Structure and Function in Single-Chain Nanoparticles

172 p.Esta tesis profundiza en la comprensión de las nanopartículas poliméricas unimoleculares (SCNPs, por sus siglas en inglés) como nano-objetos blandos complejos con aplicaciones potenciales en varios campos, especialmente catálisis y detección. En particular, este trabajo cubre diferentes aspectos relacionados con la estructura y propiedades de las SCNPs como: i) el desarrollo de un modelo teórico de SCNPs elásticas que permita comprender el comportamiento de estas nanopartículas en solución y sobre superficies; ii) el diseño de SCNPs como nanoreactores avanzados para la síntesis de un polímero intrínsecamente conductor como el poli(etilendioxitiofeno) (PEDOT); iii) el diseño de SCNPs altamente fluorescentes por fotoactivación de copolímeros que contienen grupos funcionales diazo y un fluoróforo de emisión inducida por agregación (AIE) decorado con grupos azida; y iv) la síntesis de SCNPs que contienen fluoróforos ortogonales para autoreportar compactación intra-molecular y, cuando está presente o es inducida externamente, agregación inter-molecular.El Capítulo 1, como capítulo introductorio, resume varios conceptos relevantes para la comprensión delos resultados reportados en los Capítulos 3-6.El Capítulo 2 explica las técnicas e instrumentos utilizados en la caracterización de polímeros, SCNPs y productos utilizados o sintetizados en la tesis.El Capítulo 3 presenta un nuevo modelo para el cálculo del tamaño, la masa molar aparente y el factor de contracción de las SCNP elásticas en función de la longitud de la cadena del polímero, la fracción de monómero reactivo y la calidad del disolvente. El modelo se utiliza para estimar el tamaño de las SCNPs en diferentes superficies.En el Capítulo 4 se reporta la síntesis de SCNPs enzimo-miméticas, solubles en agua, compuestas por copolímeros de poli(oligo(etilenglicol)-metil-éter metacrilato-co-2-acetoacetoxietil metacrilato) [poli(OEGMA-co-AEMA)], por coordinación con iones ferrosos (Fe2+). Estas SCNPs se utilizan -por primera vez- como catalizadores para la síntesis de dispersiones de polímero intrínsecamente conductor PEDOT: PSS mediante polimerización oxidativa.El Capítulo 5 muestra la síntesis y caracterización de SCNPs fluorescentes mediante la fotoactivación de copolímeros de poli(metil metacrilato-co-2-acetoacetoxietil metacrilato) [poli (MMA-co-AEMA)] funcionalizados con grupos funcionales diazo. La emisión de fluorescencia se produce mediante la adición de un fluoróforo de emisión inducida por agregación (AIE) con grupos azida. El entrecruzamiento en las SCNPs se logra mediante la activación de los grupos azida y diazo al irradiarlos con luz ultravioleta.El Capítulo 6 presenta una ruta para la síntesis y caracterización de SCNPs de poli(MMA-co-AEMA) fluorescentes basada en la reacción de síntesis de dehidropiridinas de Hantzsch. El empleo de fluoróforos ortogonales permite reportar tanto el plegado, como la agregación de estas SCNPs. La compactación intramolecular que da lugar a las SCNPs se identifica mediante la formación de ésteres de Hantzsch fluorescentes, mientras que la detección de agregación intermolecular se produce a través de la incorporación de luminóforos AIE con fluorescencia en longitudes de onda desplazadas al rojo.Las principales conclusiones de la tesis se recogen en el Capítulo 7

Pain-motor integration in the primary motor cortex in Parkinson's disease

In Parkinson's disease (PD), the influence of chronic pain on motor features has never been investigated. We have recently designed a technique that combines nociceptive system activation by laser stimuli and primary motor cortex (M1) activation through transcranial magnetic stimulation (TMS), in a laser-paired associative stimulation design (Laser-PAS). In controls, Laser-PAS induces long-term changes in motor evoked potentials reflecting M1 long-term potentiation-like plasticity, arising from pain-motor integration

Enzymatic and Bioinspired Systems for Hydrogen Production

The extraordinary potential of hydrogen as a clean and sustainable fuel has sparked the interest of the scientific community to find environmentally friendly methods for its production. Biological catalysts are the most attractive solution, as they usually operate under mild conditions and do not produce carbon-containing byproducts. Hydrogenases promote reversible proton reduction to hydrogen in a variety of anoxic bacteria and algae, displaying unparallel catalytic performances. Attempts to use these sophisticated enzymes in scalable hydrogen production have been hampered by limitations associated with their production and stability. Inspired by nature, significant efforts have been made in the development of artificial systems able to promote the hydrogen evolution reaction, via either electrochemical or light-driven catalysis. Starting from small-molecule coordination compounds, peptide- and protein-based architectures have been constructed around the catalytic center with the aim of reproducing hydrogenase function into robust, efficient, and cost-effective catalysts. In this review, we first provide an overview of the structural and functional properties of hydrogenases, along with their integration in devices for hydrogen and energy production. Then, we describe the most recent advances in the development of homogeneous hydrogen evolution catalysts envisioned to mimic hydrogenases

Unravelling the structure of the tetrahedral metal-binding site in METP3 through an experimental and computational approach

Understanding the structural determinants for metal ion coordination in metalloproteins is a fundamental issue for designing metal binding sites with predetermined geometry and activity. In order to achieve this, we report in this paper the design, synthesis and metal binding properties of METP3, a homodimer made up of a small peptide, which self assembles in the presence of tetra-hedrally coordinating metal ions. METP3 was obtained through a redesign approach, starting from the previously developed METP molecule. The undecapeptide sequence of METP, which dimerizes to house a Cys4 tetrahedral binding site, was redesigned in order to accommodate a Cys2His2 site. The binding properties of METP3 were determined toward different metal ions. Successful assem-bly of METP3 with Co(II), Zn(II) and Cd(II), in the expected 2:1 stoichiometry and tetrahedral geometry was proven by UV-visible spectroscopy. CD measurements on both the free and metal-bound forms revealed that the metal coordination drives the peptide chain to fold into a turned conformation. Finally, NMR data of the Zn(II)-METP3 complex, together with a retrostructural analysis of the Cys-X-X-His motif in metalloproteins, allowed us to define the model structure. All the results establish the suitability of the short METP sequence for accommodating tetrahedral metal binding sites, regardless of the first coordination ligands

Human ESCs predisposition to karyotypic instability: Is a matter of culture adaptation or differential vulnerability among hESC lines due to inherent properties?

<p>Abstract</p> <p>Background</p> <p>The use of human embryonic stem cells (hESCs) in research is increasing and hESCs hold the promise for many biological, clinical and toxicological studies. Human ESCs are expected to be chromosomally stable since karyotypic changes represent a pitfall for potential future applications. Recently, several studies have analysed the genomic stability of several hESC lines maintained after prolonged <it>in vitro </it>culture but controversial data has been reported. Here, we prompted to compare the chromosomal stability of three hESC lines maintained in the same laboratory using identical culture conditions and passaging methods.</p> <p>Results</p> <p>Molecular cytogenetic analyses performed in three different hESC lines maintained in parallel in identical culture conditions revealed significant differences among them in regard to their chromosomal integrity. In feeders, the HS181, SHEF-1 and SHEF-3 hESC lines were chromosomally stable up to 185 passages using either mechanical or enzymatic dissection methods. Despite the three hESC lines were maintained under identical conditions, each hESC line behaved differently upon being transferred to a feeder-free culture system. The two younger hESC lines, HS181 (71 passages) and SHEF-3 (51 passages) became chromosomally unstable shortly after being cultured in feeder-free conditions. The HS181 line gained a chromosome 12 by passage 17 and a marker by passage 21, characterized as a gain of chromosome 20 by SKY. Importantly, the mosaicism for trisomy 12 gradually increased up to 89% by passage 30, suggesting that this karyotypic abnormality provides a selective advantage. Similarly, the SHEF-3 line also acquired a trisomy of chromosome 14 as early as passage 10. However, this karyotypic aberration did not confer selective advantage to the genetically abnormal cells within the bulk culture and the level of mosaicism for the trisomy 14 remained overtime between 15%–36%. Strikingly, however, a much older hESC line, SHEF-1, which was maintained for 185 passages in feeders did not undergo any numerical or structural chromosomal change after 30 passages in feeder-free culture and over 215 passages in total.</p> <p>Conclusion</p> <p>These results support the concept that feeder-free conditions may partially contribute to hESC chromosomal changes but also confirm the hypothesis that regardless of the culture conditions, culture duration or splitting methods, some hESC lines are inherently more prone than others to karyotypic instability.</p

High growth rate 4H-SiC epitaxial growth using dichlorosilane in a hot-wall CVD reactor

Thick, high quality 4H-SiC epilayers have been grown in a vertical hot-wall chemical vapor deposition system at a high growth rate on (0001) 80 off-axis substrates. We discuss the use of dichlorosilane as the Si-precursor for 4H-SiC epitaxial growth as it provides the most direct decomposition route into SiCl2, which is the predominant growth species in chlorinated chemistries. A specular surface morphology was attained by limiting the hydrogen etch rate until the system was equilibrated at the desired growth temperature. The RMS roughness of the grown films ranged from 0.5-2.0 nm with very few morphological defects (carrots, triangular defects, etc.) being introduced, while enabling growth rates of 30-100 \mum/hr, 5-15 times higher than most conventional growths. Site-competition epitaxy was observed over a wide range of C/Si ratios, with doping concentrations < 1x1014 cm-3 being recorded. X-ray rocking curves indicated that the epilayers were of high crystallinity, with linewidths as narrow as 7.8 arcsec being observed, while microwave photoconductive decay (\muPCD) measurements indicated that these films had high injection (ambipolar) carrier lifetimes in the range of 2 \mus

Luminescent conjugates between dinuclear rhenium complexes and 17&#945;-ethynylestradiol: synthesis, photophysical characterization, and cell imaging

Three new luminescent conjugates between dinuclear rhenium complexes and an estradiol, namely E2-Re, are described. The derivatives have the general formula [Re2(\u3bc-Cl)2(CO)6(\u3bc-R-pydz-17\u3b1-ethynylestradiol)] (R-pydz = functionalized 1,2-pyridazine), where the estradiol moiety is covalently bound to the \u3b2 position of the pyridazine ligand. Different synthetic pathways are investigated, including the inverse-type [4 + 2] Diels Alder cycloaddition reaction between the electron poor 1,2,4,5-tetrazine and 17\u3b1-ethynylestradiol for the synthesis of E2-Re1. The three E2-Re conjugates are purified on silica gel and isolated in a spectroscopically pure form in moderate to good yields (28-50%). All the E2-Re conjugates are comprehensively characterized from the spectroscopic and photophysical points of view. Cellular internalization experiments on human MCF-7 and 231 cells are also reported, displaying interesting staining differences depending on the nature of the spacer linking the estradiol unit to the organometallic fragment. Furthermore, the suitability of these conjugates to also stain simple multicellular organisms, i.e. Ciona intestinalis embryos and larvae at different stages of development, is reported here for the first time

A case report of IgG4-related disease: an insidious path to the diagnosis through kidney, heart and brain

BACKGROUND: IgG4-related disease, described around the years 2000 as a form of autoimmune pancreatitis, is now increasingly accepted as a systemic syndrome. The diagnosis is based on both comprehensive and organ-specific criteria. For the kidney, Mayo clinic classification and the guidelines of the Japanese Nephrology Society are used. Ultimately, together with parameters that characterize every organ or apparatus involved, the key element is the confirmation of growing levels of IgG4 in blood or in tissues. CASE PRESENTATION: We describe a male patient with chronic renal failure associated to hypertension without proteinuria. IgG4-related disease was diagnosed through renal biopsy. After an initial positive response to steroids, he presented tinnitus, and histological assessment showed cerebral and subsequently cardiac damage, both IgG4-related. This case appears unique for the type of histologically documented cardiac and neurological parenchymal involvement, and at the same time, exemplifies the subtle and pernicious course of the disease. Frequently, blurred and non-specific signs prevail. Here, kidney damage was associated with minimal urinary findings, slowly progressive renal dysfunction and other factors that can be equivocated in the differential diagnosis. Neurological involvement was represented by tinnitus alone, while cardiac alterations were completely asymptomatic. CONCLUSIONS: This report is representative of the neurological and cardiac changes described in the literature for IgG4-related disease, which may be correlated or not with the renal form and highlights the need, in some cases, of targeted therapeutic approaches. In addition to glucocorticoids, as in this case, rituximab may be necessary

Klebsiella pneumoniae is able to trigger epithelial-mesenchymal transition process in cultured airway epithelial cells

The ability of some bacterial pathogens to activate Epithelial-Mesenchymal Transition normally is a consequence of the persistence of a local chronic inflammatory response or depends on a direct interaction of the pathogens with the host epithelial cells. In this study we monitored the abilities of the K. pneumoniae to activate the expression of genes related to EMT-like processes and the occurrence of phenotypic changes in airway epithelial cells during the early steps of cell infection. We describe changes in the production of intracellular reactive oxygen species and increased HIF-1α mRNA expression in cells exposed to K. pneumoniae infection. We also describe the upregulation of a set of transcription factors implicated in the EMT processes, such as Twist, Snail and ZEB, indicating that the morphological changes of epithelial cells already appreciable after few hours from the K. pneumoniae infection are tightly regulated by the activation of transcriptional pathways, driving epithelial cells to EMT. These effects appear to be effectively counteracted by resveratrol, an antioxidant that is able to exert a sustained scavenging of the intracellular ROS. This is the first report indicating that strains of K. pneumoniae may promote EMT-like programs through direct interaction with epithelial cells without the involvement of inflammatory cells

Cooling the skin for assessing small-fibre function

In this clinical and neurophysiological study using a novel cold stimulator we aim at investigating whether cold evoked potentials may prove to be a reliable diagnostic tool to assess trigeminal small-fibre function.Using a novel device consisting of micro-Peltier elements, we recorded cold evoked potentials after stimulating the supraorbital and perioral regions and the hand dorsum in 15 healthy participants and in two patients with exemplary facial neuropathic pain conditions. We measured peripheral conduction velocity at the upper arm and studied the brain generators using source analysis. In healthy participants and patients, we also compared cold evoked potentials with laser evoked potentials.In the healthy participants, cold stimulation evoked reproducible scalp potentials, similar to those elicited by laser pulses, though with a latency of about 30 ms longer. The mean peripheral conduction velocity, estimated at the upper arm, was 12.7 m/s. The main waves of the scalp potentials originated from the anterior cingulate gyrus and were preceded by activity in the bilateral opercular regions and bilateral dorso-lateral frontal regions. Unlike laser stimulation, cold stimulation evoked scalp potential of similar amplitude across perioral, supraorbital and hand dorsum stimulation. In patients with facial neuropathic pain, cold evoked potential recording showed the selective damage of cold pathways providing complementary information to laser evoked potential recording.Our clinical and neurophysiological study shows that this new device provides reliable information on trigeminal small-fibres mediating cold sensation, and might be useful for investigating patients with facial neuropathic pain associated with a distinct damage of cold-mediating fibres