Divergent modulation of nociception by glutamatergic and GABAergic neuronal subpopulations in the periaqueductal gray

The ventrolateral periaqueductal gray (vlPAG) constitutes a major descending pain modulatory system and is a crucial site for opioid-induced analgesia. A number of previous studies have demonstrated that glutamate and GABA play critical opposing roles in nociceptive processing in the vlPAG. It has been suggested that glutamatergic neurotransmission exerts antinociceptive effects, whereas GABAergic neurotransmission exert pronociceptive effects on pain transmission, through descending pathways. The inability to exclusively manipulate subpopulations of neurons in the PAG has prevented direct testing of this hypothesis. Here, we demonstrate the different contributions of genetically defined glutamatergic and GABAergic vlPAG neurons in nociceptive processing by employing cell type-specific chemogenetic approaches in mice. Global chemogenetic manipulation of vlPAG neuronal activity suggests that vlPAG neural circuits exert tonic suppression of nociception, consistent with previous pharmacological and electrophysiological studies. However, selective modulation of GABAergic or glutamatergic neurons demonstrates an inverse regulation of nociceptive behaviors by these cell populations. Selective chemogenetic activation of glutamatergic neurons, or inhibition of GABAergic neurons, in vlPAG suppresses nociception. In contrast, inhibition of glutamatergic neurons, or activation of GABAergic neurons, in vlPAG facilitates nociception. Our findings provide direct experimental support for a model in which excitatory and inhibitory neurons in the PAG bidirectionally modulate nociception

Application of Two-Photon Absorbing Fluorene-Containing Compounds in Bioimaging and Photodyanimc Therapy

Two-photon absorbing (2PA) materials has been widely studied for their highly localized excitation and nonlinear excitation efficiency. Application of 2PA materials includes fluorescence imaging, microfabrication, 3D data storage, photodynamic therapy, etc. Many materials have good 2PA photophysical properties, among which, the fluorenyl structure and its derivatives have attracted attention with their high 2PA cross-section and high fluorescence quantum yield. Herein, several compounds with 2PA properties are discussed. All of these compounds contain one or two fluorenyl core units as part of the conjugated system. The aim of this dissertation is to discuss the application of these compounds according to their photophysical properties. In chapters 2 to 4, compounds were investigated for cell imaging and tissue imaging. In chapter 5, compounds were evaluated for photodynamic therapy effects on cancer cells. Chapters 2 and 3 detail compounds with quinolizinium and pyran as core structures, respectively. Fluorene was introduced into structures as substituents. Quinolizinium structures exhibited a large increase in fluorescence when binding with Bovine Serum Albumin (BSA). Further experiments in cell imaging demonstrated a fluorescence turn-on effect in cell membranes, indicating the possibility for these novel compounds to be promising membrane probes. Pyran structures were conjugated with arginylglycylaspartic acid peptide (RGD) to recognize integrin and introduced in cells and an animal model with tumors. Both probes showed specific targeting of tumor vasculature. Imaging reached penetration as deep as 350 µm in solid tumors and exhibited good resolution. These results suggest the RGD-conjugated pyran structure should be a good candidate probe for live tissue imaging. Chapter 4 applied a fluorene core structure conjugated with RGD as well. Application of this fluorenyl probe compound is in wound healing animal models. Fluorescence was collected from vasculature and fibroblasts up to ≈ 1600 µm within wound tissue in lesions made on the skin of mice. The resolution of images is also high enough to recognize cell types by immunohistochemical staining. This technology can be applied for reliable quantification and illustration of key biological processes taking place during tissue regeneration in the skin. Chapter 5 describes three fluorenyl core structures with photoacid generation properties. One of the structures showed excellent photo-induced toxicity. Cancer cells underwent necrotic cell death due to pH decrease in lysosomes and endosomes, suggesting a new mechanism for photodynamic therapy

ENZYME BIOPROSPECTING OF MICROBIAL GLYCOSIDASES AND DIOXYGENASES FOR BIOCATALYSIS.

Main aim of this PhD project is the bioprospecting of different microbial enzymatic activities, to evaluate their biotechnological potential in different bioconversion processes. The characterization of four extradiol ring cleavage dioxygenases (ERCDs) and of a α-L-rhamnosidase isolated from Novosphingobium sp. PP1Y (N. sp. PP1Y) is described. In the last part of the project, gut microorganisms have been used for the identification and characterization of novel glycosyl hydrolases able to degrade the arabinogalactan polysaccharide (AG) of M. tuberculosis cell wall. In this work, the marine microorganism N. sp. PP1Y and the gut microorganism Bacteroides finegoldii were used as source for oxygenases and glycosyl hydrolases. More in detail, the optimization of recombinant expression and purification of four novel ERCDs from strain PP1Y allowed to obtain in good yields the corresponding proteins and to carry out their characterization. The activity screening using different catecholic substrates confirmed that these enzymes are able to catalyze the ring cleavage of a variety of mono- and polycyclic aromatic hydrocarbons with different sizes and conformation. In addition, the bioconversion of catechol estrogens, which could be used as precursors in the production of different steroid families and hormones, was evaluated. The results described in this thesis confirmed that these enzymes can be foreseen as a valuable tool for the modification of complex hydroxylated heterocyclic aromatic compounds, which are a starting point in the production pipeline of many pharmacologically active molecules, such as steroid-like molecules. Noteworthy, site-specific cleavage and subsequent modification of aromatic substrates, obtained by enzymatic biocatalysts, is of great advantage for industrial applications when compared to the complex mixture of products that are released instead during chemical modification procedures. In the second part of the PhD project, the biochemical characterization of RHA-P, a bacterial α-L-rhamnosidase isolated from the microorganism Novosphingobium sp. PP1Y, was performed. The active site topology and substrate specificity of RHA-P were investigated by homology modeling. The enzyme, whose recombinant expression and purification was optimized, resulted to be appealing from a biotechnological point of view for the bioconversion and de-rhamnosylation of natural flavonoids. The biotechnological use of either wild-type or mutant rhamnosidases is currently a need for food and beverages industry to improve the organoleptic properties of processed vegetal products. Moreover, the possibility of using efficient whole cells biocatalysts for expressing RHA-P has been described. This is particularly interesting because whole cells biocatalysts have numerous advantages in industrial bioconversion processes, allowing costs and process steps reduction. Finally, the bioprospecting of novel GHs from microorganisms belonging to the human gut microbiome (HGM), able to degrade the AG of M. tuberculosis cell wall, have been carried out. The isolation and characterization of different galactofuranosidases is described, which are able to completely degrade the mycobacteria galactan moiety of AG. Moreover, evidences for the presence of arabinofuranosidases were found for another HGM bacterium, which may be used as biocatalysts for the complete degradation of mycobacteria AG polysaccharide

Entfernung von Fotolacken mit DI/O3 in der Halbleiterfertigung : Prozesschemie und analytische Aspekte

In the production of integrated circuits (ICs), photolithography plays a key role in wafer structuring. The basic principle of photolithography is the selective processing of areas (etching, implantation, metallisation etc.) while the others are covered and therefore protected by the resist. After each process step the resist, now modified, has to be removed. In the history of semiconductor manufacturing this has been accomplished with a mixture of H2SO4 and H2O2, H2SO4 and O3 or a plasma etch. As the structure sizes decreased they reached a stage where they had to be exposed to light of shorter wavelengths for the photolithography, going from i-line (365 nm) to DUV (248 nm and 193 nm). This change in wavelength now requires new resists and therewith new stripping methods. Beside the changes in the resist the finer structures are also more sensitive to damages caused by the resist strip. Along with this the demand for cost reduction and environment-friendliness poses a big challenge for modern resist stripping. In this study ozone in deionised water (DI/O3) was the basic chemistry investigated as it is cost efficient in production and disposal as well as environment friendly. Furthermore it is a chemistry known to cause no damage to the wafers. DI/O3 has been successfully applied to strip i-line resists. The challenge now is to find ways and means to make DI/O3 strip even highly implanted DUV resists which currently can only be removed by a plasma etch. To achieve this a detailed understanding of the behaviour of ozone in DI water and the influence of factors both chemical and physical on the stripping efficiency at the different stages in the process is necessary. Along with this, methods which enable the elucidation of resist structures and the changes they undergo during the process of photolithography as well as during the ozone strip have to be developed. This will enable us to understand the mechanisms involved and hence, ideally, develop ozone-based stripping solutions customized for each resist and process step. For this purpose the ozone decomposition in DI water with and without additives was studied via UV-Vis spectroscopy. Radicals generated within the ozone decomposition were trapped and quantified, the resists were studied directly on the wafer with IR and Raman spectroscopy and stripped with DI/O3-mixtures and different setups to find optimum conditions for a complete and damage free resist strip. UV-Vis spectroscopy at 260 nm was used to study ozone decomposition and the factors, both chemical and physical, which influence it. These factors are pH, different additives at the same pH, temperature and mixing of the solution. For the radical determination trapping reactions with MeOH and DMSO both forming CH2O which is further converted to DDL as the detectable species were compared with a variation of the classical iodometric titration acting as an absolute method without the need of calibration. IR spectroscopy proved to be a suitable method for the structural characterisation of the resists and the tracking of the changes undergone during the various processing steps as well as the ozone based stripping. For the stripping with DI/O3 IR spectroscopy delivered well-defined spectra. These displayed significant peak changes which support the assumption of classical ozonolysis as the decomposition mechanism for the unimplanted resist. For the study of the resist crust originating from ion implantation IR was fundamentally unsuitable and was replaced by Raman spectroscopy and microscopy. Raman spectra showed the crust to be of a highly carbon containing structure. Regrettably, the peak assignable to the crust was too broad for the exact composition of the crust to be determined. The wavelength region of the peak corresponds to that of peaks of glassy carbon and highly ordered and conventional graphite. Such a broad peak suggests that the structure of the crust is not uniform but contains more than one carbon modification. As the purpose of all these studies is to enable or improve DI/O3 based resist stripping on unimplanted as well as high-dose implanted resists the removal efficiency of DI/O3 spiked with different additives that alter the pH was studied. For these unimplanted resists the maximum efficiency could be achieved at pH = 5 – 7. Lowering or increasing the pH beyond this range gave poor results. The stripping of highly implanted resists could be achieved only at harsh conditions with a high pH-level of 12 - 13 with a narrow process window showing no stripping at lower pHs and severe damages at higher levels. The principle application of DI/O3 stripping chemistry could be proved but the currently required process time unfortunatelly is too long for commercial application and needs further optimisation.Im Prozess der Herstellung integrierter Schaltkreise spielt die Fotolithographie eine entscheidende Rolle bei der Strukturierung der Wafer. Das Prinzip der Fotolithographie beruht dabei auf der selektiven Prozesssierung einiger Bereiche des Wafers (Ätzen, Ionenimplantation, Metallisierung usw.) während andere Bereiche durch den Fotolack geschützt werden. Dieser, nun durch die Prozesssierung modifizierte, Fotolack muß im Anschluß wieder entfernt werden. In der Geschichte der Halbleiterfertigung geschah dies mit H2SO4/H2O2, H2SO4/O3, DI/O3 oder durch Plasmaveraschung. Seitdem die Strukturgröße immer mehr abnimmt, werden immer kürzere Belichtungswellenlängen benötigt, die von i-line (365 nm) bis DUV (248 nm, 193 nm) reichen. Einhergehend mit diesen kürzeren Wellenlängen ist eine zwingende Veränderung der Fotolack-Struktur und damit die Notwendigkeit neuer Techniken zur Fotolack-Entfernung. Hinzu kommt, dass diese, nun kleineren Strukturen, ihrerseits empfindlicher gegenüber Schäden aus dem Entfernungsprozeß sind. Als zusätzliche Herausforderung ist der dauerhafte Druck zur Kostenreduzierung sowie zur Umweltverträglichkeit anzusehen. Diese Dissertation beschäftigt sich daher mit der Fotolack-Entfernung basierend auf ozoniertem Wasser (DI/O3), da es kostengünstig zu erzeugen und entsorgen ist, gleichzeitig umweltfreundlich und zu guter Letzt dafür bekannt die erzeugten Strukturen nicht zu beschädigen. Da in der Vergangenheit DI/O3 schon erfolgreich bei i-line Lacken eingesetzt wurde, besteht nun die Herausforderung darin diese Chemie auch auf moderne und dabei vor allem hochimplantierte DUV-Lacke anzuwenden, die bisher nur mittels Plasmaveraschung entfernbar sind. Um dieses Ziel erreichen zu können, ist ein detailliertes Verständnis der Ozonchemie in Wasser sowie ihrer Beeinflussung durch Additive wichtig, sowohl in Bezug auf das Ozon selbst als auch den Einfluß auf dessen Lack Entfernungspotential bei verschiedenen Prozeßschritten des Lacks. Einhergehend damit bedarf es Methoden zur Untersuchung der Lackstrukturen und zur Verfolgung ihrer Änderung während bestimmter Prozeßschritte sowie während der Entfernung mittels DI/O3. Das Ziel ist es das Verhalten von Ozon und Lack zu verstehen, um auf diese Weise Möglichkeiten zur maßgeschneiderten Lackentfernung zu erhalten. Zu diesem Zweck wurde der Ozonzerfall mittels UV-Vis-Spektroskopie unter verschiedenen Bedingungen untersucht. Ferner wurden die, bei der Zersetzung entstehenden, Radikale chemisch abgefangen und quantifiziert. Der Fotolack selbst wurde direkt auf dem Wafer mittels IR- und Raman-Spektroskopie untersucht und zu guter Letzt dessen Entfernung mit DI/O3 und verschiedensten Zusätzen sowie Versuchsaufbauten in Angriff genommen. Die Quantifizierung der bei der Ozon-Zersetzung entstehenden Radikale wurde mit 3 Verfahren durchgeführt. Die ersten beiden Verfahren beruhen auf chemischen abfangreaktionen der Radikale zu einem stabilen Produkt, hier in beiden Fällen das CH2O. Sie unterschieden sich lediglich im Abfangreagenz, das einmal DMSO und im anderen Fall MeOH ist. In beiden Fällen wird das gebildete CH2O weiter zu einem, mittels UV-Vis detektierbaren, Farbstoff (DDL) umgesetzt. Zur Kontrolle wurde eine Variante der Iodometrie verwendet, die als Absolutmethode fungiert. Alle Untersuchungen über den relevanten pH-Bereich zeigen dabei einen Anstieg der Radikalkonzentration mit dem pH-Wert an. Die Strukturaufklärung mittels IR-Spektroskopie erweist sich für die meisten Prozeßschritte des Photolacks als geeignet, versagt allerdings bei implantierten Lacken. Die Auswertung der Spektren weist für den Entfernungsprozeß mittels DI/O3 auf eine klassische Ozonolyse des Aromaten in den untersuchten, nicht implantierten Polyhydroxystyrollacken (PHS) hin. Die Anwendung von Raman-Mikroskopie hat sich als geeignet zur Untersuchung der Kruste bei implantierten Lacken erwiesen. Mit ihr konnte die Kruste dem Bereich der hochvernetzten Kohlenstoffmodifikationen (verschiedene Graphitmodifikationen, glasartiger Kohlenstoff) zugeordnet werden. Eine weitere Eingrenzung war bisher nicht möglich. Als Grund kommt eine Inhomogenität der Kruste in Frage, die wahrscheinlich aus einem Gemisch der verschiedenen Modifikationen besteht. Während sich bei der Entfernung nicht implantierter PHS-Lacke ein neutraler bis leicht saurer pH-Bereich von 5 bis 7 als optimal erwiesen hat, bedarf es für hochimplantierte PHS-Lacke eines deutlich basischen Niveaus mit einem bisherigen Optimum bei pH-Wert von etwas über 12, wobei ein sehr enges Prozeßfenster zwischen 12 und 13 festgestellt wurde. pH-Werte darunter zeigte keine Reinigungswirkung, darüber kam es zu Schädigungen des Si-Substrats. Die prinzipielle Anwendbarkeit von DI/O3 zur Lackentfernung konnte somit zwar gezeigt werden, allerdings ist die aktuell notwendige Prozesszeit zu lang für einen kommerziellen Einsatz und bedarf weitere Optimierung

Creating movable interfaces by micro-powder injection moulding

This paper presents a novel in situ technique to produce articulated components with high-precision, micro-scale movable interfaces by micro-powder injection moulding (μPIM). The presented process route is based on the use of micro-scale sacrificial layer between the movable subcomponents which is eliminated during the debinding step, creating a dimensionally-controlled, micro-scale mobile interface. The fabrication technique combines the advantages of micro-powder overmoulding, catalytic debinding and sintering. The demonstrated example was a finger bone prosthesis joint consisting of two sub-components with an interface between components of 200 μm in size. The geometries of the sub-components were designed such that they are inseparable throughout the process whilst allowing them to move relative to each other after the debinding stage. The components produced showed the feasibility of the process route to produce readily-assembled meso-, and potentially micro-, scale articulated system

Development of nanostructured supported photocatalysts for hydrogen production and inorganic pollutants removal

Semiconductor photocatalysis has emerged as one of the most promising approach to exploit a renewable energy source (i.e. sunlight irradiation) for several environmental purposes such as the production of clean energy (e.g. photocatalytic H2 evolution), the removal of organic and inorganic pollutants in natural water, purification of air and antibacterial activity. In view of these recent trends, the focus of this thesis was directed towards the study of different supported photo(electro)catalytic materials for topical environmental applications: i) Photocatalytic hydrogen gas evolution from aqueous solutions under UV light irradiation (365 nm) over highly ordered TiO2 nanotubes decorated through a sputtering/dewetting approach with a well-defined stacked co-catalyst (a WO3 layer decorated with Pt NPs); ii) Photocatalytic hydrogen gas evolution from aqueous solutions under UV light irradiation (365 nm) over highly ordered TiO2 nanotubes decorated through a sputtering/dewetting approach with dewetted-alloyed NiCu nanoparticles; iii) Photocatalytic reduction/scavenging of inorganic mercury (Hg(II)) from water under solar light irradiation over templated-dewetted Au on TiO2 nanotubes; iv) Photoelectrocatalytic oxidation/abatement of inorganic arsenic (As(III)) over hematite-based photoanodes under solar light irradiation. After a general introduction about photocatalytic processes and materials, each chapter of this dissertation contains the outcomes of the above listed studies

Sustainable use of marine biodiversity as source of novel anti-biofilm agents in industrial and clinical settings

Amongst marine bacteria, cold-adapted microorganisms represent an untapped reservoir of biodiversity endowed with an interesting chemical repertoire able to synthesize a broad range of potentially valuable bioactive compounds, including antimicrobial activity. The rapid emergence of resistant bacteria is occurring worldwide, endangering the efficacy of antibiotics. One of the main causes of antibiotic resistance is the capability of microorganisms to associate into communities of cells called biofilms. These complex structures provide protection from potential stressors, including the lack of water, high or low pH, or the presence of substances toxic to microorganisms such as antibiotics, antimicrobials or heavy metals. Therefore, coordinated efforts to implement the arsenal of novel anti-infective treatments are greatly needed. In this contest, my PhD project aimed to the sustainable exploitation of Polar marine biodiversity in an attempt to find viable sources of novel anti-biofilm agents, in particular acting against Staphylococcus epidermidis, one of the most common causes of infections associated with medical devices. In detail, during the first part of my project, I focused on the study of the Antarctic marine bacterium Pseudoalteromonas haloplanktis TAC125 and of its ability to produce anti-biofilm molecules, then, on the purification, identification and characterization of the active molecule produced. By setting up of a strategy for the large scale biofilm cultivation of the Antarctic bacterium, the production yield of P. haloplanktis TAC125 anti-biofilm agent was improved, so as to allow the purification and the identification of the active molecule, the pentadecanal. However, as the pentadecanal is a chemically reactive agent, it could easily undergo oxidation reactions, therefore it could not be suitable for all possible anti-biofilm strategies. Therefore, some chemical analogues were synthesized and characterized for their anti-biofilm activity and their possible use in combination with antibiotics were investigated. Then, as a possible clinical application, an anti-biofilm coating system, active against S. epidermidis, was developed, by physical adsorption of pentadecanal and its analogues on polydimethylsiloxane (PDMS), a silicon-based material commonly used for the manufacturing of medical devices. Finally, some physiological studies were dedicated to P. haloplanktis TAC125 biofilm formation in relation with environmental adaptations, with the purpose to explore the potentiality of P. haloplanktis TAC125 in biotechnological field. In the second part of my PhD project, given their only partially explored potential, I have also studied other Polar bacteria belonging to different genera, looking for novel anti-biofilm agents against S. epidermidis. Through the screening of small metabolites and proteins/peptides libraries designed starting from planktonic cultures of Polar bacteria, some promising producer strains were identified and their anti-biofilm activities were characterized. Preliminary purification protocols were set up for each kind of molecules, according to their physico-chemical characteristics. Further studies are still ongoing to identify the structure of the active molecules

Optical Absorption and Emission of Nanomaterials Integrated in One Dimensional Photonic Crystals

Department of Physics of Condensed Matter/University of Seville Institute of Materials Science of SevilleTese arquivada ao abrigo da Portaria nº227/2017 de 25 de julho