Optical Microring Resonators based on ion-implanted LiNbO3 ridge waveguides

The growing interest for Integrated Optics for sensing, telecommunications and even electronics is driving research to find solutions to the new challenges issued by a more and more fast, connected and smart world. This thesis deals with the design, the fabrication and the characterisation of the first prototypes of Microring Resonators realised using ion implanted Lithium Niobate (LiNbO3) ridge waveguides. Optical Resonator is one among the most important devices for all tasks described above. LiNbO3 is the substrate commonly used to fabricate optical modulators thanks to its electro-optic characteristics. Since it is produced in high quantity, good quality and large wafers its price is low compared to other electro-optic substrate. We propose to use ion implantation as fabrication technology because in the other way standard optical waveguides realised in LiNbO3 by Proton Exchange (PE) or metal diffusion do not allow small bending radii, which are necessary to keep the circuit footprint small. We will show in fact that this approach allows to fabricate waveguides on Lithium Niobate that are better than PE or metal diffused waveguides as it allows smaller size devices and tailoring of the refractive index profile controlling the implantation parameters. Moreover, we will show that the ridge technology based on enhanced etching rate via ion implantation produces a waveguide with roughness lower than a dry etched one. Finally it has been assessed a complete technological process for fabrication of Microring Resonator devices in Lithium Niobate by ion implantation and the first prototypes have been produced.

Modeling, design and experimental characterization of Micro-Electro-Mechanical-Systems for gas chromatographic applications

Design parameters, process flows, electro-thermal-fluidic simulations and experimental characterizations of Micro-Electro-Mechanical-Systems (MEMS) suited for gas-chromatographic (GC) applications are presented and thoroughly described in this thesis, whose topic belongs to the research activities the Institute for Microelectronics and Microsystems (IMM)-Bologna is involved since several years, i.e. the development of micro-systems for chemical analysis, based on silicon micro-machining techniques and able to perform analysis of complex gaseous mixtures, especially in the field of environmental monitoring. In this regard, attention has been focused on the development of micro-fabricated devices to be employed in a portable mini-GC system for the analysis of aromatic Volatile Organic Compounds (VOC) like Benzene, Toluene, Ethyl-benzene and Xylene (BTEX), i.e. chemical compounds which can significantly affect environment and human health because of their demonstrated carcinogenicity (benzene) or toxicity (toluene, xylene) even at parts per billion (ppb) concentrations. The most significant results achieved through the laboratory functional characterization of the mini-GC system have been reported, together with in-field analysis results carried out in a station of the Bologna air monitoring network and compared with those provided by a commercial GC system. The development of more advanced prototypes of micro-fabricated devices specifically suited for FAST-GC have been also presented (silicon capillary columns, Ultra-Low-Power (ULP) Metal OXide (MOX) sensor, Thermal Conductivity Detector (TCD)), together with the technological processes for their fabrication. The experimentally demonstrated very high sensitivity of ULP-MOX sensors to VOCs, coupled with the extremely low power consumption, makes the developed ULP-MOX sensor the most performing metal oxide sensor reported up to now in literature, while preliminary test results proved that the developed silicon capillary columns are capable of performances comparable to those of the best fused silica capillary columns. Finally, the development and the validation of a coupled electro-thermal Finite Element Model suited for both steady-state and transient analysis of the micro-devices has been described, and subsequently implemented with a fluidic part to investigate devices behaviour in presence of a gas flowing with certain volumetric flow rates.

Mitochondrial dysfunction in hereditary optic neuropathies

MITOCHONDRIAL DYSFUNCTION IN HEREDITARY OPTIC NEUROPATHIES Mitochondrial pathologies are a heterogeneous group of clinical manifestations characterized by oxidative phosphorylation impairment. At the beginning of their recognition mitochondrial pathologies were regarded as rare disorders but indeed they are more frequent than originally thought. Due to the unique mitochondria peculiarities mitochondrial pathologies can be caused by mutations in both mitochondrial and nuclear genomes. The poor knowledge of pathologic mechanism of these disorders has not allowed a real development of the “mitochondrial medicine”, that is currently limited to symptoms mitigation. Leber hereditary optic neuropathy (LHON) was the first pathology to be linked to a point mutation in the mtDNA. The mechanism by which point mutations in mitochondrial gene encoding Complex I subunits leads to optic nerve degeneration is still unknown, although is well accepted that other genetic or environmental factors are involved in the modulation of pathology, where a pivotal role is certainly played by oxidative stress. We studied the relationship between the Ala16Val dimorphism in the mitochondrial targeting sequence of nuclear gene SOD2 and the 3460/ND1 LHON mutation. Our results show that, in control population, the heterozygous SOD2 genotype is associated to a higher activity and quantity of MnSOD, particularly with respect to Val homozygotes. Furthermore, we demonstrated that LHON patients harboring at least one Ala allele are characterized by an increased MnSOD activity with respect to relative control population. Since the ATP synthesis rate – severely reduced in LHON patients lymphocytes - is not affected by the SOD2 genotype, we concluded that SOD2 gene could modulate the pathogenicity of LHON mutations through a mechanism associated to an increase of reactive oxygen species production. Autosomal dominant optic atrophy (ADOA) is a pathology linked to mutations in nuclear gene encoding Opa1, a dynamin-related protein localized in the mitochondrial matrix. Although the clinical course is slightly different, the endpoint of ADOA is exactly the same of LHON: optic nerve degeneration with specific involvement of retinal ganglion cells. Opa1 is a relatively new protein, whose major role is the regulation of mitochondrial fusion. Mitochondrial morphology is the results of the equilibrium between two opposite force: fusion and fission, two processes that have to be finely regulated in order to preserve mitochondrial and cellular physiology. We studied fibroblasts deriving from ADOA patients characterized by a new deletion in the GTPase domain of the OPA1 gene. The biochemical characterization of ADOA and control fibroblasts has concerned the evaluation of ATP synthesis rate, mitochondrial membrane potential in different metabolic conditions and the morphological status of mitochondria. Regarding ATP synthesis rate we did not find significant differences between ADOA and control fibroblasts even though a trend toward increased reduction in ADOA samples is observed when fibroblasts are grown in absence of glucose or in the medium containing gramicidin. Furthermore, we found that also in ADOA fibroblasts membrane potential is actively maintained by proton pumping of fully functional respiratory chain complexes. Our results indicate that the mutation found in the pedigree analyzed acts primary impairing the mitochondrial fusion without affecting the energy production, supporting the notion that cell function is tightly linked to mitochondrial morphology. Mitochondrial dysfunctions are acquiring great attention because of their recognized relevance not only in aging but also in age-related pathologies including cancer, cardiovascular disease, type II diabetes, and neurodegenerative disorders. The involvement of mitochondria in such detrimental pathologies that, currently, have become so common enhances the necessity of standardization of therapeutic strategies capable of rescuing the normal mitochondrial function. In order to propose an alternative treatment for energy deficiency-disorders we tested the effect of substrates capable to stimulate the substrate-level phosphorylation on viability and energy availability in different experimental models grown under different metabolic conditions. In fibroblasts, the energy defect was achieved by culturing cells in presence of oligomycin, an inhibitor of ATP synthase complex. NARP cybrids have been used as model of mitochondrial pathology. Cell viability and ATP content have been considered as parameters to assay the capability of exogenous substrate to rescue energy failure. Our results suggest that patients suffering for some forms of ATP synthase deficiency, or characterized by a deficiency in energy production, might benefit from dietary or pharmacological treatment based on supplementation of α-ketoglutarate and aspartate.

Molecular bases, pathogenic mechanisms and possible therapeutic approach in Leber's Hereditary Optic Neuropathy

Leber’s hereditary optic neuropathy (LHON) is a mitochondrial disease characterized by a rapid loss of central vision and optic atrophy, due to the selective degeneration of retinal ganglion cells. The age of onset is around 20, and the degenerative process is fast and usually the second eye becomes affected in weeks or months. Even if this pathology is well known and has been well characterized, there are still open questions on its pathophysiology, such as the male prevalence, the incomplete penetrance and the tissue selectivity. This maternally inherited disease is caused by mutations in mitochondrial encoded genes of NADH ubiquinone oxidoreductase (complex I) of the respiratory chain. The 90% of LHON cases are caused by one of the three common mitochondrial DNA mutations (11778/ND4, 14484/ND6 and 3460/ND1) and the remaining 10% is caused by rare pathogenic mutations, reported in literature in one or few families. Moreover, there is also a small subset of patients reported with new putative pathogenic nucleotide changes, which awaits to be confirmed. We here clarify some molecular aspects of LHON, mainly the incomplete penetrance and the role of rare mtDNA mutations or variants on LHON expression, and attempt a possible therapeutic approach using the cybrids cell model. We generated novel structural models for mitochondrial encoded complex I subunits and a conservation analysis and pathogenicity prediction have been carried out for LHON reported mutations. This in-silico approach allowed us to locate LHON pathogenic mutations in defined and conserved protein domains and can be a useful tool in the analysis of novel mtDNA variants with unclear pathogenic/functional role. Four rare LHON pathogenic mutations have been identified, confirming that the ND1 and ND6 genes are mutational hot spots for LHON. All mutations were previously described at least once and we validated their pathogenic role, suggesting the need for their screening in LHON diagnostic protocols. Two novel mtDNA variants with a possible pathogenic role have been also identified in two independent branches of a large pedigree. Functional studies are necessary to define their contribution to LHON in this family. It also been demonstrated that the combination of mtDNA rare polymorphic variants is relevant in determining the maternal recurrence of myoclonus in unrelated LHON pedigrees. Thus, we suggest that particular mtDNA backgrounds and /or the presence of specific rare mutations may increase the pathogenic potential of the primary LHON mutations, thereby giving rise to the extraocular clinical features characteristic of the LHON “plus” phenotype. We identified the first molecular parameter that clearly discriminates LHON affected individuals from asymptomatic carriers, the mtDNA copy number. This provides a valuable mechanism for future investigations on variable penetrance in LHON. However, the increased mtDNA content in LHON individuals was not correlated to the functional polymorphism G1444A of PGC-1 alpha, the master regulator of mitochondrial biogenesis, but may be due to gene expression of genes involved in this signaling pathway, such as PGC-1 alpha/beta and Tfam. Future studies will be necessary to identify the biochemical effects of rare pathogenic mutations and to validate the novel candidate mutations here described, in terms of cellular bioenergetic characterization of these variants. Moreover, we were not able to induce mitochondrial biogenesis in cybrids cell lines using bezafibrate. However, other cell line models are available, such as fibroblasts harboring LHON mutations, or other approaches can be used to trigger the mitochondrial biogenesis.

Development and characterization of non-oxide ceramic composites for mechanical and tribological applications

The main reasons for the attention focused on ceramics as possible structural materials are their wear resistance and the ability to operate with limited oxidation and ablation at temperatures above 2000°C. Hence, this work is devoted to the study of two classes of materials which can satisfy these requirements: silicon carbide -based ceramics (SiC) for wear applications and borides and carbides of transition metals for ultra-high temperatures applications (UHTCs). SiC-based materials: Silicon carbide is a hard ceramic, which finds applications in many industrial sectors, from heat production, to automotive engineering and metals processing. In view of new fields of uses, SiC-based ceramics were produced with addition of 10-30 vol% of MoSi2, in order to obtain electro conductive ceramics. MoSi2, indeed, is an intermetallic compound which possesses high temperature oxidation resistance, high electrical conductivity (21·10-6 Ω·cm), relatively low density (6.31 g/cm3), high melting point (2030°C) and high stiffness (440 GPa). The SiC-based ceramics were hot pressed at 1900°C with addition of Al2O3-Y2O3 or Y2O3-AlN as sintering additives. The microstructure of the composites and of the reference materials, SiC and MoSi2, were studied by means of conventional analytical techniques, such as X-ray diffraction (XRD), scanning electron microscopy (SEM) and energy dispersive spectroscopy (SEM-EDS). The composites showed a homogeneous microstructure, with good dispersion of the secondary phases and low residual porosity. The following thermo-mechanical properties of the SiC-based materials were measured: Vickers hardness (HV), Young’s modulus (E), fracture toughness (KIc) and room to high temperature flexural strength (σ). The mechanical properties of the composites were compared to those of two monolithic SiC and MoSi2 materials and resulted in a higher stiffness, fracture toughness and slightly higher flexural resistance. Tribological tests were also performed in two configurations disco-on-pin and slideron cylinder, aiming at studying the wear behaviour of SiC-MoSi2 composites with Al2O3 as counterfacing materials. The tests pointed out that the addition of MoSi2 was detrimental owing to a lower hardness in comparison with the pure SiC matrix. On the contrary, electrical measurements revealed that the addition of 30 vol% of MoSi2, rendered the composite electroconductive, lowering the electrical resistance of three orders of magnitude. Ultra High Temperature Ceramics: Carbides, borides and nitrides of transition metals (Ti, Zr, Hf, Ta, Nb, Mo) possess very high melting points and interesting engineering properties, such as high hardness (20-25 GPa), high stiffness (400-500 GPa), flexural strengths which remain unaltered from room temperature to 1500°C and excellent corrosion resistance in aggressive environment. All these properties place the UHTCs as potential candidates for the development of manoeuvrable hypersonic flight vehicles with sharp leading edges. To this scope Zr- and Hf- carbide and boride materials were produced with addition of 5-20 vol% of MoSi2. This secondary phase enabled the achievement of full dense composites at temperature lower than 2000°C and without the application of pressure. Besides the conventional microstructure analyses XRD and SEM-EDS, transmission electron microscopy (TEM) was employed to explore the microstructure on a small length scale to disclose the effective densification mechanisms. A thorough literature analysis revealed that neither detailed TEM work nor reports on densification mechanisms are available for this class of materials, which however are essential to optimize the sintering aids utilized and the processing parameters applied. Microstructural analyses, along with thermodynamics and crystallographic considerations, led to disclose of the effective role of MoSi2 during sintering of Zrand Hf- carbides and borides. Among the investigated mechanical properties (HV, E, KIc, σ from room temperature to 1500°C), the high temperature flexural strength was improved due to the protective and sealing effect of a silica-based glassy phase, especially for the borides. Nanoindentation tests were also performed on HfC-MoSi2 composites in order to extract hardness and elastic modulus of the single phases. Finally, arc jet tests on HfC- and HfB2-based composites confirmed the excellent oxidation behaviour of these materials under temperature exceeding 2000°C; no cracking or spallation occurred and the modified layer was only 80-90 μm thick.

Development and characterization of micromachined devices for separation techniques

Nowadays microfluidic is becoming an important technology in many chemical and biological processes and analysis applications. The potential to replace large-scale conventional laboratory instrumentation with miniaturized and self-contained systems, (called lab-on-a-chip (LOC) or point-of-care-testing (POCT)), offers a variety of advantages such as low reagent consumption, faster analysis speeds, and the capability of operating in a massively parallel scale in order to achieve high-throughput. Micro-electro-mechanical-systems (MEMS) technologies enable both the fabrication of miniaturized system and the possibility of developing compact and portable systems. The work described in this dissertation is towards the development of micromachined separation devices for both high-speed gas chromatography (HSGC) and gravitational field-flow fractionation (GrFFF) using MEMS technologies. Concerning the HSGC, a complete platform of three MEMS-based GC core components (injector, separation column and detector) is designed, fabricated and characterized. The microinjector consists of a set of pneumatically driven microvalves, based on a polymeric actuating membrane. Experimental results demonstrate that the microinjector is able to guarantee low dead volumes, fast actuation time, a wide operating temperature range and high chemical inertness. The microcolumn consists of an all-silicon microcolumn having a nearly circular cross-section channel. The extensive characterization has produced separation performances very close to the theoretical ideal expectations. A thermal conductivity detector (TCD) is chosen as most proper detector to be miniaturized since the volume reduction of the detector chamber results in increased mass and reduced dead volumes. The microTDC shows a good sensitivity and a very wide dynamic range. Finally a feasibility study for miniaturizing a channel suited for GrFFF is performed. The proposed GrFFF microchannel is at early stage of development, but represents a first step for the realization of a highly portable and potentially low-cost POCT device for biomedical applications.