A steady state level of negative supercoiling is essential for chromosome condensation, initiation of replication and subsequent elongation step. DNA gyrase, found in every eubacteria, serves the essential housekeeping function of maintenance of the negative supercoiling status of the genome. The functional holoenzyme is a heterotetramer, comprising of two GyrA and two GyrB subunits. DNA gyrase is an indispensable enzyme and serves as a readily susceptible target for natural antibacterial agents. The enzymatic steps of topoisomerisation by gyrase involve transient double strand break and rejoining of the strands after intact duplex transfer. Corruption of its catalytic cycle can lead to the generation of cytotoxic double-strand DNA breaks. Most of the anti-gyrase agents achieve their objective by targeting the vulnerable step of the reaction cycle i.e. DNA cleavage step. Bacteria on their part must have evolved and adopted strategies to counter the action of external agents and prevent the generation of double strand breaks thereby safeguarding their genome. In the present thesis, attempts have been made to understand the role of three endogenous gyrase interacting proteins in gyrase modulation and cellular defense against anti-gyrase agents. The thesis is divided into six chapters. Chapter 1 introduces the wonder enzymes “DNA topoisomerases” starting with a brief classification of these enzymes and their physiological functions. In the next section, DNA gyrase has been discussed in greater detail. The structural aspects as well as the mechanism of the topoisomerisation reaction catalyzed by gyrase have been discussed. Final section gives an overview of different gyrase modulators known till date focusing on their source, structure and mode of action. The scope and objectives of the present study is presented at the end of this chapter. In Chapter 2 is aimed at understanding the physiological role of GyrI. GyrI, originally identified in Escherichia coli as an inhibitor of DNA gyrase, has been previously shown in the laboratory to render protection against gyrase poisons and also various other DNA damaging agents (mitomycin C, MNNG). Abolishing GyrI expression renders the cell hypersensitive to these cytotoxic agents. Interestingly, GyrI exhibits contrasting behavior towards two plasmid encoded proteinaceous poisons of DNA gyrase. It reduces microcin B17-mediated double-strand breaks in vivo, imparting protection to the cells against the toxin. However, a positive cooperation between GyrI and F plasmid encoded toxin CcdB, results in enhanced DNA damage and cell death. These results suggest a more complex functional interplay and physiological role for GyrI. Search for other chromosomally encoded gyrase inhibitors led to YacG, a small zinc finger protein (7.3kDa) from E. coli, shown to be a member of DNA gyrase interactome, in a protein-protein interaction network described recently. Chapter 3 deals with the detailed characterization of YacG. It is shown that YacG inhibits DNA gyrase by binding to GyrB subunit and preventing DNA binding activity of the enzyme. More importantly, it protects against the cytotoxic effects of other gyrase inhibitors like ciprofloxacin, novobiocin, microcin B17 and CcdB. Further investigations revealed that YacG and its homologues are found only in proteobacteria. Hence, it appears to be a defense strategy developed by gram-negative bacteria to fight against the gyrase targeting cytotoxic agents. Inhibition by YacG appears to be specific to E. coli gyrase as mycobacterial enzyme is refractile to YacG action. GyrB, only in gram-negative organisms, possesses extra stretch of 165 amino acids, indispensable for DNA binding. Biochemical experiments with the truncated GyrB lacking the extra stretch reveal the importance of this stretch for stable YacG-GyrB interaction. E. coli topoisomerase IV is also resistant to YacG mediated inhibition, probably due to the absence of the extra stretch in ParE subunit, which is otherwise highly similar to GyrB. Further, YacG homologues from other proteobacterial members (Sinorhizobium meliloti and Haemophilus influenzae homologues sharing 35% and 63 % identity with E. coli YacG respectively ) also inhibits E. coli DNA gyrase at comparable levels. YacG thus emerges as a proteobacteria specific inhibitor of DNA gyrase. The occurrence of both YacG and the gyrase extra stretch only in proteobacteria, suggest co-evolution of interacting partners in proteobacteria. In Chapter 4, the study of endogenous gyrase modulators is extended to Mycobacterium sp. glutamate racemase (MurI) from E. coli has been shown earlier to be an inhibitor of DNA gyrase. However, nothing much was known about its mode of action. MurI is an important enzyme in the cell wall biosynthesis pathway, which catalyses the conversion of L-glutamate to D-glutamate, an integral component of the bacterial cell wall. In this chapter, it is demonstrated that M. tuberculosis MurI inhibits DNA gyrase activity, in addition to its precursor independent racemization function. The inhibition is not species specific as E. coli gyrase is also inhibited. However, it is gyrase specific as topoisomerase I activity remains unaltered. The mechanism of inhibition by MurI has been elucidated for the first time and it is shown that MurI binds to GyrA subunit of the enzyme leading to a decrease in DNA binding of the holoenzyme. The sequestration of the gyrase by MurI results in inhibition of all reactions catalyzed by DNA gyrase. Chapter 5 is the extension of the studies on glutamate racemase into another species, i.e. Mycobacterium smegmatis. DNA gyrase inhibition seems to be an additional attribute of some of the glutamate racemases, but not all, as Glr isozyme from B. subtilis has no effect on gyrase activity in spite of sharing a high degree of similarity with the gyrase inhibitory glutamate racemases. It is shown that like the M. tuberculosis MurI, M. smegmatis enzyme is also a bifunctional enzyme. It inhibits DNA gyrase in addition to its racemization activity. Further, overexpression of the enzyme in M. smegmatis provides protection to the organism against fluoroquinolones. DNA gyrase inhibitory property thus appears to be a typical characteristic of these MurI and seems to have evolved to either modulate the function of the essential housekeeping enzyme or to provide protection to gyrase against gyrase inhibitors, which cause double strand breaks in the genome. In the above chapters, it is shown that besides its crucial role in cell wall biosynthesis, mycobacterial MurI moon lights as DNA gyrase inhibitor. That the two activities exhibited by M. tuberculosis MurI are unlinked and independent of each other is demonstrated in Chapter 6. Racemization function of MurI is not essential for its gyrase inhibitory property as mutants compromised in racemization activity retain gyrase inhibition property. MurI- DNA gyrase interaction influences gyrase activity but has no effect on racemization activity of MurI. MurI expression in mycobacterial cells provides protection against the action of ciprofloxacin, thereby suggesting a role of MurI in countering external agents targeting DNA gyrase. Further M. tuberculosis MurI overexpressed in near homologous expression system of M. smegmatis yields highly soluble enzyme which can be further used for structural and functional studies. In conclusion, the studies reveal that the endogenous inhibitors essentially influence the enzyme activity by sequestering the enzyme away from DNA. None of them cause cytotoxicity, which usually arises as a result of DNA damage caused by accumulation of gyrase-DNA covalent intermediate. On the contrary they provide protection against such gyrase poisons. Comparative analysis of these proteinaceous inhibitors, however, does not reveal a common motif or structural fold, required for their ability to inhibit DNA gyrase. Based on these studies, it can be proposed that these endogenous proteins exist to serve as cellular defense strategies against external abuse and also to modulate the intracellular activity of DNA gyrase as and when required, for accurate division, functioning and survival of the cells.

O trabalho tem por objetivo o estudo e o desenvolvimento de interferômetros recuperadores com baixa tensão de meia onda utilizando moduladores eletro-ópticos para serem aplicados em sistemas Interferométricos de luz branca. Ele dá continuidade às pesquisas do autor em seu mestrado, em que foi desenvolvido e testado um sistema de sensoriamento eletro-óptico capaz de medir diretamente tensões de até 69,4 kVRMS. Desta forma aperfeiçoa-se o sistema de processamento de sinais ópticos desenvolvendo um novo interferômetro recuperador, baseado em óptica integrada. Para o desenvolvimento do tema proposto, inicialmente foi feito uma revisão da literatura/bibliografia, baseada em livros, artigos e teses, visando identificar o \"estado da arte\" relacionado aos moduladores eletro-ópticos para definir o tipo de modulador mais adequado à aplicação em vista. O estudo resultou na escolha de um componente em óptica integrada que foi aplicado numa configuração inédita em um protótipo de transformador de potencial óptico para medição de elevados níveis de tensão elétrica. As características de desempenho deste protótipo foram comparadas com as do protótipo previamente construído. Como resultado deste trabalho, amplia-se o conhecimento e fixa-se em âmbito nacional o domínio sobre as técnicas de construção de interferômetros recuperadores baseados em óptica integrada aplicáveis à recuperação de sinais ópticos em sistemas interferométricos para medição de altas tensões. / This work has as objective the study and development of low half-wave voltage recovery interferometers using electro-optical modulators to be applied to white light interferometric systems. This work is a continuation in the research carried out by the author to obtain his master degree, in which it was developed and tested an electro- optic sensing system capable to measure direct voltage to 69.4 kVrms. In the present work the optical signals processing system is improved by developing a new recovery interferometer based on integrated optics. To develop the proposed subject, initially a review of the literature, based on books, articles and thesis, has been done aiming to identify the State of the Art related to electro-optic modulators and helping to define the most suitable type of modulator for the desired application. The study resulted in the selection of an integrated optical device arranged in an unpublished configuration that was applied to a prototype of optical voltage transformer, intended to measure high voltage levels. The performance of this prototype was compared with a previous version. The results of this work increase the knowledge of the construction techniques of recovery interferometers based on integrated optic devices applicable to the recovering of optical signals in interferometric systems for high voltage measurement.

Une des caractéristiques histopathologiques de la maladie d'Alzheimer (AD) est la présence de plaques amyloïdes formées par les peptides amyloïdes β (Aβ) de 40 et 42 résidus, qui sont les produits de clivage par des protéases de l'APP. Afin de comprendre le rôle des variations structurelles du TM dans le traitement de l'APP, les peptides APP_TM4K ont été étudiés dans la bicouche lipidique en utilisant l’ATR-FTIR et ssNMR. Tandis que la structure secondaire globale du peptide APP_TM4K est hélicoidale, hétérogénéité de conformation et d'orientation a été observée pour le site de clivage γ et , que peuvent avoir des implications dans le mécanisme de clivage et donc dans la production d’Aβ. Les peptides Aβ s'agrègent pour produire des fibrilles et aussi de manière transitoire d'oligomères neurotoxiques. Nous avons constaté qu'en présence de Ca2+, l’Aβ (1-40) forme de préférence des oligomères, tandis qu'en absence de Ca2+ l'Aβ (1-40) s’agrège sous forme de fibrilles. Dans les échantillons sans Ca2+, l’ATR-FTIR révèle la conversion des oligomères en feuillets β antiparallèles en la conformation caractéristique des fibrilles en feuillets β parallèles. Ces résultats nous ont amené à conclure que les Ca2+ stimulent la formation d'oligomères d'Aβ (1-40), qui sont impliqués dans l’AD. Les positions et une orientation précise de deux nouveaux médicaments puissants modulateurs de la γ-sécrétase - le benzyl-carprofen et le sulfonyl-carprofen  dans la bicouche lipidique, ont été obtenus à partir des expériences des ssNMR. Ces résultats indiquent que le mécanisme probable de modulation du clivage par la y-sécrétase est une interaction directe avec le domaine TM de l’APP. / A histopathological characteristic of Alzheimer’s disease (AD) is the presence of amyloid plaques formed by amyloid β(A) peptides of 40 and 42 residues-long, which are the cleavage products of APP by proteases. To understand the role of structural changes in the TM domain of APP, APP_TM4K peptides were studied in the lipid bilayer using ATR-FTIR and ssNMR. While the overall secondary structure of the APP_TM4K peptide is helical, conformational and orientational heterogeneity was observed for the y- and for the -cleavage sites, which may have implications for the cleavage mechanism and therefore the production of Aβ. Starting from its monomeric form, Aβ peptides aggregate into fibrils and / or oligomers, the latter being the most neurotoxic. We found that in the presence of Ca2 +, Aβ (1-40) preferably forms oligomers, whereas in the absence of a2 + Aβ (1-40) aggregates into fibrils. In samples without Ca2 +, ATR-FTIR shows conversion from antiparallel β sheet conformation of oligomers into parallel β sheets, characteristic of fibrils. These results led us to conclude that Ca2 +stimulates the formation of oligomers of Aβ (1-40), that have been implicated in the pathogenesis of AD. Position and precise orientation of two new drugs  powerful modulators of γ-secretase  benzyl-carprofen and carprofen sulfonyl  in the lipid bilayer were obtained from neutron scattering and ssNMR experiments. These results indicate that carprofen-derivatives can directly interact with APP. Such interaction would interfere with proper APP-dimer formation, which is necessary for the sequential cleavage by β -secretase, diminishing or greatly reducing Aβ42 production.

The thesis deals with an influence of the errors caused by utilization of the switched-current (SI) approach in the delta-sigma modulators. The basic block of the SI technique is current memory cell (CMC). The analysis of the errors starts with the design of the CMC using CADENCE software and AMIS CMOS 0.7 um technology. Based upon analysis of the CMC no ideal transfer function and advanced techniques of their behavior modeling are depicted. The mathematical models were made and implemented using MATLAB SIMULINK software. The models are very universal. Therefore, it is possible to analyze various structures of the delta-sigma modulators using demanded technology. The influence of the SI technique approach errors sources, on the modulators behavior with the CMC of the 1st and 2nd generation, is concluded at the end.

Les maladies à expansion de polyglutamine sont un ensemble de troubles neurodégénératives héréditaires se développant lorsqu’il y a répétitions de trinucléotides CAG dans les gènes causatifs au-delà d’un certain seuil. L’expansion des répétitions de trinucléotides CAG entraîne des désordres neurologiques héréditaires précoces, dont de multiples formes d’ataxie spinocérébelleuse (SCA). Parmi celles-ci, le type le plus commun et dominant est l’ataxie spinocérébelleuse de type 3 (SCA3), aussi connue sous le nom de la maladie de Machado-Joseph (MMJ). Ce dernier est un désordre neurologique progressif autosomique dominant. Le gène causatif de MMJ est ATXN3 (ATAXINE-3). Plusieurs études récentes suggèrent une association entre ce gène et la modulation du stress du réticulum endoplasmique (RE). Lors de ce travail de maîtrise, des souches transgéniques de C. elegans exprimant les formes sauvage et mutante du gène ATXN3 humain ont été générées. Les résultats suggèrent des phénotypes importants chez la souche transgénique mutante associés à la pathologie humaine: défaut de motilité, longévité réduite et profil neurodégénératif considérable. Ceci dit, ces résultats nous ont poussé à vouloir déterminer si l’utilisation des composés chimiques, connus en tant que modulateurs du stress du RE et possédant des rôles neuroprotecteurs, sont capables de restaurer les phénotypes notés. Les composés utilisés, c’est-à-dire le Bleu de Méthylène, le Salubrinal et le Guanabenz, ont démontré une capacité de corriger les phénotypes rapportés dans la souche transgénique mutante. De plus, ces composés ont aussi été en mesure de prévenir une augmentation du niveau du stress oxydatif et de la réponse au stress du RE exhibé chez les vers mutants. Par le développement de nouveaux modèles C. elegans pour la MMJ, où il y a expression du gène ATXN3 complet dans les motoneurones, il a été possible de trouver qu’une modulation chimique du stress du RE peut réduire considérablement la neurodégénérescence et par conséquent, être une possible nouvelle approche thérapeutique pour traiter cette pathologie. / Polyglutamine expansion diseases are a class of dominantly inherited neurodegenerative disorders that develop when a CAG repeat in the causative genes is unstably expanded above a certain threshold. The expansion of trinucleotide CAG repeats causes hereditary adult-onset neurodegenerative disorders such as multiple forms of spinocerebellar ataxia (SCA). The most common dominantly inherited spinocerebellar ataxia is the type 3 (SCA3) also known as Machado-Joseph disease (MJD), an autosomal dominant, progressive neurological disorder. The gene causing MJD is ATXN3 (ATAXIN-3): MJD is caused by an abnormal CAG trinucleotide repeat expansion in the ATXN3 gene. Several recent studies have shown that this gene is associated with endoplasmic reticulum (ER) stress. In this study, we generated transgenic C. elegans strains expressing wild type or mutant human ATXN3 genes and tested them for recovery of locomotor phenotype, lifespan and neurodegeneration phenotypes upon treatment with compounds known to modulate ER stress and having neuroprotective roles. We observed differences between both transgenic lines and found that the motility defects, the reduced lifespan and the neurodegeneration can be rescued by methylene blue, guanabenz and salubrinal. These compounds were also able to prevent the oxidative stress and the ER stress response induced by mutant transgenic worms. We introduce novel C. elegans models for MJD based on the expression of full-length ATXN3 in GABAergic motor neurons. Using these models we discovered that chemical modulation of the ER unfolded protein response reduced neurodegeneration and could be a new therapeutic approach for the treatment of MJD.

Indiana University-Purdue University Indianapolis (IUPUI) / 17-β estradiol (E2) exerts protective effects in hypoxia-induced pulmonary hypertension (HPH) via endothelial cell estrogen receptor (ER)-dependent mechanisms. However, the effects of hypoxia on ER expression in the pulmonary-right ventricle (RV) axis remain unknown. Based on previous data suggesting a role of ER-β in mediating E2 protection, we hypothesized that hypoxia selectively up-regulates ER-β in the lung and pulmonary endothelial cells. In our Male Sprague-Dawley rat model, chronic hypoxia exposure (10% FiO2) resulted in a robust HPH phenotype associated with significant increases in ER- β but not ER-α protein in the lung via western blotting. More importantly, this hypoxia-induced ER-β increase was not replicated in the RV, left ventricle (LV) or in the liver. Hence, hypoxia-induced ER-β up-regulation appears to be lung-specific. Ex vivo, hypoxia exposure time-dependently up-regulated ER-β but not ER-α in cultured primary rat pulmonary artery endothelial cells (RPAECs) exposed to hypoxia (1% O2) for 4, 24 or 72h. Furthermore, the hypoxia induced ER-β protein abundance, while not accompanied by increases in its own transcript, was associated with ER-β nuclear translocation, suggesting increase in activity as well as post-transcriptional up-regulation of ER-β. Indeed, the requirement for ER-β activation was indicated in hypoxic ER-βKO mice where administration of E2 failed to inhibit hypoxia-induced pro-proliferative ERK1/2 signaling. Interestingly, HIF-1α accumulation was noted in lung tissue of hypoxic ER-βKO mice; consistent with previously reported negative feedback of ER-β on HIF-1α protein and transcriptional activation. In RAPECs, HIF-1 stabilization and overexpression did not replicate the effects of ER- β up-regulation seen in gas hypoxia; suggestive that HIF-1α is not sufficient for ER-β up- regulation. Similarly, HIF-1 inhibition with chetomin did not result in ER-β down-regulation. HIF-1α knockdown in RPAECs in hypoxic conditions is currently being investigated. Hypoxia increases ER- β, but not ER-α in the lung and lung vascular cells. Interpreted in context of beneficial effects of E2 on hypoxic PA and RV remodeling, our data suggest a protective role for ER-β in HPH. The mechanisms by which hypoxia increases ER-β appears to be post-transcriptional and HIF-1α independent. Elucidating hypoxia-related ER-β signaling pathways in PAECs may reveal novel therapeutic targets in HPH.

Tesis por compendio / [ES] La fotónica de silicio es una tecnología emergente clave en redes de comunicación e interconexiones de centros de datos de nueva generación, entre otros. Su éxito se basa en la utilización de plataformas compatibles con la tecnología CMOS para la integración de circuitos ópticos en dispositivos pequeños para una producción a gran escala a bajo coste. Dentro de este campo, los interferómetros integrados juegan un papel crucial en el desarrollo de diversas aplicaciones fotónicas en un chip como sensores biológicos, moduladores electro-ópticos, conmutadores totalmente ópticos, circuitos programables o sistemas LiDAR, entre otros. Sin embargo, es bien sabido que la interferometría óptica suele requerir caminos de interacción muy largos, lo que dificulta su integración en espacios muy compactos. Para mitigar algunas de estas limitaciones de tamaño, surgieron varios enfoques, incluyendo materiales sofisticados o estructuras más complejas, que, en principio, redujeron el área de diseño pero a expensas de aumentar los pasos del proceso de fabricación y el coste. Esta tesis tiene como objetivo proporcionar soluciones generales al problema de tamaño típico de los interferómetros ópticos integrados, con el fin de permitir la integración densa de dispositivos basados en silicio. Para ello, aunamos los beneficios tanto de las guías de onda bimodales como de las estructuras periódicas, en términos de la mejora del rendimiento y la posibilidad para diseñar interferómetros monocanal en áreas muy reducidas. Más específicamente, investigamos los efectos dispersivos que aparecen en estructuras menores a la longitud de onda y en las de cristal fotónico, para su implementación en diferentes configuraciones interferométricas bimodales. Además, demostramos varias aplicaciones potenciales como sensores, moduladores y conmutadores en tamaños ultra compactos de unas pocas micras cuadradas. En general, esta tesis propone un nuevo concepto de interferómetro integrado que aborda los requisitos de tamaño de la fotónica actual y abre nuevas vías para futuros dispositivos basados en funcionamiento bimodal. / [CA] La fotònica de silici és una tecnologia emergent clau en xarxes de comunicació i interconnexions de centres de dades de nova generació, entre altres. El seu èxit es basa en la utilització de plataformes compatibles amb la tecnologia CMOS per a la integració de circuits òptics en dispositius diminuts per a una producció a gran escala a baix cost. Dins d'aquest camp, els interferòmetres integrats juguen un paper crucial en el desenvolupament de diverses aplicacions fotòniques en un xip com a sensors biològics, moduladors electro-òptics, commutadors totalment òptics, circuits programables o sistemes LiDAR, entre altres. No obstant això, és ben sabut que la interferometría òptica sol requerir camins d'interacció molt llargs, la qual cosa dificulta la seua integració en espais molt compactes. Per a mitigar algunes d'aquestes limitacions de grandària, van sorgir diversos enfocaments, incloent materials sofisticats o estructures més complexes, que, en principi, van reduir l'àrea de disseny però a costa d'augmentar els processos de fabricació i el cost. Aquesta tesi té com a objectiu proporcionar solucions generals al problema de grandària típica dels interferòmetres òptics integrats, amb la finalitat de permetre la integració densa de dispositius basats en silici. Per a això, combinem els beneficis tant de les guies d'ones bimodals com de les estructures periòdiques, en termes de funcionament d'alt rendiment per a dissenyar interferòmetres monocanal compactes en àrees molt reduïdes. Més específicament, investiguem els efectes dispersius que apareixen en estructures menors a la longitud d'ona i en les de cristall fotònic, per a la seua implementació en diferents configuracions interferomètriques bimodals. A més, vam demostrar diverses aplicacions potencials com a sensors, moduladors i commutadors en grandàries ultres compactes d'unes poques micres cuadrades. En general, aquesta tesi proposa un nou concepte d'interferòmetre integrat que aborda els requisits de grandària de la fotònica actual i obri noves vies per a futurs dispositius basats en funcionament bimodal. / [EN] Silicon photonics is a key emerging technology in next-generation communication networks and data centers interconnects, among others. Its success relies on the ability of using CMOS-compatible platforms for the integration of optical circuits into small devices for a large-scale production at low-cost. Within this field, integrated interferometers play a crucial role in the development of several on-chip photonic applications such as biological sensors, electro-optic modulators, all-optical switches, programmable circuits or LiDAR systems, among others. However, it is well known that optical interferometry usually requires very long interaction paths, which hinders its integration in highly compact footprints. To mitigate some of these size limitations, several approaches emerged including sophisticated materials or more complex structures, which, in principle, reduced the design area but at the expense of increasing fabrication process steps and cost. This thesis aims at providing general solutions to the long-standing size problem typical of optical integrated interferometers, in order to enable the densely integration of silicon-based devices. To this end, we combine the benefits from both bimodal waveguides and periodic structures, in terms of high-performance operation and compactness to design single-channel interferometers in very reduced areas. More specifically, we investigate the dispersive effects that arise from subwavelength grating and photonic crystal structures for their implementation in different bimodal interferometric configurations. Furthermore, we demonstrate various potential applications such as sensors, modulators and switches in ultra-compact footprints of a few square microns. In general, this thesis proposes a new concept of integrated interferometer that addresses the size requirements of current photonics and open up new avenues for future bimodal-operation-based devices. / Financial support is also gratefully acknowledged through postdoctoral FPI grants from Universitat Politècnica de València (PAID-01-18). European Commission through the Horizon 2020 Programme (PHC-634013 PHOCNOSIS project). The authors acknowledge funding from the Generalitat Valenciana through the AVANTI/2019/123, ACIF/2019/009 and PPC/2020/037 grants and from the European Union through the operational program of the European Regional Development Fund (FEDER) of the Valencia Regional Government 2014–2020. / Torrijos Morán, L. (2021). Photonic Applications Based on Bimodal Interferometry in Periodic Integrated Waveguides [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/172163 / Compendio

No / The orally active microtubule-disrupting agent (S)-1-ethyl-3-(2-methoxy-4-(5-methyl-4-((1-(pyridin-3-yl)butyl)amino)pyrimidin-2- yl)phenyl)urea (CYT997), reported previously by us (Bioorg Med Chem Lett 19:4639-4642, 2009; Mol Cancer Ther 8:3036-3045, 2009), is potently cytotoxic to a variety of cancer cell lines in vitro and shows antitumor activity in vivo. In addition to its cytotoxic activity, CYT997 possesses antivascular effects on tumor vasculature. To further characterize the vascular disrupting activity of CYT997 in terms of dose and temporal effects, we studied the activity of the compound on endothelial cells in vitro and on tumor blood flow in vivo by using a variety of techniques. In vitro, CYT997 is shown to potently inhibit the proliferation of vascular endothelial growth factor-stimulated human umbilical vein endothelial cells (IC(50) 3.7 +/- 1.8 nM) and cause significant morphological changes at 100 nM, including membrane blebbing. Using the method of corrosion casting visualized with scanning electron microscopy, a single dose of CYT997 (7.5 mg/kg i.p.) in a metastatic cancer model was shown to cause destruction of tumor microvasculature in metastatic lesions. Furthermore, repeat dosing of CYT997 at 10 mg/kg and above (intraperitoneally, b.i.d.) was shown to effectively inhibit development of liver metastases. The time and dose dependence of the antivascular effects were studied in a DLD-1 colon adenocarcinoma xenograft model using the fluorescent dye Hoechst 33342. CYT997 demonstrated rapid and dose-dependent vascular shutdown, which persists for more than 24 h after a single oral dose. Together, the data demonstrate that CYT997 possesses potent antivascular activity and support continuing development of this promising compound.

Background: Sphingosine 1-phosphat receptor modulators (S1PRMs) have been linked to attenuated immune response to SARS-CoV-2 vaccines. Objective: To characterize differences in the immune response to SARS-CoV-2 vaccines in patients on selective versus unselective S1PRMs. Methods: Monocentric, longitudinal study on people with multiple sclerosis (pwMS) on fingolimod (FTY), siponimod (SIP), ozanimod (OZA), or without disease-modifying therapy (DMT) following primary and booster SARS-CoV-2 vaccination. Anti-SARS-CoV-2 antibodies and T-cell response was measured with electro-chemiluminescent immunoassay and interferon-γ release assay. Results: Primary vaccination induced a significant antibody response in pwMS without DMT while S1PRM patients exhibited reduced antibody titers. The lowest antibodies were found in patients on FTY, whereas patients on OZA and SIP presented significantly higher levels. Booster vaccinations induced increased antibody levels in untreated patients and comparable titers in patients on OZA and SIP, but no increase in FTY-treated patients. While untreated pwMS developed a T-cell response, patients on S1PRMs presented a diminished/absent response. Patients undergoing SARS-CoV-2 vaccination before onset of S1PRMs presented a preserved, although attenuated humoral response, while T-cellular response was blunted. Conclusion: Our data confirm differential effects of selective versus unselective S1PRMs on T- and B-cell response to SARS-CoV-2 vaccination and suggest association with S1PRM selectivity rather than lymphocyte redistribution.

Η πληθώρα των εφαρμογών που μπορεί να εξυπηρετήσει η τεχνολογία Υπερευρείας Ζώνης (UWB), από τα ασύρματα προσωπικά δίκτυα υψηλών ταχυτήτων, μέχρι τα ασύρματα δίκτυα αισθητήρων με δυνατότητες ακριβούς εντοπισμού θέσης, και τα ασύρματα δίκτυα ιατρικών αισθητήρων, έχει προκαλέσει έντονο ερευνητικό ενδιαφέρον γύρω από τις υλοποιήσεις UWB συστημάτων. Η ασυνήθιστα μεγάλη περιοχή συχνοτήτων που έχει ανατεθεί στο UWB, από τα 3.1-10.6 GHz, επιτρέπει την επίτευξη υψηλών ταχυτήτων με απλά σχήματα διαμόρφωσης, ωστόσο, λόγω της διαμοίρασης του φάσματος με τις υφιστάμενες τεχνολογίες ασύρματης δικτύωσης, οι UWB εκπομπές πρέπει να περιορίζονται σε ισχύ κάτω από το κατώφλι των -41.3 dBm/MHz, ικανοποιώντας πολύ αυστηρές μάσκες εκπομπής που εισάγουν έντονες προκλήσεις στη σχεδίαση των πομπών. Η υλοποίηση αναδιατάξιμων UWB πομπών σε σύγχρονες CMOS τεχνολογίες, με υψηλή φασματική ευελιξία, ταχύτητα και ποιότητα διαμόρφωσης, καθώς και με χαμηλή κατανάλωση, αποτέλεσε το αντικείμενο της συγκεκριμένης διατριβής. Υιοθετώντας την αρχιτεκτονική Multi-Band Impulse-Radio (MB-IR) σε συνδυασμό με την τεχνική Direct Sequence BPSK, η έρευνα προσανατολίστηκε προς την ανάπτυξη καινοτόμων μονάδων βασικής ζώνης, με στόχο την ενεργειακά αποδοτική αντιστροφή Γκαουσιανών μορφοποιημένων παλμών υψηλής ποιότητας φάσματος και διάρκειας μικρότερης ακόμα και από 1 nsec. Προς αυτή την κατεύθυνση, αναπτύχθηκε μια καινοτόμα γεννήτρια Γκαουσιανών παλμών με πολύ χαμηλούς πλευρικούς λοβούς στο φάσμα, τυπικά κάτω από -40 dB, ώστε να υποστηρίζονται οι αυστηρότερες μάσκες εκπομπής ή και μελλοντικές. Η σχεδίασης της προτεινόμενης γεννήτριας είχε ως κριτήριο την ευέλικτη ρύθμιση της διάρκειας των παραγόμενων παλμών, και αξιοποίησε τη χαρακτηριστική μεταφοράς τάσης ενός ωμικά φορτωμένου, ασύμμετρου CMOS αντιστροφέα. Η γεννήτρια βασίζεται κυρίως σε ψηφιακά κυκλώματα πολύ χαμηλής τάσης και, σε σύγκριση με τις υφιστάμενες υλοποιήσεις, παρουσιάζει σημαντικό προβάδισμα στον τομέα της ταχύτητας, καθώς και στο πλάτος εξόδου, η μεγάλη τιμή του οποίου χαλαρώνει σημαντικά τη σχεδίαση του RF front end. Η γεννήτρια μελετήθηκε διεξοδικά, διεξήχθη ανάλυση κλιμάκωσης, έγινε εξαγωγή σχεδιαστικών εξισώσεων και αναπτύχθηκαν εργαλεία λογισμικού για την αυτοματοποιημένη σχεδίασή της. Για περαιτέρω αύξηση της ταχύτητας των παλμικών σημάτων εφαρμόσθηκε ειδική σχεδίαση, η οποία αντιπραγματεύεται την ταχύτητα με το επίπεδο των λοβών του φάσματος. Για την αποδοτική BSPK διαμόρφωση των Γκαουσιανών παλμών αναπτύχθηκε ειδική τοπολογία “Μεταγωγής Σήματος Πυροδότησης Πλήρους Ισορροπίας με Up-Conversion”. Η τοπολογία αυτή, σε αντίθεση με τις ανταγωνιστικές τοπολογίες, αποφεύγει την αντιστροφή του παλμού με αναλογικά κυκλώματα υψηλής κατανάλωσης, αλλά και την αναλογική μεταγωγή, καθώς η διαμόρφωση λαμβάνει χώρα πριν από την παραγωγή των παλμών. Παράλληλα, επιτυγχάνονται υψηλοί ρυθμοί, καθώς και υψηλή ποιότητα διαμόρφωσης λόγω των ισορροπημένων μονοπατιών της τοπολογίας. Η γεννήτρια μαζί με το διαμορφωτή αποτελούν τις καινοτόμες παρεμβάσεις στη μονάδα Βασικής Ζώνης του προτεινόμενου πομπού. Για την ολοκλήρωση της λειτουργικότητας του πομπού, αναπτύχθηκε ένα RF front end, το οποίο αποτελείται από έναν διπλά ισορροπημένο μίκτη, έναν LO buffer, ένα μετατροπέα διαφορικού σήματος σε απλό, και έναν ενισχυτή ισχύος, ο οποίος είναι προσαρμοσμένος στα 50 Ohms, χωρίς να απαιτεί κανένα εξωτερικό στοιχείο. Το RF front end ολοκληρώθηκε μαζί με τη μονάδα βασικής ζώνης, και ο ολοκληρωμένος πομπός κατασκευάστηκε σε τεχνολογία CMOS 130 nm. Το ολοκληρωμένο προσαρτήθηκε στην RF πλακέτα συστήματος με την τεχνική Chip on Board. Για την επιτυχία του συστήματος με την πρώτη προσπάθεια έγινε συσχεδίαση σε επίπεδο IC-Package-PCB, δίνοντας ιδιαίτερη έμφαση στα ζητήματα Signal/Power Integrity. Ο πομπός παρουσίασε την υψηλότερη ταχύτητα από τις ανταγωνιστικές MB-IR UWB υλοποιήσεις, ίση με 1.5 Gbps, με αντίστοιχη ενεργειακή αποδοτικότητα 21 pJoule/bit και μέτρο διανυσματικού σφάλματος 5.5%. Ο πομπός βελτίωσε τους πλευρικούς λοβούς στο φάσμα περισσότερο από 10 dB, ενώ η διατριβή, εκμεταλλευόμενη την αναδιαταξιμότητα του πομπού, παρουσιάζει, επιπλέον, τις πρώτες μετρήσεις σε ταχύτητες εκατοντάδων Mbps για ικανοποίηση της χαμηλής ζώνης της πρόσφατα θεσμοθετημένης, και εξαιρετικά αυστηρής, ευρωπαϊκής μάσκας εκπομπής. / The multitude of applications that Ultra-Wideband (UWB) technology can serve, from high-speed Wireless Personal Area Networks, to Wireless Sensor Networks with precision Geolocation abilities, and Wireless Medical Networks, has attracted intense research interest in the implementation of UWB systems. The unusually wide range of frequencies assigned to UWB, from 3.1-10.6 GHz, allows UWB systems employing low order modulation schemes to enjoy high throughput at low power consumption. However, since UWB shares the spectrum with existing wireless networking technologies, UWB emissions must be limited to a power spectral density below the threshold of -41.3 dBm/MHz, satisfying very stringent emission masks and introducing great challenges in the design of UWB transmitters. The subject of this thesis is the design of low power, fully integrated, reconfigurable CMOS UWB transmitters, with high spectral flexibility, high speed and high modulation quality. Adopting the Multi-Band Impulse-Radio architecture, in conjunction with the Direct Sequence BPSK modulation, the research focused on the development of a baseband unit, able to precisely invert Gaussian shaped, subnanosecond pulses. The key contributions of this thesis are a CMOS Gaussian Pulse Generator and a BSPK modulation topology, which jointly constitute the proposed baseband unit. The Pulse Generator (PG) is based on non-linear shaping, so as to facilitate the configurability of the output pulse duration, and exploits the voltage transfer characteristic of a Resistive Loaded Asymmetrical CMOS Inverter, which results in spectral sidelobes typically better than -40 dB. The PG incorporates mostly-digital low voltage circuits, while the MOSFET devices that undertake the pulse shaping avoid exclusive operation in weak inversion, in contrast to previous implementations. Consequently, the proposed CMOS PG is able to support higher throughput, as well as higher output amplitude, which relaxes considerably the design of the RF front end. This thesis presents a systematic design procedure and a scaling analysis of the non-linear pulse shaper. Moreover, in order to further increase the speed, a special PRF boost technique is proposed, which trades off speed and spectral efficiency for the spectral sidelobes level. Regarding the BPSK modulator, this work introduces the “Trigger Switching Fully Balanced Up-Conversion” topology, which avoids the use of power-hungry and distortion-prone analog circuits for the accurate inversion of the subnanosecond shaped pulses, as well as avoids the application of analog waveform switching to the baseband pulses, since the baseband modulation takes place before the generation of the pulses. The digital nature of the switching lends itself to high data rates, while the balanced paths of the topology ensure high modulation quality with minimal design effort. Wafer probing measurements confirmed the high performance of the baseband unit. The functionality of the transmitter was completed by the development of an RF front end which consists of a double balanced mixer, an LO buffer, a differential to single-ended (DtoSE) converter, and a power amplifier which is ready to drive a 50 Ohms load without requiring any off-chip components. The integrated transmitter, which incorporates the proposed baseband unit and the RF front end, was fabricated in 130 nm CMOS technology. The transmitter RFIC was directly attached to the system RF PCB using the Chip-on-Board packaging option. The First-Pass success of the system was ensured by paying particular attention to Signal/Power Integrity issues and following an IC-Package-PCB co-design procedure. The transmitter was measured up to 1.5 Gbps, which, to the author’s knowledge, was the highest speed amongst the competitive Multi-Band Impulse-Radio UWB implementations in the literature. The corresponding energy efficiency was 21 pJoule/bit and the Error Vector Magnitude (EVM) 5.5%, while the proposed transmitter improved the spectral sidelobes by over 10 dB. Exploiting the reconfigurability of the transmitter, this thesis presents the first measurements at multi-Mbps speeds that completely meet the final version of the European spectrum emission mask.