Proefschrift--Amsterdam. / "Theses" (4 p.).

No description available.

Lysis of the host by bacteriophage 21 requires two proteins: the pinholin S21 (forms pinholes in the cytoplasmic membrane and controls lysis timing) and the endolysin (degrades the cell wall). S21 has a dual-start motif, encoding a holin, S2168, and a weak antiholin, S2171. Both proteins have two transmembrane domains (TMD) and adopt an N-in, C-in topology. The topology of S2168 is dynamic because TMD1 is a signal-anchor-release (SAR) domain which, while initially integrated into the cytoplasmic membrane, is eventually released into the periplasm. TMD1 is dispensable because the truncated protein, S2168?TMD1, retains the holin function. Adding two positive charges to N-terminus of S2168 by an irs tag (RYIRS) prevents the release of TMD1. The irsS2168 protein not only has lost its holin function, but is a potent antiholin and blocks the function of S2168. In this dissertation, the structure of S2168 was suggested by incorporating electron-microscopy, biochemical, and computational approaches. The results suggest that S2168 forms a symmetric heptamer, with the hydrophilic side of TMD2 lining the channel of ~ 15 A in diameter. This model also identifies two interacting surfaces, A and B, of TMD2. A model for the pinhole formation pathway was generated from analyzing phenotypes of an extensive collection of S21 mutants. In this model, the individually folded and inserted S21 molecules first form the inactive dimer, with the membrane-inserted TMD1 inhibiting the lethal function of TMD2 both inter- and intra-molecularly. A second inactive dimer may form, with one TMD1 released. When both TMD1s are released, the activated dimer is formed, with the homotypic interfaces A:A interaction of the TMD2s. However, this interaction might not be stable, which will shift to heterotypic A:B interactions, allowing TMD2 to oligomerize. Finally, the pinhole forms, possibly driven by the hydration of lumenal hydrophilic residues. In addition, the localization of pinholes was visualized by fusing the green fluorescent protein (GFP) to the C-terminus of pinholins. The results showed that pinholins form numerous small aggregates, designated as rafts, spread all over the cell body. The antiholin irsS2168 not only inhibits the triggering of S2168GFP, but inhibits the rafts formation as well.

SAR endolysins are a recently discovered class of muralytic enzymes that are regulated by dynamic membrane topology. They are synthesized as enzymatically inactive integral membrane proteins during the phage infection cycle and then are activated by conformational remodeling upon release from the membrane. This topological duality depends on N-terminal SAR (Signal-Anchor-Release) domains, which are enriched in weakly hydrophobic residues and require the proton motive force to be maintained in the bilayer. The first SAR endolysin to be characterized was P1 Lyz, of phage P1. Its activation requires a disulfide bond isomerization involving its catalytic Cys initiated by a free cysteine thiol from the newly-liberated SAR domain. A second mode of disulfide bond regulation, as typified by Lyz103 of the Erwinia Amylovora phage ERA103, has been demonstrated. In its membrane bound form, Lyz103 is inactivated by a disulfide that is formed between cysteine residues flanking a catalytic glutamate. A second class of SAR endolysins, typified by R21, the lysozyme of the lambdoid phage 21, does not require disulfide bond isomerization for activation. Rather, these proteins are dependent on the release of the SAR domain for proper folding of the catalytic cleft. Bioinformatic analysis indicates that the regulatory theme of R21 is common in the SAR endolysins of dsDNA phages. Furthermore, bioinformatic study of endolysins of dsDNA phage of Gram-negative hosts revealed two new classes of SAR endolysins that are not homologous to T4 gpe, as all SAR endolysins were once thought to be. SAR endolysins were found in nearly 25% of sequenced dsDNA phages of Gram- negative hosts including 933W, which is involved in the release of Shiga toxin from EHEC strain EDL933. An inhibitor study against the SAR endolysin of 933W, R933W, was performed using a custom compound library in a high through-put, in vivo lysis assay. Of nearly 8,000 compounds screened, one compound, designated 67-J8, inhibited lysis but not growth. In vivo and in vitro experiments show that the compound has no effect on R933W activity, accumulation, or secretion. In vivo experiments suggest that 67-J8 increases the proton motive force, thereby presumably retaining the SAR domain in the membrane.

No description available.

Lysis in cells infected with the ssRNA phage Qbeta is effected by the A2 protein. It was previously shown that a single copy of A2 assembled on the surface of the Qbeta virion inhibited the activity of MurA, which catalyzes the first committed step of murein biosynthesis. This led to a model for lysis timing in which A2 is not active as a MurA inhibitor until assembled into virion particles. Here we report that MurA inactivates purified Qbeta particles. Moreover, over-expression of MurA does not inactivate particles during the Qbeta infection cycle; thus, casting doubt on the notion that completed virions could be the lytic agent in vivo and also that the MurA-virion interaction does not occur in the infected cell. Furthermore, RNA released from particles was found to protect virions from inactivation by MurA in vitro, suggesting that Qbeta RNA might serve as the protective element during the infection cycle. Comparison of A2 accumulation between Qbeta and Qbeta^por mutants, which are Qbeta A2 mutants with a shorter infection cycle and reduced burst size, reveals that a delicate balance between assembled and unassembled A2 levels regulates lysis timing. A new model is proposed in which "free", unassembled A2 inhibits MurA. From in vitro binding studies and genetic analyses it was determined that A2 binds MurA in a closed conformation with UDP-N-acetylglucosamine bound.

The aim of this project was to work towards developing a droplet-based microfluidic device which can perform cell lysis, Whole Genome Amplification (WGA) and storage of the amplified DNA. This would provide an automated biobanking device capable of high-throughput sample processing whilst shielding the samples from the sample loss and contamination commonly experienced by conventional, isolated sample handling methods.WGA has been examined using two commercially available WGA kits (GenomiPhi V2 and HY) to produce a continuous flow device that is capable of amplifying both human genomic DNA (gDNA) and bacterial plasmid DNA samples in nanolitre volume droplets. A positive effect of reducing reaction volumes on the amplification of bacterial plasmid DNA was shown by obtaining an increase in yield with decreasing volumes. It was shown, however, that a reduction in the volume of the WGA reaction has a negative impact on the amplification of human gDNA, in terms of both reduced yield and copy number variation (CNV). Furthermore, a novel method for reducing this CNV has been achieved by pooling the products of multiple reaction volumes. Finally, a cell lysis device has been developed which can perform rapid lysis of a human neuroblastoma cell line in continuously flowing droplets through addition of an alkaline solution.These devices provide an advantage over previously developed methods, displaying cell lysis of a human cell line and amplification of both human gDNA and plasmid DNA, while the continuous flow design of the devices allows for both high-throughput processing of samples and the future integration of the devices to form a μTAS biobanking device.

Cet essai se veut une étude approfondie du Lysis de Platon. Le but que nous nous donnons à travers lui est de mettre en valeur l'interrogation du philosophe par rapport au fondement de la relation privilégiée à autrui que constitue l'amitié. Une analyse serrée du texte nous permettra d'abord de nous familiariser avec les conceptions régnant à l'époque du penseur à propos justement de ce rapport à l'autre, de constater ensuite avec lui l'insuffisance de celles-ci et, finalement, de comprendre la nécessité d'innover pour faire avancer la recherche sur l'essence même de l'amitié. Des thèmes aussi importants que celui de la privation, du désir, de la convenance existant entre l'amant et l'aimé ainsi que celui de notre quête d'un prôton philon se trouveront alors développés. Ils annonceront les traits essentiels d'œuvres postérieures telles que le Banquet et le Phèdre.

A primary objective of modern biology is to understand the molecular mechanisms which underlie cellular functions and a crucial part of this task is the ability to manipulate and analyze individual cells. As a result of interdisciplinary research, microfluidics may become the forefront of analytical methods used by biologists. This technology can be used to gain unprecedented opportunities for cell handling, lysis and investigation on a single cell basis. This thesis presents the development of a microfluidic device capable of selecting individual cells and performing selective electrical lysis of the plasma membrane, while verifying intactness of the nuclear membrane. The device is fabricated by an improved photolithography method and integrates molten solder as electrodes for lysis by a DC electric field. Quantification of lysis is accomplished by video and image analysis, and measurement of the rate of ion diffusion from the cell.

A primary objective of modern biology is to understand the molecular mechanisms which underlie cellular functions and a crucial part of this task is the ability to manipulate and analyze individual cells. As a result of interdisciplinary research, microfluidics may become the forefront of analytical methods used by biologists. This technology can be used to gain unprecedented opportunities for cell handling, lysis and investigation on a single cell basis. This thesis presents the development of a microfluidic device capable of selecting individual cells and performing selective electrical lysis of the plasma membrane, while verifying intactness of the nuclear membrane. The device is fabricated by an improved photolithography method and integrates molten solder as electrodes for lysis by a DC electric field. Quantification of lysis is accomplished by video and image analysis, and measurement of the rate of ion diffusion from the cell.