Le Sarcome d'Ewing est un cancer pédiatrique rare, principalement dû à l'expression de l'oncogène de jonction EWS-Fli1, et dont les traitements médicamenteux ont peu évolué au cours des dernières décennies. Nous nous intéressons à une nouvelle approche thérapeutique utilisant des siRNA, ciblant spécifiquement l'oncogène EWS-Fli1, et permettant l'inhibition de la croissance tumorale. Durant mon travail de thèse, j'ai utilisé des nanocristaux de diamant issus soit de détonation (DND), soit de synthèse haute pression-haute température (NDHPHT) pour vectoriser les siRNA, accrochés par interaction électrostatique. Pour ce faire, les NDs ont été rendus cationiques par différentes méthodes: (i) hydrogénation assistée par plasma, (ii) par recuit thermique, ou (iii) par traitement chimique pour les DNDs, ou (iv) greffage covalent d'un polymère cationique sur des NDHPHT (COP-NDHPHT).Mes travaux ont comporté deux axes: (i) étude in vitro des complexes ND:siRNA (caractérisations physico-chimiques des NDs et étude de l'efficacité d'inhibition de l'oncogène par les complexes); (ii) distribution tissulaire de COP-NDHPHT, injectés dans des souris, grâce à des NDHPHT fluorescents, contenant des défauts azote-lacune. Pour les détecter individuellement dans des coupes d'organes de souris portant une tumeur xénogreffée sous-cutanée, nous avons développé un système d'imagerie en épifluorescence à grande ouverture numérique, et résolu en temps afin de rejeter l'autofluorescence tissulaire (de durée de vie plus courte que celle des NDs). Nous avons quantifié le nombre, l'état d'agrégation et la localisation cellulaire de ces vecteurs (grâce à un marquage histopathologique imagé simultanément) 24h après injection. Les NDs ont été clairement détectés dans les différents organes, dont la tumeur, ouvrant la voie à un contrôle de la progression tumorale grâce au siRNA. / Ewing Sarcoma is a rare pediatric cancer, caused in the majority of the cases by the expression of the fusion oncogene EWS-Fli1. Current treatments have not much evolved over the past decades. We are investigating a new therapy based on siRNA specifically targeting the oncogene and inhibiting the tumor growth. During my PhD thesis, I have tested different types of synthetic nanodiamonds (ND) used to vectorize siRNA electrostatically bound at their surface: ND produced by detonation (DND) or by High Pressure-High Temperature synthesis (NDHPTH). Their surfaces have been cationized by various processes: (i) plasma or (ii) thermal hydrogenation, (ii) chemical treatment, or (iv) covalent grafting of a copolymer (COP-NDHPHT).My PhD work included two main axis: (i) in vitro study of ND:siRNA complexes (NDs physico-chemical characterization and oncogene inhibition efficacy by the complexes); (ii) tissue distribution of COP-NDHPHT, injected into mice, using fluorescent NDHPHT containing nitrogen-vacancy defects. To detect them individually in sections of mouse organs carrying a subcutaneous xenograft tumor, we developed an epifluorescence imaging system with large numerical aperture and resolved in time to reject tissue autofluorescence (of a shorter lifetime than NDs). We quantified the number, the aggregation state and the cell localization (thanks to simultaneous histopathological imaging) of these vectors 24 hours after injection. NDs have been clearly detected in different organs, including the tumor, paving the way for tumor progression control with siRNA.

Syftet med denna uppsats är att analysera hur människors val av boplats påverkar segregationen i Stockholm ur ett antropolgiskt perspektiv. Jag har valt att använda den välkänt fashionabla Stockholmsförorten Djursholm som exempel. Vad utgör exklusiviteten? Hur ser Djursholmarna på sig själva? Jag har använt mig av antropologisk litteratur och exempel från intervjuer ur professor Mikael Holmqvists bok Djursholm: Sveriges ledarsamhälle. I samband med detta har jag gjort egna observationer och fotograferat. Djursholm bygger på tradition, förutsägbarhet och en symbolisk gemenskap. Många villor har stora parkliknande trädgårdar omgärdade av häckar, staket och grindar. Min tes är att Djursholm kan betraktas som ett grindsamhälle – en översättning från det kända engelska begreppet "Gated Community" – och bidrar på så vis till segregationen vilket jag kommer att utforska.

Att sträva efter en hållbar samhällsutveckling sett till ekonomiska, ekologiska och sociala aspekter är en målsättning som ges stor tonvikt inom svensk politik och planering. Den påtagliga boendesegregationen som präglar det svenska samhället utgör dock ett problem i arbetet för att nå den sociala hållbarheten, vilket inte minst tydliggörs genom att det varit en fråga på den politiska agendan sedan 1970-talet. För att försöka minska segregationen eftersträvar kommuner ofta att skapa blandade bostadsområden, planera för mötesplatser samt bygga bort barriärer mellan olika stadsdelar för att öka integrationen och minska både geografiska och sociala avstånd mellan människor. Sedan avregleringen på bostadsmarknaden har de privata aktörernas roll i bostadsförsörjningen ökat, vilket resulterat i att nya bostadskoncept etablerats i Sverige. Undersökningsobjektet för denna studie, nämligen gated communities, utgör ett av de allra senaste koncepten. Det finns många varianter av gated communities runt om i världen, men en generell beskrivning är att det rör sig om bostadsområden som hägnas in med hjälp av fysiska barriärer så som murar, stängsel eller grindar. Bostadskonceptet är väletablerat i exempelvis USA och Sydamerika, men på den svenska bostadsmarknaden har det ännu inte slagit igenom. Under andra halvan av 2000-talet har det dock byggts ett antal inhägnade bostadsområden runt om i landet, vilket skapat en medial debatt kring huruvida gated communities kan komma att se en framtida utveckling även i Sverige.  Detta masterarbete syftar till att undersöka och analysera vad gated communities kan komma att innebära för den socialt hållbara bostads- och samhällsutveckling som politik och planering strävar efter att uppnå. Syftet är också att undersöka hur ett antal kommuner i deras strategiska arbete ser på bostadskonceptet och vilka konsekvenser som en ökad utveckling kan medföra. Detta har undersökts genom en flerfallstudie, där två bostadsområden och två kommuner utgör fallen i studien. Empirin i studien, vilken i huvudsak kommer från intervjuer med politiker, planerare och byggherrar samt genom dokumentstudier av olika planhandlingar, har analyserats genom en kvalitativ innehållsanalys. Studien visar att inhägnade bostadsområden i en svensk kontext existerar i två former, nämligen inhägnade bostadsrättsområden och exklusiva gated communities där boende ges privat tillgång till funktioner och service. Studiens huvudsakliga slutsats är att båda formerna skapar ytterligare en dimension till den rådande boendesegregationen genom att fysiska barriärer förs upp och förtydligar gränser i rummet som tidigare endast varit mentala. Exklusiva gated communities som riktas åt socioekonomiskt starka grupper och erbjuder funktioner som parker, lekplatser och andra fritidsaktiviteter riskerar att minska dessa gruppers incitament till att nyttja de offentliga mötesplatserna som kommunerna skapar i förhoppning om att få olika befolkningsgrupper att mötas. En ökad utveckling av gated communities riskerar därmed att skapa ännu tydligare ”vi och dom” – uppdelningar i samhället och utmana de svenska strategierna som använts för att försöka minska boendesegregationen sedan 1970-talet. Trots detta visar studien även att det finns ett visst kommunalt intresse i bostadskonceptet, vilket kan bero på att kommuner kan öka sina skatteintäkter och göra kostnadsbesparingar genom etableringen av gated communities.

[ES] La presente tesis doctoral titulada "Nanomateriales con puertas moleculares como plataforma de liberación controlada de fármacos para el tratamiento de desórdenes inflamatorios" se centra la preparación y evaluación de nanomateriales híbridos orgánico-inorgánicos, basados en nanopartículas mesoporosas de sílice, para la liberación controlada de fármacos en aplicaciones biomédicas, en concreto en el campo de la inflamación. En primer lugar se describe un nanomaterial para la liberación controlada del inhibidor de caspasa-1, VX-765, aprovechando la acumulación preferencial de las nanopartículas en las zonas inflamadas. En concreto, se han preparado nanopartículas mesoporosas de sílice, cargadas con el fármaco VX-765 y funcionalizadas covalentemente con ¿-poli-L-lisina que actúa como puerta molecular. La actividad anti-inflamatoria del material se ha comprobado tanto in vitro, en el modelo celular de monocitos THP-1, como in vivo en ratones en un modelo de inflamación de bolsa de aire. Los resultados muestran la acumulación preferente de las nanopartículas en las zonas inflamadas así como un aumento del efecto terapéutico del fármaco que se atribuye a las ventajas que ofrece la encapsulación. Se concluye que las nanopartículas mesoporosos de sílice con puertas moleculares podrían ser una herramienta importante para el desarrollo de nuevas estrategias terapéuticas en el campo de la inflamación. Basándonos en los resultados obtenidos, en el capítulo cuatro se describe un sistema de liberación controlada para el tratamiento de la inflamación pulmonar aguda como terapia alternativa que permita la administración directa de fármacos a los pulmones. Se ha preparado un nanosistema basado en nanopartículas mesoporosas de sílice cargadas con el glucocorticoide dexametasona y funcionalizadas covalentemente con una puerta molecular peptídica que reconoce el receptor del factor de necrosis tumoral 1 (TNFR1), que a su vez actúa como agente diana para la acumulación preferente en macrófagos pro-inflamatorios. La actividad terapéutica del sistema se ha corroborado en ensayos in vitro en macrófagos pro-inflamatorios, e in vivo en un modelo de ratón de inflamación pulmonar aguda. Se ha comprobado la acumulación preferente de las nanopartículas en los pulmones inflamados, así como la mejora del efecto terapéutico de la dexametasona en la reducción del daño pulmonar, minimizando los efectos adversos asociados a la administración del fármaco libre. Con todo ello se concluye que las nanopartículas mesoporosas de sílice pueden ser utilizadas para el tratamiento de la inflamación pulmonar aguda pudiendo ser una herramienta útil para superar las limitaciones de los tratamientos actuales. Finalmente, se describe otro sistema de liberación controlada de fármacos para inflamación pulmonar aguda. En este caso, se aborda el uso de un nuevo inhibidor del inflamasoma, QM-378, como terapia farmacológica alternativa. Con el objetivo de potenciar la administración directa en los pulmones inflamados, el QM-378 se encapsula en nanopartículas mesoporosas de sílice funcionalizadas con la puerta molecular peptídica que reconoce TNFR1. La acumulación preferente de las nanopartículas en los pulmones inflamados queda demostrada a través de los ensayos de biodistribución, así como la mejora del efecto terapéutico del QM-378 en la reducción del daño pulmonar, debido a las ventajas de la encapsulación en un nanosistema dirigido. Con todo ello se concluye que el QM-378 es un buen candidato para el tratamiento de la inflamación pulmonar aguda, y que su encapsulación en las nanopartículas ofrece una administración pulmonar directa y controlada, consiguiéndose así una mejora en el perfil terapéutico del fármaco. La conclusión principal de la presente tesis doctoral es que el desarrollo de nanomateriales mesoporosos de sílice para la liberación controlada de fármacos se presenta como un / [CAT] La present tesi doctoral titulada "Nanomaterials amb portes moleculars com a plataforma d'alliberament controlat de fàrmacs per al tractament de desordres inflamatoris" se centra en la preparació i avaluació de nanomaterials híbrids orgànic-inorgànics, basats en nanopartícules mesoporoses de sílice, per a l'alliberament controlat de fàrmacs en aplicacions biomèdiques, en concret en el camp de la inflamació. En primer lloc, es presenta un nanomaterial per a l'alliberament controlat de l'inhibidor de caspasa-1, VX-765, aprofitant que les nanopartícules s'acumulen preferencialment en les zones inflamades. S'han preparat nanopartícules mesoporoses de sílice, carregades amb VX-765 i funcionalitzades covalentment amb ¿-poli-L-lisina com a porta molecular. L'activitat anti-inflamatòria del material s'ha comprovat tant in vitro, en el model cel·lular de THP-1, com in vivo en ratolins en un model d'inflamació de bossa d'aire. Els resultats mostren la acumulació preferent de les nanopartícules en les zones inflamades així com un augment de l'efecte terapèutic del fàrmac, atribuÏt als avantatges que ofereix l'encapsulació. Es conclou que les nanopartícules mesoporoses de sílice amb porta molecular podrien ser una eina important per al desenvolupament de noves estratègies terapèutiques en el camp de la inflamació. Basant-nos en els resultats obtinguts, en el capítol quatre es presenta un sistema d'alliberament controlat per al tractament de la inflamació pulmonar aguda com a teràpia alternativa que permet l'administració directa de fàrmacs als pulmons. S'ha preparat un nanosistema basat en nanopartícules mesoporoses de sílice carregades amb el glucocorticoide dexametasona i funcionalitzades amb la unió covalent de una porta molecular peptídica que reconeix el receptor del factor de necrosi tumoral 1 (TNFR1), que al seu torn actua com a agent diana per a la acumulació preferent en macròfags pro-inflamatoris. L'activitat terapèutica del sistema dissenyat s'ha corroborat en assajos in vitro en macròfags pro-inflamatoris, i in vivo en un model de ratolí d'inflamació pulmonar aguda. S'ha comprovat la acumulació preferent de les nanopartícules en els pulmons inflamats a través d'assajos de biodistribució, així com la millora de l'efecte terapèutic de la dexametasona en la reducció de la lesió pulmonar minimitzant els efectes adversos associats a l'administració del fàrmac lliure. Amb tot això es conclou que les nanopartícules mesoporoses de sílice poden ser utilitzades per al tractament de la inflamació pulmonar aguda ja que poden ajudar a superar les limitacions dels tractaments actuals. Finalment es mostra també un sistema d'alliberament controlat de fàrmacs per a inflamació pulmonar aguda. En aquest cas, es descriu l'ús d'un nou inhibidor de l'inflamasoma, QM-378, com a teràpia farmacològica alternativa al tractament de la inflamació descontrolada en la inflamació pulmonar aguda. Amb l'objectiu de potenciar l'administració directa en els pulmons inflamats, el QM-378 s'encapsula en les nanopartícules mesoporoses de sílice funcionalitzades amb la porta molecular péptidica que reconeix TNFR1. La acumulació preferent de les nanopartícules en els pulmons inflamats queda demostrada a través dels assajos de biodistribució, així com la millora de l'efecte terapèutic del QM-378 en la reducció de la inflamació pulmonar, atribuït als avantatges de l'encapsulació en un nanosistema dirigit. Amb tot això es conclou que el QM-378 és un bon candidat per al tractament de la inflamació pulmonar aguda, i que la seua encapsulació en les nanopartícules ofereix una administració pulmonar directa i controlada aconseguint-se així una millora en el perfil terapèutic del fàrmac. La conclusió principal és que el desenvolupament de nanomaterials mesoporosos de sílice per a l'alliberament controlat de fàrmacs es presenta com una estratègia amb molt potencial en el camp de les / [EN] This PhD thesis entitled "Gated nanomaterials as delivery platform to manage inflammatory disorders" is focused on the design, synthesis and evaluation of hybrid organic-inorganic nanomaterials using mesoporous silica nanoparticles, for controlled drug release in biomedical applications, specifically in the field of inflammation. In a fist step, we present a new nanodevice for the controlled delivery of VX-765, a caspase 1 inhibitor, which takes advantage of the intrinsic passive targeting effect of the nanoparticles to inflamed tissues. In particular, mesoporous silica nanoparticles loaded with the drug VX-765 and functionalized with ¿-poly-L-lysine (acting as gatekeeper) have been prepared. The anti-inflammatory activity of the prepared nanodevice has been evaluated both in vitro, in the cellular model of monocytes THP-1, and in vivo using air pouch mouse as model of inflammation. The results showed the preferential accumulation of the nanoparticles in the inflamed tissue, as well as an increase in the therapeutic effect of the entrapped drug. As conclusion, gated mesoporous silica nanoparticles constitute an important tool for the development of new therapeutic strategies in the inflammatory field. Based on the previous results presented, a drug delivery system for the treatment of acute lung injury is described in chapter four as alternative therapy that allow the direct delivery of drugs into the lungs. Mesoporous silica nanoparticles has been prepared, loaded with the glucocorticoid dexamethasone and capped with a peptide gatekeeper that recognizes the receptor of tumour necrosis factor 1 (TNFR1), which also targets the pro-inflammatory macrophages. The therapeutic activity of the designed nanoparticles has been studied in vitro in pro-inflammatory macrophages, and in vivo in an acute lung injury mouse model. The preferential accumulation of the nanoparticles in the inflamed lungs has been corroborated through biodistribution assays, as well as the ability to enhance the dexamethasone therapeutic effect by the reduction of lung injury and minimizing the undesired side effects associated of the free drug administration. As conclusion, gated mesoporous silica nanoparticles can be used for the treatment of acute lung injury and represent a potential tool to overcome the limitations of current treatments. Finally, a drug delivery system for acute lung injury is also presented. In this case, we use the novel inflammasome inhibitor QM-378 as pharmacological alternative therapy to the treatment of uncontrolled inflammation in acute lung injury. With the aim of enhancing the direct drug delivery in lungs, QM-378 is encapsulated in mesoporous silica nanoparticles capped with a peptidic gate that recognizes TNFR1. The preferential accumulation of nanoparticles to inflamed lungs has been also corroborated through biodistribution assays. An enhancement of the therapeutic effect of QM-378 by reducing lung inflammation is demonstrated, due to the advantages of drug encapsulation in a targeted-lung nanosystem. As conclusion, the QM-378 is a suitable candidate for acute lung injury treatment, and its encapsulation in mesoporous silica nanoparticles offers a direct lung drug delivery thus improving the therapeutic profile of the drug. The principal conclusion from this PhD thesis is that the preparation of mesoporous silica nanoaprticles for drug delivery is presented as potential strategy in the field of inflammatory disorders. / García Fernández, A. (2019). Gated nanomaterials as delivery platform for the treatment of inflammatory disorders [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/132694 / TESIS

Today, we are living in a data-driven world. Due to a surge in data generation, there is a need for efficient and accurate techniques to analyze data. One such kind of data which is needed to be analyzed are text reviews given for movies. Rather than classifying the reviews as positive or negative, we will classify the sentiment of the reviews on the scale of one to ten. In doing so, we will compare two recurrent neural network algorithms Long short term memory(LSTM) and Gated recurrent unit(GRU). The main objective of this study is to compare the accuracies of LSTM and GRU models. For training models, we collected data from two different sources. For filtering data, we used porter stemming and stop words. We coupled LSTM and GRU with the convolutional neural networks to increase the performance. After conducting experiments, we have observed that LSTM performed better in predicting border values. Whereas, GRU predicted every class equally. Overall GRU was able to predict multiclass text data of movie reviews slightly better than LSTM. GRU was computationally expansive when compared to LSTM.

Pentameric ligand-gated ion channels are critical mediators of electrochemical signal transduction in neurons and other excitable cells, causing them to be important targets of psychoactive drugs. Structural data for these complex proteins are limited, particularly among eukaryotic family members and for the functionally critical open state. These data limitations cause knowledge gaps regarding the mechanisms of ion channel opening, gating, and modulation. However, a newly discovered bacterial family member, known as sTeLIC, shares numerous structural features with its eukaryotic relatives in our central nervous system. A recently solved electron microscopy structure depicts sTeLIC in an apparent open state with binding pockets in its extracellular domain, compatible with binding a drug with structural similarities to amphetamines, like the 4-bromoamphetamine. This project aims to provide the first structure-function evidence for direct modulation of a pentameric ligand-gated ion channel by an amphetamine. The two most essential tools used in this project to examine the effects of 4-bromoamphetamine on sTeLIC were Xenopus laevis oocytes and two-electrode voltage-clamp. These tools were necessary for the collection of gating and modulation data. Ion channel activities can be analysed by clamping sTeLIC injected Xenopus laevis oocytes into the two-electrode voltage-clamp since it can artificially control the membrane voltage of oocytes. Modulation data show that 4-bromoamphetamine is a bimodal allosteric potentiator, as well as an allosteric agonist. Residues Y104 and W75, located in the binding pocket, were selected by comparing the published open state model with an AlphaFold-generated non-conducting model. Mutating these into valine or alanine reduces the potentiation. One explanation may be that removing tyrosine's aromatic ring complicates retaining essential interactions in the binding pocket while swapping tryptophan for smaller residues could make it easier for the drug to stabilise the closed state.

<p>The miniaturization of a MOSFET is the constant driving force in semiconductor technology over the decades. This scaling enables the realization of the ever complex and functional integration on a single chip where over tens of billions of transistors densely packed. Silicon (Si) is always the golden performer until recent years when the shrinking of a transistor becomes more and more difficult, due to phenomena such as short channel effect and mobility degradation, which is a challenge especially for atomic level scaling. The dawning of low dimensional materials, such as graphene, transition metal dichalcogenides (TMDs), black phosphorus (BP), with their natural atomically thin two-dimension (2D) layered structure and other novel properties, might serve as an alternative solution for ultimate scaling. However, the understanding of the electronic transport in these Van der Waals materials is still lacking. </p><p> In this research, the exploration of this material was first initiated on the vertical heterojunctions where two materials’ interfaces meet. Many previous literatures claimed this hetero-interface creates a P/N junction that results in a diode-like rectification. Yet, by careful analysis and comparing with our “real” vertical structures where the lateral components were eliminated, it is proved this rectification is a direct result from the contact region. The Schottky barrier on the drain side together with the gate effect is the true culprit.</p><p> Realizing how the Schottky barrier could be dominating in these 2D FETs, the second study is the Schottky barrier effect on the contact resistances and furthermore the mobility of the device. Because of the existence of the Schottky barrier between the channel and contact, the contact resistance is not negligible, unlike the ohmic contact for conventional Si MOSFETs. By comparing the intrinsic and extrinsic mobilities of TMD materials, It is found that the contact resistance’s response to the back gate, namely, the rate of how it changes with the back gate has a huge factor in determining whether the extrinsic field-effect mobility underestimates or overestimates its intrinsic mobility. This opens a new insight on the understanding of the transport mechanism under contacts for different TMDs.</p> With the understanding of the Schottky barrier FETs, lastly, the flexibility of these 2D materials is utilized to create high performance three-dimensionally stacked multi-channel FETs, from the inspiration of the Si gate-all-around nanosheet structure. A first-ever 3D integrated high performance MoS₂ device with two channels on top of each other was designed and fabricated, where the current is doubled with an extra layer of channel. The potential of these novel material to be implemented on the future generations of high-performance devices is demonstrated, shedding light on the prospect for extending the Moore’s Law with proper assistance from new materials.

The brain network is a multiscale hierarchical organization from neurons and local circuits to macroscopic brain areas. The precise synaptic transmission at each synapse is therefore crucial for neural communication and the generation of orchestrated behaviors. Activation of presynaptic voltage-gated calcium channels (CaV2) initiates synaptic vesicle release and plays a key role in neurotransmission. In this dissertation, I have aimed to uncover how CaV2 activity affects synaptic transmission, circuit function and behavioral outcomes using Caenorhabditis elegans as a model. The C. elegans genome encodes an ensemble of highly conserved neurotransmission machinery, providing an opportunity to study the molecular mechanisms of synaptic function in a powerful genetic system. I identified a novel gain of function CaV2α1 mutation that causes CaV2 channels to activate at a lower membrane potential and slow the inactivation. Cell-specific expression of these gain-of-function CaV2 channels is sufficient to hyper-activate neurons of interest, offering a way to study their roles in a given circuit. CaV2(gf) mutants display behavioral hyperactivity and an excitation-dominant synaptic transmission. Imbalanced excitation and inhibition of the nervous system have been associated with several neurological disorders, including Familial Hemiplegic Migraine type 1 (FHM1) which is caused by gain- of-function mutations in the human CaV2.1α1 gene. I showed that animals carrying C. elegans CaV2α1 transgenes with corresponding human FHM1 mutations recapitulate the hyperactive behavioral phenotype exhibited by CaV2(gf) mutants, strongly suggesting the molecular function of CaV2 channels is highly conserved from C. elegans to human. Through performing a genome-wide forward genetic screen looking for CaV2α(gf) suppressors, we isolated new alleles of genes that required for CaV2 trafficking, localization and function. These regulators include subunits of CaV2 channel complex, components of synaptic and dense core vesicle release machinery as well as predicted extracellular proteins. Taken together, this work advances the understanding of CaV2 malfunction at both cellular and circuit levels, and provides a genetically amenable model for neurological disorders associated with excitation-inhibition imbalance. Additionally, through identifying regulators of CaV2, this research provides new avenues for understanding the CaV2 channel mediated neurotransmission and potential pharmacological targets for the treatments of calcium channelopathies.

Philosophiae Doctor - PhD / Neurodegenerative disorders are characterised by progressive loss of the brain’s physiological functions as a result of gradual degeneration of neurons in the central nervous system. Even though they are classified as diseases of the elderly, occurrence earlier in life is possible, but that would suggest the influence of genetic and/or environmental factors. Due to the continuous rise in modernisation and industrialisation over the years, there has been an increase in incidence and prevalence of neurodegenerative disorders. With the advances in technology and life expectancy, the rates of the common forms (Alzheimer’s disease and Parkinson’s disease), are expected to increase exponentially by 2050. Unfortunately, there is still no clinically approved treatment or therapy to slow down or halt the degenerative process as most registered drugs only offer symptomatic relief. Confounding this issue is the lack of definite mechanism of neurodegeneration, which is still poorly defined and not completely understood. Nonetheless, the pathology of most neurodegenerative disorders is believed to be a combination of interrelated processes that eventually leads to neuronal cell death. Among the postulated processes, the impact of excitotoxicity mediated by NMDA receptor over-activation is prominent and it is implicated in virtually all neurodegenerative disorders. With this basic insight, it is believed that molecules capable of inhibiting NMDA receptors and associated calcium channels, without affecting the normal physiological functions of the brain, could potentially serve as good neuroprotective drugs. Competitive and uncompetitive blockers (MK-801 and ketamine) have been explored, but none were clinically accepted due to undesirable side effects such as hallucinations, sedation and depression. However, NGP1-01, a polycyclic cage molecule, has been shown to be neuroprotective through modulation of NMDA receptors and voltage gated calcium channels and attenuation of MPP+ -induced toxicity. A similar approach could be useful in the design and development of new neuroprotective drugs. The aim of this study was to synthesise a series of open and rearranged cage-like molecules and explore their neuroprotective potential in neuroblastoma SH-SY5Y cells. The proposed structures, with norbornane scaffolds that contained different moieties, were designed to structurally resemble NGP1-01 and MK-801. Once synthesised, the compounds were purified and characterised, and were evaluated for their biological activities. Compounds were first screened for cytotoxicity at different concentrations. Thereafter, they were evaluated for neuroprotective effects against MPP+ -induced excitotoxicity and for calcium flux modulatory effects on NMDA receptor and voltage gated calcium channels. The norbornane derivatives were synthesised and characterised, and all final products were afforded in sufficient yields. All compounds with the exception of two compounds displayed good cytotoxic profiles towards the SH-SY5Y neuroblastoma cells at 10 µM, 50 µM and 100 µM concentrations as they demonstrated percentage cell viabilities close to 100% (control treated cells). Only two compounds showed percentage cell viability of 51% and 59% at 100 µM. Utilising the same cell line, all compounds, tested at 10 µM, attenuated MPP+ -induced toxicity after 24 hours of exposure to a neurotoxin. This was evident in the 23% to 53% enhancement (significant with p < 0.05) in cell viability when compared to the MPP+ only treated cells. In comparison to known NMDA receptor and/or voltage gated calcium channel blockers (MK-801, NGP1-01 or nimodipine), the synthesised compounds demonstrated mono or dual inhibition of calcium channels as they effectively attenuated calcium influx by blocking NMDA receptors and/or voltage gated calcium channels expressed in neuroblastoma SHSY5Y cells. This group of compounds were found to be more potent NMDA receptor inhibitors, probably due to similarities with MK-801 and memantine, than voltage gated calcium channel inhibitors. All compounds demonstrated moderate to good calcium inhibitory effects at NMDA receptors in the range of 23% to 70% while a selected few displayed very little or no activity at the voltage gated calcium channels. In conclusion, 27 compounds with norbornane scaffolds were successfully synthesised and evaluated for cytotoxicity and neuroprotection. The abilities of the synthesised compounds to protect neurons from the neurotoxin MPP+ and reduce calcium flux into neuronal cells were successfully demonstrated. These characteristics are essential in neuroprotection as they may prove significant in halting or slowing down the disease progression. The compounds showing a good cytotoxicity profile, neuroprotective effects and ability to reduce calcium overload, could potentially act as neuroprotective agents with good safety profiles or contribute as lead structures to the development and design of structurally related molecules that could clinically benefit people with neurodegenerative disorders.

This dissertation focuses on the development of analytical methods based on Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) and their applications for separation and structural characterization of oligosaccharides. Porous graphitized carbon liquid chromatography (PGC-LC), gated-trapped ion mobility spectrometry (Gated-TIMS), and electronic excitation dissociation tandem mass spectrometry (EED MS/MS) are three essential techniques employed here. First, the EED method was optimized to generate more informative glycan tandem mass spectra for accurate structural analysis. Glycans were reduced and permethylated or labeled with a reducing-end fixed charge to increase sensitivity, avoid gas-phase structural rearrangement, and facilitate spectral interpretation. EED of glycans produced nearly complete series of Z-, Y- and 1,5X-ions, that appear in the spectra as triplets with characteristic spacing, thus facilitating accurate determination of the glycan topology. Additional radical-driven dissociation pathways were identified, from which different types of linkage-diagnostic ions (cross-ring, secondary, or internal fragments) were generated. The results demonstrated that linkage analysis can be accomplished by utilizing one or a combination of several linkage-diagnostic fragments. EED MS/MS was then implemented, in conjunction with PGC-LC or Gated-TIMS, for on-line separation and characterization of complex mixtures of glycans. These two methods were successfully applied for high-throughput and detailed structural analysis of N-glycans released from human serum, O-glycans released from bovine submaxillary mucin and free oligosaccharides. The performance of these methods was tested and improved through analysis of different types of glycans from a variety of biological sources. Finally, in collaboration with bioinformaticians, a spectral interpretation algorithm, GlycoDeNovo, has been developed for automated and de novo glycan topology reconstruction from their tandem mass spectra. A large number of EED tandem spectra of glycan standards generated in house were used as the training dataset to establish appropriate IonClassifiers for candidate ranking. GlycoDeNovo is capable of identifying correct topologies from MS/MS spectra of glycans in different derivatized forms. Several aspects of this collaborative project were covered in this thesis, including glycan derivatization, data acquisition and manual spectral interpretation to guide the development and evaluate the performance of the automated approach. In this thesis research, integrated approaches utilizing PGC-LC–EED-MS/MS and Gated-TIMS–EED-MS/MS, and the appropriate bioinformatics software, have been established for structural analysis of glycan mixtures. They hold great potential for comprehensive, automated, and de novo glycome characterization.