Die Matrix-Proteine von Vertretern der Ordnung Mononegavirales sind essentiell für späte Schritte im viralen Lebenszyklus, insbesondere der Knospung und Partikelmorphogenese. Die Abschnürung und Freisetzung umhüllter RNA-Viren ist dabei abhängig von dem Transport des viralen Matrix-Proteins und seiner Interaktion mit Wirtsproteinen wie dem ESCRT-System (endosomal sorting complex required for transport), welches in die Sortierung zellulärer Proteine involviert ist. Im Verlauf der Masernvirus (MV)-Infektion interagiert das M-Protein einerseits mit dem viralen Nukleoproteinkomplex und andererseits mit den viralen Glykoproteinen an der Oberfläche. Die Bedeutung des MV-M-Proteins für die Partikelproduktion und sein intrazellulärer Transport wurden bislang kaum untersucht. Bisher ist nur bekannt, dass das M-Protein oligomerisiert, teilweise monoubiquitiniert vorliegt und in Zellen, wenn alleine exprimiert, die Produktion von Virus-like-particle (VLP) vermittelt (Pohl et al., 2007). In dieser Studie wird gezeigt, dass das MV-M-Protein ähnlich wie das VP40-Protein des Ebolavirus (EBOV) mit Lipid Rafts und TEMs (tetraspanin enriched microdomain) assoziiert ist, wobei aber das M-Protein weniger effizient an der Plasmamembran akkumuliert. Beide Proteine unterscheiden sich nicht wesentlich in ihrer Assoziation mit Kompartimentmarkern. Interessant ist jedoch, dass das VP40-Protein und das M-Protein an der Plasmamembran mit dem Adaptor Protein-3 (AP-3) kolokalisieren. Die Kolokalisation des M-Proteins mit AP-3 wird aber nur in infizierten Zellen und nicht in Zellen, in denen das M-Protein allein exprimiert wird, beobachtet. Im Gegensatz zum VP40-Protein, welches die ESCRT-Komponenten über seine N-terminale L-Domäne rekrutiert und diese für die Partikelproduktion benutzt, geschieht dies beim M-Protein ESCRT-unabhängig, da die Mutation der Motive, die Ähnlichkeiten zu den bekannten L-Domänen zeigen, keine Auswirkungen auf die VLP-Produktion haben. Zudem rekrutierte das M-Protein weder Tsg101, Aip-1 oder Vps4 an die Plasmamembran, noch wird die VLP- oder die Virusproduktion durch dominant negatives Vps4 inhibiert. Der Transfer der VP40 L-Domäne in das MV-M-Protein hatte weder einen Einfluss auf die Assoziation mit Tetraspaninen noch auf die ESCRT-Abhängigkeit der VLP-Produktion. Damit wurde gezeigt, dass die VLP-Freisetzung des MV-M-Proteins ESCRT-unabhängig ist. Die Freisetzung erfolgt beim MV durch einen grundsätzlich anderen Weg, der noch untersucht werden muss. / The matrix proteins of the order Mononegarivales are essential for late steps in the viral life cycle, especially for budding process and viral morphogenesis. Budding and release of enveloped RNA-viruses depend on transport of the viral matrix protein and its interaction with host proteins, which are involved in sorting of cellular proteins, e.g. the ESCRT system. During the course of measles virus infection, the M-protein interacts with the viral nucleocapsid-complex and the viral glycoproteins. The impact of MV-M-protein for the particle production and intracellular transport is barely known yet. So far, it has been shown that the M-protein forms oligomers, is partially mono-ubiquinated and has the capability to promote VLP-production. In this study it could be shown that the MV-M-protein and EBOV-VP40-protein associate with detergent-resistant and tetraspanin-enriched microdomains. However, compared to the VP40-protein the M-protein accumulates less efficiently at the plasma membrane. Both proteins do not essentially vary in their association with compartment markers. Interestingly, VP40- and M-protein, the latter only when expressed in the context of MV infection (but not from plasmid), efficiently co-localised with adaptor protein 3 (AP-3) at the plasma membrane indicating its importance for membrane targeting. In contrast to VP40, which recruits ESCRT components via its N-terminal late (L) domain and exploits them for particle production, M-protein promotes particle production independently of this pathway since 1) ablation of motifs bearing similarity to canonical L domains did not affect VLP production, 2) it did not redistribute Tsg101, AIP-1, or Vps4 to the plasma membrane, and 3) neither VLP nor infectious virus production were sensitive to inhibition by dominant negative Vps4. In addition, transfer of the VP40 L domain into the MV-M-protein did not alter trafficking or ESCRT dependence for VLP production to that of VP40 suggesting that budding activity of MV-M-protein follows an ESCRT independent pathway.

Ho Kin Chuen Matthew. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 153-157). / Abstract also in Chinese. / Chapter 1 --- Introduction --- p.15 / Chapter 1.1 --- The U-line Balancing Problem --- p.15 / Chapter 1.2 --- Configuration of an U-line --- p.17 / Chapter 1.3 --- Feasible subsets and sequences --- p.19 / Chapter 1.4 --- Assignment of tasks to stations --- p.21 / Chapter 1.5 --- Costs --- p.22 / Chapter 1.6 --- Formulation of The U-line Balancing Problem --- p.23 / Chapter 1.7 --- Design of computational study --- p.25 / Chapter 1.7.1 --- Input parameters --- p.25 / Chapter 1.7.2 --- Output variables --- p.26 / Chapter 1.7.3 --- Problems solved --- p.27 / Chapter 1.7.3.1 --- Problem Set One --- p.28 / Chapter 1.7.3.2 --- Problem Set Two --- p.28 / Chapter 1.7.3.3 --- Problem Set Three --- p.29 / Chapter 1.7.3.4 --- Problem Set Four --- p.29 / Chapter 1.8 --- Contributions --- p.29 / Chapter 1.9 --- Organization of thesis --- p.30 / Chapter 2 --- Literature Review --- p.31 / Chapter 2.1 --- Introduction --- p.31 / Chapter 2.2 --- The Straight-line Balancing Problem --- p.32 / Chapter 2.2.1 --- Single-model Assembly Line Balancing with deterministic task time (SMD) --- p.34 / Chapter 2.2.2 --- Single-model Assembly Line Balancing with stochastic task times (SMS) --- p.36 / Chapter 2.2.3 --- Multi/Mixed-model Assemble Line Balancing with deterministic task times (MMD) --- p.37 / Chapter 2.2.4 --- Multi/Mixed-model Assembly Line Balancing with stochastic task times (MMS) --- p.38 / Chapter 2.3 --- The U-line Balancing Problem --- p.39 / Chapter 2.4 --- Conclusions --- p.45 / Chapter 3 --- Heuristic Methods --- p.47 / Chapter 3.1 --- Introduction --- p.47 / Chapter 3.2 --- Single-pass heuristic methods --- p.47 / Chapter 3.3 --- Computational results --- p.50 / Chapter 3.3.1 --- Problem Set One --- p.50 / Chapter 3.3.2 --- Problem Set Two --- p.52 / Chapter 3.3.3 --- Problem Set Three --- p.54 / Chapter 3.3.4 --- Problem Set Four --- p.55 / Chapter 3.4 --- Discussions --- p.57 / Chapter 3.5 --- Conclusions --- p.59 / Chapter 4 --- Genetic Algorithm --- p.60 / Chapter 4.1 --- Introduction --- p.60 / Chapter 4.2 --- Application of GA to The Straight-line Balancing Problem --- p.61 / Chapter 4.3 --- Application of GA to The U-line Balancing Problem --- p.62 / Chapter 4.3.1 --- Coding scheme --- p.63 / Chapter 4.3.2 --- Initial population --- p.64 / Chapter 4.3.3 --- Fitness function --- p.65 / Chapter 4.3.4 --- Selection scheme --- p.66 / Chapter 4.3.5 --- Reproduction --- p.67 / Chapter 4.3.6 --- Replacement scheme --- p.68 / Chapter 4.3.7 --- Elitism --- p.68 / Chapter 4.3.8 --- Termination criteria --- p.68 / Chapter 4.4 --- Repair method --- p.69 / Chapter 4.5 --- Crossover operators --- p.71 / Chapter 4.5.1 --- Sequence and configuration infeasible crossover operators --- p.72 / Chapter 4.5.1.1 --- Partially Mapped Crossover (PMX) --- p.72 / Chapter 4.5.1.2 --- Order Crossover #1 (ORD#l) --- p.74 / Chapter 4.5.1.3 --- Order Crossover #2 (ORD#2) --- p.74 / Chapter 4.5.1.4 --- Position Based Crossover (POS) --- p.75 / Chapter 4.5.1.5 --- Cycle Crossover (CYC) --- p.76 / Chapter 4.5.1.6 --- Edge Recombination Crossover (EDG) --- p.77 / Chapter 4.5.1.7 --- Enhanced Edge Recombination Crossover (EEDG) --- p.80 / Chapter 4.5.1.8 --- Uniform-order Based Crossover (UOX) --- p.81 / Chapter 4.5.2 --- Sequence feasible but configuration infeasible crossover operators --- p.82 / Chapter 4.5.2.1 --- One-point Crossover (1PX) --- p.82 / Chapter 4.5.2.2 --- Two-point Crossover (2PX) --- p.84 / Chapter 4.5.2.3 --- Uniform Crossover (UX) --- p.85 / Chapter 4.6 --- Mutation operators --- p.86 / Chapter 4.6.1 --- Sequence infeasible mutation operators --- p.87 / Chapter 4.6.1.1 --- Inversion (INV) --- p.87 / Chapter 4.6.1.2 --- Insertion (INS) --- p.87 / Chapter 4.6.1.3 --- Displacement (DIS) --- p.88 / Chapter 4.6.1.4 --- Reciprocal Exchange (RE) --- p.88 / Chapter 4.6.2 --- Sequence and configuration feasible mutation operators --- p.89 / Chapter 4.6.2.1 --- Scramble Mutation (SCR) --- p.89 / Chapter 4.6.2.2 --- Feasible Insertion (FINS) --- p.90 / Chapter 4.7 --- Computational study --- p.91 / Chapter 4.7.1 --- Comparison of crossover operators --- p.91 / Chapter 4.7.2 --- Comparison of mutation operators --- p.95 / Chapter 4.7.2.1 --- Order crossover#2 and mutation operators --- p.95 / Chapter 4.7.2.2 --- Position based crossover and mutation operators --- p.97 / Chapter 4.7.3 --- Parameters setting --- p.99 / Chapter 4.7.4 --- Computational results --- p.104 / Chapter 4.7.5 --- Comparative results --- p.105 / Chapter 4.7.5.1 --- Problem Set One --- p.105 / Chapter 4.7.5.2 --- Problem Set Two --- p.105 / Chapter 4.7.5.3 --- Problem Set Three --- p.107 / Chapter 4.7.5.4 --- Problem Set Four --- p.107 / Chapter 4.8 --- Conclusions --- p.109 / Chapter 5 --- Dynamic Programming and Lower Bounds --- p.110 / Chapter 5.1 --- Dynamic Programming (DP) --- p.110 / Chapter 5.1.1 --- Introduction --- p.110 / Chapter 5.1.2 --- Modified Dynamic Programming algorithm --- p.112 / Chapter 5.1.3 --- Comparison between optimal solution and heuristics --- p.120 / Chapter 5.1.4 --- Comparison between optimal solution and the GA --- p.123 / Chapter 5.2 --- Lower Bounds --- p.123 / Chapter 5.2.1 --- Introduction --- p.123 / Chapter 5.2.2 --- The U-line Balancing Problem and The Bin Packing Problem --- p.127 / Chapter 5.2.3 --- Martello and Toth's lower bounds for The BPP --- p.128 / Chapter 5.2.3.1 --- Bound L1 --- p.128 / Chapter 5.2.3.2 --- Bound L2 --- p.128 / Chapter 5.2.3.3 --- Dominances and reductions --- p.129 / Chapter 5.2.3.3.1 --- Dominance criterion --- p.129 / Chapter 5.2.3.3.2 --- Reduction procedure --- p.130 / Chapter 5.2.3.4 --- Lower Bound LR --- p.131 / Chapter 5.2.4 --- Chen and Srivastava's lower bounds for The BPP --- p.131 / Chapter 5.2.4.1 --- A unified lower bound --- p.132 / Chapter 5.2.4.2 --- Improving Lm --- p.133 / Chapter 5.2.4.3 --- "Computing a lower bound on N(1/4,1]" --- p.134 / Chapter 5.2.5 --- Lower bounds for The U-line Balancing Problem --- p.137 / Chapter 5.2.5.1 --- Lower bounds on number of stations required --- p.137 / Chapter 5.2.5.2 --- Lower bounds on total cost --- p.139 / Chapter 5.2.6 --- Computational results --- p.140 / Chapter 5.2.6.1 --- Results for different Problem Sets --- p.140 / Chapter 5.2.6.2 --- Comparison between lower bounds and optimal solutions --- p.143 / Chapter 5.2.6.3 --- Comparison between lower bounds and heuristics --- p.145 / Chapter 5.2.6.4 --- Comparison between lower bounds and GA --- p.147 / Chapter 5.3 --- Conclusions --- p.149 / Chapter 6 --- Conclusions --- p.150 / Chapter 6.1 --- Summary of achievements --- p.150 / Chapter 6.2 --- Future works --- p.151

Over the past few decades, it has become increasingly apparent that host-associated microbial communities play an integral role in the development, physiology, and health of their host organisms. All hosts have evolved mechanisms to filter the microbial taxa that comprise their resident intestinal microbial community, or gut microbiota. Utilizing the zebrafish as a model host organism, we documented the development of the gut microbiota through time, and found a significant shift in the composition of the gut microbiota after the onset of adaptive immunity. This led us to hypothesize that adaptive immunity is an important determinant of gut microbiota composition. We tested this hypothesis using wild type and rag1-/- zebrafish, which lack a functional adaptive immune system. Additionally we tested the robustness of the effects of adaptive immunity to dispersal of microbes between immune-compromised and immune-competent genotypes. We found that adaptive immunity had less of an effect on the composition of the gut microbiota than we expected, although there were intriguing differences in the nature of selection imposed when adaptive immunity was present than when it was absent. Because “westernization”, or market-integration, has been associated with significant changes in the human microbiota and certain health risks, we used similar analyses to those we applied to the zebrafish system to determine whether market-integration alters the filtering effects of inflammation and intestinal helminth parasites on the intestinal microbial community. We found that market-integration increased inter-subject dissimilarity and reduced inter-subject dispersal. Even small changes in the inflammation marker, CRP, were associated with differences in the gut microbiota, but these effects were reduced in the presence of helminth infection, which has been hypothesized to affect the microbiota by reducing inflammation. In total, this dissertation provides evidence for the nature and importance of host filters of the gut microbiota across two vertebrate species, as well as providing a framework for future studies of the effects of such filters on the assembly of the gut microbiota. This dissertation includes previously published, and unpublished, co-authored material.

Synthetic Laponite-clay particles with a platelet-like shape display strong gelation when dispersed in aqueous solutions because of their positively charged rims and negatively charged flat surfaces. In this thesis, my aim was to modify the surfaces of these clay particles such that we can both access their liquid crystalline (LC) discotic phase and further build transparent and mechanically resilient coatings with a 3D "brick-and-mortar" structure that is similar to that observed in natural mother of pearl (nacre). I first introduce a simple strategy that successfully suppresses Laponite's ageing phenomenon and enables the system's isotropic-to-LC phase transition. By grafting Laponite particle surfaces with comb-like polymers, poly (L-lysine)-g-poly (ethylene glycol) (PLL-PEG), I was able to screen negative surface charges and ensure steric stabilisation. Besides using long-chain polymers, I also coated the positively charged Laponite rims with small, barrel-shaped molecules cucurbit[7]uril (CB[7]). By carefully tuning the ratio between CB[7] and Laponite, the system experienced a macroscopic phase separation into a Laponite-poor suspension and a birefringent LC gel. Inspired by the hierarchical structure of nacre, here I also demonstrate a simple approach to fabricate polymer-clay hybrid films via a water-evaporation process. In this third method, Laponite platelets were bridged by natural abundant polymers (carboxymethyl cellulose) through hydrogen bonding. This hybrid material possesses high transparency, flexibility and an outstanding fire-retardant property. After Ca2+ ion-coordination of these cellulose-Laponite composite films, the interface between the polymers and clays was further strengthened, leading to enhanced mechanical properties along with improved thermal- and water-resistance. I also present that using Dextran as a depletant, sterically stabilised Laponite can access its liquid crystal phase under low clay concentration. Finally, I show that Laponite can be coated with various polymers (PEO, chitosan, sodium alginate) for the purpose of obtaining LC gels and hybrid films. I believe that our findings on surface-modification of clay particles can open new routes to large-scale and inexpensive production of bio-inspired functional materials.

Thesis. 1979. M.S. cn--Massachusetts Institute of Technology. Dept. of Mechanical Engineering. / MICROFICHE COPY AVAILABLE IN ARCHIVES AND ENGINEERING. / Includes bibliographical references. / by Craig C. Selvage. / M.S.cn

Thesis (M.B.A.)--Boston University / PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / 2031-01-01

Thesis (Ph.D.)--University of Delaware, 2007. / Principal faculty advisor: Joel P. Schneider, Dept. of Chemistry & Biochemistry. Includes bibliographical references.

Elastin is a polymeric protein of the extracellular matrix that imparts the characteristics of extensibility and elastic recoil to tissues. Recombinant polypeptides based on the domain structures and sequences of human elastin self-assemble into organized fibrous structures, with physical properties similar to those of native polymeric elastin. Elastin self-assembly is initiated by a temperature-induced phase separation, called coacervation. Previous to this work, coacervation temperature had been the only parameter available to measure propensity for self-assembly. A variety of techniques were developed using spectrophotometry, microscopy, and rheometry to differentiate the stages of self-assembly, thereby enabling independent observation and quantitation of each stage, and allowing investigations into properties of polypeptides and solution conditions affecting these stages. Kinetic analysis of self-assembly yielded two additional parameters: coacervation velocity and maturation velocity. Examining the effects of agitation, salt concentration, temperature, polypeptide concentration, size of a polypeptide, hydrophobic domain sequence, and cross-linking domain structure on the kinetics demonstrated that coacervation and maturation are independent stages of self-assembly involving distinct mechanisms. Microscopic observations showed that protein-rich droplets of coacervate grew by coalescence to a stable droplet size, which correlated to differences in maturation velocities between polypeptides. Coacervate droplet growth appeared limited by the formation of organized polypeptide at the surface of the droplets, decreasing surface fluidity. Many of the general principles of the physical chemistry of colloids and emulsions appeared to apply to the formation, growth and stabilization of coacervates of the elastin-like polypeptides. Self-assembly in the presence of non-elastin, matrix-associated proteins showed that these proteins maintained the coacervate as small droplets, which sometimes flocculated into fibre-like structures. Rheometry demonstrated a second temperature-induced transition above the coacervation temperature, which resulted in gelation and viscoelastic characteristics similar to microgels. Together, these observations have resulted in a greater level of understanding of the entire self-assembly process, and provided a comprehensive model of elastin-like polypeptide self-assembly that relates to in vivo assembly of elastic fibres.

The atomic force microscope (AFM) was used to study a series of self-assembled systems: alkanethiol self-assembled monolayer (SAM), diblock copolymer thin film, solid supported lipid bilayer membrane, and microgel with double interpenetrating polymer network. In the first system, packing and restructuring of self-assembled monolayers as exhibited by several alkanethiol systems (1-hexanethiol, 1-decanethiol, 11-ferrocenyl-1-undecanethiol) is demonstrated using conducting probe AFM (CP-AFM). Pressure is induced by an AFM tip, and simultaneously, electrical behavior is measured via detection of tunneling currents between metallic tip and substrate. The behavior is fit using a mechanical model that attempts to predict the observed junction resistance as a function of applied force with consideration for mechanical restructuring of the monolayer at higher loads. CP-AFM is also used to study self-assembled thin film of the diblock copolymer polystyrene- block-polyferrocenylsilane (PS-b-PFS) on gold substrate. Simultaneous height and electrical current imaging verify the phase separation of the two blocks of the polymer and additionally, distinguish each block due to differential conductivity. The phase separation of multi-component phospholipid bilayers (phosphatidylcholine/ sphingomyelin/ cholesterol) on supporting substrate into liquid-ordered and liquid-disordered phases is demonstrated using both topographical imaging, and the use of force map analysis through tip indentation and rupture measurements. The segregation and differential mechanical stiffness of the phases help to understand the important role of mechanical stability and rigidity membranes. An automated batch analysis process was implemented to facilitate the procedure. The mechanical properties of microfluidically produced microgels (cross-linked sodium alginate and poly(N-isopropylacrylamide)) are measured using indentation experiments, to evaluate the suitability of these gels as cell-mimics. Nanoscale heterogeneities were avoided by using a tipless cantilever. This body of work shows that the alginate content of these microgels can be varied to tune their mechanical properties and that a platform for mechanical measurement of cell and cell-mimics is possible.

Elastin is a polymeric protein of the extracellular matrix that imparts the characteristics of extensibility and elastic recoil to tissues. Recombinant polypeptides based on the domain structures and sequences of human elastin self-assemble into organized fibrous structures, with physical properties similar to those of native polymeric elastin. Elastin self-assembly is initiated by a temperature-induced phase separation, called coacervation. Previous to this work, coacervation temperature had been the only parameter available to measure propensity for self-assembly. A variety of techniques were developed using spectrophotometry, microscopy, and rheometry to differentiate the stages of self-assembly, thereby enabling independent observation and quantitation of each stage, and allowing investigations into properties of polypeptides and solution conditions affecting these stages. Kinetic analysis of self-assembly yielded two additional parameters: coacervation velocity and maturation velocity. Examining the effects of agitation, salt concentration, temperature, polypeptide concentration, size of a polypeptide, hydrophobic domain sequence, and cross-linking domain structure on the kinetics demonstrated that coacervation and maturation are independent stages of self-assembly involving distinct mechanisms. Microscopic observations showed that protein-rich droplets of coacervate grew by coalescence to a stable droplet size, which correlated to differences in maturation velocities between polypeptides. Coacervate droplet growth appeared limited by the formation of organized polypeptide at the surface of the droplets, decreasing surface fluidity. Many of the general principles of the physical chemistry of colloids and emulsions appeared to apply to the formation, growth and stabilization of coacervates of the elastin-like polypeptides. Self-assembly in the presence of non-elastin, matrix-associated proteins showed that these proteins maintained the coacervate as small droplets, which sometimes flocculated into fibre-like structures. Rheometry demonstrated a second temperature-induced transition above the coacervation temperature, which resulted in gelation and viscoelastic characteristics similar to microgels. Together, these observations have resulted in a greater level of understanding of the entire self-assembly process, and provided a comprehensive model of elastin-like polypeptide self-assembly that relates to in vivo assembly of elastic fibres.