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The thesis addresses the legal regulation of the freedom of assembly in the Czech Republic, aiming to analyse the current version of the Assembly Act, point out certain problematic aspects and suggest improvements de lege ferenda, critically assess the relevant case law and provide a comparison with legal regulations in some other countries. Part Two analyses the meaning of the freedom of assembly and the term "assembly", summarising and assessing different opinions on what constitutes an assembly. It addresses the constitutional principle that no permission may be required for organising public assemblies and its implication for so called "accesory activities" during assemblies. Part Three examines the requirement to notify the council about planned assemblies. The time requirement prescribed by the Czech law is compared to the requirements in 30 other - mostly European - countries. The formal requirements of the notifications are analysed in detail. Part Four concerns the power of the council to prohibit an assembly in advance. A considerable part is devoted to the critical evaluation of the case law regarding prohibition of assemblies and the issue of large-scale false notifications, where both constitutionally conform interpretation and amendments of the law are suggested. Conditions under which the...

Influenza A virus (IAV) is one of the most common viruses circulating in the human population and is responsible for seasonal epidemics that affect millions of individuals worldwide. The need to develop new drugs and vaccines against IAVs led scientists to study the main IAV surface antigens hemagglutinin (HA) and neuraminidase (NA). In contrast to HA, which facilitates cell binding and entry of IAVs, NA plays a critical role in the release and spreading of the viral particles. The aim of this thesis was to study how the enzymatic head domain, the stalk and transmembrane domains have evolved to facilitate NA assembly into an enzymatically active homotetramer, and to determine how these regions have evolved together over time. Initially, we observed that the NA transmembrane domain (TMD) assists in the assembly of the head domain by tethering the stalk to the membrane in a tetrameric conformation. Upon examination of the available sequences for NA, we found that the subtype 1 (N1) TMDs have become more polar since 1918 while the subtype 2 (N2) TMDs have consistently retained the expected hydrophobicity of a TMD. Further analysis of the amino-acid sequences revealed a characteristic indicative of an amphipathic assembly for the N1 TMDs that were absent in the TMDs from N2. The function of the amphipathic assembly was examined by creating two viral chimeras, where the original TMD was replaced by another more polar or an engineered hydrophobic TMD. In both cases the viruses carrying the NA TMD chimeras showed reduced growth indicating that the TMD changes created an incompatibility with the head domain of NA. After prolonged passaging of these viruses, natural occurring mutations were observed in the TMD that were able to rescue the defects in viral growth, head domain folding and budding by creating a TMD with the appropriate polar or hydrophobic assembly properties. Interestingly, we observed that N1 and N2 have a great difference in the localization and length of amino-acid deletions occurring in the stalk region. In line with this observation, our data suggests that N1 supports large stalk deletions due to its strong TMD association, whereas N2 requires the presence of a strong oligomerizing stalk region to compensate for its weak TMD interaction. These results have demonstrated how important the NA TMD is for viral infectivity and how the three different domains have evolved in a cooperative manner to promote proper NA assembly / Influensa är en av de mest smittsamma sjukdomarna som drabbar människor och de flesta kan räkna med att bli infekterade många gånger under sin livstid. Influensaviruset attackerar främst luftvägarna, men kan även leda till t.ex. lunginflammation. De enskilda viruspartiklarna (virionerna) kan komma i olika former, men den vanligaste formen som används för att beskriva viruset är den sfäriska. På en virions yta så finns det två olika typer av membranproteiner, som kan liknas med två olika sorters spikar som sticker ut från viruset. Den ena ”spiken” kallas neuraminidas, eller bara kort för NA, och den andra för hemagglutinin (HA). När man har andats in ett influensavirus så kan viruset ta sig till de övre luftvägarna och vidare ner i luftstrupen för att där använda sig av HA för att ta sig in i en cell. Viruset använder sig sedan av cellen för att skapa många nya virioner, som tar sig ut ur cellen för att infektera fler celler. NA är det protein som virionerna använder sig av för att klyva sig loss från modercellen. Målet för avhandlingen var att studera NA och beskriva hur proteinet måste vara ihopsatt för att vara aktivt. NA har en uppbyggnad liknande en trädklunga, där fyra stycken identiska träd (med tillhörande rötter, stammar och trädkronor) går ihop och bildar en enda aktiv enhet, en s.k. tetramer. ”Rötterna” hos NA är den transmembrana domänen (TMD), den del av proteinet som sitter fast i influenaviruskroppen. ”Stammen”, eller stjälkdelen av NA, binder samman TMD med den största delen, huvuddomänen som motsvarar ”trädkronan”. Det är just huvuddomänen som är ansvarig för att klyva loss viruspartiklar från en modercell. Vi har i våra studier sett att det kan vara väldigt viktigt att TMD-domänerna går ihop i grupper om fyra för att hela NA ska kunna gå ihop i en tetramer och aktivt kunna klyva loss viruspartiklarna. När vi studerade TMD från olika influensavirus så märkte vi att vissa egenskaper hos TMD krävs för att de skulle kunna gå ihop, men också att dessa egenskaper inte fanns hos alla influensavirus. Virusen har evolverat över lång tid och har anpassat sig efter värdorganismerna (inklusive människan) och har hittat olika lösningar på problemet med att behöva bilda en tetramer. När vi gjorde ändringar i en TMD som vanligtvis gick ihop till en tetramer, och därmed förhindrade detta, så noterade vi att huvuddomänens funktion påverkades vilket ledde till att influensaviruset hade svårt att spridas. Vidare så har våra pågående studier på stjälkdelen visat att även denna del kan ha stor betydelse för tetrameriseringen av NA, speciellt i de fall där TM-domänen saknar egenskaper för att gå ihop. Avhandlingen tillför inte bara ny och viktig information till influensaforskningen, utan även potentiellt för framställandet av nya influensavacciner/-mediciner. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.</p>

Thesis advisor: Larry W. McLaughlin / There are two major goals for my project. The first is to create and characterize metal-ligand-DNA complexes that could be synthesized using traditional organic methods followed by solid phase techniques. The second is to demonstrate that these complexes with complementary DNA sequences could self-assemble into higher-ordered structures. In order to generate supramolecular DNA-metal structures such as cubic lattices, it is necessary to create an octahedral metal-ligand center tethering six DNA arms as a building block. The Iron/Ru (II) bis(2,2':6',2''terpyridine) derivatives were chosen because: (i) the complex is well known to present octahedral geometry; (ii) the coordination is very stable; and (iii) while previous work required the solid-phase synthesis of six DNA arms simultaneously--an inefficient process--by using terpyridine ligands we need only extend three arms at once. Thus, several terpyridine-linker compounds were synthesized via two different routes. A DNA 14mer was synthesized afterwards by "Reverse Coupling Protocol" on a solid phase synthesizer and the terpyridine was connected to it followed by elongation of the rest two DNA arms. The DNA-terpyridine complexes were evaluated by stepwise hybridization tests and gel electrophoresis with or without the assistance of radio labeling. In addition, the assembly of metal with the terpyridine-DNA complex was also characterized by adding different metal ions such as Iron (II) and Ru (II) to the complex. Various buffer conditions were applied in constructing those conjugates in order to help forming branched DNA-ligand-metal complexes with higher molecular weight. / Thesis (MS) — Boston College, 2010. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

The communities of microorganisms associated with humans and other animals are characterized by a large degree of diversity and unexplained variation across individual hosts. While efforts to explain this variation in host-associated systems have focused heavily on the effects of host selection, community assembly theory emphasizes the role of dispersal and stochastic demographic processes, otherwise known as ecological drift. In this dissertation, I characterize the communities of microorganisms associated with the zebrafish, Danio rerio, intestine, and assess the importance of microbial dispersal and drift to their assembly. First, I describe changes in the composition and diversity of the zebrafish intestinal microbiome over zebrafish development and show that while host development is a major driver of community composition over time, there remains a large amount of unexplained variation among similar hosts of the same age. I go on to show that random dispersal and ecological drift alone in the absence of host selection are sufficient to explain a substantial amount of this variation, but the ability of these processes to predict the distribution of microorganisms across hosts decreases over host development. Finally, I present an experimental test of dispersal in host-associated systems, and show that not only does dispersal among individual zebrafish hosts have a large impact on the composition and diversity of associated microbial communities, but it can also overwhelm the effects of important host factors, such as the innate immune system. As a whole, this work demonstrates that the composition and diversity of microbial communities associated with animal hosts are not solely the result of selection by the host environment, but rather dispersal and stochastic processes have important and often overwhelming effects on their assembly. To fully understand the assembly of host-microbe systems, we must broaden our focus to include scales beyond that of an individual host and their associated microorganisms. / 10000-01-01

Actuellement, les technologies de séquençage ne permettent de lire la séquence d'un génome entier d'un individu, mais donnent les séquences de portions courtes de ce génome avec des erreurs. On doit ensuite procéder à un assemblage de ces séquences (que l'on appelle lectures ou "read" en anglais) pour retrouver la séquence du génome complet. Une version théorique de cette problématique est le problème de la plus courte superchaîne: étant donné un ensemble de mots (notre ensemble de lectures), on cherche à trouver le plus petit mot qui contient tous les autres comme sous-chaîne (le génome d'origine). Ce problème étudié depuis les années 60 est notoirement difficile à résoudre de manière exacte et approchée.L'assemblage nécessite certains pré-traitements des lectures, comme par exemple la correction des erreurs dues au séquençage dans les lectures (au sens où on cherche à enlever les erreurs). Certains logiciels de correction (ou d'autres pré-traitements) utilisent une structure d'indexation des séquences pour repérer les erreurs. Or, après la correction, cette structure de données est perdue et l'assemblage n'utilise plus que les lectures corrigées. Dans cette thèse, on se demande comment utiliser les structures d'indexation pour faciliter ou améliorer la qualité de l'assemblage.Dans un premier temps, on a montré qu'à partir d'une structure d'indexation, on pouvait rapidement reconstruire les graphes utilisés dans les algorithmes d'assemblage (graphe de Bruijn, graphe de Bruijn contracté, graphe de chevauchements). De plus, on a mis en évidence un nouveau graphe, le graphe hiérarchique de chevauchements ou "Hierarchical Overlap Graph", qui résume les informations des graphes classiques de l'assemblage.Dans un deuxième temps, on s'est demandé comment une structure d'indexation pouvait aider à résoudre directement le problème théorique de la plus courte superchaîne. Pour cela, on a étudié les solutions que l'algorithme glouton donnait à ce problème (leur approximation, leur combinatoire, ...) et à plusieurs de ces variantes (cas des mots renversés et complémentaires, cas de superchaîne cyclique, cas de couverture par un ensemble de superchaînes). Ceci a permis de résoudre plusieurs questions concernant la complexité et l'approximabilité de ces problèmes. En particulier, l'algorithme glouton permet de résoudre en temps linéaire la question de la plus petite couverture par des chaînes cycliques. Même si l'algorithme glouton est le plus simple et un des plus étudiés pour ces problèmes, il n'en reste pas moins un mystère. Notre étude a permis de mettre en évidence un nouveau graphe, le graphe des superchaînes ou "Superstring Graph", qui correspond à un plongement des solutions de l'algorithme glouton dans la structure d'indexation qu'est l'arbre des suffixes. Autrement dit, le graphe des superchaînes synthétise l'ensemble des solutions gloutonnes dans un espace linéaire.Enfin, on s'est intéressé aux algorithmes des meilleurs assembleurs utilisés en pratique (IDBA, SPAdes) qui ont permis d'améliorer l'assemblage de lectures courtes en utilisant plusieurs graphes d'assemblage. Nous avons montré tout d'abord que le graphe des superchaînes permet de stocker plus d'informations que ces assembleurs et avec une complexité en espace bien plus faible. Ensuite, il ressort que l'algorithme glouton pour une variante du problème de plus courte superchaîne donne des séquences qui incluent les contigs trouvés pour ces algorithmes. Ces résultats permettent de lier l'assemblage pratique et les problèmes de superchaînes, et donnent un cadre théorique fort pour étudier ces algorithmes heuristiques. / Whole genome can not be read by the current sequencing technologies. Instead, the output is short sequences which are portions with errors of the whole genome. One must then proceed to an assembly of these sequences (called read) to find the sequence of the complete genome. A theoretical version of this problem is the problem of the shortest superstring: given a set of words (own set of reads), we try to find the shortest string that contains all others as substring (the genome of origin). Studied since the 60s, this problem is notoriously difficult to solve by both exactly and approximate methods.Genome assembly requires some reads preprocessing, such as the correction of errors introduced by the sequencing. Some correction softwares (or other pre-treatments) use an indexing data structure of the sequences to localize errors. However, after the correction, this data structure is lost and the assembly uses only the corrected reads. In this thesis, we wonder how to use indexing structures to facilitate or to improve the quality of the genome assembly.First, we show that the graphs used in assembly algorithms could quickly rebuild from an indexing structure (de Bruijn graph, contracted de Bruijn graph and overlap graph). In addition, we present a new graph which summarizes the information of conventional assembly graphs and that we call the hierarchical overlap graph.Secondly, we wondered how an indexing data structure could directly help to solve the theoretical problem of the shortest superstring. For this purpose, we study the solutions that the greedy algorithm gives to this problem (their approximation, their combinatorics, ...) and many of these variants (reverse complement case, cyclic superstring case, case cover by a set of superstrings). This has solved several questions about the complexity and the approximation of these problems. In particular, the greedy algorithm solves in linear time the question of the shortest cyclic cover of strings. Although the greedy algorithm is the simplest and one of the most studied of these problems, it remains a mystery. Our study has highlighted a new graph, the superstring graph, which corresponds to a dip from solutions of the greedy algorithm in the index structure that is the suffix tree. In other words, the superstring graph summarizes all the greedy solutions in a linear space.Finally, attention has turned to the algorithms of the best assemblers used in practice (IDBA, Spades), which have improved the assembly of short reads using several assembly graphs. We show firstly that the superstring graph can store more information than these assemblers and with a complexity in much smaller space. Then, it is apparent that the greedy algorithm for a variant of the shorter superstring problem provides sequences which include the contigs found for these algorithms. These results link the assembly in practice and the superstring problems, and give a strong theoretical framework for studying these heuristic algorithms.

The skin microbiome is a critical component of human health, however, little is understood about the daily dynamics of skin microbiome community assembly and the skin’s potential to acquire microorganisms from the external environment. I performed a series of microbial transfers using three skin habitat types (dry, moist, sebaceous) on human subject volunteers. Microbial communities were transferred to recipient skin using a sterile swab 1) from other skin sites on the same individual, 2) from other skin sites on a different individual, 3) and from two environmental donor sources (plant leaf surfaces and farm soil). With these experiments I was able to test for the presence of initial transfer effects and for the persistence of those effects over the time period sampled (2-, 4-, 8-, and 24-hours post-transfer). The sebaceous skin community was associated with the strongest initial effect of transfer and persistence on the moist recipient skin site, and to a lesser extent the dry skin site. The soil donor community when transferred to dry skin resulted in the strongest initial transfer effect and was persistent over 8- and even 24-hours post-transfer. These experiments are the first in scope and scale to directly demonstrate that dispersal from other human or environmental microbial communities are plausible drivers of community dynamics in the skin microbiome.

This thesis describes the synthesis and coordination chemistry of self-assembled multidentate iron(II) phosphine complexes. Chapter 1 introduces the background to phosphine ligands, their properties, interactions with transition metals and applications. The chapter then discusses macrocyclic and medium ring P,N-containing ligands, as well as some water soluble phosphines. The chapter also introduces the novel self-assembled macrocyclic phosphine complex [FeL1(H2O)2]SO4 (1) and its tetradentate cyclic phosphine ligand L1. Chapter 2 describes the synthesis of [FeL1(H2O)2]SO4 (1) and its coordination chemistry with a variety of ligands, including halides, pseudo-halides, and CO. 57Fe labelled versions of complex 1 and the related dicarbonyl complex [FeL1(H2O)2]SO4 (9) were synthesised as models for the hydrogenase protein Hmd in a Nuclear Resonance Vibrational Spectroscopy study. Reactions were also undertaken to functionalise the hydroxymethyl groups in order to alter the properties of the complexes. The reaction of 1 with acetic anhydride afforded complex [Fe(L2)(k2-O2SO2)] (13), possessing the acylated ligand L2 and a coordinated sulfate ligand. The coordination chemistry of 13 was explored with a variety of neutral and anionic ligands, including halides, pseudohalides, carbonate, and CO. Electrochemical cyclic voltammetric investigations of L1 and L2 complexes were also explored. Chapter 3 reports the investigations carried out to explore the effect of altering the reagents of the self-assembly reaction. The self-assembly reaction to synthesise complex 1 was also attempted with copper(II), nickel(II), copper(II) and zinc(II) salts, as well as in the absence of a metal template, which all did not lead to the formation of any isolable species. The syntheses of the novel iron(II) complexes [Fe(L3)2(SO4)] (23) and cis-[Fe(L3)2Cl2] (24a) containing the new bidentate phosphine ligand L3 are also reported, as well as the coordination chemistry of 24a with a variety of ligands. The reaction of 24a with NaBH4 gave the trans hydride-chloride complex trans-[Fe(L3)2(H)Cl] (29). Electrochemical investigations of the L3 complexes were also carried out. Chapter 4 provides the experimental details for the reactions described in chapters 2 and 3.

The use of low-molecular weight peptide-based hydrogelators (LMWGs) for the immobilisation of enzymes is presented in this thesis. Low-molecular weight hydrogelators are a class of materials which are highly suitable for increasing enzyme lifetimes as they create a suitable biomimetic environment. Immobilised enzymes can be utilised in enzyme fuel cells, providing low-energy conversion routes for chemical processes. The hydrogels also possess tunable properties which allow their structure to be manipulated to give desirable properties. This work begins with an exploration of dipeptide hydrogelators by investigating the effect of varying salt solutions and concentrations of dipeptide on final hydrogel structures. A wide range of characterisation techniques are employed to provide information about the micro- and macro-structure of the hydrogels. The creation of dipeptide hydrogel materials via an electrochemical method is explored, which allows the production of nanometre thick, membrane-like materials. These layers are measured using Surface Plasmon Resonance techniques. The electrochemical technique for dipeptide gelation is expanded in later chapters to produce a range of novel materials. Finally, an exploration into the effect of additives on dipeptide hydrogels is conducted, where the effect of adding chiral molecules is investigated. This provides interesting information regarding the self-assembly processes involved with hydrogelation processes, which has important implications for studying the folding and unfolding processes of peptides.

Small diameter vascular grafts comprised entirely from cells and cell-derived extracellular matrix (ECM) have shown promise in clinical trials and may have potential advantages as in vitro vascular tissue models. A challenge with current cell-derived tissue engineering approaches is the length of time required to generate strong, robust tissue. There is a lack of alternative methods to rapidly assemble cells into a 3D format without the support of a scaffold. Toward the goal of engineering a new approach to rapidly synthesizing vascular tissue constructs entirely from cells, we have developed and characterized a strategy for creating cell-derived tissue rings by cellular self-assembly. The focus of this thesis was to develop the system to rapidly generate engineered tissue rings, and to evaluate their structural and functional properties. To generate tissue rings, rat smooth muscle cells (SMCs) were seeded into round-bottomed, ring-shaped agarose wells with varying inner post diameters (2, 4, and 6 mm). Within 24 hours of seeding, cells aggregated, contracted, and formed robust tissue that could be removed from their wells and handled. If kept in culture, the thickness of these tissue rings increased with time. Mechanical analysis of the tissue showed that it was stronger after only 8 days in culture than engineered tissues generated by other approaches (such as seeding cells in biopolymer gels) cultured and tested at similar time points. Histological staining of the tissue rings revealed high cell densities throughout, along with the presence of glycosaminoglycans and some collagen. We also found that we could use the tissue rings as building blocks to generate larger tubular structures. Briefly, tissue rings were removed from the agarose wells and transferred onto silicone tubing mandrels. Once the rings were placed in contact with each other on the mandrel, they were cultured to allow the rings to fuse together. We found that the ability of tissue rings to fuse decreased with increasing ring “pre-culture� duration, and that we were able to generate fully fused tissue tubes in as little as 8 days (with only one day of ring pre-culture and seven days of fusion). In the last section of this thesis, we established the feasibility of using primary human SMCs to generate self-assembled tissue rings, similar to the self-assembled rings generated with rat SMCs. Compared to the rat SMC rings, human SMC rings were stronger, stiffer and appeared to contain increased levels of collagen. These data showed that human SMCs are capable of self-assembling into tissue rings similar to rat SMCs, and may therefore be used to create engineered human vascular tissue. Overall, we have developed a platform technology that can be used to screen the effects of culture parameters on the structure, mechanics, and function of vascular tissue. We anticipate that through the use of this technology, we can further improve vascular grafts by better understanding factors which promote ECM synthesis and SMC contraction. We can use these results directly toward the generation of vascular grafts by fusing self-assembled cell rings together to form tissue tubes. These novel bioengineered vascular tissues may also serve as a method to produce in vitro models to help further our understanding of vascular diseases, as well as facilitate pre-clinical screening of vascular tissue responses to pharmacologic therapies.