Biomimetic topography in orthopaedic ceramic

The primary objective of this research was to perform an in vitro assessment of the ability of microscale topography to alter cell behaviour, with specific regard to producing favourable topography in an orthopaedic ceramic material suitable for implantation in the treatment of arthritis. Topography at microscale and nanoscale alters the bioactivity of the material. This has been used in orthopaedics for some time as seen with optimal pore size in uncemented hip and knee implants. This level of topography involves scale in hundreds of micrometres and allows for the ingrowth of tissue. Topography at smaller scale is possible thanks to progressive miniaturisation of technology. A topographic feature was created in a readily available clinically licensed polymer, Polycaprolcatone (PCL). The effect of this topography was assessed in vitro. The same topography was transferred to the latest generation composite orthopaedic ceramic, zirconia toughened alumina (ZTA). The fidelity of reproduction of the topography was examined using scanning electron microscopy (SEM) and atomic force microscopy (AFM). These investigations showed more accurate reproduction of the topography in PCL than ZTA with some material artefacts in the ZTA. Cell culture in vitro was performed on the patterned substrates. The response of osteoprogenitor cells was assessed using immunohistochemistry, real-time polymerase chain reaction and alizarin staining. These results showed a small effect on cell behaviour. Finally metabolic comparison was made of the effects created by the two different materials and the topography in each. The results have shown a reproducible topography in orthopaedic ceramics. This topography has demonstrated a positive osteogenic effect in both polycaprolactone and zirconia toughened alumina across multiple assessment modalities.

Asfixia perinatal y proteína S100B

La asfixia perinatal es la principal causa de muerte en la primera semana de vida la nivel mundial, los niños que sufren esta complicación y sobreviven pueden presentar trastornos neurológicos de diferente nivel de compromiso que inciden en su desarrollo personal y social. Las cifras de muerte por este problema de salud han disminuido de manera importante, sin embargo en el reporte de la Organización Mundial de Salud (OPS) del 2010, la asfixia perinatal es causa del 29% de muertes infantiles en los países de América Latina y el Caribe 2. Es necesario conocer además la extensión del daño neurológico que sufren estos niños, con este fin se desarrolló un estudio piloto en el Hospital Universitario Mayor Mederi de Bogotá, en el cual se determinó la concentración de un marcador metabólico de daño cerebral, la proteína S100B en suero de 60 recién nacidos sanos, con el objetivo de analizar la asociación del mismo con el peso al nacer, la edad gestacional y el diagnóstico. Los resultados no mostraron diferencias significativas entre este marcador y las variables analizadas que puede asociarse al pequeño número de pacientes, sin embargo han sentado las bases para el desarrollo de un estudio que incluya varios hospitales de Bogotá y sobre todo la determinación del mismo en recién nacidos con diagnóstico de hipoxia en el período perinatal, lo cual aportará información del grado de la alteración que puedan tener a nivel cerebral y contribuya al mejor manejo evolutivo con la aplicación de medidas de intervención en estadios tempranos de la vida.

First report on Meloidogyne chitwoodi hatching inhibition activity of essential oils and essential oils fractions

The Columbia root-knot nematode (CRKN), Meloidogyne chitwoodi, is an EPPO A2 type quarantine pest since 1998. This nematode causes severe damage in economically important crops such as potato and tomato, making agricultural products unacceptable for the fresh market and food processing. Commonly used nematicidal synthetic chemicals are often environmentally unsafe. Essential oils (EOs) may constitute safer alternatives against RKN. EOs, isolated from 56 plant samples, were tested against CRKN hatching, in direct contact bioassays. Some of the most successful EOs were fractionated and the hydrocarbon molecules (HM) and oxygen-containing molecules (OCM) fractions tested separately. 24 EOs displayed very strong hatching inhibitions (≥90 %) at 2 µL mL−1 and were further tested at lower concentrations. Dysphaniaambrosioides, Filipendula ulmaria, Ruta graveolens, Satureja montana and Thymbra capitata EOs revealed the lowest EC50 values (<0.15 µL mL−1). The main compounds of these EOs, namely 2-undecanone, ascaridol, carvacrol, isoascaridol, methyl salicylate, p-cymene and/or γ-terpinene, were putatively considered responsible for CRKN hatching inhibition. S. montana and T. capitata OCM fractions showed hatching inhibitions higher than HM fractions. The comparison of EO and corresponding fractions EC50 values suggests interactions between OCM and HM fractions against CRKN hatching. These species EOs showed to be potential environmentally friendly CRKN hatching inhibitors; nonetheless, bioactivity should be considered globally, since its HM and OCM fractions may contribute, diversely, to the full anti-hatching activity.

Dedifferentiation of Leaf Cells and Growth Pattern of Calluses of Capsicum annuumcv. Etna.

Background:In vitrocell suspension cultivation systems have been largely reported assafe and standardized methods for production of secondary metabolites with medicinaland agricultural interest.Capsicum annuumis one of the most widely grown vegetablein the world and its biological activities have been demonstrated against insects, fungi,bacteria and other groups of organisms. The determination of procedures for thededifferentiation of cells into callus cells and the subsequent study of the callus growthpattern are necessary for the establishment of cellsuspensions and also to subsidizestudies regarding the bioactivity of its secondarymetabolites. To date, no study hasdescribed the development of protocols for callus induction inC. annuumL. cv. Etna. Objective:The objective of this study was to establish a protocol for dedifferentiationof leaf cells of the cultivarC. annuumcv. Etna and to determine the growth pattern ofthe calluses with a focus on the deceleration phase, when the callus cells must besubcultured into a liquid medium in order to establish cell suspension cultivationsaiming at the production of secondary metabolites.Results:The treatment that resultedin the highest %CI, ACCC and callus weight was thecombination of 4.52 μ M 2,4-D +0.44 μ M BA. The calluses produced were friable andwhitish and their growth patternfollowed a sigmoid shape. The deceleration phase started on the 23rdday of cultivation.Conclusion:Callus induction in leaf explants ofC. annuumcv. Etnacan be achieved inMS medium supplemented with 4.52 μ M 2,4-D + 0.44 μ MBA, which results in highcellular proliferation; in order to start a cell suspension culture, callus cells on the 23rdday of culture should be used.

Multifarious tumor-oriented small molecules: development of synthetic methodologies and application for diagnostic platforms

Cancer represents one of the most relevant and widespread diseases in the modern age. In this context, integrin receptors are important for the interactions of cells with extracellular matrix and for the development of both inflammation and carcinogenic phenomena. There are many tricks to improve the bioactivity and receptor selectivity of exogenous ligands; one of these is to integrate the amino acid sequence into a cyclic peptide to restrict its conformational space. Another approach is to develop small peptidomimetic molecules in order to enhance the molecular stability and open the way to versatile synthetic strategies. Starting from isoxazoline-based peptidomimetic molecules we recently reported, in this thesis we are going to present the synthesis of new integrin ligands obtained by modifying or introducing appendages on already reported structures. Initially, we are going to introduce the synthesis of linear and cyclic α-dehydro-β-amino acids as scaffolds for the preparation of bioactive peptidomimetics. Subsequently, we are going to present the construction of small molecule ligands (SMLs) based delivery systems performed starting from a polyfunctionalised isoxazoline scaffold, whose potency towards αVβ3 and α5β1 integrins has already been established by our research group. In the light of these results and due to the necessity to understand the behaviour of a single enantiomer of the isoxazoline-based compounds, the research group decided to synthesise the enantiopure heterocycle using a 1,3-dipolar cycloaddiction approach. Subsequently, we are going to introduce the synthesis of a Reporting Drug Delivery System composed by a carrier, a first spacer, a linker, a self-immolative system, a second spacer and a latent fluorophore. The last part of this work will describe the results obtained during the internship abroad in Prof. Aggarwal’s laboratory at the University of Bristol. The project was focused on the Mycapolyol A synthesis.

Contaminants of emerging concern in drinking water: assessment of the effectiveness of removal by drinking water treatments and possible risks to human health

Contaminants of emerging concern are increasingly detected in the water cycle, with endocrine-disrupting chemicals (EDCs) receiving attention due to their potential to cause adverse health effects even at low concentrations. Although the EU has recently introduced some EDCs into drinking water legislation, most drinking water treatment plants (DWTPs) are not designed to remove EDCs, making their detection and removal in DWTPs an important challenge. The aim of this doctoral project was to investigate hormones and phenolic compounds as suspected EDCs in drinking waters across the Romagna area (Italy). The main objectives were to assess the occurrence of considered contaminants in source and drinking water from three DWTPs, characterize the effectiveness of removal by different water treatment processes, and evaluate the potential biological impact on drinking water and human health. Specifically, a complementary approach of target chemical analysis and effect-based methods was adopted to explore drinking water quality, treatment efficacy, and biological potential. This study found that nonylphenol (NP) was prevalent in all samples, followed by BPA. Sporadic contamination of hormones was found only in source waters. Although the measured EDC concentrations in drinking water did not exceed threshold guideline values, the potential role of DWTPs as an additional source of EDC contamination should be considered. Significant increases in BPA and NP levels were observed during water treatment steps, which were also reflected in estrogenic and mutagenic responses in water samples after the ultrafiltration. This highlights the need to monitor water quality during various treatment processes to improve the efficiency of DWTPs. Biological assessments on finished water did not reveal any bioactivity, except for few treated water samples that exhibited estrogenic responses. Overall, the data emphasize the high quality of produced drinking water and the value of applying integrated chemical analysis and in vitro bioassays for water quality assessment.

Novel functionalized calcium phosphates: structures, morphologies and potential applications.

The stable increase in average life expectancy and the consecutive increase in the number of cases of bone related diseases has led to a growing interest in the development of materials that can promote bone repair and/or replacement. Among the best candidates are those materials that have a high similarity to bones, in terms of composition, structure, morphology and functionality. Biomineralized tissue, and thus also bones, have three main components: water, an organic matrix and an inorganic deposit. In vertebrates, the inorganic deposit consists of what is called biological apatite, which slightly differ from stoichiometric hydroxyapatite (HA) both in crystallographic terms and in the presence of foreign atoms and species. This justifies the great attention towards calcium phosphates, which show excellent biocompatibility and bioactivity. The performances of the material and the response of the biological tissue can be further improved through their functionalization with ions, biologically active molecules and nanostructures. This thesis focuses on several possible functionalizations of calcium phosphates, and their effects on chemical properties and biological performances. In particular, the functionalizing agents include several biologically relevant ions, such as Cobalt (Co), Manganese (Mn), Strontium (Sr) and Zinc (Zn); two organic molecules, a flavonoid (Quercetin) and a polyphenol (Curcumin); and nanoparticles, namely tungsten oxide (WO3) NPs. Functionalization was carried out on various calcium phosphates: dicalcium phosphate dihydrate (DCPD), dicalcium phosphate anhydrous (DCPA) and hydroxyapatite (HA). Two different strategies of functionalization were applied: direct synthesis and adsorption from solution. Finally, a chapter is devoted to a preliminary study on the development of cements based on some of the functionalized phosphates obtained.