Winding Modes and Large Extra Dimensions

We review briefly the main features of the Large Extra Dimensions scenario in the framework of weakly coupled Type I string theory. Kaluza-Klein (KK) excitations of the graviton are expected, whereas no KK modes for the gauge bosons arise if the gauge group is tied to a D3-brane. In this scenario, typical signatures such as direct production of KK modes of the graviton at high-energy colliders could test the size of the compactified dimensions. We point out that contrary to what considered in the literature on the subject, in the general case of anisotropic compactification Winding Modes of the Standard Model gauge bosons could also be directly observable, thus further constraining the model

Molecular cloning and Biochemical characterization of pyridoxal 5’-phosphate dependent enzymes of unknown function

General introduction The main topic of this Ph.D. thesis is the characterization of gene products identified as PLP-dependent enzymes with unknown function. A complete and detailed description on function and evolution of PLP-dependent enzymes is provided in chapter 1. Chapter 2 describes the molecular cloning as well as the recombinant expression and the initial biochemical characterization of a threonine synthase homolog from mouse. Finally, in chapter 3 I describe the study of two recombinant human proteins homolog of alanine-glyoxylate amino transferase 2. The last part, Appendix, reports the evaluation of small RNA-cleaving DNAs (ribozymes), chemically modified by means of introduction of monomers of locked nucleic acids (LNA), in order to improve to cleavage of long and structured mRNAs.Introduzione generale Argomento principale di questa tesi di dottorato è la caratterizzazione di prodotti genici identificati come enzimi PLP-dipendenti, ma aventi funzione ignota. Nel capitolo 1 viene data una introduzione completa e dettagliata sull’evoluzione e le caratteristiche degli enzimi PLP-dipendenti. Nel capitolo 2 sono descritti il clonaggio, l’espressione in forma ricombinante e l’iniziale caratterizzazione biochimica, di una protiena di topo omologa della treonina sintasi microbica. Nel capitolo 3 è illustrato lo studio di due proteine umane, omologhe dell’alanina-gliossilato amino transferasi 2. Nell’ultima parte, l‘Appendice, sono descritti studi su piccoli DNA ad attività ribonucleasica (ribozimi) modificati attraverso l’introduzione di monomeri di locked nucleic acid (LNA), con lo scopo di migliorare il taglio di mRNA dotati di struttura secondaria

Hybrid 1D Plasmonic/Photonic Crystals are Responsive to Escherichia Coli

Photonic crystal-based biosensors hold great promise as valid and low-cost devices for real-time monitoring of a variety of biotargets. Given the high processability and easiness of read-out even for unskilled operators, these systems can be highly appealing for the detection of bacterial contaminants in food and water. Here, we propose a novel hybrid plasmonic/photonic device that is responsive to Escherichia coli, which is one of the most hazardous pathogenic bacterium. Our system consists of a thin layer of silver, a metal that exhibits both a plasmonic behavior and a well-known biocidal activity, on top of a solution processed 1D photonic crystal. We attribute the bio-responsivity to the modification of the dielectric properties of the silver film upon bacterial contamination, an effect that likely stems from the formation of polarization charges at the Ag/bacterium interface within a sort of bio-doping mechanism. Interestingly, this triggers a blue-shift in the photonic response. This work demonstrates that our hybrid plasmonic/photonic device can be a low-cost and portable platform for the detection of common contaminants in food and water

The advantages of being locked. Assessing the cleavage of short and long RNAs by locked nucleic acid-containing 8-17 deoxyribozymes.

RNA-cleaving deoxyribozymes can be used for the sequence-specific knockdown of mRNAs. It was previously shown that activity of these deoxyribozymes is enhanced when their substrate-binding arms include some locked nucleic acid (LNA) residues, but the mechanistic basis of this enhancement was not explored. Here we dissected the kinetics and thermodynamics underlying the reaction of LNA-containing 8-17 deoxyribozymes. Four 8-17 constructs were designed to target sequences within the E6 mRNA from human papillomavirus type 16. When one of these deoxyribozymes (DNAzymes) and the corresponding LNA-armed enzyme (LNAzyme) were tested against a minimal RNA substrate, they showed similar rates of substrate binding and similar rates of intramolecular cleavage, but the LNAzyme released its substrate more slowly. The superior thermodynamic stability of the LNAzyme-substrate complex led to improved performances in reactions carried out at low catalyst concentrations. The four DNAzymes and the corresponding LNAzymes were then tested against extended E6 transcripts (>500 nucleotides long). With these structured substrates, the LNAzymes retained full activity, whereas the DNAzymes cleaved extremely poorly, unless they were allowed to pre-anneal to their targets. These results imply that LNAzymes can easily overcome the kinetic barrier represented by local RNA structure and bind to folded targets with a faster association rate as compared with DNAzymes. Such faster annealing to structured targets can be explained by a model whereby LNA monomers favor the initial hybridization to short stretches of unpaired residues ("nucleation"), which precedes disruption of the local mRNA structure and completion of the binding process

Oxyresveratrol-Loaded PLGA Nanoparticles Inhibit Oxygen Free Radical Production by Human Monocytes: Role in Nanoparticle Biocompatibility

Oxyresveratrol, a polyphenol extracted from the plant Artocarpus lakoocha Roxb, has been reported to be an antioxidant and an oxygen-free radical scavenger. We investigated whether oxyresveratrol affects the generation of superoxide anion (O2 ) by human monocytes, which are powerful reactive oxygen species (ROS) producers. We found that oxyresveratrol inhibited the O2 production induced upon stimulation of monocytes with -glucan, a well known fungal immune cell activator. We then investigated whether the inclusion of oxyresveratrol into nanoparticles could modulate its effects on O2 release. We synthesized poly(lactic-co-glycolic acid) (PLGA) nanoparticles, and we assessed their effects on monocytes. We found that empty PLGA nanoparticles induced O2 production by resting monocytes and enhanced the formation of this radical in -glucan-stimulated monocytes. Interestingly, the insertion of oxyresveratrol into PLGA nanoparticles significantly inhibited the O2 production elicited by unloaded nanoparticles in resting monocytes as well as the synergistic effect of nanoparticles and -glucan. Our results indicate that oxyresveratrol is able to inhibit ROS production by activated monocytes, and its inclusion into PLGA nanoparticles mitigates the oxidative effects due to the interaction between these nanoparticles and resting monocytes. Moreover, oxyresveratrol can contrast the synergistic effects of nanoparticles with fungal agents that could be present in the patient tissues. Therefore, oxyresveratrol is a natural compound able to make PLGA nanoparticles more biocompatible

Porous silicon microparticles as efficient carriers for immunologic adjuvants

In this work we report a first-time combination of porous silicon (pSi) particles with the immunologic adjuvant Pam3CSK4, a TLR 1/2 agonist, as a tool for immunotherapy. pSi is a sponge-like biocompatible and biode gradable nanomaterial with high porosity, large surface-to-volume ratio and tunable surface, suitable for drug delivery applications. This study provides, by means of live-cell confocal microscopy, an insight about the time course of the interaction of free Pam3CSK4 vs vectorized by pSi microparticles with human dendritic cells (DCs). We found a delay in the ingestion of the agonist when carried by pSi microparticles. These findings were sup ported by the observation of the morphological changes related to the activation of DCs that occurred with a 5 h difference when treated with the vectorized ligand. These results provide the first demonstration of pSi as a conceivable candidate to deliver Pam3CSK4 to DCs paving the way towards immunotherapy practice

Oxyresveratrol Inhibits R848-Induced Pro-Inflammatory Mediators Release by Human Dendritic Cells Even When Embedded in PLGA Nanoparticles

Oxyresveratrol, a stilbene extracted from the plant Artocarpus lakoocha Roxb., has been reported to provide a considerable anti-inflammatory activity. Since the mechanisms of this therapeutic action have been poorly clarified, we investigated whether oxyresveratrol affects the release of the pro-inflammatory cytokines IL-12, IL-6, and TNF-\u3b1 by human dendritic cells (DCs). We found that oxyresveratrol did not elicit per se the release of these cytokines, but inhibited their secretion induced upon DC stimulation with R848 (Resiquimod), a well-known immune cell activator en-gaging receptors recognizing RNA viruses. We then investigated whether the inclusion of ox-yresveratrol into nanoparticles promoting its ingestion by DCs could favor its effects on cytokine release. For this purpose we synthesized and characterized poly(lactic-co-glycolic acid) (PLGA) nanoparticles, and we assessed their effects on DCs. We found that bare PLGA nanoparticles did not affect cytokine secretion by resting DCs, but increased IL-12, IL-6, and TNF-\u3b1 secretion by R848-stimulated DCs, an event known as \u201cpriming effect\u201d. We then loaded PLGA nanoparticles with oxyresveratrol and we observed that oxyresveratrol-bearing particles did not stimulate the cytokine release by resting DCs and inhibited the PLGA-dependent enhancement of IL-12, IL-6, and TNF-\u3b1 secretion by R848-stimulated DCs. The results herein reported indicate that oxyresveratrol suppresses the cytokine production by activated DCs, thus representing a good anti-inflammatory and immune-suppressive agent. Moreover, its inclusion into PLGA nanoparticles mitigates the pro-inflammatory effects due to cooperation between nanoparticles and R848 in cytokine release. Therefore, oxyresveratrol can be able to contrast the synergistic effects of nanoparticles with microorganisms that could be present in the patient tissues, therefore overcoming a condition unfavorable to the use of some nanoparticles in biological systems

Biohybrid Electrospun Membrane for the Filtration of Ketoprofen Drug from Water

A current challenge in materials science and biotechnology is to express a specific and controlled functionality on the large interfacial area of a nanostructured material to create smart biohybrid systems for targeted applications. Here, we report on a biohybrid system featuring poly(vinyl alcohol) as the supporting synthetic polymer and bovine serum albumin as the biofunctional element. The optimal processing conditions to produce these self-standing composite membranes are determined, and the composition and distribution of the bioactive agent within the polymeric matrices are analyzed. A post-processing cross-linking using glutaraldehyde enables this functional membrane to be used as a chemical filter in aqueous environments. By demonstrating that our mats can remove large amounts of ketoprofen from water, we show that the combination of a BSA-induced biofunctionality with a nanostructured fibrous material allows for the development of an efficient biohybrid filtering device for the large and widely used family of nonsteroidal anti-inflammatory drugs (NSAIDs). The crystal structure of the complex between BSA and ketoprofen is determined for the first time and confirms the interaction between the two species

Monocytes of Patients with Systemic Sclerosis (Scleroderma) Spontaneously Release In Vitro Increased Amounts of Superoxide Anion

It has been suggested that toxic oxygen free radicals can be involved in the pathogenesis of systemic sclerosis (scleroderma) (SSc). Because the cells that contribute to the generation of free radicals are not known, our aim was (i) to evaluate the ability of unmanipulated and phorbol 12-myristate 13-acetate-stimulated monocytes and polymorphonucleate neutrophils of SSc patients to generate superoxide anion (O2·–); and (ii) to investigate whether the O2·– produced by these cells involved the activation of nicotinamide-adenine dinucleotide diphosphate oxidase biochemical pathway. Employing the superoxide dismutase-inhibitable reduction of cytochrome c to evaluate the generation of O2·–, unmanipulated monocytes of SSc patients generated more O2·– than primary Raynaud’s phenomenon patients and normal control monocytes (p= 0.0001), and the release was higher in patients with diffuse cutaneous involvement and 5 y or less disease duration (p = 0.02). The involvement of nicotinamide-adenine dinucleotide diphosphate oxidase in the enhanced O2·– production was demonstrated by the finding that the cytosolic components of the enzyme, p47phox and p67phox, were both translocated to the plasma membrane of enriched but otherwise unmanipulated monocytes of SSc patients. The involvement of mitochondrial oxidases was excluded by the lack of inhibition of O2·– production when monocytes were incubated in the presence of rotenone, a mitochondrial oxidase inhibitor. Upon stimulation with phorbol 12-myristate 13-acetate, monocytes of SSc patients produced more O2·– than controls. In SSc patients untreated polymorphonucleate neutrophils generated significantly less O2·– than monocytes (p = 0.0001) and only slightly more than polymorphonucleate neutrophils of primary Raynaud’s phenomenon patients and normal controls (p = 0.03). In conclusion, we demonstrate that in patients with scleroderma, unmanipulated and phorbol 12-myristate 13-acetate-stimulated monocytes release in vitro increased amounts of superoxide anion through the activation of nicotinamide-adenine dinucleotide diphosphate oxidase and, thus, contribute to the oxidative stress found in this disease

Probiotics Modulate Intestinal Expression of Nuclear Receptor and Provide Counter-Regulatory Signals to Inflammation-Driven Adipose Tissue Activation

BACKGROUND: Adipocytes from mesenteric white adipose tissue amplify the inflammatory response and participate in inflammation-driven immune dysfunction in Crohn's disease by releasing proinflammatory mediators. Peroxisome proliferator-activated receptors (PPAR)-α and -γ, pregnane x receptor (PXR), farnesoid x receptor (FXR) and liver x-receptor (LXR) are ligand-activated nuclear receptor that provide counter-regulatory signals to dysregulated immunity and modulates adipose tissue. AIMS: To investigate the expression and function of nuclear receptors in intestinal and adipose tissues in a rodent model of colitis and mesenteric fat from Crohn's patients and to investigate their modulation by probiotics. METHODS: Colitis was induced by TNBS administration. Mice were administered vehicle or VSL#3, daily for 10 days. Abdominal fat explants obtained at surgery from five Crohn's disease patients and five patients with colon cancer were cultured with VSL#3 medium. RESULTS: Probiotic administration attenuated development of signs and symptoms of colitis, reduced colonic expression of TNFα, IL-6 and IFNγ and reserved colonic downregulation of PPARγ, PXR and FXR caused by TNBS. Mesenteric fat depots isolated from TNBS-treated animals had increased expression of inflammatory mediators along with PPARγ, FXR, leptin and adiponectin. These changes were prevented by VSL#3. Creeping fat and mesenteric adipose tissue from Crohn's patients showed a differential expression of PPARγ and FXR with both tissue expressing high levels of leptin. Exposure of these tissues to VSL#3 medium abrogates leptin release. CONCLUSIONS: Mesenteric adipose tissue from rodent colitis and Crohn's disease is metabolically active and shows inflammation-driven regulation of PPARγ, FXR and leptin. Probiotics correct the inflammation-driven metabolic dysfunction