz de predizer a resposta à quimioterapia neoadjuvante com doxorrubicina e ciclofosfamida em pacientes com câncer de mama. No presente estudo, avaliamos se a expressão destes genes é reprodutível na identificação de pacientes responsivas e não-responsivas através de RT-PCR em tempo real, que representa uma técnica mais acessível. Avaliamos inicialmente amostras de 28 pacientes anteriormente estudadas (grupo de validação técnica = 23 responsivas e cinco não-responsivas) e a seguir um grupo de 14 novas pacientes (grupo de validação biológica = 11 responsivas e três não-responsivas). Dentre os trios de genes inicialmente identificados, a expressão de RPL37A + XLHSRF-1 + NOTCH1 e RPL37A + XLHSRF-1 + NUP210 classificou corretamente 86% (24/28) das amostras do grupo de validação técnica e 71% (10/14) das amostras do grupo de validação biológica, através de análise de classificação discriminante. Desse modo, esses trios não demonstraram a mesma precisão em comparação com resultados de cDNA microarray. Uma nova análise combinatória foi realizada na procura do melhor modelo preditivo utilizando valores de expressão obtidos por RT-PCR em tempo real. Identificamos então um novo trio, composto pelos genes RPL37A, SMYD2 e MTSS1, cuja expressão classificou corretamente 93% das amostras do grupo de validação técnica (22/23 responsivas e 4/5 não-responsivas) e 79% do grupo de validação biológica (8/11 responsivas e 3/3 não-responsivas). Portanto, o teste apresentou 88% de sensibilidade e especificidade em detectar pacientes responsivas para o total de amostras analisadas. Ao verificarmos o poder de classificação do mesmo grupo de genes, utilizando os valores de expressão pela análise de cDNA microarray, observamos um resultado semelhante (91% de sensibilidade e especificidade em reconhecer as amostras responsivas). Dessa forma, demonstramos que o perfil de expressão gênica obtido com cDNA microarray é reprodutível através do uso de RT-PCR em tempo real. Um estudo integrando um maior número de pacientes e uma plataforma de cDNA microarray mais abrangente pode auxiliar na identificação de um modelo preditivo baseado em grupos de genes mais acurado para antever a resposta ao tratamento com quimioterapia baseada em doxorrubicina. / Patients with locally advanced breast cancer are submitted to primary chemotherapy as an attempt to reduce tumor dimension and increase breast conserving surgery rates. Our group has previously identified through cDNA microarray technology gene trios, including BZRP, CLPTM1, MTSS1, NOTCH1, NUP210, PRSS11, RPL37A, SMYD2 and XLHSRF-1, whose expression was capable of predicting response to neoadjuvant chemotherapy with doxorubicin and cyclophosphamide in breast cancer patients. In the current study, it was evaluated whether expression of these genes is reproducible in the identification of responsive and non-responsive patients by real time RT-PCR, which represents a more accessible technique. We initially evaluated samples from 28 patients earlier studied (technical validation group = 23 responsive and 5 non-responsive) and subsequent to a new 14 patients set (biological validation group = 11 responsive and three non-responsive). Among the initially identified gene trios, RPL37A + XLHSRF-1 + NOTCH1 and RPL37A + XLHSRF-1 + NUP210 expression correctly classify 86% (24/28) samples from the technical validation group and 71% (10/14) samples from the biological validation group, through discriminant classification analysis. Therefore, these trios didnt demonstrate the same precision as compared with cDNA microarray results. A new combinatorial analysis was also performed in search of the best predictive model using real time RT-PCR expression values. A new trio was identified, represented by RPL37A, SMYD2 and MTSS1 genes, whose expression correctly classified 93% samples from technical validation group (22/23 responsive and 4/5 non-responsive) and 79% samples from biological validation group (8/11 responsive samples and 3/3 non-responsive samples). Therefore, the test presented 88% sensibility and specificity in identifying responsive patients for all samples analyzed. By means of verifying the classification strength of the same gene group, using cDNA microarray expression values, we observed a similar result (91% sensibility and specificity in recognizing responsive samples). Thus, we demonstrated that gene expression profile obtained by cDNA microarray is reproducible through real time RT-PCR. A study integrating a larger number of patients and a more comprehensive cDNA microarray platform may help the identification of a more accurate predictive model based on gene groups to foresee response to doxorubicin-based chemotherapy treatment.

Ionisierende Strahlung verursacht in Abhängigkeit von den strahlenphysikalischen Eigenschaften der Radionuklide Zellschäden unterschiedlicher Komplexität. An humanen Lymphozyten wurde untersucht, ob die biologische Wirksamkeit von Alpha- und Betastrahlung sowie der Einfluss von Doxorubicin der Qualität des Strahlenschadens zugewiesen werden kann. Die DNA-Schäden und deren Reparatur wurden mit zellbiologischen Methoden quantifiziert.

Η φασματοσκοπία Raman θεωρείται αξιόπιστη μέθοδος χαρακτηρισμού της μοριακής δομής της ύλης. Τελευταία δε καταβάλλεται μεγάλη προσπάθεια να αναδειχθεί και ως μη επεμβατική τεχνική ανίχνευσης ουσιών φαρμακευτικού και βιολογικού ενδιαφέροντος. Επειδή όμως το φαινόμενο Raman είναι ασθενές και τα όρια ανίχνευσης των ουσιών αυτών ιδιαίτερα χαμηλά, η συμβατική φασματοσκοπία Raman ανταποκρίνεται με μεγάλη δυσκολία στις απαιτήσεις μιας μη επεμβατικής τεχνικής. Τα τελευταία χρόνια με την ανακάλυψη της επιφανειακής ενίσχυσης της σκέδασης Raman (Surface Enhanced Raman Scattering) και την ανάπτυξη της ομώνυμης τεχνικής SERS αυξήθηκε η πιθανότητα του φαινομένου κατά τάξεις μεγέθους. Στο πλαίσιο αυτό, έχουν αναφερθεί και μελέτες SERS με ανίχνευση σκέδασης Raman ακόμη και από ένα μόνο μόριο (single molecule detection). Προϋπόθεση όμως ανάπτυξης φαινομένου SERS είναι η γειτνίαση της εξεταζόμενης ουσίας με νανοσωματίδια αργύρου (Ag) ή χρυσού (Au). Τα νανοσωματίδια αυτά χρησιμοποιούνται κυρίως είτε υπό μορφή κολλοειδών διαλυμάτων είτε υπό την μορφή νανοδομημένων επιφανειών. Μια επιπλέον παράμετρος που πρέπει να συνυπολογισθεί ώστε η μέθοδος αυτή να χαρακτηριστεί αναλυτική είναι η δυνατότητα πραγματοποίησης με SERS ποσοτικών μετρήσεων. Στην παρούσα εργασία ως “υπόστρωμα” εμφάνισης του φαινομένου SERS χρησιμοποιήθηκαν κολλοειδή διαλύματα Au και Ag (~2 mL), στα οποία προστίθεντο 25-100 μL διαλύματος της προς ανίχνευσης ουσίας. Η χρησιμοποίηση της γεωμετρίας σκέδασης 90ο, της πλέον ενδεδειγμένης για λήψη φασμάτων Raman από υγρά και κολλοειδή διαλύματα νανομετρικής κλίμακας (10-100 nm), έδωσε τη δυνατότητα ανίχνευσης συγκεντρώσεων ουσιών-στόχων ακόμη και της τάξης των fg/mL. Επιπλέον, η χρησιμοποίηση μιας κυψελίδας με συνεχή ταλάντωση σε κάθετη ως προς το επίπεδο σκέδασης διεύθυνση (oscillating or shaking cell), επέτρεψε την πραγματοποίηση ποσοτικών μετρήσεων SERS σε συγκεντρώσεις της τάξης των (sub)-ng & (sub)-pg/mL. Η διάταξη αυτή φωταψίας δείγματος και συλλογής σκεδαζόμενης ακτινοβολίας εφαρμόστηκε σε αντικαρκινικά φάρμακα (Novantrone® & Doxorubicin), αντιβιοτικό (Ciproxin®) και βάσεις του DNA. Στην περίπτωση του αντιβιοτικού με ενεργή ουσία το Ciprofloxacin ελήφθησαν ποσοτικά αποτελέσματα σε διαλύματα τεχνητών δακρύων της τάξης των ng/mL, κάτω από την συνολική κλίμακα της μέσης ανασταλτικής πυκνότητας (minimum inhibitory concentration range) του φαρμάκου. Στόχος και φιλοδοξία είναι η τεχνική αυτή να αναδειχθεί ως μια ταχεία μη επεμβατική αναλυτική μέθοδος μέτρησης φαρμακευτικών ή/και βιολογικών ουσιών σε σωματικά υγρά (π.χ. σίελος, δάκρυα, …), για την μελέτη της φαρμακοκινητικής, την έγκαιρη διάγνωση ασθενειών καθώς και τη μελέτη ελεγχόμενης αποδέσμευσης ενεργών ουσιών από πολυμερικές μήτρες σε πολύ μικρές συγκεντρώσεις. Επιπλέον, έγινε προσπάθεια μελέτης/ανίχνευσης μορίων Ε18 primary rat cortical cells με την χρήση του SERS σε συνθήκες προσομοίωσης ηλεκτροχημικού εμφυτεύματος. Για τον λόγο αυτό σχεδιάστηκε και αναπτύχθηκε ένας μικρο-επωαστής (micro-incubator) που επιτρέπει αφ’ ενός μεν τη συντήρηση των κυττάρων σε περιβάλλον κατάλληλο για τη μελέτη τους (layer of Neurobasal-culture medium with a 95% air & 5% CO2 gas flow at 37oC), αφ’ ετέρου δε την προσαρμογή του σε χώρο υποδοχής δείγματος ενός φασματοφωτόμετρου μικρο-Raman για τη λήψη φασματοηλεκτροχημικών μετρήσεων Raman/SERS. Το κύριο σημείο ενδιαφέροντος στο οποίο εστιάσαμε στην παρούσα εργασία αναφέρεται στην διερεύνηση ανίχνευσης βασικών/θεμελιωδών μορίων των κυττάρων στo θρεπτικό υγρό του πειράματος για την ταχεία εκτίμηση ενδεχόμενης αποδέσμευσης DNA που θα αντικατόπτριζε την κατάσταση των κυττάρων. / Laser Raman spectroscopy plays an increasing important role in polymer science, biophysics/biochemistry and life science. Based on vibrational transitions, it has long been regarded as a valuable non destructive tool for the identification of chemical and biological samples as well as the elucidation of molecular structure, surface processes and interface reactions. Spontaneous Raman scattering is however an inherently weak process characterized by extremely small cross sections. Even so, the Raman signal can be highly enhanced when the analyte is placed on or near either to nano-rough noble-metal substrates or to nano-structured colloidal clusters of noble metals. This nano-enhanced scattering process is known as Surface Enhanced Raman Scattering, SERS. With SERS, extremely small amounts of substances can be detected; even single molecule detection has been reported. This constitutes a challenge of applying SERS to extremely low concentration measurements. Nevertheless, the quantitative evaluation by means of SERS was proved difficult, due largely to lack of nano-sized noble metal structures with analytically suitable stability and reproducibility. The commonly used SERS substrates are nanostructured colloids or roughened surfaces of mainly silver and gold undergoing visible or NIR excitation. A note is made of the fact that the Raman scattering geometry that is actually largely used is the backscattering geometry. For solids this is better achieved via a microscope objective, while for liquids via a macro lens. However, the best scattering geometry for Raman light collection from liquids and nanostructured colloids is the right angle scattering; that is, the scattered light is collected at 90o from the excitation light. Having all above in mind, in order to perform quantitative SERS measurements we have designed and developed an oscillating cell making use of the right angle Raman scattering geometry. Originally, the development of the oscillating or “shaking” cell allows large sample sectional surface monitoring, better mixing and homogeneity giving rise to SERS signal reproducibility. In addition, the application of the advantageous for nanocolloidal solutions right angle Raman scattering collection geometry enables SERS measurements at extremely low concentrations. We demonstrated the use this new surface enhanced Raman scattering excitation/collection configuration to monitor the level of the antitumor drugs mitoxantrone and doxorubicin as well as the antibiotic ciprofloxacin at very low concentrations performing fast SERS & SERRS measurements. We have also quantified the concentration of the active agents in aqueous solutions as well as in artificial tears by a partial least-squares (PLS) chemo metric regression algorithm. All above reveal the potential of this technique in the monitoring of the controlled release of active agents from polymeric matrices as well as in the quantitative analysis of drugs in corporal fluids. The only need is to add in a test tube containing 0.5 mL of a nanocolloidal noble metal solution3 about 25-100 μL of a sampling fluid. Finally, in an attempt to detect “living” cells by SERS, to identify DNA components released in a cell culture medium as a “touch mark” of cell death and to perform even single molecule detection, a home-made micro-incubator was designed to allow Raman measurements of neuron cells under the microscope. Commercial micro incubators feature higher optical path between coverslips than the required (<1-1,5 cm) for typical micro-Raman systems even bearing long working distance objectives. It was at that sense a prerequisite to design, construct and optimize a micro-incubator to fit a conventional micro-Raman configuration. The designed micro-incubator meets well-defined stable conditions (temperature, culture solution and atmosphere).