This thesis covers a range of methodologies to provide an account of the current (2010-2014) state of the art and to develop new methods for solubility prediction. We focus on predictions of intrinsic aqueous solubility, as this is a measure commonly used in many important industries including the pharmaceutical and agrochemical industries. These industries require fast and accurate methods, two objectives which are rarely complementary. We apply machine learning in chapters 4 and 5 suggesting methodologies to meet these objectives. In chapter 4 we look to combine machine learning, cheminformatics and chemical theory. Whilst in chapter 5 we look to predict related properties to solubility and apply them to a previously derived empirical equation. We also look at ab initio (from first principles) methods of solubility prediction. This is shown in chapter 3. In this chapter we present a proof of concept work that shows intrinsic aqueous solubility predictions, of sufficient accuracy to be used in industry, are now possible from theoretical chemistry using a small but diverse dataset. Chapter 6 provides a summary of our most recent research. We have begun to investigate predictions of sublimation thermodynamics. We apply quantum chemical, lattice minimisation and machine learning techniques in this chapter. In summary, this body of work concludes that currently, QSPR/QSAR methods remain the current state of the art for solubility prediction, although it is becoming possible for purely theoretical methods to achieve useful predictions of solubility. Theoretical chemistry can offer little useful additional input to informatics models for solubility predictions. However, theoretical chemistry will be crucial for enriching our understanding of the solvation process, and can have a beneficial impact when applied to informatics predictions of properties related to solubility.

The rationalisation of drug potency using three-dimensional structures of protein-ligand complexes is a central paradigm in medicinal research. For over two decades, a major goal has been to find the rules that accurately relate the structure of any protein-ligand complex to its affinity. Addressing this problem is of great concern to the pharmaceutical industry, which uses virtual screens to computationally assay up to many millions of compounds against a protein target. A fast and trustworthy affinity estimator could potentially streamline the drug discovery process, reducing reliance on expensive wet lab experiments, speeding up the discovery of new hits and aiding lead optimization. Water plays a critical role in drug-protein interactions. To address the often ambiguous nature of water in binding sites, a water placement method was developed and found to be in good agreement with X-ray crystallography, neutron diffraction data and molecular dynamics simulations. The method is fast and has facilitated a large scale study of the statistics of water in ligand binding sites, as well as the creation of models pertaining to water binding free energies and displacement propensities, which are of particular interest to medicinal chemistry. Structure-based scoring functions employing the explicit water models were developed. Surprisingly, these attempts were no more accurate than the current state of the art, and the models suffered from the same inadequacies which have plagued all previous scoring functions. This suggests a unifying cause behind scoring function inaccuracy. Accordingly, mathematical analyses on the fundamental uncertainties in structure-based modelling were conducted. Using statistical learning theory and information theory, the existence of inherent errors in empirical scoring functions was proven. Among other results, it was found that even the very best generalised structure-based model is significantly limited in its accuracy, and protein-specific models are always likely to be better. The theoretical framework developed herein hints at modelling strategies that operate at the leading edge of achievable accuracy.

O microrganismo gram-negativo Aggregatibacter actinomycetemcomitans, fortemente associado a quadros de periodontite agressiva, coloniza a cavidade oral, aderindo e invadindo as células epiteliais e participando da formação de biofilme nas superfícies do hospedeiro. A. actinomycetemcomitans expressa Aae, uma proteína de superfície, relacionada com a adesão às células epiteliais. Este estudo avaliou o papel de Aae na adesão a outros substratos tais como proteínas extracelulares colagenosas e não-colagenosas, hidroxiapatita recoberta por saliva (SHA) e na formação de biofilme. Um mutante nulo em aae foi construído e o fenótipo comparado com o da linhagem selvagem (VT1169). O mutante nulo exibiu significativa redução na adesão às células epiteliais e a SHA, na formação de biofilme, além de apresentar-se menos hidrofóbico que a linhagem parental. A capacidade de adesão ao colágeno V e a fibronectina foi fracamente afetada pela interrupção do gene aae. Entretanto, o mutante nulo em aae exibiu uma capacidade diminuída na adesão à laminina e colágenos I, III e IV. Estes dados sugerem que Aae desempenha um importante papel na colonização da cavidade oral, não somente promovendo sua adesão às células epiteliais, mas também a outros substratos. / The gram-negative organism Aggregatibacter actinomycetemcomitans, associated with aggressive periodontitis, colonizes the oral cavity by binding to and invading epithelial cells and by participating in biofilms formed in host surfaces. A. actinomycetemcomitans express Aae, a surface protein, implicated in the adhesion to epithelial cells. This study evaluated the role of Aae in adhesion to other substrates such as collagen and non-collagen extracellular matrix proteins and saliva coated hydroxyapatite (SHA) and in biofilm formation. A null mutant in aae was constructed and its behavior was compared with the wild type strain VT1169. The null mutant exhibited a decreased ability to bind to epithelial cells and to SHA, formed less biofilm and was less hydrophobic than the parental strain. The abilities to bind to collagen V and fibronectin were very poorly affected by aae interruption. However, the null aae mutant exhibited a decreased ability to adhere to laminin, collagen I, III and IV. These data suggest that Aae may play an important role in the colonization of the oral cavity by A. actinomycetemcomitans, not only by promoting its adhesion to epithelial cells, but by mediating adhesion to other substrates.

Made available in DSpace on 2017-07-21T20:42:46Z (GMT). No. of bitstreams: 1 Mauricio Marlon Mazur.pdf: 3613957 bytes, checksum: 0b5adb9ffe8a242c568978a647c5e384 (MD5) Previous issue date: 2014-04-16 / Instituto de Tecnologia para o Desenvolvimento / Ceramic electric insulators are fundamental support devices for electric energy distribution due to its mechanical resistance and dielectric properties. The presence of atmospheric humidity and pollutants can reduce the dielectric property. To achieve an auto cleaning surface or hydrophobic properties, the insulator must have low contact angle and low hysteresis with water. Although, the insulator vitreous surface layer possess great interaction affinity with environmental water. At the present work, aluminum nitride thin films were deposited on the vitreous protective layer of ceramic electric insulators. For this, a pulsed magnetron sputtering plasma was employed, with direct current (DC), and moisture of argon and nitrogen gases. An aluminum target with 99.999% purity was used as precursor. Continuous films deposited produced with Ar and N2 presented about 2 μm thickness, while the films produced with 100% N2 presented around 1 μm, both for the same treatment time. Low exposure times lead to depositions on surface defects and porosity of the insulator vitreous body and showed nanosize island growth (166, 155, 100 nm). The films thickness and surface characterization was performed with field emission scanning electron microscopy (FEG) and atomic force microscopy (AFM). Raman spectroscopy and X-ray diffraction showed the formation of an amorphous aluminum nitride film. The continuous film produced with 100% N2 presented 82.7° water drop contact angle, while the non-continuous film presented in average 67°. According to ASTM D7334-08, these films are hydrophobic. Roughness was not a determinative parameter for surface wettability, but the deposited film surface energy. To measure the contact angle a goniometer was developed. A four point probe was used to measure the electric resistivity for a semi-infinite surface. The Ar and N2 continuous film surface resistivity was 656/. For the nanosize films the surface resistivity was about M/. / Isoladores elétricos cerâmicos são dispositivos de suporte fundamentais para distribuição de energia elétrica, devido a sua resistência mecânica e a sua propriedade dielétrica. A presença tanto de umidade, como de poluentes na atmosfera, reduz essa capacidade. Com o intuito de se obter uma propriedade autolimpante ou hidrofóbica a superfície deve apresentar um baixo ângulo de contato com a água e possuir baixa histerese. No entanto, o vidrado do isolador apresenta uma grande afinidade com a água resultante da interação da superfície do vidro com a atmosfera. No presente trabalho, foi realizada a deposição de filmes de nitreto de alumínio sobre o vidrado de proteção do isolador elétrico cerâmico. Para isto, um plasma magnetron sputtering pulsado, de corrente contínua (CC), foi utilizado com a mistura dos gases argônio e nitrogênio. Como precursor de alumínio foi utilizado um alvo de 99,999% de pureza. Os filmes contínuos produzidos com Ar e N2 apresentaram espessura de, aproximadamente, 2 μm, enquanto os filmes produzidos com 100% de N2 possuem espessura de aproximadamente 1μm, ambos com o mesmo tempo de tratamento. Os filmes depositados sobre a superfície do vidrado com baixos tempos de exposição apresentaram crescimento em ilhas com tamanho nanométrico (166, 155, 100 nm) sobre os defeitos e as porosidades do corpo do isolador. A caracterização da espessura do filme e da superfície foi realizada com técnicas de microscopia eletrônica de varredura de efeito de campo (FEG) e microscopia de força atômica (AFM). Análises de espectroscopia Raman e de difração de raios X indicaram a formação de um filme de nitreto de alumínio (AlN) amorfo. O filme contínuo utilizando apenas N2 apresentou um ângulo de contato com a gota de água de 82,7°, enquanto os filmes não contínuos apresentaram em média 67°. Em acordo com a norma ASTM D7334-08 os filmes apresentam propriedade hidrofóbica. A rugosidade superficial não foi um fator determinante na molhabilidade superficial e sim a energia de superfície do filme depositado. Para a medição do ângulo de contato (Ɵ) foi desenvolvido um goniômetro. As propriedades da resistividade dos filmes para superfícies semi-infinitas foi adquirida pela técnica de sonda quatro pontas. A resistividade superficial do filme contínuo com Ar e N2 foi de 656/. Para os filmes nanoestruturados a resistividade superficial foi da ordem de M/.

La photothérapie dynamique (PDT) consiste en la destruction d’une tumeur par l’association de l’administration d’un photosensibilisateur et de l’exposition à la lumière visible. Ce travail comporte : i) une étude de l’ionisation et de la lipophilie d’une série de photosensibilisateurs, des méso-tétraphénylporphyrines (TPP) glycoconjuguées, ii) une évaluation de l’impact de ces deux propriétés sur la distribution plasmatique et la localisation subcellulaire du photosensibilisateur.La protonation des azotes tétrapyrroliques a été étudiée par spectroscopie électronique, combinéeà une analyse chimiométrique, tandis que la lipophilie a été évaluée par chromatographie liquide haute performance. L’impact de différents effets de substitution (position, nombre ou nature du substituant) sur ces deux propriétés physico-chimiques a été mis en évidence.Dans le plasma, les TPP glycoconjuguées se lient principalement aux lipoprotéines de haute densité. La lipophilie de ces dérivés permet d'expliquer leur affinité pour les lipoprotéines, mais pas pour l'albumine. L’étude de localisation subcellulaire, combinant approche expérimentale et modélisation, a conduit à proposer une hypothèse expliquant la localisation de la TPP(pODEGOαManOH)3 au niveau du réticulum endoplasmique, hypothèse accordant un rôle central à la lipophilie de la TPP . A l'issue de ce travail, avant d'appliquer nos hypothèses à la synthèse de nouvelles molécules, il apparaît nécessaire de mieux explorer l'impact de la distribution plasmatique et de la localisation subcellulaire sur l'efficacité PDT. / Photodynamic Therapy (PDT) is based on the destruction of a tumor tissue through a combinationof administration of a photosensitizer and exposure to visible light. This work includes : i) a study of ionization and hydrophobicity of a series of candidate sensitizers, glycoconjugated mesotetraphenylporphyrins (TPP), ii) an evaluation of the impact of those two physico-chemicalproperties on sensitizer's plasma distribution and subcellular localization. Protonation of tetrapyrrolic nitrogens has been studied by electronic spectroscopy combined with chemometric analysis whereas hydrophobicity has been evaluated by high-performance liquid chromatography. The effect of substitution modalities (position, number and nature of pendantgroups) on both physico-chemical properties has been evidenced.In human plasma, glycoconjugated TPPs mainly bind to high density lipoproteins. Hydrophobicity accounts for differences in affinities towards lipoproteins, but not towards albumin. Subcellular localization studies, combining computational and experimental approaches, led to formulate some assumptions explaining localization of TPP(pODEGOαManOH)3 in endoplasmic reticulum,assumptions centered on a major role of sensitizer's hydrophobicity. At the end of this work, before trying to use our conclusions for the design of new sensitizers, it remains necessary to better explore the effect of plasma distribution and subcellular localization on sensitizer's photo-efficiency.

Due to increasing problems with bacterial resistance development, there is a growing need for identifying new types of antibiotics. Antimicrobial peptides constitute an interesting group of substances for this purpose, since they are believed to act mainly by disrupting the bacterial membrane, which is a fast and non-specific mechanism. In order to understand the details on this action simplified phospholipid model membranes based on liposomes, monolayers and bilayers, were employed in this thesis. By in situ ellipsometry studies on supported lipid bilayers in combination with leakage from liposomes it was found that peptide-induced membrane rupture to a great extent is related to peptide adsorption. The peptide activity and mechanism of action is highly dependent on peptide properties such as length, topology, charge, and hydrophobicity. Electrostatic interactions are crucial for peptide adsorption, whereas α-helix formation is of less importance, demonstrated by the dominating peptide conformation being random coil both in absence and presence of membranes, as investigated by circular dichroism. Comparable effects were observed in both mono- and bilayer systems, showing that formation of transmembrane structures is no prerequisite for membrane rupture by complement-derived peptides. Electrochemical studies on these peptides further demonstrated that hydrophobic interactions facilitate peptide penetration into the membrane, causing defects in close proximity to the peptides, while strong electrostatic interactions arrest the peptide in the headgroup region. Increasing the peptide hydrophobicity, by e.g., tryptophan end-tagging, also increases salt resistance. Good correlations were found between model membrane investigations and antibacterial activity towards both Gram-negative and Gram-positive bacteria, showing that membrane rupture is a key mechanism of action for the peptides investigated. In addition, for all peptides investigated cell toxicity is low.

Hydrophobic interactions guide important molecular self-assembly processes such as protein folding. On the macroscale, hydrophobic interactions consist of the aggregation of "oil-like" objects in water by minimizing the interfacial energy. However, the hydration mechanism of small hydrophobic molecules on the nanoscale (~1 nm) differs fundamentally from its macroscopic counterpart. Theoretical studies over the last two decades have pointed to an intricate dependence of molecular hydration mechanisms on the length scale. The microscopic-to-macroscopic cross-over length scale is critically important to hydrophobic interactions in polymers, proteins and other macromolecules. Accurate experimental determination of hydration mechanisms and their interaction strengths are needed to understand protein folding. This thesis reports the development of experimental and analytical techniques that allow for direct measurements of hydrophobic interactions in a single molecule. Using single molecule force spectroscopy, the mechanical unfolding of a single hydrophobic homopolymer was identified and modeled. Two experiments examined how hydrophobicity at the molecular scale differ from the macroscopic scale. The first experiment identifies macroscopic interfacial tension as a critical parameter governing the molecular hydrophobic hydration strength. This experiment shows that the solvent conditions affect the microscopic and macroscopic hydrophobic strengths in similar ways, consistent with theoretical predictions. The second experiment probes the hydrophobic size effect by studying how the size of a non-polar side-chain affects the thermal signatures of hydration. Our experimental results reveal a cross-over length scale of approximately 1 nm that bridges the transition from entropically driven microscopic hydration mechanism to enthalpically driven macroscopic hydration mechanism. These results indicate that hydrophobic interactions at the molecular scale differ from macroscopic scale, pointing to potential ways to improve our understanding and predictions of molecular interactions. The system established in this thesis forms the foundation for further investigation of polymer hydrophobicity.

Hemp seed proteins (HSP) were investigated for physicochemical and functional properties in model food systems. In addition, the HSP were enzymatically digested and the released peptides investigated as potential therapeutic agents. Membrane isolated HSP (mHPC) were the most soluble with >60% solubility at pH 3-9 when compared to a maximum of 27% for isoelectric pH-precipitated proteins (iHPI). However, iHPI formed emulsions with smaller oil droplet sizes (<1 µm) while mHPI formed bigger oil droplets. The iHPI was subjected to enzymatic hydrolysis using different concentrations (1-4%) of six proteases (pepsin, pancreatin, flavourzyme, thermoase, papain and alcalase) to produce various HSP hydrolysates (HPHs). HPHs had strong in vitro inhibitions of angiotensin converting enzyme (ACE) and renin activities, the two main enzyme systems involved in hypertension. Oral administration of the HPHs to spontaneously hypertensive rats led to fast and persistent reductions in systolic blood pressure. The HPHs also inhibited in vitro activities of acetylcholinesterase (AChE), a serine hydrolase whose excessive activities lead to inadequate level of the cholinergic neurotransmitter, acetylcholine (ACh). Inadequate ACh level in the brain has been linked to neurodegenerative diseases such as dementia and Alzheimer’s disease (AD); therefore, AChE inhibition is a therapeutic target. The 1% pepsin HPH was the most active with up to 54% AChE inhibition at 10 µg/mL peptide concentration. The 1% pepsin HPH (dominated by <1 kDa) was subjected to reverse-phase HPLC peptide purification coupled with tandem mass spectrometry, which led to identification of several peptide sequences. Some of the peptides inhibited activities of both animal and human AChE forms with LYV being the most potent against human AChE (IC50 = 7 µg/ml). Thus the LYV peptide may serve as a useful template for the development of future potent AChE-inhibitory peptidomimetics. In conclusion, several novel AChE-inhibitory peptides were discovered and their amino acid sequences elucidated for the first time. Results from this work identified HSP products that could serve as functional ingredients in the food industry. The work also produced and confirmed the in vitro AChE-inhibitory activities of several new peptide sequences that may serve as therapeutic agents for AD management. / October 2015

Most of the processes in a living cell are carried out by proteins. Depending on the needs of the cell, different proteins will interact and form the molecular machines demanded for the moment. A subset of proteins called integral membrane proteins are responsible for the interchange of matter and information across the biological membrane, the lipid bilayer enveloping and defining the cell. Most of these proteins are co-translationally integrated into the membrane by the Sec translocation machinery. This thesis addresses two questions that have emerged during the last decade. The first concerns membrane proteins: a number of α-helices have been observed to span the membrane in the obtained three-dimensional structures even though these helices are predicted not to be hydrophobic enough to be recognized by the translocon for integration. We show for a number of these marginally hydrophobic protein segments that they indeed do not insert well outside of their native context, but that their local sequence context can improve the level of integration mediated by the translocon. We also find that many of these helices are overlapped by more hydrophobic segments. We propose, supported by experimental results, that the latter are initially integrated into the membrane, followed by post-translational structural rearrangements. Finally, we investigate whether the integration of the marginally hydrophobic TMHs of the lactose permease of Escherichia coli is facilitated by the formation of hairpin structures. However our combined efforts of computational simulations and experimental investigations find no evidence for this. The second question addressed in this thesis is that of the interpretation of the large datasets on which proteins that interact with each other in a cell. We have analyzed the results from several large-scale investigations concerning protein interactions in yeast and draw conclusions regarding the biases, strengths and weaknesses of these datasets and the methods used to obtain them. / At the time of the doctoral defense the following publications were not published and had a status as follows: Paper 2: In press; Paper 4 Manuscript.

Most membrane proteins are inserted into the membrane co-translationally utilizing the translocon, which allows a sufficiently long and hydrophobic stretch of amino acids to partition into the membrane. However, X-ray structures of membrane proteins have revealed that some transmembrane helices (TMHs) are surprisingly hydrophilic. These marginally hydrophobic transmembrane helices (mTMH) are not recognized as TMHs by the translocon in the absence of local sequence context. We have studied three native mTMHs, which were previously shown to depend on a subsequent TMH for membrane insertion. Their recognition was not due to specific interactions. Instead, the presence of basic amino acids in their cytoplasmic loop allowed membrane insertion of one of them. In the other two, basic residues are not sufficient unless followed by another, hydrophobic TMH. Post-insertional repositioning are another way to bring hydrophilic residues into the membrane. We show how four long TMHs with hydrophilic residues seen in X-ray structures, are initially inserted as much shorter membrane-embedded segments. Tilting is thus induced after membrane-insertion, probably through tertiary packing interactions within the protein. Aquaporin 1 illustrates how a mTMH can shape membrane protein folding and how repositioning can be important in post-insertional folding. It initially adopts a four-helical intermediate, where mTMH2 and TMH4 are not inserted into the membrane. Consequently, TMH3 is inserted in an inverted orientation. The final conformation with six TMHs is formed by TMH2 and 4 entering the membrane and TMH3 rotating 180°. Based on experimental and computational results, we propose a mechanism for the initial step in the folding of AQP1: A shift of TMH3 out from membrane core allows the preceding regions to enter the membrane, which provides flexibility for TMH3 to re-insert in its correct orientation. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: Manuscript.</p>