Foot-and-mouth disease virus (FMDV) is an economically devastating and highly contagious livestock pathogen. It exists as seven serotypes, comprising numerous antigenically distinct subtypes. The large amount of antigenic heterogeneity has confounded attempts at developing broadly reactive vaccines. In order to overcome this issue the fundamentals of the interactions between the virus and the host humoral immune response must first be understood. Previous work in this area using monoclonal antibody (mAb) escape mutants has identified five antigenic sites for the O serotype and efforts have been made to quantify their relative importance. However, this does not represent a complete picture of serotype O antigenicity. The work conducted in this thesis demonstrates the role of a limited number of dominant substitutions in mediating the antigenic diversity of serotype O Foot-and-Mouth disease virus. Two alternative but complementary methods for identifying epitopes were developed. The first used a mathematical model to analyse newly generated serological and sequence data from 105 viruses, cultured for this purpose (and cross-reacted to 5 reference antisera), in the context of an existing crystallographic structure to identify and quantify the antigenic importance of sites on the surface of the virus. The second approach was purely structural, using existing B cell epitope prediction tools to develop a method for predicting FMDV epitopes using existing crystallographic structures of FMDV. These techniques were validated by the use of reverse genetics, which confirmed the impact on cross reactivity of two predicted novel serotype O antigenic residues, with a further four novel residues identified by looking in depth at the interactions between two genetically close, but antigenically distant viruses. This increased knowledge of the antigenic composition of serotype O FMDV contributes to our understanding of the nature of vaccine efficacy and the breadth of protection, which, in the longer term, will aid in the goal of developing vaccines to better protect livestock from such a highly antigenically variable disease.

Deregulation of protein kinases is associated with many diseases making them important targets for therapeutic intervention. Kinases can switch between active and inactive conformations that can be targeted by type 1 or type 2 inhibitors respectively. One of the most relevant conformational switches is the ‘in’ and ‘out’ movement of the ATP/Mg2+ binding motif DFG. Factors modulating the conformational equilibria such as the residue environment of regulatory motifs remain poorly understood despite their importance for drug discovery. In this thesis, the first model system tested the hypothesis that accessibility of the DFG-out conformation is restricted by the energetic cost of transition between the in and out states. CDK2 was chosen as a target that was thought to have an inaccessible DFG-out conformation, and several point mutations were introduced to promote this conformational transition. Detailed biochemical and biophysical characterisation illustrated that the mutants bound type 2 inhibitors more potently than the wild type. In addition, the wild-type CDK2 was shown to bind type 2 inhibitors in the absence, but not in the presence, of cyclin. The first known CDK2 co-crystal structure in the DFG-out conformation was solved, opening the door to a new class of CDK2 inhibitors. In the second project, site-directed mutagenesis was used to explore the residues determining inhibitor selectivity between PIM1 and PIM2. Evaluation of ligand binding to the variants and comparison of PIM1 and PIM2 crystal structures showed that flexibility of the phosphate-binding loop was the dominant factor determining the differences in their affinities for ATP and small molecule inhibitors. These studies illustrate that residues contributing to kinase conformational equilibria can be just as important for inhibitor binding as contact residues formed in the ligand complex.

There is a strong need to develop a mechanical method to apply external torque to the bacterial flagellar motor. Such a method will allow us to probe the behaviour of the motor at a range of different speeds under different external conditions. In this thesis, I explored various methods to deliver torque at the single-molecule level, in particular the use of angular optical trapping and magnetic tweezers. I have identified rutile particles as suitable handles for use in angular optical trapping due to their high birefringence. Further progress was not achieved using angular optical trapping due to the lack of a suitable method to attach birefringent particles to the bacterial flagellar motor. On the other hand, I was able to make further progress using magnetic tweezers. A highly-reproducible and high-yielding magnetic bead assay was developed along with electromagnets capable of generating fast-rotating magnetic fields at magnitudes on the order of tens of mT. Using the system of delivering magnetic torque developed, I was able to stall and rotate the motor forward at speeds up to 220 Hz and in the reverse direction. Stalling experiments carried out on the motor revealed the stator mechanosensing depends on torque and not rotation. Signatures of stators dropping out at low load experiments further confirm the load dependence of stators.

Made available in DSpace on 2014-06-11T19:30:54Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-03-28Bitstream added on 2014-06-13T18:41:06Z : No. of bitstreams: 1 moreno_fbmb_dr_sjrp.pdf: 4373891 bytes, checksum: f5d8f544b8871aeb85c60a9d3288cd99 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Esta tese tem como foco o estudo estrutural de uma lectina presente em sementes da espécie vegetal Lotus tetragonolobus (LTA). Inicialmente a LTA, previamente purificada, foi cristalizada. Os cristais foram obtidos a uma temperatura constante de 20ºC, durante 30 dias, utilizando a técnica de cristalização por difusão de vapor. Dois conjuntos de dados foram coletados a 2.00 e 2.35 Å de resolução, através da fonte de Raios X do Laboratório Nacional de Luz Síncrotron (Campinas – Brasil). Os cristais são monoclínicos e apresentam simetria do tipo P21, com parâmetros de cela de a=68.89, b=65.83 e c=102.53 Å. A substituição molecular foi feita utilizando-se o monômero da lectina presente na espécie Arachis Hypogae (Peanut). O homotetrâmero, produto da substituição, foi utilizado como ponto de partida para o refinamento cristalográfico. Diversos ciclos de refinamento por satisfação das restrições parciais foram feitos até a conversão total para valores satisfatórios de Rfree e Rfactor. A análise da estrutura revelou que a LTA possui uma forma tetramérica não identificada dentre outras lectinas de leguminosas. Sua estrutura é composta por dois dímeros, um deles semelhante ao dímero GS4 presente na lectina de Griffonia simplicifolia e outro que é único, caracterizado por ser um dímero do tipo LTA-dímero. Diversos fatores são responsáveis pela forma de tetramerização diferenciada da LTA, dentre os quais podemos destacar a influência da glicosilação no resíduo ASN4. Para investigarmos a estrutura da LTA em meio aquoso foi utilizada a técnica de espalhamento de Raios X a baixo ângulo (SAXS), que mostrou que a LTA possui a mesma forma tetramérica em solução da que foi observada no retículo cristalino... / The lectin, LTA, an agglutinin found in Lotus tetragonolobus seeds have been crystalized. Crystals grew at a temperature of 20º C during a month and were obtained using the vapor diffusion method. Two data sets were collected at 2.00 and 2.35 Å resolution using a sincrotron radiation source at Laboratório Nacional de Luz Síncrotron (Campinas – Brasil). The LTA crystals are monoclinic belonging to the P21 space group with a=68.89, b=65.83 and c=102.53 Å. The Molecular replacement was performed using the Arachis Hypogae lectin monomer (Peanut) that yielded an homotetramer which was used to initiate the crystallographic refinement. Several steps of restrained refinement were performed to obtain the best values for Rfree and Rfactor. Structural analysis of the LTA showed that its tetramer adopts a new structural oligomerization in contrast of that observed for others legume lectins. Its structure contains two GS4-like dimers disposed in an unusual dimer-interface, named as LTA-dimer. Several properties are involved in the new mode of tetramerization adopted for LTA, which includes the glycosilation at ASN4. The LTA tetramer investigation at aqueous solution was perfomed by Small Angle XRay Scattering (SAXS), showing that LTA behaves as a tetramer in solution which corroborates with the crystalline structure. Our investigations suggest that the L-fucose binding sites of LTA are disposed in a conformation that permits to perform two dimensional type-2 cross-linking interaction with divalent L-fucosyloligosaccharides.

Made available in DSpace on 2014-06-11T19:30:54Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-08-14Bitstream added on 2014-06-13T19:00:59Z : No. of bitstreams: 1 silva_ras_dr_sjrp.pdf: 752309 bytes, checksum: 4052fa1214621cd77a1b7e3ebe94146c (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O objetivo deste trabalho é investigar um modelo mecânico para a molécula de DNA. O modelo consiste de duas cadeias de osciladores harmônicos representando as tas do DNA. Esses osciladores são ligados por um potencial de Morse que simula as interações tipo pontes de hidrogênio, como no modelo original de Peyrard e Bishop. Entretanto, neste trabalho, os movimentos de rotação e de vibração de cada par de base podem ocorrer ao mesmo tempo. Neste contexto, propriedades estruturais e termodinâmicas são discutidas. / The objective of this work is to investigate a mechanical model for the DNA molecule. The model consist in two chains of harmonic oscillators representing the ribbons of DNA linked by a Morse potential which represents the hydrogen bonds as in the Peyrard-Bishop's model. However, in this work, the rotation and vibration motion of each base pairs can be occur at the same time. In this context, thermodynamic and structural properties are discussed.

Made available in DSpace on 2014-06-11T19:30:54Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-06-25Bitstream added on 2014-06-13T19:00:53Z : No. of bitstreams: 1 carvalho_sj_dr_sjrp.pdf: 2475593 bytes, checksum: 23a8255d4493f96e6f105ed3d095798a (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O entendimento da formação de complexos compostos por polieletrólitos e macroíons é fundamental para o estudo de vários processos biológicos e tecnológicos. Neste trabalho foram investigados os aspectos eletrostásticos da complexação, verificando os efeitos da força iônica, da carga macromolecular e do grau de polimerização do políımero na estabilidade do complexo e nas características conformacionais da cadeia. Com o objetivo de obter conclusões gerais para este tipo de sistema, um modelo simplificado foi utilizado. O políımero foi representado por um conjunto de esferas ríıgidas carregadas conectadas por um potencial harmônico, enquanto o macroíıon seguiu um modelo esférico com distribuição de carga homogênea. A interação entre as espécies carregadas foi tratada de acordo com a aproximação de Debye-H¨uckel. Utilizando simulaçãoes Monte Carlo no ensemble canônico, verificou-se que a dissociação do complexo possui características de transição de fase. Condições críticas foram obtidas em concordância com o comportamento observado empiricamente. Resultados da interação de polieletrólitos com proteínas no seu ponto isoelétrico mostraram que a contribuição devido apenas a regulação de carga é suficiente para estabilizar o complexo e pode ser aumentada através de mutações que diminuam a heterogeneidade de carga da proteína. / The understanding of macroíon–polielectrolyte complex formation is fundamental for the biological and technological process studies. In this work the electrostatic issues on the complexation were investigated, verifying the effects of ionic strenght, macromolecular charge, and degree of polimerization on the complex stability and the chain conformational features. With the purpose of obtain general conclusions for this system, a simplified model was adopted. The polymer was represented by a set of rigid charged spheres connected by a harmonic potential, and the macroion was represented by a rigid sphere with homogeneous charge distribution. The interaction among macromolecules was described by the Debye-H¨uckel theory. Using Monte Carlo simulations within the canonical ensemble, it was verified that complex dissociation has phase–like transition behaviour. Numerical values for critical conditions were obtained in agreement with the ones experimentally observed. Results of interaction among polyeletrolyte and proteins on their isoelectric point showed that the charge regulation contribuition only is sufficient to make the complex stable. Is is also shown that charge regulation contribution is increased by mutations which promote a lower protein charge density heterogenity. 14

The complexity of multi cellular organisms demands systems that facilitate communicationbetween cells. The neurons in our brains for instance are specialized in this cell-cellcommunication. The flow of ions, through their different ion channels, across the membrane, isresponsible for almost all of the communication between neurons in the brain by changing theneurons membrane potentials. Voltage-gated ion channels open when a certain thresholdpotential is reached. This change in membrane potential is detected by voltage-sensors in the ionchannels. In this licentiate thesis the Homo sapiens voltage- and calcium-gated BK potassiumchannel (HsapBK) has been studied. The NMR solution structure of the voltage-sensor ofHsapBK was solved to shed light upon the voltage-gating in these channels. Structures of othervoltage-gated potassium channels (Kv) have been determined by other groups, enablingcomparison among different types of Kv channels. Interestingly, the peptide-lipid interactions ofthe voltage-sensor in HsapBK are crucial for its mechanism of action.Uni cellular organisms need to sense their environment too, to be able to move towardsmore favorable areas and from less favorable ones, and to adapt their gene profiles to currentcircumstances. This is accomplished by the two-component system, comprising a sensor proteinand a response regulator. The sensor protein transfers signals across the membrane to thecytoplasm. Many sensor proteins contain a HAMP domain close to the membrane that isinvolved in transmitting the signal. The mechanism of this transfer is not yet revealed. Ourstudies show that HAMP domains can be divided into two groups based on the membraneinteraction of their AS1 segments. Further, these two groups are suggested to work by differentmechanisms; one membrane-dependent and one membrane-independent mechanism.Both the voltage-gating mechanism and the signal transduction carried out by HAMPdomains in the membrane-dependent group, demand peptide-lipid interactions that can be readilymodulated. This modulation enables movement of peptides within membranes or within thelipid-water interface. These conditions make these peptides especially suitable for NMR studies.

Livet tas för givet av de flesta. Det finns däremot många som ägnar stora delar av sitt liv för att försöka lösa dess mysterier. En del av lösningen ligger i att förstå hur alla molekyler är sammanlänkade i det gigantiska nätverk som definierar den levande organismen. Under det senaste seklet har en hel del forskning utförts för att kartlägga dessa nätverk. Resultatet av dessa mödor kan vi se i de läkemedel som vi har idag och som har utvecklats för att bota eller åtminstone lindra olika sjukdomar och tillstånd. Dessvärre finns det fortfarande många sjukdomar som är obotliga (t.ex. cancer) och mycket arbete krävs för att förstå dem till fullo och kunna designa framgångsrika behandlingar. Arbetet i denna avhandling beskriver en analytisk plattform som kan användas för att effektivisera kartläggningsprocessen; protein-mikroarrayer. Mikroarrayer är ytor som har mikrometerstora (tusendels millimeter) strukturer i ett regelbundet mönster med möjligheten att studera många interaktioner mellan biologiska molekyler samtidigt. Detta medför snabbare och fler analyser - till en lägre kostnad. Protein-mikroarrayer har funnits i ungefär ett decennium och har följt i fotspåren av de framgångsrika DNA-mikroarrayerna. Man bedömer att protein-mikroarrayerna har en minst lika stor potential som DNA mikroarrayerna då det egentligen är mer relevant att studera proteiner, som är de funktionsreglerande molekylerna i en organism. Vi har i detta arbete tillverkat modellytor för stabil inbindning av proteiner, som lämnar dem intakta, funktionella och korrekt orienterade i ett mikroarray format. Därmed har vi adresserat ett stort problem med protein mikroarrays, nämligen att proteiner är känsliga molekyler och har i många fall svårt att överleva tillverkningsprocessen av mikroarrayerna. Vi har även studerat en metod att tillverka mikroarrayer av proteiner bundna till strukturer, som modellerats att efterlikna cellytor. Detta är särkilt viktigt eftersom många (hälften) av dagens (och säkerligen framtidens) läkemedel är riktade mot att påverka denna typ av proteiner och att studera dessa i sin naturliga miljö är därför väldigt relevant. I ett annat projekt har vi använt protein mikroarrayer för att detektera fyra vanliga droger (heroin, amfetamin, ecstasy och kokain). Detektionen baseras på användandet av antikroppar som lossnar från platser på ytan när de kommer i kontakt med ett narkotikum. Detta koncept kan enkelt utvecklas till att detektera mer än bara fyra droger. Vi har även lyckats att parallellt mäta förekomsten av en annan typ av förening på mikroarray ytan, nämligen det explosiva ämnet trinitrotoluen (TNT). Detta visar på en mångsidig plattform för detektionen av i princip vilken typ av farlig eller olaglig substans som helst - och på en yta! Vi föreställer oss därför att möjliga tillämpningsområden finns inom brottsbekämpning, i kampen mot terrorism och mot narkotikamissbruk etc. Mikroarrayerna har i denna avhandling utforskats med optiska metoder som tillåter studie av omärkta proteiner, vilket resulterar i så naturliga molekyler som möjligt. / Life is a thing taken for granted by most. However, it is the life-long quest of many to unravel the mysteries of it. Understanding and characterizing the incomprehensively complex molecular interaction networks within a biological organism, which defines that organism, is a vital prerequisite to understand life itself. Already, there has been a lot of research conducted and a large knowledge has been obtained about these pathways over, especially, the last century. We have seen the fruits of these labors in e.g. the development of medicines which have been able to cure or at least arrest many diseases and conditions. However, many diseases are still incurable (e.g. cancer) and a lot more work is still needed for understanding them fully and designing successful treatments. This work describes a generic analytical tool platform for aiding in more efficient (bio)molecular interaction mapping analyses; protein microarray chips. Microarray chips are surfaces with micrometer sized features with the possibility of studying the interactions of many (thousands to tens of thousands) (bio)molecules in parallel. This allows for a higher throughput of analyses to be performed at a reduced time and cost. Protein microarrays have been around for approximately a decade, following in the footsteps of the, so far, more successfully used DNA microarrays (developed in the 1990s). Microarrays of proteins are more difficult to produce because of the more complex nature of proteins as compared to DNA. In our work we have constructed model surfaces which allow for the stable, highly oriented, and functional immobilization of proteins in an array format. Our capture molecules are based on multivalent units of the chelator nitrilotriacetic acid (NTA), which is able to bind histidine-tagged proteins. Furthermore, we have explored an approach for studying lipid membrane bound systems, e.g. receptor-ligand interactions, in a parallelized, microarray format. The approach relies on the addressable, DNA-mediated adsorption of tagged lipid vesicles. In an analogous work we have used the protein microarray concept for the detection of four common narcotics (heroin, amphetamine, ecstasy, and cocaine). The detection is based on the displacement of loosely bound antibodies from surface array positions upon injection of a specific target analyte, i.e. a narcotic substance. The proof-of-concept chip can easily be expanded to monitor many more narcotic substances. In addition, we have also been able to simultaneously detect the explosive trinitrotoluene (TNT) along with the narcotics, showing that the chip is a versatile platform for the detection of virtually any type of harmful or illegal compound. This type of biosensor system is potentially envisaged to be used in the fight against crime, terrorism, drug abuse etc. Infrared reflection absorption spectroscopy together with ellipsometry has been used to characterize molecular layers used in the fabrication processes of the microarray features. Imaging surface plasmon resonance operating in the ellipsometric mode is subsequently used for functional evaluation of the microarrays using a well-defined receptor-ligand model system. This approach allows simultaneous and continuous monitoring of binding events taking place in multiple regions of interest on the microarray chip. A common characteristic of all the instrumentation used is that there is no requirement for labeling of the biomolecules to be detected, e.g. with fluorescent or radioactive probes. This feature allows for a flexible assay design and the use of more native proteins, without any time-consuming pretreatments.

This thesis describes the use of Nuclear Magnetic Resonance (NMR) for characterizing two host-pathogen interactions: The behavior of a regulatory RNA of the Hepatitis B virus (HBV) and the attachment of Helicobacter pylori (H. pylori) to the gastric mucosa. NMR is a powerful tool in biomedicine, because molecules ranging from small ligands to biomacromolecules can be studied with atomic resolution. Different NMR experiments are designed to determine structures, or to monitor interactions, folding, stability or motion. Paper I describes the analysis of the motions of a regulatory RNA of HBV. The NMR structure of the RNA had revealed before that several well-conserved nucleotides adopt multiple conformations. Therefore an analysis of possible underlying motions was undertaken using two different NMR techniques, one of which (off-resonance ROESY) was applied to nucleic acids for the first time. The observed motions suggest an explanation why the structurally poorly defined nucleotides are highly conserved. In paper II we improved the ROESY NMR experiment, which is used to measure internuclear distances for structure determination of medium-sized molecules. Using a small protein and an organometallic complex as examples, we demonstrated that the new EASY ROESY experiment yields clean spectra that can directly be integrated to derive interatomic distances. H. pylori, the bacterium involved in peptic ulcer disease and gastric cancer, survives in the harsh acidic environment of the stomach. It possesses many membrane proteins which mediate adherence, raising the question, if their activity is related to membrane composition. In paper III & IV we analyzed therefore the phospholipid composition of H. pylori membranes. In paper III, an advanced method for the analysis of the phospholipid composition of biological membranes was developed. The two-dimensional semi-constant-time 31P,1H-COSY experiment combines information from phosphorus and hydrogen atoms of phospholipids for their unambiguous identification. Furthermore, the high resolution of the two-dimensional experiment allows the quantification of phospholipids where conventional methods fail. In paper IV we applied the new experiment to analyze the lipid composition of whole H. pylori cells, their inner and outer membranes, and of vesicles shed by the bacterium. The goal of this study was to characterize the vesicles which are suggested to play a role in the inflammation process. We established that the outer membrane and the vesicles have similar phospholipid compositions, suggesting that the vesicles are largely derived from the outer membrane. The NMR results presented here elucidate details of molecular systems engaged in pathogenicity, as basis for therapeutic strategies against these pathogens.

This thesis addresses gaps that exist in the theory and knowledge of ancient DNA (aDNA). Much of the underlying basis of the field has been neglected in the excitement that followed the first aDNA studies. Therefore the results of many studies have been based on untested assumptions about the nature of post mortem DNA damage, sample preservation, contamination, and the efficacy of sample decontamination techniques. The validity of such results is questionable if the assumptions prove false. Hydrolytic post mortem DNA damage may modify recovered aDNA sequences. This thesis reports new insights into the biochemical basis of, predisposition of certain sequences and nucleotide positions towards, and subsequent effects of, such damage. Parallels of post mortem damage with in vivo mutation also enable insights into DNA sequence evolution. The long-term survival of DNA, and contamination of samples with exogenous DNA are two related problems characteristic to aDNA. The survival of endogenous DNA within bone, teeth and hair samples, the susceptibility of such samples to contamination, and the efficacy of decontamination techniques used to remedy such problems are investigated. The results highlight serious flaws in using bone and teeth as a DNA source. In contrast, the results demonstrate that hair may present a valuable DNA source for future studies. Numerous studies have reported the retrieval of ancient pathogen DNA from human samples. Analyses of the DNA content within teeth extracted from putative victims of the 2^nd plague argue that such studies are at great risk from DNA degradation, and contamination arising due to environmental microorganisms. An extrapolation of these results using basic physical and chemical theory is used to evaluate the potential survival of aDNA in ancient Egyptian remains. This suggests that positive results from such samples are unlikely.