An Intertemporal Model of Rational Criminal Choice

This research presents a dynamic model of crime in which agents anticipate future consequences of their actions. Current period decisions affect future outcomes by a process of capital accumulation. While investigating the role of human capital, the focus of our study is on a form of capital that has received somewhat less attention in the literature, social capital. Social capital is an index of one's 'stock' in society. Introduction of social capital into the utility function results in an intertemporally nonseparable preference structure which admits state dependence in the decision to participate in crime. Our model is empirically implemented using panel data on a sample from the 1958 Philadelphia Birth Cohort Study. In estimation, we take account of unobserved choices in states not realized, which potentially depend on individual specific heterogeneity, by using simulation techniques. Our results provide evidence of state dependence in the decision to participate in crime. We also find that the initial level of social capital stock is important in determining the pattern of criminal involvement in adulthood.

Engineering RNA phage MS2 virus-like particles for peptide display

Phage display is a powerful and versatile technology that enables the selection of novel binding functions from large populations of randomly generated peptide sequences. Random sequences are genetically fused to a viral structural protein to produce complex peptide libraries. From a sufficiently complex library, phage bearing peptides with practically any desired binding activity can be physically isolated by affinity selection, and, since each particle carries in its genome the genetic information for its own replication, the selectants can be amplified by infection of bacteria. For certain applications however, existing phage display platforms have limitations. One such area is in the field of vaccine development, where the goal is to identify relevant epitopes by affinity-selection against an antibody target, and then to utilize them as immunogens to elicit a desired antibody response. Today, affinity selection is usually conducted using display on filamentous phages like M13. This technology provides an efficient means for epitope identification, but, because filamentous phages do not display peptides in the high-density, multivalent arrays the immune system prefers to recognize, they generally make poor immunogens and are typically useless as vaccines. This makes it necessary to confer immunogenicity by conjugating synthetic versions of the peptides to more immunogenic carriers. Unfortunately, when introduced into these new structural environments, the epitopes often fail to elicit relevant antibody responses. Thus, it would be advantageous to combine the epitope selection and immunogen functions into a single platform where the structural constraints present during affinity selection can be preserved during immunization. This dissertation describes efforts to develop a peptide display system based on the virus-like particles (VLPs) of bacteriophage MS2. Phage display technologies rely on (1) the identification of a site in a viral structural protein that is present on the surface of the virus particle and can accept foreign sequence insertions without disruption of protein folding and viral particle assembly, and (2) on the encapsidation of nucleic acid sequences encoding both the VLP and the peptide it displays. The experiments described here are aimed at satisfying the first of these two requirements by engineering efficient peptide display at two different sites in MS2 coat protein. First, we evaluated the suitability of the N-terminus of MS2 coat for peptide insertions. It was observed that random N-terminal 10-mer fusions generally disrupted protein folding and VLP assembly, but by bracketing the foreign sequences with certain specific dipeptides, these defects could be suppressed. Next, the suitability of a coat protein surface loop for foreign sequence insertion was tested. Specifically, random sequence peptides were inserted into the N-terminal-most AB-loop of a coat protein single-chain dimer. Again we found that efficient display required the presence of appropriate dipeptides bracketing the peptide insertion. Finally, it was shown that an N-terminal fusion that tended to interfere specifically with capsid assembly could be efficiently incorporated into mosaic particles when co-expressed with wild-type coat protein

Quantum brane cosmology

This thesis deals with the interaction of quantum mechanical models and cosmologies based on brane universes, an area of active theoretical speculation over the last five years.For convenience, the material has been split into two parts. Part 1 deals with a selection of background topics which are necessary and relevant to the original research. This research is presented in Part 2. In addition, some auxiliary topics, both more elementary and more advanced, are described in the appendices. The selection of background topics has been influenced by the various techniques, physical theories and mathematical technologies which play a major role in the work presented in Part 2. Although the exposition is ad hoc, an attempt has been made to systematically develop portions where the technique (or use of it) may be unfamiliar.A fairly complete treatment of the necessary mathematical scaffolding is supplied. Although important, this material is familiar or strongly mathematical, and is deferred to the appendices. This includes an elementary survey of functional analysis in Appendix A, sufficient to support a discussion of the path integral. The path integral formalism is used extensively throughout this thesis, and, where available, constitutes our preferred representation of quantum mechanics. The discussion is limited to the relevant portions of the theory: functions in Banacli spaces, and the Sturm-Liouville basis (technology which appears many times in Part 2); direct evaluation of Gaussian functional integrals, ubiquitous in field theory calculations; and ((-function regularization of the operator determinants to which such Gaussian integrals give rise, which has a direct application in Chapter 9. In Appendix B we describe the necessary framework of differential geometry which supports general relativity, and low-energy discussions of string theory. All calculations in metric gravity are based on differential geometry, together with a good proportion of the technology which buttresses quantum field theory on curved space time, string theory, and some more advanced representations of quantum mechanics (see below). All of this is used extensively throughout both parts of the thesis. We include some more advanced topological technology which supports the discussion of string compactification. General results from compactification theory, when appropriately interpreted in the brane context, contribute important stability results for zero-modes of the Kaluza—Klein fields, and provide a natural home for the spectral KK technology used (in one form or another) throughout Part 2, but most especially in Chapter 7 and Chapter 8. Einstein gravity and Yang-Mills theory are set in context as examples of connexions on fibre bundles

Structural studies of apolipoprotein A-I and ATP-binding cassette A1 and their roles in nascent high density lipoprotein biogenesis

Apolipoprotein A-I (apoA-I) and ATP-Binding Cassette A1 (ABCA1) transporter play important roles in nascent high density lipoprotein (nHDL) biogenesis – the first step in the reverse cholesterol transport pathway. Based on the crystal structure of a C-terminally truncated form of apoA-I (apoA-I(1-184)) determined in the laboratory, structurally designed and naturally occurring mutants of apoA-I were conformationally characterized in solution. The function of these mutants in nHDL formation was assessed in ABCA1-transfected HEK293 cells. An apoA-I mutant designed to destabilize the N-terminal helical bundle at the first hinge region, 38/40G, exhibited a locally reduced α-helical content, destabilized overall structure, and increased lipid binding ability in solution, indicating a destabilized N-terminal helical bundle. In the cellular system, 38/40G showed significantly enhanced nHDL forming ability, suggesting that a destabilized N-terminal bundle will facilitate nHDL formation. Other designed N-terminal mutants (Q41A, P66G, G65A, V67P, T68P, 65/67/68P) and the naturally occurring mutants (R153P, L178P, and insertion mutant apoA-INashua) all showed either unchanged or destabilized overall structure, unchanged lipid binding abilities in solution and unchanged nHDL formation and cholesterol efflux promotion from the cells. Mutants designed to progressively extend the C-terminus (1-184, 1-198, 1-209, 1-220, 1-231) yielded progressively increased nHDL formation and cholesterol efflux, suggesting that the C-terminus of apoA-I is critical for these two activities. Central Helix 5 triple glycine mutation (H5 3xG) designed to lock the monomer conformation of apoA-I resulted in reduced nHDL formation but unaffected cholesterol efflux, suggesting that hindering apoA-I monomer to dimer conversion could retard nHDL formation. Remarkably, studies of cholesterol efflux and nHDL particle formation indicated that the two processes might be two uncoupled events. Analysis of the nHDL particles revealed the presence of ganglioside (GM1) in the complexes. Cross-linking data demonstrated binding of apoA-I to ABCA1-expressing cells. The binding level of apoA-I mutants to ABCA1-expressing cells was positively correlated with nHDL forming ability of these mutants. ABCA1 was isolated from FreeStyle™ HEK293-F cells in suspension by detergent solubilization and was shown to have ATPase activity. A direct interaction between apoA-I and amphipol solubilized- ABCA1 in solution was detected for the first time. Furthermore, the successful purification of ABCA1 has laid the foundation of structure determination of this protein in the future

Structural studies of apolipoprotein A-I and ATP-binding cassette A1 and their roles in nascent high density lipoprotein biogenesis

Apolipoprotein A-I (apoA-I) and ATP-Binding Cassette A1 (ABCA1) transporter play important roles in nascent high density lipoprotein (nHDL) biogenesis – the first step in the reverse cholesterol transport pathway. Based on the crystal structure of a C-terminally truncated form of apoA-I (apoA-I(1-184)) determined in the laboratory, structurally designed and naturally occurring mutants of apoA-I were conformationally characterized in solution. The function of these mutants in nHDL formation was assessed in ABCA1-transfected HEK293 cells. An apoA-I mutant designed to destabilize the N-terminal helical bundle at the first hinge region, 38/40G, exhibited a locally reduced α-helical content, destabilized overall structure, and increased lipid binding ability in solution, indicating a destabilized N-terminal helical bundle. In the cellular system, 38/40G showed significantly enhanced nHDL forming ability, suggesting that a destabilized N-terminal bundle will facilitate nHDL formation. Other designed N-terminal mutants (Q41A, P66G, G65A, V67P, T68P, 65/67/68P) and the naturally occurring mutants (R153P, L178P, and insertion mutant apoA-INashua) all showed either unchanged or destabilized overall structure, unchanged lipid binding abilities in solution and unchanged nHDL formation and cholesterol efflux promotion from the cells. Mutants designed to progressively extend the C-terminus (1-184, 1-198, 1-209, 1-220, 1-231) yielded progressively increased nHDL formation and cholesterol efflux, suggesting that the C-terminus of apoA-I is critical for these two activities. Central Helix 5 triple glycine mutation (H5 3xG) designed to lock the monomer conformation of apoA-I resulted in reduced nHDL formation but unaffected cholesterol efflux, suggesting that hindering apoA-I monomer to dimer conversion could retard nHDL formation. Remarkably, studies of cholesterol efflux and nHDL particle formation indicated that the two processes might be two uncoupled events. Analysis of the nHDL particles revealed the presence of ganglioside (GM1) in the complexes. Cross-linking data demonstrated binding of apoA-I to ABCA1-expressing cells. The binding level of apoA-I mutants to ABCA1-expressing cells was positively correlated with nHDL forming ability of these mutants. ABCA1 was isolated from FreeStyle™ HEK293-F cells in suspension by detergent solubilization and was shown to have ATPase activity. A direct interaction between apoA-I and amphipol solubilized- ABCA1 in solution was detected for the first time. Furthermore, the successful purification of ABCA1 has laid the foundation of structure determination of this protein in the future

Towards higher predictability in enzyme engineering : investigation of protein epistasis in dynamic ß-lactamases and Cal-A lipase

L'ingénierie enzymatique est un outil très avantageux dans l'industrie biotechnologique. Elle permet d'adapter les enzymes à une activité ou à une condition de réaction spécifique. En outre, elle peut permettre de déchiffrer les éléments clés qui ont facilité leur modification. Bien que l'ingénierie enzymatique soit largement pratiquée, elle comporte encore plusieurs goulets d'étranglement. Certains de ces goulets d'étranglement sont techniques, comme le développement de méthodologies pour la création de banques de mutations ciblées ou la réalisation de criblages à haut débit, et d'autres sont conceptuels, comme le déchiffrage des caractéristiques clés pertinentes d'une protéine cible pour la réussite d'un projet d'ingénierie. Parmi ces défis, l'épistasie intra-génique, ou la non-additivité des effets phénotypiques des mutations, est une caractéristique qui entrave grandement la prévisibilité. L'amélioration de l'ingénierie enzymatique nécessite une approche multidisciplinaire qui inclut une meilleure compréhension des relations structure-fonction-évolution. Cette thèse vise à contribuer à l'avancement de l'ingénierie enzymatique en étudiant deux systèmes modèles. Premièrement, des variantes dynamiques de la ß-lactamase TEM-1 ont été choisies pour étudier le lien entre la dynamique des protéines et l'évolution. La ß-lactamase TEM-1 a été largement caractérisée dans la littérature, ce qui s'est traduit par des connaissances approfondies sur son mécanisme de réaction, ses caractéristiques structurelles et son évolution. Les variantes de la ß-lactamase TEM-1 utilisées comme système modèle dans cette thèse ont été largement caractérisées, montrant une dynamique accrue à l'échelle temporelle pertinente pour la catalyse (µs à ms) mais maintenant la reconnaissance du substrat. Dans cette thèse, l'évolution in vitro de ces variantes dynamiques a été réalisée par des cycles itératifs de mutagenèse et de sélection aléatoires pour permettre une exploration impartiale du paysage de ‘fitness’. Nous démontrons que la présence de ces mouvements particuliers au début de l'évolution a permis d'accéder à des voies de mutations connues. De plus, des interactions épistatiques connues ont été introduites dans les variantes dynamiques. Leur caractérisation in silico et cinétique a révélé que les mouvements supplémentaires sur l'échelle de temps de la catalyse ont permis d'accéder à des conformations conduisant à une fonction améliorée, comme dans le TEM-1 natif. Dans l'ensemble, nous démontrons que l'évolution de la b-lactamase TEM-1 vers une nouvelle fonction est compatible avec divers mouvements à l'échelle de temps µs à ms. Il reste à savoir si cela peut se traduire par d'autres enzymes ayant un potentiel biotechnologique. Deuxièmement, la lipase Cal-A, pertinente sur le plan industriel, a été choisie pour identifier les caractéristiques qui pourraient faciliter son ingénierie. La lipase Cal-A présente des caractéristiques telles que la polyvalence du substrat et une grande stabilité thermique et réactivité qui la rendent attrayante pour la modification des triglycérides ou la synthèse de molécules pertinentes dans les industries alimentaire et pharmaceutique. Contrairement à TEM-1, la plupart des études d'évolution in vitro de la lipase Cal-A ont été réalisées dans un but industriel, avec une exploration limitée de l'espace de mutation. Par conséquent, les caractéristiques qui définissent la fonction de la lipase Cal-A restent insaisissables. Dans cette thèse, nous faisons état de la mutagenèse ciblée de la lipase Cal-A, confirmant l'existence d'une région clé pour la reconnaissance du substrat. Cela a été fait en combinant une nouvelle méthodologie de création de bibliothèque basée sur l'assemblage Golden-gate avec une visualisation structurelle basée sur des scripts pour identifier et cartographier les mutations sélectionnées dans la structure 3D. La caractérisation et la déconvolution de deux des plus aptes ont révélé l'existence d'une épistasie dans l'évolution de la lipase Cal-A vers une nouvelle fonction. Dans l'ensemble, nous démontrons que l’identification d'une variété de propriétés suite à la mutagenèse ciblée peut grandement améliorer la connaissance d'une enzyme. Cette information peut être appliquée pour améliorer l'efficacité de l'ingénierie dirigée.Enzyme engineering is a tool with great utility in the biotechnological industry. It allows to tailor enzymes to a specific activity or reaction condition. In addition, it can allow to decipher key elements that facilitated their modification. While enzyme engineering is extensively practised, it still entails several bottlenecks. Some of these bottlenecks are technical such as the development of methodologies for creating targeted mutational libraries or performing high-throughput screening and some are conceptual such as deciphering the key relevant features in a target protein for a successful engineering project. Among these challenges, intragenic epistasis, or the non-additivity of the phenotypic effects of mutations, is a feature that greatly hinders predictability. Improving enzyme engineering needs a multidisciplinary approach that includes gaining a better understanding of structure-function-evolution relations. This thesis seeks to contribute in the advancement of enzyme engineering by investigating two model systems. First, dynamic variants of TEM-1 ß-lactamase were chosen to investigate the link between protein dynamics and evolution. TEM-1 ß-lactamase has been extensively characterized in the literature, which has translated into extensive knowledge on its reaction mechanism, structural features and evolution. The variants of TEM-1 ß-lactamase used as model system in this thesis had been extensively characterized, showing increased dynamics at the timescale relevant to catalysis (µs to ms) but maintaining substrate recognition. In this thesis, in vitro evolution of these dynamic variants was done by iterative rounds of random mutagenesis and selection to allow an unbiased exploration of the fitness landscape. We demonstrate that the presence of these particular motions at the outset of evolution allowed access to known mutational pathways. In addition, known epistatic interactions were introduced in the dynamic variants. Their in silico and kinetic characterization revealed that the additional motions on the timescale of catalysis allowed access to conformations leading to enhanced function, as in native TEM-1. Overall, we demonstrate that the evolution of TEM-1 b-lactamase toward new function is compatible with diverse motions at the µs to ms timescale. Whether this can be translated to other enzymes with biotechnological potential remains to be explored. Secondly, the industrially relevant Cal-A lipase was chosen to identify features that could facilitate its engineering. Cal-A lipase presents characteristics such as substrate versatility and high thermal stability and reactivity that make it attractive for modification of triglycerides or synthesis of relevant molecules in the food and pharmaceutical industries. Contrary to TEM-1, most in vitro evolution studies of Cal-A lipase have been done towards an industrially-specified goal, with limited exploration of mutational space. As a result, features that define function in Cal-A lipase remain elusive. In this thesis, we report on focused mutagenesis of Cal-A lipase, confirming the existence of a key region for substrate recognition. This was done by combining a novel library creation methodology based on Golden-gate assembly with script-based structural visualization to identify and map the selected mutations into the 3D structure. The characterization and deconvolution of two of the fittest revealed the existence of epistasis in the evolution of Cal-A lipase towards new function. Overall, we demonstrate that mapping a variety of properties following mutagenesis targeted to specific regions can greatly improve knowledge of an enzyme that can be applied to improve the efficiency of directed engineering

Synuclein plasticity: the Achilles’ heel of nerve function linked to the onset of Parkinson’s disease

Lewy bodies – the hallmarks of Parkinson’s disease – are majorly constituted of aggregates of the presynaptic protein alpha-synuclein. The molecular mechanism of alpha-synuclein aggregation through which it changes dramatically from a soluble disordered monomer to insoluble structured fibrils remains unknown. As an intrinsically disordered protein, alpha-synuclein does not have a specific three-dimensional structure, but rather behaves mostly as a meta-stable ensemble of highly dynamic conformers, and as such undergoes rapid kinetics, making it almost impossible to measure its conformational changes with most techniques. Millisecond amide hydrogen exchange can provide valuable insights on the dynamic behaviour of proteins, especially at flexible regions. Thus, the work in this thesis reports on the development of methods and tools for hydrogen/deuterium-exchange mass spectrometry (HDX-MS) and the application of these for the study of aSyn under physiological conditions. In the first part of this thesis, high resolution on the alpha-synuclein monomer was achieved over two dimensions: time and space. Using a novel in-house rapid- mixing quench-flow instrument, hydrogen/deuterium-exchange mass spectrometry data on alpha-synuclein on the millisecond timescale was attained. Furthermore, using a ‘soft’ gas-phase mass spectrometry fragmentation technique called Electron Transfer Dissociation, structural resolution in the protein increased. The second part of this work focuses on the development of a software, HDfleX, in an effort to primarily automate the HDX-MS workflow and allow the merging of HDX-MS data at different levels: bottom-up, middle-down and top-down. The rest of the thesis uses the tools and methods developed earlier on to explore the effects of different solution conditions (cellular compartments and salt cations) on the monomeric conformations of aSyn, and how these correlate to the different stages of aggregation and the ensuing fibril polymorphs. Altogether, the achievements in this work will allow us to better understand the plasticity of the alpha-synuclein monomer as it cycles through different local environments

Adnectin Solubility and Dynamics

Rapid growth of the global market for monoclonal antibodies (mAbs) has generated considerable interest in the development of alternative molecules that facilitate rapid discovery and manufacturing, while replicating the low toxicity/immunogenicity and tight, specific binding of mAbs. One such molecule is the tenth human fibronectin type III domain (10Fn3), which has solvent accessible loops resembling the VH complementarity-determining regions H1, H2, and H3 of immunoglobulin. 10Fn3-based binding proteins called Adnectins have been engineered to bind with high affinity to diverse targets using in vitro evolution methods such as mRNA display, yeast display, and phage display. Adnectins are known to vary in aggregation propensity, sometimes despite exceptionally high amino acid sequence identity, and have been used as a basis for protein aggregation/solubility research. Aggregation of therapeutic proteins can provoke a protein-specific immune response, and the solubility of Adnectins is therefore of immediate practical interest. The aggregation of proteins in general is a complicated and incompletely understood phenomenon, the study of which we advance using Adnectins as a model system. We also investigate protein dynamics (which can be related to protein aggregation, but additionally has enormous impact on how we think about protein structure and function) through nuclear magnetic resonance (NMR) spectroscopic and computational study of Adnectins. Here, we first present solubility data for a reference set of 41 Adnectins and use them to screen computational solubility/aggregation prediction methods. On the basis of these results, we select the CamSol prediction method for use in a protein engineering project that applies the principles of consensus design to enhance the solubility of the Adnectin scaffold. Furthermore, we demonstrate that hydrogen/deuterium exchange by hydrogen bonded amides in the C-terminal β-strand of the original scaffold is induced by transient inter-Adnectin association, and that equivalent exchange is not observed in our solubility-enhanced scaffold. Next, we describe the results of variable-temperature solution NMR experiments that probe Adnectin dynamics. Each resonance observable by NMR spectroscopy is composed of contributions from structurally equivalent nuclei in a vast number of proteins, the conformations of which may both differ from each other at any particular instant and evolve over the timescale of the experiment. The temperature dependences of amide proton and nitrogen chemical shifts are due to differences in the conformations sampled and their probabilities of occupation. Empirically, these temperature dependences are well-approximated by fits to a linear model, the slopes of which (known as temperature coefficients) may report on protein dynamics in the vicinity of each backbone amide. We explore possible determinants of amide proton and nitrogen temperature coefficients using a combination of molecular dynamics simulations and quantum chemical (density functional theory) calculations. In the directly detected (high resolution) proton dimension, we also analyze deviations from linearity, which may be attributable to fast exchange between protein conformations with distinct chemical shifts and a temperature-dependent difference in free energy