Structural and functional characterization of Group B Streptococcus pilus 2b

Group B Streptococcus (GBS) is a Gram-positive human pathogen representing one of the most common causes of life-threatening bacterial infections such as sepsis and meningitis in neonates. Covalently polymerized pilus-like structures have been discovered in GBS as important virulence factors as well as vaccine candidates. Pili are protein polymers forming long and thin filamentous structures protruding from bacterial cells, mediating adhesion and colonization to host cells. Gram-positive bacteria, including GBS, build pili on their cell surface via a class C sortase-catalyzed transpeptidation mechanism from pilin protein substrates that are the backbone protein forming the pilus shaft and two ancillary proteins. Also the cell-wall anchoring of the pilus polymers made of covalently linked pilin subunits is mediated by a sortase enzyme. GBS expresses three structurally distinct pilus types (type 1, 2a and 2b). Although the mechanisms of assembly and cell wall anchoring of GBS types 1 and 2a pili have been investigated, those of pilus 2b are not understood until now. Pilus 2b is frequently found in ST-17 strains that are mostly associated with meningitis and high mortality rate especially in infants. In this work the assembly mechanism of GBS pilus type 2b has been elucidated by dissecting through genetic, biochemical and structural studies the role of the two pilus-associated sortases. The most significant findings show that pilus 2b assembly appears “non-canonical”, differing significantly from current pilus assembly models in Gram-positive pathogens. Only sortase-C1 is involved in pilin polymerization, while the sortase-C2 does not act as a pilin polymerase, but it is involved in cell-wall pilus anchoring. Our findings provide new insights into pili biogenesis in Gram-positive bacteria. Moreover, the role of this pilus type during host infection has been investigated. By using a mouse model of meningitis we demonstrated that type 2b pilus contributes to pathogenesis of meningitis in vivo.

Tailoring properties and functionalities of TiO2 and Ag nanoparticles involved in surfaces engineering processes

The functionalization of substrates through the application of nanostructured coatings allows to create new materials, with enhanced properties. In this work, the development of self-cleaning and antibacterial textiles, through the application of TiO2 and Ag based nanostructured coatings was carried out. The production of TiO2 and Ag functionalized materials was achieved both by the classical dip-padding-curing method and by the innovative electrospinning process to obtain nanofibers doped with nano-TiO2 and nano-Ag. In order to optimize the production of functionalized textiles, the study focused on the comprehension of mechanisms involved in the photocatalytic and antibacterial processes and on the real applicability of the products. In particular, a deep investigation on the relationship between nanosol physicochemical characteristics, nanocoating properties and their performances was accomplished. Self-cleaning textiles with optimized properties were obtained by properly purifying and applying commercial TiO2 nanosol while the studies on the photocatalytic mechanism operating in self-cleaning application demonstrated the strong influence of hydrophilic properties and of interaction surface/radicals on final performance. Moreover, a study about the safety in handling of nano-TiO2 was carried out and risk remediation strategies, based on “safety by design” approach, were developed. In particular, the coating of TiO2 nanoparticles by a SiO2 shell was demonstrated to be the best risk remediation strategy in term of biological response and preserving of photoreactivity. The obtained results were confirmed determining the reactive oxygen species production by a multiple approach. Antibacterial textiles for biotechnological applications were also studied and Ag-coated cotton materials, with significant anti-bacterial properties, were produced. Finally, composite nanofibers were obtained merging biopolymer processing and sol-gel techniques. Indeed, electrospun nanofibers embedded with TiO2 and Ag NPs, starting from aqueous keratin based formulation were produced and the photocatalytic and antibacterial properties were assessed. The results confirmed the capability of electrospun keratin nanofibers matrix to preserve nanoparticle properties.

Synthesis of constrained peptidomimetics for therapeutic, diagnostic and theranostic applications

This thesis work deals, principally, with the development of different chemical protocols ranging from environmental sustainability peptide synthesis to asymmetric synthesis of modified tryptophans to a series of straightforward procedures for constraining peptide backbones without the need for a pre-formed scaffold. Much efforts have been dedicated to the structural analysis in a biomimetic environment, fundamental for predicting the in vivo conformation of compounds, as well as for giving a rationale to the experimentally determined bioactivity. The conformational analyses in solution has been done mostly by NMR (2D gCosy, Roesy, VT, titration experiments, molecular dynamics, etc.), FT-IR and ECD spectroscopy. As a practical application, 3D rigid scaffolds have been employed for the synthesis of biological active compounds based on peptidomimetic and retro-mimetic structures. These mimics have been investigated for their potential as antiflammatory agents and actually the results obtained are very promising. Moreover, the synthesis of Amo ring permitted the development of an alternative high effective synthetic pathway for obtaining Linezolid antibiotic. The final section is, instead, dedicated to the construction of a new biosensor based on zeolite L SAMs functionalized with the integrin ligand c[RGDfK], that has showed high efficiency for the selective detection of tumor cells. Such kind of sensor could, in fact, enable the convenient, non-invasive detection and diagnosis of cancer in early stages, from a few drops of a patient's blood or other biological fluids. In conclusion, the researches described herein demonstrate that the peptidomimetic approach to 3D definite structures, allows unambiguous investigation of the structure-activity relationships, giving an access to a wide range bioactive compounds of pharmaceutical interest to use not only as potential drugs but also for diagnostic and theranostic applications.

Synthesis, properties and application of polyphenylene phosphonium salts

The central objective of this work was to generate weakly coordinating cations of unprecedented molecular size providing an inherently stable hydrophobic shell around a central charge. It was hypothesized that divergent dendritic growth by means of thermal [4+2] Diels-Alder cycloaddition might represent a feasible synthetic method to circumvent steric constraints and enable a drastic increase in cation size.rnThis initial proposition could be verified: applying the divergent dendrimer synthesis to an ethynyl-functionalized tetraphenylphosphonium derivative afforded monodisperse cations with precisely nanoscopic dimensions for the first time. Furthermore, the versatile nature of the applied cascade reactions enabled a throughout flexible design and structural tuning of the desired target cations. The specific surface functionalization as well as the implementation of triazolyl-moieties within the dendrimer scaffold could be addressed by sophisticated variation of the employed building block units (see chapter 3). rnDue to the steric screening provided by their large, hydrophobic and shape-persistent polyphenylene shells, rigidly dendronized cations proved more weakly coordinating compared to their non-dendronized analogues. This hypothesis has been experimentally confirmed by means of dielectric spectroscopy (see chapter 4). It was demonstrated for a series of dendronized borate salts that the degree of ion dissociation increased with the size of the cations. The utilization of the very large phosphonium cations developed within this work almost achieved to separate the charge carriers about the Bjerrum length in solvents of low polarity, which was reflected by approaching near quantitative ion dissociation even at room temperature. In addition to effect the electrolyte behavior in solution, the steric enlargement of ions could be visualized by means of several crystal structure analyses. Thus an insight into lattice packing under the effect of extraordinary large cations could be gathered. rnAn essential theme of this work focused on the application of benzylphosphonium salts in the classical Wittig reaction, where the concept of dendronization served as synthetic means to introduce an exceptionally large polyphenylene substituent at the -position. The straightforward influence of this unprecedented bulky group on the Wittig stereochemistry was investigated by NMR-analysis of the resulting alkenes. Based on the obtained data a valuable explanation for the origin of the observed selectivity was brought in line with the up-to-date operating [2+2] cycloaddition mechanism. Furthermore, a reliable synthesis protocol for unsymmetrically substituted polyphenylene alkenes and stilbenes was established by the design of custom-built polyphenylene precursors (see chapter 5).rnFinally, fundamental experiments to functionalize a polymer chain with sterically shielded ionic groups either in the pending or internal position were outlined within this work. Thus, inherently hydrophobic polysalts shall be formed so that future research can invesigate their physical properties with regard to counter ion condensation and charge carrier mobility.rnIn summary, this work demonstrates how the principles of dendrimer chemistry can be applied to modify and specifically tailor the properties of salts. The numerously synthesized dendrimer-ions shown herein represent a versatile interface between classic organic and inorganic electrolytes, and defined macromolecular structures in the nanometer-scale. Furthermore the particular value of polyphenylene dendrimers in terms of a broad applicability was illustrated. This work accomplished in an interdisciplinary manner to give answer to various questions such as structural modification of ions, the resulting influence on the electrolyte behavior, as well as the stereochemical control of organic syntheses via polyphenylene phosphonium salts. rn

Intracellular imaging of nanoparticles: is it an elemental mistake to believe what you see?

In order to understand how nanoparticles (NPs <100 nm) interact with cellular systems, potentially causing adverse effects, it is important to be able to detect and localize them within cells. Due to the small size of NPs, transmission electron microscopy (TEM) is an appropriate technique to use for visualizing NPs inside cells, since light microscopy fails to resolve them at a single particle level. However, the presence of other cellular and non-cellular nano-sized structures in TEM cell samples, which may resemble NPs in size, morphology and electron density, can obstruct the precise intracellular identification of NPs. Therefore, elemental analysis is recommended to confirm the presence of NPs inside the cell. The present study highlights the necessity to perform elemental analysis, specifically energy filtering TEM, to confirm intracellular NP localization using the example of quantum dots (QDs). Recently, QDs have gained increased attention due to their fluorescent characteristics, and possible applications for biomedical imaging have been suggested. Nevertheless, potential adverse effects cannot be excluded and some studies point to a correlation between intracellular particle localization and toxic effects. J774.A1 murine macrophage-like cells were exposed to NH2 polyethylene (PEG) QDs and elemental co-localization analysis of two elements present in the QDs (sulfur and cadmium) was performed on putative intracellular QDs with electron spectroscopic imaging (ESI). Both elements were shown on a single particle level and QDs were confirmed to be located inside intracellular vesicles. Nevertheless, ESI analysis showed that not all nano-sized structures, initially identified as QDs, were confirmed. This observation emphasizes the necessity to perform elemental analysis when investigating intracellular NP localization using TEM.

Proliferation, differentiation and gene expression of osteoblasts in boron-containing associated with dexamethasone deliver from mesoporous bioactive glass scaffolds

Boron is one of the trace elements in the human body which plays an important role in bone growth. Porous mesopore bioactive glass (MBG) scaffolds are proposed as potential bone regeneration materials due to their excellent bioactivity and drug-delivery ability. The aims of the present study were to develop boron-containing MBG (B-MBG) scaffolds by sol-gel method and to evaluate the effect of boron on the physiochemistry of B-MBG scaffolds and the response of osteoblasts to these scaffolds. Furthermore, the effect of dexamethasone (DEX) delivery in B-MBG scaffold system was investigated on the proliferation, differentiation and bone-related gene expression of osteoblasts. The composition, microstructure and mesopore properties (specific surface area, nano-pore volume and nano-pore distribution) of B-MBG scaffolds have been characterized. The effect of boron contents and large-pore porosity on the loading and release of DEX in B-MBG scaffolds were also investigated. The results have shown that the incorporation of boron into MBG scaffolds slightly decreases the specific surface area and pore volume, but maintains well-ordered mesopore structure and high surface area and nano-pore volume compared to non-mesopore bioactive glass. Boron contents in MBG scaffolds did not influence the nano-pore size distribution or the loading and release of DEX. B-MBG scaffolds have the ability to maintain a sustained release of DEX in a long-term span. Incorporating boron into MBG glass scaffolds led to a controllable release of boron ions and significantly improved the proliferation and bone-related gene expression (Col I and Runx2) of osteoblasts. Furthermore, the sustained release of DEX from B-MBG scaffolds significantly enhanced alkaline phosphatase (ALP) activity and gene expressions (Col I, Runx2, ALP and BSP) of osteoblasts. These results suggest that boron plays an important role in enhancing osteoblast proliferation in B-MBG scaffold system and DEX-loaded B-MBG scaffolds show great potential as a release system to enhance osteogenic property for bone tissue engineering application.

Barriers to front propagation in ordered and disordered vortex flows

We present experiments on reactive front propagation in a two-dimensional (2D) vortex chain flow (both time-independent and time-periodic) and a 2D spatially disordered (time-independent) vortex-dominated flow. The flows are generated using magnetohydrodynamic forcing techniques, and the fronts are produced using the excitable, ferroin-catalyzed Belousov-Zhabotinsky chemical reaction. In both of these flows, front propagation is dominated by the presence of burning invariant manifolds (BIMs) that act as barriers, similar to invariant manifolds that dominate the transport of passive impurities. Convergence of the fronts onto these BIMs is shown experimentally for all of the flows studied. The BIMs are also shown to collapse onto the invariant manifolds for passive transport in the limit of large flow velocities. For the disordered flow, the measured BIMs are compared to those predicted using a measured velocity field and a three-dimensional set of ordinary differential equations that describe the dynamics of front propagation in advection-reaction-diffusion systems.

The Body of Margaret Atwood: Sex Work and Prostitution within Margaret Atwood's The Handmaid's Tale, Oryx and Crake, and The Year of the Flood

In this paper, I will argue that Canadian author Margaret Atwood uses fiscal and socially conservative dystopias to show how sex work and prostitution are choices that women would never have to make in a world with true gender equality. In these radically different worlds, women have no agency beyond their sexuality and no ability to express themselves as equals within either society. And while the structures of both societies, the society of The Handmaid’s Tale and that of both Oryx and Crake and The Year of the Flood, are inherently different, they both stem from modern conservative philosophies: for example, the country of Gilead in The Handmaid’s Tale holds Christian conservative beliefs on the role of religion in the state and the culturally designated roles of women. I define social conservatism as the idea that government organizations are used to pursue an agenda promoting traditional religious values such as “public morality” and opposing “immoralities” such as abortion, prostitution, and homosexuality. I define fiscal conservatism as an agenda promoting privatization of the market, deregulation and lower taxes. In this paper I argue that because these philosophies are incompatible with gender equality, they drive women to occupations such as sex work. Women find that they have no choices and sex work provides something to “trade.” For Offred, this “trading” is more limited, because she is a sex slave. For Oryx, this trading allows her to travel to the West, yet not before her childhood is marked by prostitution and pornography. Sex work allows for Ren to reclaim some agency over her life, yet she only chooses sex work because she is presented with few other options. All of these issues stem from the philosophies that define these dystopias.

Tenovaginoscopic approach to the common digital flexor tendon sheath of adult cattle: technique, normal findings and preliminary results in four clinical cases

The aim of this study was to describe the tenovaginoscopic approach to the bovine common digital flexor tendon sheath (CDFTS). A comparative anatomical, ultrasonographic and endoscopic study was undertaken using 26 healthy cadaver feet from adult dairy cows. Tenovaginoscopy was performed using a rigid, 30 degrees arthroscope (length 18 cm; outer diameter 4mm) enabling a direct view of the synovial cavity and the following structures: digital flexor tendons, digital annular ligaments, lateral and medial pouches, three mesotendons, the vinculum of the superficial digital flexor tendon, and a slot-shaped opening in the manicaflexoria of the hind feet. Additionally, four clinical cases of septic tenosynovitis treated with lavage under tenovaginoscopic control were examined. Tenovaginoscopy represents a feasible, minimally invasive method for the diagnosis and treatment of septic tenosynovitis of the CDFTS, which allows the degree of alterations of the normal structures to be evaluated.

Simply a nest? Effects of different enrichments on stereotypic and anxiety-related behaviour in mice

Improving the home cages of laboratory mice by environmental enrichment has been widely used to reduce cage stereotypies and anxiety-related behaviour in behavioural tests. However, enrichment studies differ substantially in type, complexity and variation of enrichments. Therefore, it is unclear whether success depends on specific enrichment items, environmental complexity, or novelty associated with enrichment. The aim of this study was therefore to dissociate the effects of environmental complexity and novelty on stereotypy development and compare these effects with the provision of nesting material alone. Thus, 54 freshly weaned male ICR (CD-1) mice were pairwise allocated to standard laboratory cages enriched in three different ways (n = 18 per group). Treatment 1 consisted of cotton wool as nesting material. Treatments 2 and 3 were structurally more complex, including a shelter and a climbing structure as additional resources. To render complexity and novelty independent of the specific enrichment items, three shelters (cardboard house, plastic tunnel, red plastic house) and three climbing structures (ladder, rope, wooden bars) were used to create nine different combinations of enrichment. In treatment 2 (complexity), each pair of mice was assigned to a different combination that remained constant throughout 9 weeks, whereas in treatment 3 (novelty), each pair of mice was exposed to all 9 combinations in turn by changing them weekly in a pseudorandom order. After 9 weeks, stereotypic behaviour in the home cage was assessed from video recordings, and anxiety-related behaviour was assessed in two behavioural tests (elevated zero-maze, open-field). However, no significant differences in stereotypy scores and no consistent differences in anxiety-related behaviours were found between the three groups. These findings indicate that within standard laboratory cages neither complexity nor novelty of simple enrichments have additional effects on stereotypic and anxiety-related behaviour beyond those of adequate nesting material. (C) 2011 Elsevier B.V. All rights reserved.

TGF-b2 induction regulates invasiveness of Theileria-transformed leukocytes and disease susceptibility

Theileria parasites invade and transform bovine leukocytes causing either East Coast fever (T. parva), or tropical theileriosis (T. annulata). Susceptible animals usually die within weeks of infection, but indigenous infected cattle show markedly reduced pathology, suggesting that host genetic factors may cause disease susceptibility. Attenuated live vaccines are widely used to control tropical theileriosis and attenuation is associated with reduced invasiveness of infected macrophages in vitro. Disease pathogenesis is therefore linked to aggressive invasiveness, rather than uncontrolled proliferation of Theileria-infected leukocytes. We show that the invasive potential of Theileria-transformed leukocytes involves TGF-b signalling. Attenuated live vaccine lines express reduced TGF-b2 and their invasiveness can be rescued with exogenous TGF-b. Importantly, infected macrophages from disease susceptible Holstein-Friesian (HF) cows express more TGF-b2 and traverse Matrigel with great efficiency compared to those from disease-resistant Sahiwal cattle. Thus, TGF-b2 levels correlate with disease susceptibility. Using fluorescence and time-lapse video microscopy we show that Theileria-infected, disease-susceptible HF macrophages exhibit increased actin dynamics in their lamellipodia and podosomal adhesion structures and develop more membrane blebs. TGF-b2-associated invasiveness in HF macrophages has a transcription-independent element that relies on cytoskeleton remodelling via activation of Rho kinase (ROCK). We propose that a TGF-b autocrine loop confers an amoeboid-like motility on Theileria-infected leukocytes, which combines with MMP-dependent motility to drive invasiveness and virulence.

Hoisting C Structures Into Clay In Device Drivers

This project addresses the unreliability of operating system code, in particular in device drivers. Device driver software is the interface between the operating system and the device's hardware. Device drivers are written in low level code, making them difficult to understand. Almost all device drivers are written in the programming language C which allows for direct manipulation of memory. Due to the complexity of manual movement of data, most mistakes in operating systems occur in device driver code. The programming language Clay can be used to check device driver code at compile-time. Clay does most of its error checking statically to minimize the overhead of run-time checks in order to stay competitive with C's performance time. The Clay compiler can detect a lot more types of errors than the C compiler like buffer overflows, kernel stack overflows, NULL pointer uses, freed memory uses, and aliasing errors. Clay code that successfully compiles is guaranteed to run without failing on errors that Clay can detect. Even though C is unsafe, currently most device drivers are written in it. Not only are device drivers the part of the operating system most likely to fail, they also are the largest part of the operating system. As rewriting every existing device driver in Clay by hand would be impractical, this thesis is part of a project to automate translation of existing drivers from C to Clay. Although C and Clay both allow low level manipulation of data and fill the same niche for developing low level code, they have different syntax, type systems, and paradigms. This paper explores how C can be translated into Clay. It identifies what part of C device drivers cannot be translated into Clay and what information drivers in Clay will require that C cannot provide. It also explains how these translations will occur by explaining how each C structure is represented in the compiler and how these structures are changed to represent a Clay structure.

Continuous, Passive Liquid-Liquid Extraction and Emulsion Separation Within Microfluidic and Millifluidic Devices

Liquid-liquid extraction (LLE) is a method used to separate compounds based on their relative activity in two immiscible phases. By significantly reducing the scale of liquid-liquid extraction to the micro- and milli-fluidic levels, this separation process can bemade suitable for low volume, high value materials.

Aporias of Trauma in Joyce's Ulysses and Faulkner's Light in August

In this thesis, I explore the relationships between trauma, memory, and narrative, particularly the ways in which trauma simultaneously disrupts and engenders narrative structures. I consider various trauma theories by authors such as Cathy Caruth, Judith Herman, Ruth Leys, and Dominick LaCapra. I also consider how psychoanalytic theory and criticism, specifically the writings of Sigmund Freud, inform the study of traumatic experience from both literary and personal perspectives. Furthermore, I consider theories regarding the relationship between trauma and narrative by authors such as Peter Brooks and John Pilkington. James Joyce¿s Ulysses and William Faulkner¿s Light in August serve, for my purposes, as trauma-texts and reflect the ways in which trauma might complicate the simultaneous destruction and creation of narrative strategies. Reading Ulysses and Light in August as trauma-texts that are both in mourning and melancholic gives us complementary, and contradictory, reasons for why we enjoy them. Mourning constructs a relationship between victim and witness, in which we can hear the voice of trauma and engage it in discourse. Conversely, melancholia creates a relationship between performer and spectator, in which we experience, and are fascinated by, the spectacle of another¿s trauma. Laughter, perversity, sorrow, and respite engage the reader in both texts, and raise questions about how one `remembers-to-forget¿ traumatic experiences. The narratives of each text¿s characters offer unique performances of mourning and melancholia. Thus, while this thesis engenders more questions than answers, I hope to argue that Ulysses and Light in August are significant literary works because each engages the reader in traumatic discourse, entertains the reader with the traumatic spectacle, and enlightens the reader on the complex relationship between trauma and narrative.

Role of FGFRL1 and other FGF signaling proteins in early kidney development

The mammalian kidney develops from the ureteric bud and the metanephric mesenchyme. In mice, the ureteric bud invades the metanephric mesenchyme at day E10.5 and begins to branch. The tips of the ureteric bud induce the metanephric mesenchyme to condense and form the cap mesenchyme. Some cells of this cap mesenchyme undergo a mesenchymal-to-epithelial transition and differentiate into renal vesicles, which further develop into nephrons. The developing kidney expresses Fibroblast growth factor (Fgf)1, 7, 8, 9, 10, 12 and 20 and Fgf receptors Fgfr1 and Fgfr2. Fgf7 and Fgf10, mainly secreted by the metanephric mesenchyme, bind to Fgfr2b of the ureteric bud and induce branching. Fgfr1 and Fgfr2c are required for formation of the metanephric mesenchyme, however the two receptors can substitute for one another. Fgf8, secreted by renal vesicles, binds to Fgfr1 and supports survival of cells in the nascent nephrons. Fgf9 and Fgf20, expressed in the metanephric mesenchyme, are necessary to maintain survival of progenitor cells in the cortical region of the kidney. FgfrL1 is a novel member of the Fgfr family that lacks the intracellular tyrosine kinase domain. It is expressed in the ureteric bud and all nephrogenic structures. Targeted deletion of FgfrL1 leads to severe kidney dysgenesis due to the lack of renal vesicles. FgfrL1 is known to interact mainly with Fgf8. It is therefore conceivable that FgfrL1 restricts signaling of Fgf8 to the precise location of the nascent nephrons. It might also promote tight adhesion of cells in the condensed metanephric mesenchyme as required for the mesenchymal-to-epithelial transition.