Models and Algorithms for Sorting Permutations with Tandem Duplication and Random Loss

A central topic of evolutionary biology is the inference of phylogeny, i. e., the evolutionary history of species. A powerful tool for the inference of such phylogenetic relationships is the arrangement of the genes in mitochondrial genomes. The rationale is that these gene arrangements are subject to different types of mutations in the course of evolution. Hence, a high similarity in the gene arrangement between two species indicates a close evolutionary relation. Metazoan mitochondrial gene arrangements are particularly well suited for such phylogenetic studies as they are available for a wide range of species, their gene content is almost invariant, and usually free of duplicates. With these properties gene arrangements of mitochondrial genomes are modeled by permutations in which each element represents a gene, i. e., a specific genetic sequence. The mutations that shape the gene arrangement of genomes are then represented by operations that rearrange elements in permutations, so-called genome rearrangements, and thereby bridge the gap between evolutionary biology and optimization. Many problems of phylogeny inference can be formulated as challenging combinatorial optimization problems which makes this research area especially interesting for computer scientists. The most prominent examples of such optimization problems are the sorting problem and the distance problem. While the sorting problem requires a minimum length sequence of rearrangements that transforms one given permutation into another given permutation, i. e., it aims for a hypothetical scenario of gene order evolution, the distance problem intends to determine only the length of such a sequence. This minimum length is called distance and used as a (dis)similarity measure quantifying the evolutionary relatedness. Most evolutionary changes occurring in gene arrangements of mitochondrial genomes can be explained by the tandem duplication random loss (TDRL) genome rearrangement model. A TDRL consists of a duplication of a consecutive set of genes in tandem followed by a random loss of one copy of each duplicated gene. In spite of the importance of the TDRL genome rearrangement in mitochondrial evolution, its combinatorial properties have rarely been studied. In addition, models of genome rearrangements which include all types of rearrangement that are relevant for mitochondrial genomes, i. e., inversions, transpositions, inverse transpositions, and TDRLs, while admitting computational tractability are rare. Nevertheless, especially for metazoan gene arrangements the TDRL rearrangement should be considered for the reconstruction of phylogeny. Realizing that a better understanding of the TDRL model is indispensable for the study of mitochondrial gene arrangements, the central theme of this thesis is to broaden the horizon of TDRL genome rearrangements with respect to mitochondrial genome evolution. For this purpose, this thesis provides combinatorial properties of the TDRL model and its variants as well as efficient methods for a plausible reconstruction of rearrangement scenarios between gene arrangements. The methods that are proposed consider all types of genome rearrangements that predominately occur during mitochondrial evolution. More precisely, the main points contained in this thesis are as follows: The distance problem and the sorting problem for the TDRL model are further examined in respect to circular permutations, a formal concept that reflects the circular structure of mitochondrial genomes. As a result, a closed formula for the distance is provided. Recently, evidence for a variant of the TDRL rearrangement model in which the duplicated set of genes is additionally inverted have been found. Initiating the algorithmic study of this new rearrangement model on a certain type of permutations, a closed formula solving the distance problem is proposed as well as a quasilinear time algorithm that solves the corresponding sorting problem. The assumption that only one type of genome rearrangement has occurred during the evolution of certain gene arrangements is most likely unrealistic, e. g., at least three types of rearrangements on top of the TDRL rearrangement have to be considered for the evolution metazoan mitochondrial genomes. Therefore, three different biologically motivated constraints are taken into account in this thesis in order to produce plausible evolutionary rearrangement scenarios. The first constraint is extending the considered set of genome rearrangements to the model that covers all four common types of mitochondrial genome rearrangements. For this 4-type model a sharp lower bound and several close additive upper bounds on the distance are developed. As a byproduct, a polynomial-time approximation algorithm for the corresponding sorting problem is provided that guarantees the computation of pairwise rearrangement scenarios that deviate from a minimum length scenario by at most two rearrangement operations. The second biologically motivated constraint is the relative frequency of the different types of rearrangements occurring during the evolution. The frequency is modeled by employing a weighting scheme on the 4-type model in which every rearrangement is weighted with respect to its type. The resulting NP-hard sorting problem is then solved by means of a polynomial size integer linear program. The third biologically motivated constraint that has been taken into account is that certain subsets of genes are often found in close proximity in the gene arrangements of many different species. This observation is reflected by demanding rearrangement scenarios to preserve certain groups of genes which are modeled by common intervals of permutations. In order to solve the sorting problem that considers all three types of biologically motivated constraints, the exact dynamic programming algorithm CREx2 is proposed. CREx2 has a linear runtime for a large class of problem instances. Otherwise, two versions of the CREx2 are provided: The first version provides exact solutions but has an exponential runtime in the worst case and the second version provides approximated solutions efficiently. CREx2 is evaluated by an empirical study for simulated artificial and real biological mitochondrial gene arrangements

Synthetic, Biochemical, X-ray Crystallographic, Computational and High-Throughput Screening Approaches Toward Anthrax Toxin Lethal Factor Inhibition

University of Minnesota Ph.D. dissertation.October 2015. Major: Medicinal Chemistry. Advisor: Elizabeth Amin. 1 computer file (PDF); xvi, 227 pages.The lethal factor (LF) enzyme secreted by Bacillus anthracis is chiefly responsible for anthrax-related cytotoxicity. In this dissertation, I present the computational design, synthesis, biochemical testing, structural biology, and virtual and high-throughput screening approaches to identify binding requirements for LF inhibition. To this end, we designed ~50 novel compounds to probe design principles and structural requirements for LF. Specifically, in Chapters 2 and 3, computational, synthetic, biochemical and structural biology methods to explore the underinvestigated LF S2′ binding subsite are described. We discovered that LF domain 3 is very flexible and results in a relatively unconstrained S2′ binding site region. Additionally, we found that the S1′ subsite can undergo a novel conformational change resulting in a previously unreported tunnel region, which we term S1′*, that we expect can further be explored to design potent and selective LF inhibitors. Using this novel LF configuration, we virtually screened ~11 million drug-like compounds for activity against LF and have identified a novel compound that inhibits LF with an IC50 of 126 μM. In the course of this work, we found that reliable representation of zinc and other transition metal centers in macromolecules is nontrivial, due to the complexity of the coordination environment and charge distribution at the catalytic center. In Chapter 7, I will present work on applying and optimizing quantum mechanical methods developed by the Truhlar group to accurately calculate bond dissociation energies at low computational cost for various representative Zn2+ and Cd2+ model systems. By analyzing errors, we developed a prescription for an optimal system fragmentation strategy for our models. With this scheme, we find that the EE-3B-CE method is able to reproduce 53 conventionally calculated bond energies with an average absolute error of only 0.59 kcal/mol. Therefore, one could use the EE 3B CE approximation to obtain accurate results for large systems and/or identify better parameters for Zn centers for use in virtual screening. Finally, we present the results of a large-scale in vitro HTS campaign of ~250,000 small-molecules against LF. After extensive validation, involving secondary assays and hit synthesis we were able to prioritize a key lead for further prosecution

Differentiating noise and modulators in artificial neural networks

Research in Computational Neural Networks is currently taking place at many different levels; from coarse-grain symbolic models to fine-grain representations of neurons and cell processes. One feature that the different approaches share, is that they are all in relative infancy. Thus, most research concentrates on gross aspects of neural communication and methods of computational simulation. Recently, some clues have been found which point to more subtle mechanisms underlying the information processing capability of neural 'nodes'. These clues are the improvement in network operation by the injection of random noise; and the neurobiological finding that neuropeptides may exist as slower Signal transmission channels between neurons. This study concerns the difference between random noise injection, and directed, low-level, activity injections which are postulated to be produced by neuromodulators such as neuropeptides. The findings of this study are that random noise does, indeed, enhance the operation of coarse-grain neural models; and that a 'neuropeptidergic' analogue also enhances operation; but to a different extent, and probably through a different mechanism. Further testing of a medium-grain computer model gives some indication of how a neuropeptidergic modulation might affect real neurons, by extending the time-course of the activation of the neuron. This appears to be a similar mechanism to that postulated for the coarse-grain 'neuropeptidergic' simulation model. Given these findings, is it possible that signal transmission in real nervous systems assume these mechanisms? If so, it may be possible that a process of concurrent propagation, through different signal channels, also occurs in real nervous systems, making the nervous system much more complex than current models allow

Computational methodologies and resources for discovery of phosphorylation regulation and function in cellular networks

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2010.Cataloged from PDF version of thesis.Includes bibliographical references (p. 145-156).Post-translational modifications (PTMs) regulate cellular signaling networks by modifying activity, localization, turnover and other characteristics of proteins in the cell. For example, signaling in receptor tyrosine kinase (RTK) networks, such as those downstream of epidermal growth factor receptor (EGFR) and insulin receptor, is initiated by binding of cytokines or growth factors, and is generally propagated by phosphorylation of signaling molecules. The rate of discovery of PTM sites is increasing rapidly and is significantly outpacing our biological understanding of the function and regulation of those modifications. The ten-fold increase in known phosphorylation sites over a five year time span can primarily be attributed to mass spectrometry (MS) measurement methods, which are capable of identifying and monitoring hundreds to thousands of phosphorylation sites across multiple biological samples. There is significant interest in the field in understanding these modifications, due to their important role in basic physiology as well as their implication in disease. In this thesis, we develop algorithms and tools to aid in analysis and organization of these immense datasets, which fundamentally seek to generate novel insights and testable hypotheses regarding the function and regulation of phosphorylation in RTK networks. We have developed a web-accessible analysis and repository resource for high-throughput quantitative measurements of post-translational modifications, called PTMScout. Additionally, we have developed a semi-automatic, high-throughput screen for unsupervised learning parameters based on their relative ability to partition datasets into functionally related and biologically meaningful clusters. We developed methods for comparing the variability and robustness of these clustering solutions and discovered that phosphopeptide co-clustering robustness can recapitulate known protein interaction networks, and extend them. Both of these tools take advantage of a new linear motif discovery algorithm, which we additionally used to find a putative regulatory sequence downstream of the highly tumorigenic EGFRvIII mutation that indicates casein kinase II (CK2) activity may be increased in glioblastoma.by Kristen M. Naegle.Ph.D

Classical And Quantum Mechanical Simulations Of Condensed Systems And Biomolecules

This work describes the fundamental study of two enzymes of Fe(II)/-KG super family enzymes (TET2 and AlkB) by applying MD and QM/MM approaches, as well as the development of multipolar-polarizable force field (AMOEBA/GEM-DM) for condensed systems (ionic liquids and water). TET2 catalytic activity has been studied extensively to identify the potential source of its substrate preference in three iterative oxidation steps. Our MD results along with some experimental data show that the wild type TET2 active site is shaped to enable higher order oxidation. We showed that the scaffold stablished by Y1902 and T1372 is required for iterative oxidation. The mutation of these residues perturbs the alignment of the substrate in the active site, resulting in “5hmC-stalling” phenotype in some of the mutants. We provided more details on 5hmC to 5fC oxidation mechanism for wild type and one of the “5hmC-stallling” mutants (E mutant). We showed that 5hmC oxidizes to 5fC in the wild type via three steps. The first step is the hydrogen atom abstraction from hydroxyl group of 5hmC, while the second hydrogen is transferred from methylene group of 5hmC through the third transition state as a proton. Our results suggest that the oxidation in E mutant is kinetically unfavorable due to its high barrier energy. Many analyses have been performed to qualitatively describe our results and we believed our results can be used as a guide for other researchers. In addition, two MD approaches (explicit ligand sampling and WHAM) are used to study the oxygen molecule diffusion into the active site of AlkB. Our results showed that there are two possible channels for oxygen diffusion, however, diffusion through one of them is thermodynamically favorable. We also applied multipolar-polarizable force field to describe the oxygen diffusion along the preferred tunnel. We showed that the polarizable force field can describe the behavior of the highly polarizable systems accurately. We also developed a new multipolar-polarizable force field (AMOEBA/GEM-DM) to calculate the properties of imidazolium- and pyrrolidinium- based ionic liquids and water in a range of temperature. Our results agree well with the experimental data. The good agreement between our results and experimental data is because our new parameters provide an accurate description of non-bonded interactions. We fit all the non-bonded parameters against QM. We use the multipoles extracted from fitted electron densities (GEM) and we consider both inter- and intra-molecular polarization. We believe this method can accurately calculate the properties of condensed systems and can be helpful for designing new systems such as electrolytes

A theoretical study of TPA-like tumour promotors and inositol polyphosphates

In this thesis, the structures and selected properties of TPA-like tumour promoters and of Myo-inositol polyphosphates are calculated and compared with their promoting and calcium releasing activities respectively. Three different levels of theory are used in the calculations, namely ab initio, semi empirical and molecular mechanics, though the majority of the calculations are performed using the MNDO derived semi-empirical methods implemented in MOPAC. A detailed description of the phorbol ester / DAG binding site is obtained from the structure / activity relationships derived for the tumour promoters and molecular dynamics simulations of the phorbol ester, TPA, in a lipid bilayer is carried out to investigate the position of the binding groups in relation to the surface of the bilayer. In order to carry out this simulation, a method (HYDRO) has been developed to produce close packed heterogeneous bilayers in which the headgroups of the components lie in random orientations. Thus the simulations are more realistic than calculations in which the lipids are placed in a regular array and interstitial spaces due to the difference in surface area of the bilayer components are kept to a minimum. The effects of the number and position of phosphate groups on the ring conformations of myo -inositol phosphates and the connection between this and calcium releasing activity are studied in chapter 5. As the molecules are particularly flexible with a large number of potential local minima, phosphate groups have been added sequentially to keep the required number of starting points as low as possible and rotation of phosphate hydroxyls has been ignored. The heats of formation, calculated with the different semi-empirical parametrisations, differ considerably, so the final calculations chapter compares energies and selected properties calculated for model organics phosphates using different methods and theory. The results using the new parametrisation, PM3, are compared with those of the earlier AMI for both the phorbol systems and the inositol phosphates to test its suitability

MATLAB

A well-known statement says that the PID controller is the "bread and butter" of the control engineer. This is indeed true, from a scientific standpoint. However, nowadays, in the era of computer science, when the paper and pencil have been replaced by the keyboard and the display of computers, one may equally say that MATLAB is the "bread" in the above statement. MATLAB has became a de facto tool for the modern system engineer. This book is written for both engineering students, as well as for practicing engineers. The wide range of applications in which MATLAB is the working framework, shows that it is a powerful, comprehensive and easy-to-use environment for performing technical computations. The book includes various excellent applications in which MATLAB is employed: from pure algebraic computations to data acquisition in real-life experiments, from control strategies to image processing algorithms, from graphical user interface design for educational purposes to Simulink embedded systems

Understanding orchestrated chemical reactions in toluene/o-xylene monooxygenase from pseudomonas sporium OX1

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2011.Vita. Cataloged from PDF version of thesis.Includes bibliographical references.Chapter 1. Geometric and Functional Versatility of Carboxylate-Bridged Nonheme- Diiron Motifs: sMMO and ToMO. Several metalloenzymes utilize a carboxylate-bridged non-heme diiron motif for dioxygen activation. Despite their conserved diiron active site structures and mechanisms of dioxygen activation, they catalyze a wide range of chemical transformations. These observations suggest that diiron-containing enzymes have distinct active sites and secondary/tertiary environments that are tuned for their dedicated biological functions. Detailed studies of two diiron-containing enzymes in the family of bacterial multicomponent monooxygenases (BMMs), soluble methane monooxygenase (sMMO) and toluene/o-xylene monooxygenase (ToMO), are described. The functions and structures of the three or four components of sMMO and ToMO are summarized. Distinctly different dioxygen activation chemistry and hydrocarbon specificity is observed for these two enzymes. A comparison of these two enzymes provides insight into the evolution of diironcontaining enzymes as well as their differing chemical mechanisms of catalysis. Chapter 2. Role of an Active Site Threonine in the Determination of Distinctive Dioxygen Reactivity in Toluene/o-Xylene Monooxygenase Hydroxylase. Dioxygen activation of toluene/o-xylene monooxygenase hydroxylase (ToMOH) exhibits the formation of a diiron(III) intermediate having unprecedented spectroscopic properties. To evaluate whether an active site threonine plays a role in the determination of the dioxygen chemistry in ToMOH, a T201S variant was prepared by site-directed mutagenesis. We reported the observation of a novel intermediate in the reaction of reduced ToMOH T201 S variant with dioxygen in the presence of its cognate regulatory protein (ToMOD). This species, T201 peroxo, is the first oxygenated intermediate of any toluene monooxygenase to display an optical band. The optical and M6ssbauer spectroscopic properties of the intermediate allowed us to assign it as a peroxodiiron(III) species, similar to Hperoxo in soluble methane monooxygenase hydroxylase (sMMOH). This result indicates that mutation of the T201 to serine altered the dioxygen chemistry of ToMOH in part to be more similar to that of sMMOH. Computational studies suggest that the T201 mutation can greatly perturb the energetics of the enzyme, which might be responsible for the distinct dioxygen reactivity of sMMOH and ToMOH. Structures of the oxygenated intermediates of ToMOH are proposed. Chapter 3. Role of an Active Site Threonine in the Kinetics of Dioxygen Activation in Toluene/o-Xylene Monooxygenase Hydroxylase. To elucidate the role of a strictly conserved T201 residue during dioxygen activation of toluene/o-xylene monooxygenase hydroxylase (ToMOH), T201S, T201G, T201C, and T201V variants of this enzyme were prepared by site-directed mutagenesis. X-ray crystal structures of all variants were obtained. Steady-state activity, regiospecificity, and single-turnover yields were also determined for the T201 mutants. Dioxygen activation by the reduced T201 variants was monitored by stopped-flow UV-vis and M6ssbauer spectroscopy. These studies demonstrated that the same dioxygen activation mechanism is preserved in the T201S, T201C, and T201G variants; however, both formation and decay kinetics of a peroxodiiron(III) intermediate, T201peroxo, were greatly altered, revealing that the T201 residue is critically involved in dioxygen activation. Rate-limiting steps in dioxygen activation of the T201S, T201C, and T201G variants were identified, revealing that T201 plays a major role in proton transfer, which is required to generate the peroxodiiron(III) intermediate. The role of the active site threonine residue in ToMOH is analogous to that of cytochrome P450 monooxygenases, suggesting it as a general threonine-dependent process in Nature to control proton transfer.(cont.) Chapter 4. Mechanistic Studies of Reactions of Peroxodiiron(III) Intermediates in the T201 Variants of Toluene/o-Xylene Monooxygenase Hydroxylase. Site-directed mutagenesis studies of a strictly conserved T201 residue in the active site of toluene/oxylene monooxygenase hydroxylase (ToMOH) revealed that a single mutation can facilitate kinetic isolation of two distinct peroxodiiron(III) species, designated T201peroxo and ToMOHperoxo, during dioxygen activation. In Chapter 2 and 3, we characterized both oxygenated intermediates by UV-vis and M6ssbauer spectroscopy, proposed structures from DFT and QM/MM computational studies, and elucidated chemical steps involved in dioxygen activation through the kinetic studies of T201peroxo formation. In Chapter 4, we investigated the kinetics of T2 0lperoxo decay to explore the reaction mechanism of the oxygenated intermediates following 02 activation. The decay rates of T201 peroxo were monitored in the absence and presence of external (phenol) or internal (tryptophan residue in I100W variant) substrates under pre-steady-state conditions. Three possible reaction models for the formation and decay of T201perX0 were evaluated, and the results demonstrate that this species is on the pathway of arene oxidation and appears to be in equilibrium with TOMOHperoxo. Chapter 5. Tracking a Defined Route of 0 2-Migration in a Dioxygen-Activating Diiron Enzyme, Toluene/o-Xylene Monooxygenase Hydroxylase. For numerous enzymes reactive toward small gaseous compounds, growing evidence indicates that these substrates diffuse into active site pockets through defined pathways in the protein matrix. Toluene/oxylene monooxygenase hydroxylase (ToMOH) is a dioxygen-activating carboxylatebridged nonheme-diiron enzyme. Structural analyses of the resting state enzyme suggest two possible pathways for dioxygen to access the c-subunit diiron center, a series of hydrophobic cavities or long solvent-exposed channel. To distinguish which pathway is utilized for dioxygen transfer, the dimensions of the cavities and channel were varied by site-directed mutagenesis and confirmed by X-ray crystallography. The rate of dioxygen access to the active site was monitored by measuring the formation rate of an oxygenated intermediate (T 2 01peroxo), a process that is dependent on 02 concentration. Altering the dimensions of the cavity but not the channel drastically changed the rate of dioxygen activation by the reduced enzyme. These results explicitly reveal that the cavities in the ToMOH a-subunit are not merely artifacts of protein packing/folding but rather programmed routes of dioxygen movement through the protein matrix. This conclusion indicates that conformational changes are required during catalysis to form a dioxygen trajectory and that the temporary opening/closing of the cavities control dioxygen transfer. Given that the cavities are present in all BMMs, the breathing motion presumably controls dioxygen consumption in all BMMs. This study represents the first approach to track kinetically a defined transient pathway by which a small gaseous molecule gains access to a diiron enzyme.(cont.) Appendix A. Insights into the Different Dioxygen Activation Pathways of Methane and Toluene Monooxygenase Hydroxylases. The methane and toluene monooxygenase hydroxylases (MMOH and TMOH, respectively) have almost identical active sites, yet the physical and chemical properties of their oxygenated intermediates, designated P*, Hperoxo, Q and Q* in MMOH, and ToMOHperoxo in toluene/o-xylene monooxygenase hydroxylase (ToMOH), are substantially different. We review and compare the structural differences in the vicinity of the active sites of these enzymes and discuss the differences that give rise to the distinct behavior of dioxygen reactivity in sMMOH and ToMOH. In particular, analysis of multiple crystal structures reveals that T213 of MMOH and analogous T201 of TMOH, located in the immediate vicinity of the active site, have different rotamer configurations. We study the rotation energy profiles of these threonine residues with the use of molecular mechanics (MM) and quantum mechanics/molecular mechanics (QM/MM) computational methods and put forward a hypothesis according to whether T201 and T213 play an important role in the formation of different types of peroxodiiron(III) species in MMOH and ToMOH. The hypothesis is indirectly supported by QM/MM calculations of the peroxodiiron(III) models of ToMOH and the theoretically computed M6ssbauer spectra. It also helps explain the formation of two distinct peroxodiiron(III) species in the T201S mutant of ToMOH. Additionally, a role for the regulatory protein (ToMOD), which is essential for oxygenated intermediate formation and the protein functioning in the ToMO system, is advanced. Appendix B. Multiple Roles of Component Proteins in Bacterial Multicomponent Monooxygenases: Phenol Hydroxylase and Toluene/o-Xylene Monooxygenase from Pseudomonas sp. OX1. Phenol hydroxylase (PH) and toluene/o-xylene monooxygenase (ToMO) from Pseudomonas sp. OXI require three or four protein components to activate dioxygen for the oxidation of aromatic substrates at a carboxylate-bridged diiron center. In this study, we investigated the influence of the hydroxylases, regulatory proteins, and electron-transfer components of these systems on substrate consumption and product generation. Single-turnover experiments revealed that only complete systems containing all three or four protein components are capable of oxidizing phenol, a major substrate for both enzymes. Under ideal conditions, the hydroxylated product yield was -50% of the diiron centers for both systems, suggesting that these enzymes operate by half-sites reactivity mechanisms. Single-turnover studies indicated that the PH and ToMO electron-transfer components exert regulatory effects on substrate oxidation processes taking place at the hydroxylase active sites, most likely through allostery. Steady state NADH consumption assays showed that the regulatory proteins facilitate the electron-transfer step in the hydrocarbon oxidation cycle in the absence of phenol. Under these conditions, electron consumption is coupled to H20 2 formation in a hydroxylase-dependent manner. Mechanistic implications of these results are discussed.by Woon Ju Song.Ph.D

Coupling of experimental and computational approaches for the development of new dendrimeric nanocarriers for gene therapy.

2013/2014Gene therapy is increasingly critical in the treatment of different types of maladies. The approach of gene therapy can be fundamental in dealing with many kinds of tumors, viral infections (e.g., HIV, HSV), and disturbs linked to genetic anomalies. However, the use of nucleic acids is limited by their ability to reach their action site—the target cell and, often, the inside of its nucleus. Dendrimers, on the other hand, are an interesting kind of polymers, the general synthetic scheme of which is relatively of recent development (∼1980). Among the many possible uses of these polymers, they revealed themselves as great nanocarriers for drugs in general, and particularly for genetic material. Many of the properties of these molecules are directly linked to their structure, and this in turn is critically influenced by their molecular composition. Exploiting in silico techniques, we can reveal many informations about the atomistic structure of dendrimers, some of which are otherwise difficult to gather. The interactions between the carrier and its cargo, and also with all the biological systems that are interposed between the administration and the reaching of the target (e.g., serum proteins, lipid membranes. . . ) are of critical importance in the development of new dendrimers for gene therapy. These interactions can be described and studied at a detail once unthinkable, thanks to the in silico simulation of these systems. In this thesis many different molecular simulation techniques will be employed to give a characterization as precise as possible of the structure and interactions of new families of dendrimers. In particular two new families of dendrimers (viologen and carbosilane) will be structurally characterized, and their interactions with albumin and two oligodeoxynucleotide, respectively, will be described. Then, the point of view of these interactions will be changed: the interactions between a fifth generation triethanolamine-core poly(amidoamine) dendrimer (G5 TEA-core PAMAM) and a sticky siRNA will be studied, varying the length and chemical compositions of the overhangs of the siRNA. Studying dendrimers the use of new molecular simulations techniques were deepened, and such techniques will be employed in other parallel projects. We’ll see the steered molecular dynamic method applied in the study of one mutation of the SMO receptor. The development of biological membranes models (that will be used in future to study the interactions of dendrimers with such membranes) was also used to refine and better characterize the σ1 receptor 3D model, previously developed by our research group. A detailed characterization of the putative binding site of this receptor will be given, employing this refined model.La terapia genica si sta rivelando sempre più importante nel trattamento di diversi tipi di malattie. Da diversi tipi di tumori alle infezioni virali, quale ad esempio da HIV, fino anche a malattie legate ad anomalie genetiche sono tutti disturbi in cui l’approccio della terapia genica può rivelarsi fondamentale. L’utilizzo di acidi nucleici quali agenti terapeutici è fortemente limitato dalla possibilità di portare queste molecole al loro sito d’azione—la cellula bersaglio e, spesso, l’interno del nucleo di quest’ultima. I dendrimeri d’altro canto sono un interessante tipo di polimero, di cui lo schema generale di sintesi è relativamente recente (∼1980). Tra i diversi loro utilizzi, questi polimeri si sono rivelati anche ottimi agenti di trasporto per farmaci, ed in particolare per materiale genetico. Molte delle proprietà di queste molecole derivano direttamente dalla loro struttura, e questa è influenzata criticamente dalla loro composizione. Mediante tecniche in silico è possibile avere molte informazioni riguardo la struttura dei dendrimeri, alcune delle quali sono altrimenti difficilmente ottenibili. L’interazione tra il trasportatore ed il suo “carico”, come anche con tutti i diversi sistemi biologici che si frappongono tra la somministrazione ed il raggiungimento dell’obbiettivo (ad es. proteine seriche, membrane lipidiche...) è un parametro chiave nello sviluppo di nuovi dendrimeri per la terapia genica. Queste interazioni possono essere descritte e studiate con un dettaglio un tempo impossibile, mediante la simulazione in silico di tali sistemi. In questo lavoro di tesi diverse tecniche di simulazione molecolare saranno utilizzate al fine di dare una caratterizzazione quanto più precisa possibile della struttura e delle intera- zioni di nuove classi di dendrimeri. In particolare sarà data una descrizione strutturale di due nuove famiglie di dendrimeri viologeni e carbosilani, e delle loro interazioni rispettivamente con albumina e due diversi oligodeossinucleotidi. Si alternerà poi il punto di vista da cui studiare tale interazione: sarà data una descrizione dell’interazione tra un dendrimero po- liammidoamminico a nucleo trietanolamminico (TEA-core PAMAM) di generazione 5 e uno sticky siRNA, al variare della lunghezza e tipo di “braccia” del siRNA. Nello studio di dendrimeri alcune nuove tecniche di simulazione molecolare sono state approfondite, e tali tecniche sono state utilizzate anche in altri progetti paralleli. Vedremo la steered molecular dynamic applicata allo studio di una mutazione del recettore SMO. Lo sviluppo di modelli di membrane biologiche (utile in futuro per lo studio dell’interazione di dendrimeri con tali membrane) è stato utilizzato per perfezionare e meglio caratterizzare il modello tridimensionale del recettore σ1, precedentemente sviluppato dal nostro gruppo di ricerca. Una caratterizzazione dettagliata del sito di binding putativo di questo recettore sarà descritta, usando tale perfezionato modello.XXVII Ciclo198