Dissertations / Theses on the topic 'MD-2'

Il toll-like receptor 4 (TLR4) rappresenta un mediatore centrale dell’immunità innata ed adattativa in mammiferi. L’attivazione di TLR4 in risposta al lipopolisaccaride (LPS) batterico induce un rapido innesco di processi pro-infiammatori essenziali per una risposta immunitaria ottimale. L’attivazione di TLR4 mediata da LPS è un meccanismo che coinvolge la partecipazione di diverse proteine e culmina con la formazione del complesso attivato (TLR4/MD-2/LPS)2. MD-2 è il co-rettore di TLR4, e svolge un importante ruolo nell’interazione con LPS e la susseguente dimerizzazione del TLR4. MD-2 è la componente che interagisce con il ligando (LPS) nel complesso recettoriale TLR4/MD-2. Il legame di LPS al complesso TLR4/MD-2 induce la dimerizzazione del TLR4; mentre gli antagonisti del TLR4 sono in grado di legare il complesso TLR4/MD-2 ma non inducono la dimerizzazione del TLR4. L’attivazione non regolata del TLR4 è correlata ad un’ampia serie di problematiche prive di un trattamento farmacologico specifico. Esse includono disordini autoimmuni, infiammazione cronica, allergie, asma, infezioni e malattie del sistema nervoso centrale, cancro, e setticemia. L’inibizione del TLR4 tramite l’uso di piccole molecole sintetiche o naturali può quindi rappresentare una via per lo sviluppo di nuove terapie contro questa vasta gamma di problematiche. Questa tesi è parte di un studio originale di relazione struttura-attività (SAR) svolto su glicolipidi monosaccaridici sintetici nel contesto della modulazione del TLR4. In particolare, essa si focalizza sulla caratterizzazione del legame in vitro di nuovi glicolipidi monosaccaridici sintetici con il recettore MD-2 purificato. Per gli studi di interazione la proteina MD-2 umana (hMD-2) purificata e funzionale è stata espressa in cellule di lievito. Due diversi sistemi di espressione per la produzione di hMD-2 ricombinante sono stati testati: mammifero (HEK293T) e cellule di lievito (Pichia pastoris). La purificazione di hMD-2 da lievito è stata ottimizzata ottenendo una concentrazione finale di hMD-2 purificato di 30 μM. Per confrontare l’attività biologica di hMD-2 espresso nei diversi microorganismi è stato sviluppato un ELISA. hMD-2 da cellule di mammifero ha ottenuto l’attività biologica più elevata, seguito da hMD-2 espresso in P. pastoris. hMD-2 da E. coli ha ottenuto l’attività biologica più bassa dei tre. Date le rese più elevate di purificazione in lievito, hMD-2 espresso in P. Pastoris è stato utilizzato nei quattro diversi tipi di esperimenti di legame per studiare l’affinità di molecole naturali e sintetiche. I test di legame comprendono due ELISA con hMD-2 immobilizzato, un saggio fluorescente di spiazzamento, e misure di risonanza plasmonica di superficie (SPR). I due test ELISA sono basati su: i) spiazzamento dose-dipendente di un anticorpo da hMD-2 immobilizzato. L’anticorpo lega hMD-2 in una regione in prossimità del sito di legame del ligando; ii) spiazzamento di LPS biotinilato da hMD-2 immobilizzato. L’esperimento di florescenza è basato sullo spiazzamento di bis-ANS da hMD-2. Mentre la tecnica SPR è stata utilizzata per studiare la diretta interazione tra le molecole e hMD-2 immobilizzato. L’affinità per hMD-2 delle molecole analizzate (che risulta essere nell’intervallo del basso μM) è in linea con i risultati di attività biologica e la produzione di citochine in vitro in modelli cellulari. I risultati ottenuti da questi studi in vitro sul recettore hMD-2 purificato evidenziano l’interazione delle molecole con la tasca idrofobica di hMD-2, presentando differenze nei valori di affinità. Questi dati generati permettono uno studio sistemico dei modulatori del TLR4, creando buone prospettive per lo sviluppo di una nuova generazione di farmaci hits e leads che agiscano direttamente sul recettore TLR4.
Toll-like receptor 4 (TLR4) represents a central mediator of innate and adaptive immune responses in mammals. TLR4 activation in response to bacterial lipopolysaccharides (LPS) results in the rapid triggering of pro-inflammatory processes essential for optimal host immune responses. TLR4 activation mediated by LPS is a complex event which involves several proteins (lipid binding protein (LBP), cluster of differentiation 14 (CD14), and myeloid differentiation 2 (MD-2)) and it ends with the formation of the activated (TLR4/MD-2/LPS)2 complex. The TLR4 co-receptor MD-2 plays an important role in the interaction with LPS and subsequent TLR4 dimerization. MD-2 alone binds to LPS, whereas TLR4 alone does not. MD-2 is the ligand-binding component of the TLR4/MD-2 receptor complex. LPS binding to TLR4/MD-2 induces TLR4 dimerization; whereas TLR4 antagonists binding to TLR4/MD-2 does not induce TLR4 dimerization. Deregulated TLR4 activation is related to an impressively broad spectrum of disorders still lacking specific pharmacological treatment. These include autoimmune disorders, chronic inflammations, allergies, asthma, infectious and central nervous system diseases, cancer, and sepsis. The TLR4 inhibition by small molecules of synthetic and natural origin provides access to new TLR-based therapeutics targeting this large array of diseases. This thesis is part of an original structure-activity relationship (SAR) study on synthetic monosaccharide glycolipids in the context of TLR4 modulation. Thesis work focuses on the in vitro binding characterization of new synthetic monosaccharide glycolipids with the purified receptor MD-2. Pure and functional human MD-2 (hMD-2) protein for binding studies has been obtained by expression in yeast cells. Two different expression systems for the production of recombinant hMD-2 were tested: mammalian (HEK293T) and yeast cells (Pichia pastoris). Recovery of hMD-2 from the medium of yeast cells was optimized, achieving a concentration of recombinant hMD-2 of 30 μM. An ELISA was developed in order to compare the biological activity of the hMD-2 expressed in different hosts. hMD-2 from mammalian cells obtained the highest biological activity, followed by the hMD-2 expressed by P. pastoris. hMD-2 expressed by E. coli presented the lowest biological activity of the three. Due to the higher yield of recovery achieved, hMD-2 expressed in P. pastoris was used in four different types of binding experiments to assess its affinity for natural and synthetic molecules. The binding tests comprise two plate based ELISA with immobilized hMD-2, a fluorescence displacement assay and surface plasmon resonance (SPR) measurements. The two ELISA tests were based on: i) dose-dependent displacement of a monoclonal antibody from immobilized hMD-2. The antibody binds to hMD-2 in a region proximal to ligand binding site; ii) displacement of biotin-LPS from immobilized hMD-2. The fluorescence experiment was based on the displacement of the bis-ANS from hMD-2, whereas the SPR technique was used to study the direct interactions between small ligands and immobilized hMD-2. The obtained binding affinities for hMD-2 of the tested molecules (which turned out to be in the low μM range) mirror their biological activity in modulating TLR4 signaling and cytokine production in vitro in cell models. The results obtained from these in vitro cell-free studies indicate that the tested molecules bind to the hMD-2 pocket, with differences in the affinity values. These data allow a systematic study on SAR for TLR4 modulators, opening the way for the development of a new generation of drug hits and leads targeting directly TLR4 signaling.

Os receptores tipo Toll desempenham um importante papel na resposta imune inata, reconhecendo tanto padrões moleculares associados a patógenos (PAMPs), quanto padrões moleculares associados a danos (DAMPs), liberados sob condições de injúria ou estresse celular. O receptor tipo Toll 4 humano (hTLR4), associado ao seu co-receptor, o fator de diferenciação mielóide 2 (MD-2), forma um heterodímero caracterizado como responsável pelo reconhecimento de lipopolissacarídeos bacterianos (LPS), derivados de bactérias Gram-negativas. Nestes casos, sabe-se que o MD-2 reconhece LPS e promove a dimerização do complexo hTLR4 - MD-2 - LPS, promovendo a sinalização intracelular. Já foi reportada a ausência da associação hTLR4 a MD-2, no reconhecimento de outros ligantes por hTLR4, e, nesses casos, pouco é conhecido a respeito das mudanças estruturais e conformacionais sofridas por este receptor. No presente estudo, empregando a técnica de simulação por dinâmica molecular, foram exploradas as propriedades dinâmicas do complexo de reconhecimento de LPS, hTLR4 - MD-2, bem como investigou-se as implicações da presença do co-receptor para a biologia estrutural do hTLR4. Os resultados mostram que o receptor apresenta um movimento do tipo pinça, o qual leva a um estado final mais aberto da estrutura em forma de ferradura. Ademais, a estabilidade desta estrutura parece ser influenciada pela presença do co-receptor, MD-2.
Toll-like receptors (TLRs) play an important role in innate immunity recognizing pathogen-associated molecular patterns (PAMPs), as also damageassociated molecular patterns (DAMPs), released after cellular injury or stress. Human Toll-Like Receptor 4 (hTLR4) and its co-receptor, myeloid differentiation factor 2 (MD-2), as a heterodimer, is a well-known complex of Gram-negative bacteria lipopolysaccharide (LPS) recognition. In this process, MD-2 recognizes LPS and promotes the dimerization of the complex hTLR4 - MD-2 - LPS, initiating an intracellular immune signaling. Moreover, it has been reported that hTLR4 can also act in the absence of MD-2, in the case of other ligands recognition, and, in these cases, little is known about the structural and conformational changes that hTLR4 structure underwent. In the current study, employing molecular dynamics simulations, we had explored the dynamical properties of the hTLR4 - MD-2 complex and investigated the implications of the co-receptor complexation to the structural biology of hTR4. We observed that the receptor showed a tweezers-like movement, leading to a more oppened final state of its horseshoe-shaped structure. Additionally, the stability of this structure seems to be influenced by the presence of the co-receptor, MD-2.

The Malingered Depression Scale (Md Scale; Steffan, Clopton, & Morgan, 2003) was recently developed for use with the MMPI-2 in attempts to distinguish individuals with genuine symptoms of depression from individuals who feign depression on the test. With respect to the Md scale, a relative lack of research and mixed findings regarding its utility are problematic; therefore, these issues were explored. The predictive and incremental validity of the Md scale were tested in this study to determine if use of the Md scale conferred a distinct predictive advantage over standard validity scales (e.g. F, FB, FP) in the differentiation between participants instructed to feign depression and participants who, prior to taking the MMPI-2, endorsed a significant number of depressive symptoms on a self-report measure. The Md scale demonstrated predictive and incremental validity in this study in distinguishing the two groups; however several limitations arose regarding use of the Md scale, most notably conceptual clarity within participant groups and problems regarding the use of cut scores.

Desarrolla la técnica de películas radiocrómicas en dosimetría de braquiterapia ocular, para un aplicador oftálmico de oro con fuentes en forma de pequeños hilos de Ir-192, teniendo en cuenta una buena precisión, sensibilidad, buena respuesta en el rango de aplicación, versatilidad en su manejo y otras cualidades. Por ello, el objetivo fundamental de esta tesis es mostrar las bondades de las películas radiocrómicas ya que poseen características apropiadas para la verificación de la dosimetría en pacientes.
Tesis

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Pineapple is a fruit widely appreciated as fresh fruit. But its chemical composition varies widely between different cultivars. This study evaluated the content of some chemical components and the activity of peroxidase and polyphenoloxidase in pineapple MD-2 and Pearl mature, comparing them with each other. The chemical components of two cultivars analyzed showed that, statistically, the Soluble Solids and Titratable Total Acidity showed higher values than MD-2 pineapple pineapples in Pearl, possibly explaining the latter being more appreciated as fresh fruit by locals while the MD-2 pineapples are more accepted for export. The optimum pH and temperature for the peroxidase activity of MD-2 pineapples were pH 5.0 at 40 ° C and less favorable pH found in this study form. In pineapples Pearl optimal conditions recorded were 50 ° C and pH 5.5 and less favorable 40 º C and pH 6.0. At 30 ° C the specific activity was recorded 3.69 and 3.92 U mg protein-1 for pineapples and Pearl MD-2, respectively. The natural pH of 3.23 MD-2 and 3.59 pH of Pearl were statistically different between the cultivars did not differ tannins and content of L-ascorbic acid was significantly higher in Pearl pineapples. However, the peroxidase activity was significantly higher in Pearl, that the MD-2 and therefore the MD-2 deterioradora less subject to the action of this enzyme.
O abacaxi é uma fruta amplamente apreciada como fruta fresca. Mas sua composição química apresenta grande variação entre cultivares distintas. Este trabalho avaliou o teor de alguns componentes químicos e a atividade da peroxidase e polifenoloxidase nos abacaxis MD-2 e Pérola maduros, comparando-os entre si. Os componentes químicos das duas cultivares analisadas demonstraram que, estatisticamente, os teores de Sólidos Solúveis e Acidez Total Titulável apresentaram valores maiores em abacaxis MD-2 que em abacaxis Pérola; possivelmente, o que explica estes últimos serem mais apreciados como fruta fresca pela população local, enquanto os abacaxis MD-2 são mais aceitos para fins de exportação. As condições ótimas de pH e temperatura para a atividade da peroxidase de abacaxis MD-2 foram pH 5,0 a 40°C e as menos favoráveis encontrados neste estudo forma pH. Em abacaxis Pérola as condições ótimas registradas foram 50ºC e pH 5,5 e as menos favoráveis 40ºC e pH 6,0. À temperatura de 30°C a atividade específica registrada foi 3,69 e 3,92 U·mg Proteína-1 para os abacaxis MD-2 e Pérola, respectivamente. O pH 3,23 natural do MD-2 e o pH 3,59 do Pérola foram estatisticamente diferentes entre os cultivares, taninos não diferiram e o teor de ácido L-ascórbico foi significativamente superior em abacaxis Pérola. Contudo, a atividade da peroxidase foi significativamente maior no Pérola, que no MD-2, sendo, portanto o MD-2 menos sujeito à ação deterioradora desta enzima.

L’objectif de ce travail de thèse était de développer des méthodologies analytiques sur un spectromètre de masse haute résolution couplée à une source plasma (HR-ICP-MS Element 2 de Finigan) pour déterminer quantitativement des éléments en traces dans des matrices de carbonate de calcium. Deux méthodes (couplage LA-ICP-MS et analyse en mode liquide) ont été mises en places afin de placer sur une échelle calendaire (± 3 jours) les variations ontogéniques des teneurs élémentaires dans les valves de P. Maximus. Les résultats d’analyses obtenus sur une même valve gauche par les deux méthodes, montrent que les profils élémentaires demeurent reproductibles quels que soient les concentrations rencontrées et le transect choisi sur la valve. Les variations de la concentration valvaire en Mo et en Li chez P. Maximus ont été détaillées. La teneur coquillière en Mo est ponctuée au printemps par un ou deux enrichissements épisodiques. L’ingestion de cellules phytoplanctoniques ayant utilisées les nitrates comme source d’azote semble l’hypothèse la plus probable (Ces cellules présenteraient une concentration forte en Mo suite au rôle de Mo lors de l’activité enzymatique de la nitrate réductase). Certaines populations de P. Maximus présentent de brusques augmentations transitoires de la teneur en Li pendant leur période de croissance. Notre étude n’a pas pu mettre en évidence un lien généralisable entre ces pics en Li et un paramètre environnemental mais ces pics semblent apparaître quelques jours après des floraisons phytoplanctoniques de diatomées. En outre, nous constatons que le bruit de fond de la teneur en Li serait majoritairement lié à la vitesse de croissance de l’animal
The elemental composition of bivalve shells is already considered, by international scientific community, as a promising tool to record environmental parameters like sea surface temperature, salinity and primary productivity. The aim of this work was to develop an analytical method on the high-resolution inductively coupled plasma-mass spectrometry (HR-ICP. MS, Finnigan Element 2) to demonstrate a quantitative detection of trace element in biocarbonates matrices (CaCO3). Two methods (LA-ICP-MS, Solution Based ICP-MS) has been developed to drawn ontogenetic profiles of elemental ratios with a 3-day temporal resolution. Element profiles obtained on the same left valve of P. Maximus by the two methods were reproducible whatever the concentrations and whatever the ribs selected on the valve. The variations of two specific element traces (Mo, Li) in P. Maximus shells were discussed. Mo concentration is characterised by a background level within sharp episodic peaks occurring in spring (may). Explanation hypothesis are discussed but the most probable is the ingestion of phytoplankton cells grown upon N03- (which have a large Mo concentration associated to the activity of nitrate reductase). Some populations of P. Maximus present sharp episodic Li peaks in the growth period. This study illustrate that there is no direct relationship between environmental parameters (salinity, temperature, water composition of Li, nitrates. . . ) and Li peaks, but Li enrichment still may be related to blooms of specific species of phytoplancton. In addition, we found that the background profile of Li seems to be mainly controlled by the growth rate

L’attivazione del Toll-Like Receptor 4 (TLR4) e il conseguente signaling intracellulare in risposta a esigue quantità di endotossine (lipopolisaccaridi di batteri Gram-negativi, LPS) presenti nel circolo sanguigno porta al rapido innesco di processi pro-infiammatori necessari per una risposta immunitaria ottimale dell’organismo ospite contro l’invasione di batteri Gram-negativi. Il TLR4 non lega però direttamente i LPS e la sua attivazione da parte dell’endotossina è un processo complesso che prevede la partecipazione di altre proteine che legano i LPS (LBP, CD14 e MD-2) e porta infine alla formazione del complesso attivato (TLR4•MD-2•LPS)2. In particolare, CD14 è stato il primo recettore che lega LPS ad essere scoperto e sembra che favorisca la formazione del complesso (TLR4•MD-2•LPS)2. CD14 è anche necessario per l’endocitosi di TLR4 indotta da LPS e la localizzazione dell’intero complesso recettoriale per l’endotossina negli endosomi, da dove parte un secondo pathway del segnale. Un’attivazione di TLR4 eccessivamente potente e sregolata causa però gravi sindromi come lo shock settico, associato a un’alta mortalità (50-70%) e a danni agli organi anche molto gravi. Sono perciò necessari degli antagonisti (inibitori) di TLR4 efficaci e selettivi con una struttura chimica più semplice di quella del LPS, per lo sviluppo di potenziali nuovi farmaci con un’ampia gamma di applicazioni mediche e farmacologiche (dalla sepsi alle patologie del sistema nervoso). In questa tesi è descritta la sintesi di monosaccaridi mimetici del LPS basati su un core di glucosammina legata a due catene di acidi grassi che portano uno o due gruppi fosfato. Di questi, i due composti con un solo fosfato hanno mostrato una certa attività come inibitori dell’attivazione di TLR4 e della produzione di citochine in cellule HEK-Blue® e in macrofagi murini. Il composto con due gruppi fosfato è risultato invece molto più efficace nell’inibire il segnale di TLR4 in entrambi i modelli cellulari. L’interazione diretta tra questo composto e il recettore MD-2 è stata investigata mediante spettroscopia NMR e analisi bioinformatiche (molecular modelling/docking). Questo composto interagisce direttamente anche con il recettore CD14, inducendone l’internalizzazione per endocitosi. Il targeting sia di CD14 che di MD-2, unito a una buona solubilità in acqua e all’assenza di tossicità, rendono il monosaccaride difosfato presentato in questa tesi un possibile lead compound per lo sviluppo di farmaci contro le sindromi collegate al TLR4. E’ stata inoltre avviata una funzionalizzazione di questo composto in modo da coniugarlo a una molecola fluorescente o a un chelante del gadolinio per studi, rispettivamente, di microscopia o imaging mediante risonanza magnetica.
Activation of Toll-like receptor 4 (TLR4) and subsequent intracellular signaling in response to minute amounts of circulating endotoxins (Gram-negative bacterial lipopolysaccharides, LPS), results in the rapid triggering of proinflammatory processes necessary for optimal host immune responses to invading Gram-negative bacteria in mammalians. TLR4 does not bind directly to LPS, and TLR4 activation by endotoxin is a complex event, involving the participation of other LPS-binding proteins — namely LBP, CD14, and MD-2 — and ending with the formation of the activated (TLR4•MD-2•LPS)2 complex. In particular, CD14 was the first identified Pattern Recognition Receptor (PRR) that binds directly to LPS, and chaperones the formation of the (TLR4•MD-2•LPS)2 complex. CD14 is also required for endotoxin-induced TLR4 endocytosis and relocalization of the entire LPS receptor complex into the endosome, where a second signaling pathway initiates. Excessively potent and deregulated TLR4 activation and signaling causes severe syndromes such as septic shock, associated with a high mortality (50–70%), and organ-specific damages. Efficient and selective TLR4 antagonists with chemical structures simpler than that of lipid A are therefore required for the development of potential new drugs with a wide array of medical and pharmacological applications (from sepsis to CNS pathologies). In this thesis, the synthesis of three monosaccharide LPS mimetics based on a glucosamine core linked to two fatty acid chains and bearing one or two phosphate groups is described. Two of them, each with one phosphate group, resulted quite active in inhibiting LPS-induced TLR4 signaling and cytokine production in HEK-Blue™ cells and murine macrophages. The compound with two phosphate groups, however, was found to be more active in efficiently inhibiting TLR4 signal in both cell types. The direct interaction between this compound and the MD-2 receptor was investigated by NMR spectroscopy and molecular modeling/docking analysis. This compound also interacts directly with the CD14 receptor, stimulating its internalization by endocytosis. The dual targeting of MD-2 and CD14, accompanied by good solubility in water and lack of toxicity, suggests the use of monosaccharide 3 as a lead compound for the development of drugs directed against TLR4-related syndromes. A derivatization of this compound was also started in order to couple it to a fluorescent moiety or to a Gadolinium-chelating agent, for microscopy and MRI studies respectively.

Améliorer notre connaissance de la structure de l'eau dans l'environnement spécial offert par une interface est essentiel pour la compréhension de nombreux phénomènes naturels et applications technologiques. Pour révéler cette structure interfaciale de l'eau, des techniques capables de fournir des informations microscopiques, de manière sélective, pour cette couche interfaciale (BIL) sont nécessaires. Dans le présent travail de thèse, nous avons donc étudié les interfaces aqueuses au niveau moléculaire, en couplant la modélisation théorique à partir de simulations DFT-MD avec les spectroscopies SFG et THz-IR. En développant de nouveaux protocoles/outils d'investigation associant simulations DFT-MD et spectroscopie SFG, en particulier pour la rationalisation plus complexe des interfaces chargées, nous avons fourni une compréhension globale de l'effet des conditions interfaciales d'hydrophilicité, de pH, de force ionique sur le réseau des liaisons-H formé dans la couche interfaciale BIL, sur ses signatures spectroscopiques et sur son impact sur les propriétés physico-chimiques. Nous avons montré pour la première fois que, dans des conditions suffisamment hydrophobes, l'eau interfaciale crée des réseaux des liaisons-H bidimensionnels, révélé expérimentalement par les spectres THz-IR. Le réseau-2D dicte la dynamique de l'eau interfaciale, le potentiel de surface, l'acidité de surface, la tension superficielle et la thermodynamique d'hydratation des solutés hydrophobes. Cet "ordre horizontal" aux interfaces hydrophobes est opposé à "l'ordre verticale" obtenu aux interfaces hydrophiles. Nous avons aussi révélé comment les ions et les conditions de pH modifient ces arrangements structuraux
Improving our knowledge on water H-Bonded networks formed in the special environment offered by an interface is pivotal for our understanding of many natural phenomena and technological applications. To reveal the interfacial water arrangement, techniques able to provide detailed microscopic information selectively for the interfacial layer are required. In the present thesis work, we have hence investigated aqueous interfaces at the molecular level, by coupling theoretical modeling from DFT-MD simulations with SFG & THz-IR spectroscopies. By developing new investigation protocols/tools, coupling DFT-MD simulations and SFG spectroscopy, in particular for the more complex rationalization of charged interfaces, we have provided a global comprehension of the effect of various interfacial conditions (hydrophilicity, pH, ionic strength) on the HB-Network formed in the interfacial layer (BIL), on its spectroscopic signatures and on its impact on physico-chemical properties. We have shown for the first time that, in sufficiently hydrophobic conditions, BIL interfacial water creates special 2-Dimensional HB-Networks, experimentally revealed by one specific THz-IR marker band. Such 2D-network dictates HBs and orientational dynamics of interfacial water, surface potential, surface acidity, water surface tension and thermodynamics of hydration of hydrophobic solutes. Such "horizontal ordering” of water at hydrophobic interfaces is found opposite to the “vertical ordering” of water at hydrophilic interfaces, while coexistence of the two orders leads to disordered interfacial water in intermediate hydrophilic/hydrophobic conditions. Both DFT-MD and SFG further revealed how ions & pH conditions alter these BIL-water orders

The work of this thesis was focused on the development of high-performance algorithms for a new generation of molecular descriptors, with many advantages with respect to its predecessors, suitable for diverse applications in the field of drug design, as well as its implementation in commercial grade scientific software (Pentacle). As a first step, we developed a new algorithm (AMANDA) for discretizing molecular interaction fields which allows extracting from them the most interesting regions in an efficient way. This algorithm was incorporated into a new generation of alignmentindependent molecular descriptors, named GRIND-2. The computing speed and efficiency of the new algorithm allow the application of these descriptors in virtual screening. In addition, we developed a new alignment-independent encoding algorithm (CLACC) producing quantitative structure-activity relationship models which have better predictive ability and are easier to interpret than those obtained with other methods.
El trabajo que se presenta en esta tesis se ha centrado en el desarrollo de algoritmos de altas prestaciones para la obtención de una nueva generación de descriptores moleculares, con numerosas ventajas con respecto a sus predecesores, adecuados para diversas aplicaciones en el área del diseño de fármacos, y en su implementación en un programa científico de calidad comercial (Pentacle). Inicialmente se desarrolló un nuevo algoritmo de discretización de campos de interacción molecular (AMANDA) que permite extraer eficientemente las regiones de máximo interés. Este algoritmo fue incorporado en una nueva generación de descriptores moleculares independientes del alineamiento, denominados GRIND-2. La rapidez y eficiencia del nuevo algoritmo permitieron aplicar estos descriptores en cribados virtuales. Por último, se puso a punto un nuevo algoritmo de codificación independiente de alineamiento (CLACC) que permite obtener modelos cuantitativos de relación estructura-actividad con mejor capacidad predictiva y mucho más fáciles de interpretar que los obtenidos con otros métodos.

Title: Security and usability of standard hash functions, in particular MD-5, SHA-1 and SHA-2 Author: Galaczová Barbora Department: Department of Algebra Supervisor: Doc. RNDr. Tůma Jiří, DrSc., Department of Algebra Consultant: Ing. Budiš Petr, Ph.D. Abstract: In the present work we try to digestedly describe standard hash functions, in particular MD-5, SHA-1 and SHA-2. We describe resume of existing attacks on these hash functions. We closely focused on MD-5 collision attacks, because the other hash function collision attacks result from these. Next we describe posibilities of practical usage of hash function collisions, in particular into the qualified certificates area and possible threats. At the end to the present work we describe new hash functions, which could replace current hash functions. This work also contains software to calculate MD-5 hash and search it`s collisions. The software is based on method invented by Czech cryptoanalytist Vlastimil Klíma. Keywords: hash function, collision, qualified certificate, security.

ML (MD-2-related lipid-recognition) domain containing proteins are recognized as immune-related molecules. They do not belong among well-studied proteins in ticks although their occurence is quite often. Generally, ML proteins are involved in innate immunity processes, lipid binding and transport. Usually, expression of tick ML domain containing proteins is induced by blood feeding. Two members of the ML protein family, ML-domain containing protein and Der-p2 allergen-like protein were isolated from Ixodes ricinus and characterized for the first time.

The term „cancer‟ encompasses a large group of diseases that are characterized by the development of abnormal cells, which divide, grow and spread without control in any part of the organism, spreading through blood or lymph vessels into surrounding tissues. Chemotherapy is a type of cancer treatment that uses drugs to kill cancer cells and is focused on stopping or slowing the growth of cancer cells, which divide abnormally and causes tumors. In most of the cases of chemotherapy prescription and selection of the specific drug, the more effective is a combination of two or more medications that, preferentially, should not interact with each other, either on their mechanism of action and/or their metabolism and elimination. Second and third generations of the existing drugs show increased bioactivity against cancer, but side effects are still a matter of concern. Recent studies show a significant interest in metal-based drugs, with some existing drugs being used already as antitumor agents, with proven effectiveness and fewer side effects, comparing to other drug treatments. Copper(II) as a metal and its complexes with various organic compounds have been reported to show cytotoxic activity at low concentrations. The aim of this work is to examine the effects of newly synthesized copper complexes in human cancer cell lines, both in terms of cytotoxicity and of mechanism of action. In this research work, two copper(II) based compounds were (copper(II)-tropolone and copper(II)-hinokitiol complexes), for their cytotoxic properties and tested in the human mammary breast cancer cell line MCF7 and MDA-MB-231 for their effect on viability, oxidative stress, apoptosis and interaction with DNA. Additionally, as a model for in vivo studies, the 3D model testing on cell viability was conducted and showed a positive result against MCF7 cell line survival. Together with that, the comparative analysis of complexes, its ligands and copper salt was performed. These compounds showed quite promising results in terms of their potential effect as antitumor drugs.
O termo „cancro‟ abrange um largo número de doenças, que são caracterizadas pelo desenvolvimento anormal de células que se dividem, crescem e propagam-se de forma descontrolada por todo o organismo, propagando-se através do sangue ou dos vasos linfáticos para os tecidos vizinhos. A quimioterapia é um tipo de tratamento anticancerígeno, que tem por base o uso de fármacos que atuam por parar ou por diminuir o crescimento de células cancerígenas, células que se dividem anormalmente causando tumores. Na maioria dos casos, ao prescrever um fármaco específico, o uso combinado de dois ou mais fármacos é preferencial, sendo que não deve haver nenhum tipo de interação entre os mesmos, quer no mecanismo de ação e/ou metabolismo, quer na eliminação. A segunda e a terceira geração de fármacos existentes, mostraram um aumento da bioatividade contra o cancro, contudo, os efeitos secundários continuam a ser uma grande preocupação. Estudos recentes têm demonstrado um interesse significativo em fármacos com compostos metálicos, sendo que alguns destes fármacos já são usados como agentes antitumorais, com eficácia e menos efeitos secundários comprovados, comparativamente com outros fármacos. O Cobre (II), tanto como metal, tanto complexado com vários compostos orgânicos, mostrou ter atividade citotóxica a baixas concentrações. O objetivo desde trabalho é analisar, em linhas celulares humanas, os efeitos de complexos de cobre recentemente sintetizados, tanto em termos de citotoxicidade como em termos de mecanismo de ação. Neste trabalho de investigação, dois compostos com base no cobre (II), (cobre(II)-tropolone) e cobra(II)-hinokitiol complexos), foram observados e avaliados quanto às suas propriedade citotóxicas, e testados na linha celular humana de cancro da mama MCF7 e MDA-MB-231, quanto ao seu efeito na viabilidade, no stress oxidativo, na apoptose e na interação com o DNA. Adicionalmente, como modelo para estudos in vivo, foi conduzido um teste modelo 3D quanto à viabilidade, onde foram observados resultados positivos contra a linha celular MCF7 sobrevivente. Simultaneamente, foi realizada uma análise comparativa dos complexos sintetizados, dos seus ligandos e do sal de cobre. Estes compostos mostraram resultados promissores com efeitos potenciais como fármacos antitumorais.