Na patogênese do diabete melito tipo 1 (DM1) vários genes e fatores ambientais, como as infecções virais, interagem para iniciar um ataque autoimune contra as células beta pancreáticas. Durante a fase inicial desse processo, as células beta desempenham um papel importante através da promoção de um “diálogo” com o sistema imune. Recentemente, o uso de técnicas de genotipagem em larga escala proporcionou um aumento significativo no número de genes conhecidos potencialmente associados ao desenvolvimento do DM1. Para compreender como esses novos genes candidatos modificam as respostas das células beta pancreáticas a mediadores inflamatórios e aos vírus, nós analisamos os dados de estudos prévios de array e um banco de dados online (www.t1dbase.org) para identificar os genes expressos nas células beta e modificados por citocinas ou pelo subproduto da replicação viral, RNA de fita dupla (RNAfd). Dois genes foram selecionados para serem estudados nesta tese, PTPN2 e MDA5. PTPN2 é uma proteína tirosina fosfatase que tem entre os seus alvos o STAT1, um fator de transcrição chave no processo de morte das células beta. Inicialmente, confirmamos a presença de PTPN2 pela quantificação do seu RNAm e produto protéico em uma linhagem de células beta (INS-1E), células beta primárias de rato purificadas por FACS e ilhotas humanas. Tratamento com citocinas ou RNAfd intracelular significativamente aumentou a sua expressão. O knockdown específico deste gene pela técnica de RNA de interferência aumentou significativamente a apoptose das células beta expostas a uma combinação de citocinas (interleucina-1! (IL-1!) + interferon-" (IFN-")) ou RNAfd intracelular, e converteu IFN-" isoladamente em um estímulo pró-apoptótico. O silenciamento do PTPN2 amplificou a fosforilação do STAT1. O duplo knockdown, PTPN2 + STAT1, protegeu as células beta contra a apoptose induzida por citocinas, sugerindo que PTPN2 age como um regulador negativo dos efeitos pró-apoptóticos do STAT1. Contudo, o silenciamento do PTPN2 não produziu nenhuma alteração maior na expressão de citocinas e quimiocinas. O segundo gene candidato, MDA5, é uma helicase associada com o reconhecimento de ácidos nucléicos virais intracelulares. A principal função do MDA5 é a detecção de infecções virais; sendo assim, esse gene foi avaliado apenas no contexto do mimético viral RNAfd. A transfecção de células INS-1E e células beta primárias de rato purificadas por FACS com RNAfd induziu um aumento significativo no RNAm codificando MDA5. O silenciamento do MDA5 e do RIG-I (outra helicase envolvida no reconhecimento do RNAfd intracelular) não modificou a frequência da apoptose induziu por RNAfd. Por outro lado, o knockdown do MDA5, mas não do RIG-I, significativamente reduziu a expressão de várias citocinas/quimiocinas produzidas pelas células beta expostas ao RNAfd intracelular. Concluindo, os dados apresentados sugerem que esses dois genes candidatos, através de suas funções nas células beta, podem ter importantes papéis no desenvolvimento do DM1. PTPN2 aparentemente previne a apoptose das células beta controlando a ativação do STAT1, enquanto MDA5 pode regular o ataque imune local através da diminuição no recrutamento e ativação das células do sistema imune. / In type 1 diabetes (T1D) several genes and environmental factors, such as viral infections, interact to trigger a chronic autoimmune assault against the insulin-producing pancreatic beta cells. During this process beta cells have an important role in maintenance/amplification of this autoimmune response via a cross-talk with the immune system. In recent years the development of high-throughput techniques for searching new genetic variants significantly increased the number of known genes potentially contributing for T1D. To clarify how these new candidate genes modify pancreatic beta responses to proinflammatory mediators and viruses, we used data from our previous array studies and an online beta cell database (www.t1dbase.org) to select candidate genes that are expressed in beta cells and modified by cytokines or the viral by-product double-stranded RNA (dsRNA). Two genes were identified, PTPN2 and MDA5, and further studied in this thesis. PTPN2 is a protein tyrosine phosphatase with several targets including STAT1, a key transcription factor involved in beta cell death. We confirmed at mRNA and protein levels the expression of PTPN2 in a beta cell linage (INS-1E), primary FACS-purified rat beta cells and human islets. Exposure to cytokines or to intracellular dsRNA increased PTPN2 expression. Knockdown of PTPN2, by using specific small interference (si)RNAs, exacerbated beta cell apoptosis after treatment with a combination of cytokines (interleukin-1! (IL-1!) + interferon-" (IFN-")) or intracellular dsRNA, and converted IFN-" alone in a pro-apoptotic stimulus. Importantly, PTPN2 silencing amplified STAT1 phosphorylation. The double knockdown of PTPN2 and STAT1 protected beta cells against cytokine-induced apoptosis, suggesting that PTPN2 acts as a negative regulator of the pro-apoptotic transcription factor STAT1. Knocking-down PTPN2, however, did not affect to any major extent the expression of cytokines and chemokines. The second candidate gene, MDA5, is an helicase involved in recognition of intracellular viral nucleic acids. Since MDA5 works as a receptor for detection of viral infection, this gene was only evaluated in the context of the viral mimetic dsRNA. Transfection of INS-1E cells and FACSpurified rat beta cells with dsRNA significantly upregulated the mRNA expression of MDA5. The silencing of MDA5 and RIG-I (another helicase involved in recognition of intracellular dsRNA) did not modify dsRNA-induced apoptosis. On the other hand, the knockdown of MDA5, but not of RIG-I, decreased the expression of several cytokines/chemokines in beta cells exposed to intracellular dsRNA. In conclusion, our data suggest that these two candidate genes may have important roles in the development of T1D via their actions at the beta cell level. PTPN2 seems to prevent beta cells apoptosis by controlling STAT1 activation, while MDA5 might regulate the local autoimmune assault via decreasing recruitment and activation of immune cells.

Dbp2p, a member of the large family of DEAD-box proteins and a yeast homolog of human p68, was shown to interact with Upf1p, an essential component of the nonsense-mediated mRNA decay pathway. Dbp2p:Upf1p interaction occurs within a large conserved region in the middle of Upf1p that is largely distinct from its Nmd2p and Sup35/45p interaction domains. Deletion of DBP2, or point mutations within its highly conserved DEAD-box motifs, increased the abundance of nonsense-containing transcripts, leading us to conclude that Dbp2p also functions in the nonsense-mediated mRNA decay pathway. Dbp2p, like Upf1p, acts before or at decapping, is predominantly cytoplasmic, and associates with polyribosomes. Interestingly, Dbp2p also plays an important role in rRNA processing. In dbp2Δ cells, polyribosome profiles are deficient in free 60S subunits and the mature 25S rRNA is greatly reduced. The ribosome biogenesis phenotype, but not the mRNA decay function, of dbp2Δ cells can be complemented by the human p68 gene. We propose a unifying model in which Dbp2p affects both nonsense-mediated mRNA decay and rRNA processing by altering rRNA structure, allowing specific processing events in one instance and facilitating dissociation of the translation termination complex in the other.

In eukaryotes, the spliceosome is a macromolecular ribonucleoprotein machine that excises introns from pre-mRNAs through two sequential transesterification reactions. The chemistry and fidelity of pre-mRNA splicing are dependent upon a series of spliceosomal rearrangements, which are mediated by trans-acting splicing factors. One key class of these factors is the DEAH-box ATPase subfamily of proteins, whose members couple ATP hydrolysis to promote RNP structural rearrangements within the spliceosome. This is typified by Prp22, which promotes release of the spliced mRNA from the spliceosome and ensures fidelity of the second step of splicing. This role is well documented through classical biochemical and yeast genetics methods. Yet very little is known regarding the comings and goings of Prp22 relative to the spliceosome. My thesis research investigated the dynamics of Prp22 during splicing by using single-molecule fluorescence methods that allowed direct observation of these events. To do this, I helped construct a toolkit that combined yeast genetics, chemical biology and Colocalization Single Molecule Spectroscopy (CoSMoS) with in vitro splicing assays. Specifically, my thesis research consisted of CoSMoS splicing experiments in which fluorescently labeled pre-mRNA, spliceosome components and Prp22 were directly visualized and analyzed. Using these methods, I found that Prp22’s interactions with the spliceosome are highly dynamic and reversible. By simultaneously monitoring Prp22 and individual spliceosome subcomplexes, I was able to frame these Prp22 binding events in context relative to specific steps in spliceosome assembly and splicing. These experiments provide insight into how Prp22 promotes mRNA release from the spliceosome and maintains splicing fidelity.

No description available.

Thesis (Ph. D.)--University of Missouri--Columbia, 2000. / Typescript. Vita. Includes bibliographical references (leaves 107-112). Also available on the Internet.

Thesis (Ph.D. in Pharmacology) -- University of Colorado Denver, 2007. / Typescript. Includes bibliographical references (leaves 82-92; 128-134). Online version available via ProQuest Digital Dissertations.

Les hélicases à ARN de la famille DEAH/RHA sont impliquées dans la plupart des processus essentiels à la vie tels que l'épissage, la biogenèse des ribosomes, la réplication, la transcription ou encore la détection d’ARN viraux. Ces enzymes sont capables de catalyser la dissociation de duplexes d'ARN, la réorganisation de structures secondaires ou de remodeler des complexes ARN-protéines. L'hélicase DEAH/RHA Prp43 présente la particularité d'être bifonctionnelle. Prp43 est impliquée dans l'épissage des Pré-ARNm, où elle assure le recyclage du spliceosome et du lasso, mais aussi dans la biogenèse des ribosomes où elle est impliquée dans la maturation des deux sous-unités. Prp43 est activée et régulée par cinq partenaires protéiques : Ntr1, Gno1, Pfa1, RBM5 et GPATCH2. Ces partenaires protéiques présentent tous un domaine G-patch et sont capables de stimuler les activités hélicase et ATPase de Prp43. La structure cristallographique de Prp43 en complexe avec l'ADP a été résolue au laboratoire. Cette structure a mis en évidence un mode de fixation du nucléotide inédit chez les autres hélicases, notamment au niveau de la base qui s'empile entre la phénylalanine 357 (F357) du domaine RecA2 et l'arginine 159 (R159) du domaine RecA1. Les déterminants de l'activation de Prp43 par les protéines à domaine G-patch demeurent méconnus. Dans ce travail, nous avons cherché à déterminer quel était le rôle de l’empilement de la base dans l’activation de Prp43. Nous présentons ici plusieurs structures cristallographiques de Prp43 en complexe avec tous les nucléotides diphosphates(NDP) et les désoxynucléotides triphosphates (dNDP). Ces structures ont permis de conclure qu'il y avait des différences dans l’empilement de la base selon le (d)NDP considéré. Des dosages d'activité NTPase de Prp43 avec et sans son partenaire protéique Pfa1 montrent que lorsque la base ne s'empile pas avec la F357 et la R159, l'activité de l'enzyme n'est pas correctement régulée par son partenaire protéique. Les dosages d’activité enzymatique sur les mutants ponctuels F357A et R159A révèlent que le résidu F357 permet de moduler l’activité de Prp43. Tous ces résultats nous ont permis de mettre en évidence un modèle de la régulation de Prp43 par les protéines à domaines G-patch et d'expliquer l'importance du mode de fixation de la base à l'enzyme dans cette régulation. / RNA helicases from the DEAH/RHA family are involved in most of essential processes of life such as pre-mRNA splicing, ribosome biogenesis, replication, transcription or viral RNA sensing. These enzymes are able to catalyze RNA unwinding, secondary structures reorganization or RNA-protein complexes remodeling. The DEAH/RHA helicase Prp43 is remarkable because it is bifunctional, as it is involved both in pre-mRNA splicing, where it is responsible of spliceosome and lariat recycling and in the biogenesis of the two ribosomal subunits. Prp43 is activated by five protein partners: Ntr1, Gno1, Pfa1, RBM5 and GPATCH2. These protein partners all possess a G-patch domain and are able to stimulate helicase and ATPase activity of Prp43. The structure of Prp43 in complex with ADP has been solved by X-ray crystallography. The structure reveals that the nucleotide is bound to the enzyme in a novel mode that has never been observed in other known helicase structures. The specific feature of this binding mode is the base, stacked between phenylalanine (F357) from RecA2 domain and an arginine (R159) from RecA1 domain. Features of the activation of Prp43 by G-patch proteins are unclear. In this work, we investigated the role of base stacking in the activation of Prp43. We present several structures of Prp43 bound to all the nucleotide diphosphates (NDP) and deoxynucleotide diphosphates (dNTP). These results indicate that there are differences in stacking according to the (d)NDP bound to the enzyme. NTPase activity assays revealed that when stacking is weakened, Prp43 activity cannot be properly regulated by its protein partner Pfa1. Moreover, point mutations F357A and R159A show that stacking of F357 permits to modulate Prp43 activity. All these results allow us to propose a model of NTPase activity activation of Prp43 by G-patch proteins and to highlight the importance of base stacking in this regulation.

Le virus de l’immunodéficience humaine de type 1 (VIH-1) est un virus à simple brin positif qui appartient au genre Lentivirus dans la famille retroviridae et qui constitue l’agent étiologique du SIDA pandémique.Pendant le cycle réplicatif du VIH-1, la traduction de protéines virales dépend exclusivement de la machinerie traductionnelle cellulaire. Pour cette raison, nous avons cherché à comprendre le rôle de quelques facteurs cellulaires qui pourraient contrôler la traduction du VIH-1 à différents nivaux. Nous avons centré nos recherches sur la traduction de l’ARN génomique (ARNg) du virus qui sert en même temps de génome pour être encapsidé et comme ARN messager pour la traduction des protéines virales Gag et Gag-Pol. 1) Le rôle de l’hélicase d’ARN DDX3 dans la traduction du VIH-1. L’ARNg du VIH-1 possède une région 5’ non traduite très structurée, raison pour laquelle nous avons spéculé sur un possible rôle de DDX3 dans la traduction du VIH-1. Nous avons utilisé une combinaison de techniques in vitro et ex vivo afin de pouvoir démontrer que DDX3 était capable de lier et faire des complexes avec l’ARN de la région 5’ non traduite pour promouvoir l’initiation de la traduction. Nous avons aussi pu démontrer que DDX3 formait des complexes avec les facteurs d’initiation de la traduction PABP, eIF4G et eIF4E. 2) Le changement programmé du cadre de lecture (PRF) dans l’ARN génomique du VIH-1. La traduction de la polyprotéine Gag-Pol du VIH-1 nécessite un décalage de phase de 1 nucléotide en arrière. Ce mécanisme permet la synthèse des protéines Gag et Gag-Pol avec des ratios de 95 et 5% respectivement à partir du même ARN. Cette proportion doit être conservée pour assurer la réplication du virus. Nous avons utilisé un système de double gène rapporteurs et un système de réplication complète du provirus pour montrer que la protéine associé aux granules de stress TIAR pouvait contrôler la réplication viral en régulant la proportion de ribosome qui assurent / Human Immunodeficiency virus type 1 (HIV-1) is a positive strand RNA virus belonging to the lentivirus genus of the retroviridae family and it is the etiological agent of the pandemic AIDS, which is a major health concern worldwide. Throughout HIV-1 replication cycle, the production of viral proteins depends exclusively on the cellular translational machinery. This is the reason why we have explored the role of some cellular factors that could control HIV-1 translation at different stages. We have focused our studies on the translation of the full length genomic RNA (gRNA), which serves both as genome for viral encapsidation and as a messenger for translation of Gag and Gag-Pol viral polyproteins.1) The role of the RNA helicase DDX3 in HIV-1 translation Initiation The fact that HIV-1 possesses a highly structured 5’ untranslated region (5’UTR) prompted us to speculate that DDX3 may be involved in HIV-1 translation. We used a combination of in vitro and ex-vivo approaches to show that DDX3 was able to bind and form complexes with the 5’-UTR of HIV-1 to assist translation initiation. We also demonstrated that DDX3 can form a complex with initiation factors such as PABP, eIF4G and eIF4E. 2) Programmed Ribosomal Frameshift (PRF) in the genomic RNA of HIV-1Translation of HIV-1 Gag-Pol polyprotein requires a -1 PRF. This mechanism allows the synthesis of Gag and Gag-Pol polyproteins, using the same mRNA template, at ratios of 95 and 5% respectively. Keeping the -1PRF ratio is important as any change leads to reduction in virus infectivity.By means of a dual reporter construct and full provirus replication system we were able to demonstrate that the stress granules associated protein TIAR, controls HIV-1 infectious progeny by regulating the ratio of the HIV-1 PRF.

All organisms exist in some sort of symbiosis with their environment. The food we eat, air we breathe, and things we touch all have their own microbiota and we interact with these microbiota on a daily basis. As such, we employ a method of compartmentalization in order to keep foreign entities outside of the protected internal environments of the body. However, as other organisms seek to replicate themselves, they may invade our sterile compartments in order to do so. To protect ourselves from unfettered replication of pathogens or from cellular damage, we have developed a series of receptors and signaling pathways that detect foreign bodies as well as abnormal signals from our own perturbed cells. The downstream effector molecules that these signaling pathways initiate can be toxic and damaging to both pathogen and host, so special care is given to the regulation of these systems. One method of regulation is the production of endogenous small ribonucleic acids that can regulate the expression of various receptors and adaptors in the immune signaling pathways. In this dissertation, I present work that establishes an important protein in small ribonucleic acid regulation, Dicer, as an essential protein for regulating the innate immune response to immuno-stimulatory nucleic acids as well as regulating the productive infection of encephalomyocarditis virus. Depleting Dicer from murine embryonic fibroblasts renders a disparate type I interferon response where nucleic acid stimulation in the Dicer null cells fails to produce an appreciable interferon response while infection with the paramyxovirus, Sendai, induces a more robust interferon response than the wild-type control. Additionally, I show that Dicer plays a vital role in controlling infection by the picornavirus, encephalomyocarditis virus. Encephalomyocarditis virus fails to grow efficiently in Dicer null cells due to the inability for the virus to bind to the outside of the cell, suggesting that Dicer has a role in modulating viral infection by affecting host cellular protein levels. Together, this work identifies Dicer as a key protein in viral innate immunology by regulating both the growth of virus and also the immune response generated by exposure to pathogen associated molecular patterns. Understanding this regulation will be vital for future development of small molecule therapeutics that can either modulate the innate immune response or directly affect viral growth.

Knowledge of the dissociation constants of the ionizable protons of weak acids in aqueous media is of fundamental importance in many areas of chemistry and biochemistry. The pKa value, or equilibrium dissociation constant, of a molecule determines the relative concentration of its protonated and deprotonated forms at a specified pH and is therefore an important descriptor of its chemical reactivity. Considerable efforts have been devoted to the determination of pKa values by different experimental techniques. Although in most cases the determination of pKa values from experimental is straightforward, there are situations where interpretation is difficult and the results ambiguous. It is, therefore, not surprising that the capability to provide accurate estimates of the pKa value has been a central goal in theoretical chemistry and there has been a large effort in developing methodologies for predicting pKa values for a variety of chemical systems by differing quantum chemical techniques. A prediction accuracy within 0.5 pKa units of experiment is the desirable level of accuracy. This is a non-trivial exercise, for an error of 1 kcal/mol in estimates of the free energy value would result in an error of 0.74 pKa units. In this thesis ab initio Car-Parrinello molecular dynamics (CPMD) has been used for investigating the Brϕnsted acid-base chemistry of weak acids in aqueous solution. A key issue in any dissociation event is how the solvating water molecules arrange themselves spatially and dynamically around the neutral and dissociated acid molecule. Ab initio methods have the advantage that all solvent water molecules can, in principle, be con- sidered explicitly. One of the factors that has inhibited the widespread use of ab initio MD methods to study the dissociation reaction is that dissociation of weak acids are rare events that require extremely long simulation times before one is observed. The metady- namics formalism provides a solution to this conundrum by preventing the system from revisiting regions of configuration space where it has been in the past. The formalism allows the system to escape the free-energy minima by biasing the dynamics with a history dependent potential (or force) that acts on select degrees of freedom, referred to as collective variables. The bias potentials, modeled by repulsive inverted Gaussians that are dropped during propagation, drive the system out of any free-energy minima and allow it to explore the configurational space by a relatively quick and efficient sampling. The the- sis deals with a detailed investigation of the Brϕnsted acid-base chemistry of weak acids in aqueous solutions by the CPMD-metadynamics procedure. In Chapter 1, current approaches for the theoretical estimation of pKa values are summarized while in Chapter 2 the simulation methodology and the metadynamics sampling techniques used in this study are described. The potential of the CPMD-metadynamics procedure to provide estimates of the acid dissociation constant (pKa) is explored in Chapter 3, using acetic acid as a test sys- tem. Using the bond-distance dependent coordination number of protons bound to the dissociating carboxylic groups as the collective variable, the free-energy profile for the dissociation reaction of acetic acid in water was computed. Convergence of the free-energy profiles and barriers for the simulations parameters is demonstrated. The free-energy profiles exhibit two distinct minima corresponding to the dissociated and neutral states of the acid and the deterrence in their values provides the estimate for pKa. The estimated value of pKa for acetic acid from the simulations, 4.80, is in good agreement with the experiment at value of 4.76. It is shown that the good agreement with experiment is a consequence of the cancellation of errors, as the pKa values are computed as the divergence in the free energy values at the minima corresponding to the neutral and dissociated state. The chapter further explores the critical factors required for obtaining accurate estimates of the pKa values by the CPMD-metadynamics procedure. It is shown that having water molecules sufficient to complete three hydration shells as well as maintaining water density in the simulation cell as close to unity is important. In Chapter 4, the CPMD-metadynamics procedure described in Chapter-3 has been used to investigate the dissociation of a series of weak organic acids in aqueous solutions. The acids studied were chosen to highlight some of the major factors that influence the dissociation constant. These include the influence of the inductive effect, the stabilization of the dissociated anion by H-bonding as well as the presence of multiple ionizable groups. The acids investigated were aliphatic carboxylic acids, chlorine-substituted carboxylic acids, cis- and trans-butenedioic, the isomers of hydroxybenzoic acid and ophthalmic acids and its isomers. It was found that in each of these examples the CPMD-metadynamics procedure correctly estimates the pKa values, indicating that the formulism is capable of capturing these influences and equally importantly indicating that the cancellation of errors is indeed universal. Further, it is shown that the procedure can provide accurate estimates of the successive pKa values of polypro tic acids as well as the subtle difference in their values for different isomers of the acid molecule. Changes in protonation-deprotonation of amino acid residues in proteins play a key role in many biological processes and pathways. It is shown that CPMD simulations in conjunction with metadynamics calculations of the free energy profile of the protonation- deprotonation reaction can provide estimates of the multiple pKa values of the 20 canonical α-amino acids in aqueous solutions in good agreement with experiment (Chapter 5). The distance-dependent coordination number of the protons bound to the hydroxyl oxygen of the carboxylic and the amine groups is used as the collective variable to explore the free energy profiles of the Brϕnsted acid-base chemistry of amino acids in aqueous solutions. Water molecules, sufficient to complete three hydration shells surrounding the acid molecule were included explicitly in the computation procedure. The method works equally well for amino acids with neutral, acidic and basic side chains and provides estimates of the multiple pKa values with a mean relative error with respect to experimental results, of 0.2 pKa units. The tripeptide Glutathione (GSH) is one of the most abundant peptides and the major repository for non-protein sulfur in both animal and plant cells. It plays a critical role in intracellular oxidative stress management by the reversible formation of glutathione disulfide with the thioldisulfide pair acting as a redox buffer. The state of charge of the ionizable groups of GSH can influences the redox couple and hence the pKa value of the cysteine residue of GSH is critical to its functioning. In Chapter 6, it has been reported that ab initio Car-Parrinello Molecular Dynamics simulations of glutathione solvated by 200 water molecules, all of which are considered in the simulation. It is shown that the free-energy landscape for the protonation - deprotonation reaction of the cysteine residue of GSH computed using metadynamics sampling provides accurate estimates of the pKa and correctly predicts the shift in the dissociation constant values as compared to the isolated cysteine amino acid. The dissociation constants of weak acids are commonly determined from pH-titration curves. For simple acids the determination of the pKa from the titration curves using the Henderson-Hasselbalch equation is relatively straightforward. There are situations, however, especially in polyprotic acids with closely spaced dissociation constants, where titration curves do not exhibit clear inflexion and equivalence stages and consequently the estimation of multiple pKa values from a single titration curve is no longer straightfor- ward resulting in uncertainties in the determined pKa values. In Chapter 7, the multiple dissociation constant of the hexapeptide glutathione disulfide (GSSG) with six ionizable groups and six associated dissociation constants has been investigated. The six pKa values of GSSG were estimated using the CPMD-metadynamics procedure from the free-energy profiles for each dissociation reaction computed using the appropriate collective variable. The six pKa values of GSSG were estimated and the theoretical pH-titration curve was then compared with the experimentally measured pH-titration curve and found to be in excellent agreement. The object of the exercise was to establish whether interpretation of pH-titration curves of complex molecules with multiple ionizable groups could be facilitated using results of ab initio molecular dynamics simulations.