Topographic guidance scaffolds for peripheral nerve interfacing

In response to high and rising amputation rates, significant advances have been made in the field of prosthetic limb design. Unfortunately, there exists a lag in the neural interfacing technology required to provide an adequate link between the nervous system and this emerging generation of advanced prosthetic devices. Novel approaches to peripheral nerve interfacing are required to establish the stable, high channel count connections necessary to provide natural, thought driven control of an external prosthesis. Here, a tissue engineering-based approach has been used to create a device capable of interfacing with a regenerated portion of amputated nerve. As part of this work, a nerve guidance channel design, in which small amounts of interior scaffolding material could be precisely positioned, was evaluated. Guidance channels containing a single thin-film sheet of aligned scaffolding were shown to support robust functional nerve regeneration across extended injury gaps by minimally supplementing natural repair mechanisms. Significantly, these "thin-film enhanced nerve guidance channels" also provided the capability to guide the course of axons regenerating from a cut nerve. This capability to control axonal growth was next leveraged to create "regenerative scaffold electrodes (RSEs)" able to interface with axons regenerating from an amputated nerve. In the RSE design, low-profile arrays of interfacing electrodes were embedded within layers of aligned scaffolding material, such that regenerating axons were topographically guided by the scaffolding through the device and directly across the embedded electrodes. Chronically implanted RSEs were successfully used to record evoked neural activity from amputated nerves in an animal model. These results demonstrate that the use of topographic cues within a nerve guidance channel might offer the potential to influence the course of nerve regeneration to the advantage of a peripheral nerve interface suitable for limb amputees.PhDCommittee Chair: Ravi Bellamkonda; Committee Member: Arthur English; Committee Member: Pamela Bhatti; Committee Member: Robert Butera; Committee Member: Robert Le

Advance in Energy Harvesters/Nanogenerators and Self-Powered Sensors

This reprint is a collection of the Special Issue "Advance in Energy Harvesters/Nanogenerators and Self-Powered Sensors" published in Nanomaterials, which includes one editorial, six novel research articles and four review articles, showcasing the very recent advances in energy-harvesting and self-powered sensing technologies. With its broad coverage of innovations in transducing/sensing mechanisms, material and structural designs, system integration and applications, as well as the timely reviews of the progress in energy harvesting and self-powered sensing technologies, this reprint could give readers an excellent overview of the challenges, opportunities, advancements and development trends of this rapidly evolving field

Nuclear import mechanism of EGFR in breast cancer cells

Receptor tyrosine kinases (RTKs), such as epidermal growth factor receptor (EGFR) are internalised from the plasma membrane by endocytosis and may be transported to the nucleus. EGFR, a receptor for EGF and other RTKs, HER-2 and HER-4 has an important role in signalling; it contains transactivational activity and can function as a transcription co-factor to activate gene promoters. High nuclear accumulation of imported full length EGFR is associated with an increased tumour proliferation and a reduced survival in cancer patients. However, little is known about the mechanism by which membrane-bound proteins, such as EGFR, translocate from the cell surface into the cell nucleus; how nuclear membrane proteins cross through the NPC to reach the INM. The mechanism of translocation for soluble proteins is also presently unclear. EGFR nuclear import is mediated by importin α/β. And it is exported from the nucleus by the exportin CRM1. Sec61β which may reside in the inner nuclear membrane (INM) is required for the release of EGFR from the INM into the nucleus. Nuclear transport involves binding of nuclear localisation sequences (NLSs) within the cargo to a transport receptor (karyopherins or importin). Karyopherins interact with certain nuclear pore complex (NPC) proteins (nucleoporins). Membrane proteins can access the INM through the NPC membrane: by diffusion, using classical nuclear transport factors (the importin/Ran system); or by an ATP dependent mechanism. EGFR may use the former mechanism. This work concentrates to show by electron microscopy and by Immuno-Fluorescence that upon EGF treatment, the biotinylated cell surface EGFR is trafficked to the INM through the NPC, yet remaining a membrane-bound protein. We also confirm that importin regulates EGFR nuclear transport to the INM and in addition, Sec61β is required for EGFR release to the nucleoplasm. Altogether, this study of the mechanism of EGFR nuclear-cytoplasmic import in breast cancer cells, further confirms previous reports and provides an understanding of the nature and regulation of the nuclear EGFR pathway and the mechanism by which cell-surface EGFR is shuttled in the cell cytoplasm and channelled through the Golgi and Endoplasmic Reticulum (ER) compartments and into the nucleus through the NPC

Mechanical investigation of wall-less plant cells using microfluidics

Each cell within a plant experiences a myriad of mechanical and chemical cues that direct growth and development. Selective application of such cues and live-imaging of the resulting cellular responses are challenging within the tissue context. For this reason, I have developed a microfluidic device, called Ψ-trap, for mechano-chemical investigations of single cells, by combining microfluidic technology and automated live-imaging. The platform allows the microscopic time-course observation of individual plant cells within a heterogeneous population, such as leaf cells, while applying precise chemical or physical stimuli. As a pilot study I have quantitatively monitored the cellular expansion of cell wall-less plant cells, called protoplasts. I have further shown that the application of cyclic compression forces to single cells inside the Ψ-trap can be used to study the mechanical volume and shape control of single protoplasts from different developmental conditions. To separate the intracellular and extracellular factors that influence the cellular shape control, I have created a complementary microfluidic shape induction device, called the Ψ-constriction trap. My results suggest wall-less shape retainment upon mechanical compression in plant cells

Minimally invasive therapies for the brain using magnetic particles

Delivering a therapy with precision, while reducing off target effects is key to the success of any novel therapeutic intervention. This is of most relevance in the brain, where the preservation of surrounding healthy tissue is crucial in reducing the risk of cognitive impairment and improving patient prognosis. Our scientific understanding of the brain would also benefit from minimally invasive investigations of specific cell types so that they may be observed in their most natural physiological environment. Magnetic particles based techniques have the potential to deliver cellular precision in a minimally invasive manner. When inside the body, Magnetic particles can be actuated remotely using externally applied magnetic fields while their position can be detected non-invasively using MRI. The magnetic forces applied to the particles however, rapidly decline with increasing distance from the magnetic source. It is therefore critical to understand the amount of force needed for a particular application. The properties of the magnetic particle such as the size, shape and magnetic content, as well as the properties of the applied magnetic field, can then be tailored to that application. The aim of this thesis was to develop magnetic particle based techniques for precise manipulation of cells in the brain. Two different approaches were explored, utilising the versatile nature of magnetic actuation for two different applications. The first approach uses magnetic nanoparticles to mechanically stimulate a specific cell type. Magnetic particles conjugated with the antibody ACSA-1 would selectively bind to astrocytes to evoke the controlled release of ATP and induce a calcium flux which are used for communication with neighbouring cells. This approach allows for the investigation into the role of astrocytes in localised brain regions using a naturally occurring actuation process (mechanical force) without effecting their natural environment. The second approach uses a millimetre sized magnetic particle which can be navigated through the brain and ablate localised regions of cells using a magnetic resonance imaging system. The magnetic particle causes a distinct contrast in MRI images, allowing for precise detection of its location so that it may be iteratively guided along a pre-determined path to avoid eloquent brain regions. Once at the desired location, an alternating magnetic field can be applied causing the magnetic particle to heat and deliver controllable, well defined regions of cell death. The forces needed for cell stimulation are orders of magnitude less than the forces needed to guide particles through the brain. Chapters 4 and 5 use external magnets to deliver forces in the piconewton range. While stimulation was demonstrated in small animals, scaling up this technique to human proportions remains a challenge. Chapters 6 and 7 use a preclinical MRI system to generate forces in the millinewton range, allowing the particle to be moved several centimetres through the brain within a typical surgical timescale. When inside the scanner, an alternating magnetic field causes the particle to heat rapidly, enabling the potential for multiple ablations within a single surgery. For clinical translation of this technique, MRI scanners would require a dedicated propulsion gradient set and heating coil

Manipulation of global chromatin architecture in the human cell nucleus and critical assessment of current model views

In spite of strong evidence that the mammalian cell nucleus is a highly organized organelle, a consensus on basic principles of global nuclear architecture has not so far been achieved. The existence of major architectural features such as an organized interchromatin compartment and higher order organization of chromatin postulated by some of the models is questioned or even refused by the others. This study was set up to test predictions of the various model views after manipulating nuclear architecture by applying the induced formation of hypercondensed chromatin (HCC). This method leads to massive but completely reversible conformational changes of chromatin arrangements in living cell nuclei, but does not affect the cells survivability. Nuclear functions like transcription, replication and cell cycling were immediately stalled when HCC formation was induced, but were rapidly recovered upon recovery of normal chromatin configurations. The emerging pattern of HCC revealed a 3D network of interconnected chromosome territories. The surface of the emerging HCC bundles was the site of preceding activity like RNA transcription or DNA replication, which confirmed the existence of a distinct topological arrangement of functional processes with respect to the architecture of chromatin. This arrangement could further be demonstrated by analyzing the topography of defined chromatin modifications, showing that active chromatin is preferentially located at the HCC bundle surfaces, whereas inactive chromatin regions are preferentially found in the HCC bundle interior. The emerging patterns of HCC were further strikingly similar in consecutively repeated cycles of HCC formation and recovery, demonstrating a non-random but pre-existing and defined chromatin and interchromatin topography. All results of this study were obtained using confocal laser scanning microscopy. A protocol for deconvolution of confocal images was established to enhance confocal image quality to an extent sufficient for subsequent image analysis. In contribution to the present model views this study demonstrates: [1] That most chromatin exists in the form of higher-order sub-compartments ('~1 Mb chromatin domains') above the level of extended 30 nm fibers and [2] That an interchromatin compartment exists as a dynamic, structurally distinct nuclear compartment, which is functionally linked with the chromatin compartment. An updated chromosome territory-interchromatin compartment model on the basis of the gained results is presented at the end of this thesis together with an attempt to provide a comprehensive view linking ultrastructural with light microscopic insights

Rakennemuunnosten ja kantajien käyttö oligonukleotidien saattamisessa vaikutuskohteeseensa : Synteesi, analyysi ja biologinen testaus

Several serious diseases remain without non-toxic curative treatments. To fill this void, one of the promising groups of medicines is that of oligonucleotides, encompassing aptamers, transcription factor decoys, and antisense therapeutics such as short interfering RNA and splice-correcting oligonucleotides. These short strands of DNA or RNA can bind to specific cellular nucleic acids or proteins and thereby inhibit or correct the function of disease-causing molecules. Extensive enzymatic degradation and poor cellular uptake are the most important obstacles for systemic oligonucleotide therapy. Numerous chemical modifications have been introduced to improve enzymatic stability, but they must be carefully optimized to avoid toxicity and to maintain target affinity. One solution is to design topological modifications, such as looped or circular oligonucleotides, which conserve the natural phosphodiester backbone but cannot be attacked by exonucleases. Cellular uptake has proven to be even more challenging. Oligonucleotides are internalized into cells by endocytosis, after which they often remain trapped in endosomes. Therefore, it would be advantageous to develop delivery vectors capable of bypassing endocytic routes of uptake or enhancing endosomal escape. Cell-penetrating peptides, for example, exploit several mechanisms of uptake, some of which lead to rapid entry without endosomal localization. In addition, encouraging results have been achieved using liposomes, gold nanoparticles, and other nanocarriers, which also shield the oligonucleotide from degrading enzymes. The aim of this work was to improve the in vitro delivery of oligonucleotides by employing chemical modifications and nanoparticle carriers. The synthesis of the compounds, their characterization by various analytical methods, and the evaluation of biological effects are described. Antisense oligonucleotides covalently linked to cell-penetrating peptides via convergent conjugation displayed improved cellular uptake but failed to inhibit reporter genes due to endosomal entrapment in cells. Circular oligonucleotides exhibited enhanced selectivity of mismatch detection and increased stability in biological fluids compared to linear oligonucleotides. Altogether 44 compounds were analyzed by electrospray ionization mass spectrometry and liquid chromatography mass spectrometry, which were found to be excellent methods for the characterization of modified oligonucleotides. Finally, we synthesized cationic gold nanoparticles modified with a Tat-related peptide, which did not adversely affect cell viability and effectively delivered short interfering RNA into cells as non-covalent complex.Monet parantumattomat sairaudet johtuvat haitallisten proteiinien syntymisestä elimistössä. Näiden proteiinien toimintaa pyritään estämään yleisimmin perinteisillä pienimolekyylisillä lääkeaineilla. Vielä tehokkaampi ja turvallisempi vaikutus voidaan saada estämällä haitallisten proteiinien syntyminen jo RNA-tasolla esimerkiksi oligonukleotidilääkkeillä. Nämä lyhyet nukleiinihappomolekyylit sitoutuvat spesifisesti kohde-RNA:han ja voivat hajottaa sen useilla eri mekanismeilla. Ne voivat myös korjata RNA:n viallista prosessointia tai sitoutua suoraan kohdeproteiiniin. Oligonukleotideja tutkitaan muun muassa syöpien ja parantumattomien geneettisten sairauksien hoitoa varten. Tähän mennessä vain yksi ei-paikallinen oligonukleotidilääke on hyväksytty kliiniseen käyttöön Yhdysvalloissa, mutta toksisuuden takia sen käyttö on tarkoin rajattu eikä sitä ole hyväksytty Euroopassa. Ongelmana hoitojen kehittämisessä on oligonukleotidien entsymaattinen hajoaminen elimistössä sekä niiden huono pääsy solujen sisään. Oligonukleotideja on muunneltu entsyyminkestäviksi muokkaamalla niiden kemiallista rakennetta, mutta tällöin toksisuus voi lisääntyä ja tehokkuus heikentyä. Oligonukleotidi voidaan myös naamioida entsyymeiltä muuttamatta luonnollista nukleotidirankaa, jos se syklisoidaan liittämällä nukleotidiketjujen päät toisiinsa. Kolmas vaihtoehto on liittää oligonukleotidi sopivaan kantajamolekyyliin, jonka läsnäolo suojaa oligonukleotidia entsyymeiltä ja lisäksi auttaa sen vaikutuspaikalleen solun sisään. Lupaavia kantajia ovat muun muassa liposomit, polymeeri- ja kultananopartikkelit sekä soluun penetroituvat peptidit, joista viimeksi mainitut on löydetty tutkittaessa virusten tehokasta pääsyä soluihin. Näiden peptidien toivotaan pystyvän ohittamaan endosytoosireitin, jonka kautta soluun kulkiessaan oligonukleotidi voi jäädä loukkuun endosomiin. Tässä väitöskirjatyössä tutkittiin kemiallisten rakennemuunnosten ja nanopartikkelikantajien mahdollisuuksia oligonukleotidien saattamisessa soluihin. Osatöissä paitsi tutkittiin muunnosten ja kantajien biologisia vaikutuksia, myös optimoitiin synteesi- ja analyysimenetelmiä. Kovalentisti liitetyt soluun penetroituvat peptidit paransivat oligonukleotidien soluunottoa, mutta antisense-vaikutusta ei silti nähty soluissa yhdisteiden juututtua endosomeihin. DNA-diagnostiikkaa varten valmistetut sykliset oligonukleotidit osoittautuivat huomattavasti lineaarisia oligonukleotideja selektiivisemmiksi emäsmutaatioiden havaitsemisessa, ja myös niiden entsyyminkestävyys oli parempi. Yhteensä 44 yhdistettä analysoitiin sähkösumutusmassaspektrometrialla ja nestekromatografia massaspektrometrialla, jotka sopivat erinomaisesti muunneltujen oligonukleotidien analysointiin. Viimeisessä osatyössä syntetisoitiin kationisia kultananopartikkeleita, joihin lisättiin peptidianalogi. Tähän kantajaan kompleksoidulla pienellä häiritsevällä RNA:lla (siRNA:lla) hiljennettiin rekombinanttisolujen ilmentämä reportterigeeni heikentämättä solujen elävyyttä

Biointerfaces based on the combination of synthetic polymers and biomolecules

Alicat embargament des de la defensa de la tesi fins al 31 de desembre de 2019Premi Extraordinari de Doctorat, promoció 2018-2019. Àmbit d’Enginyeria IndustrialDuring the last decades, research focused on the preparation of highly selective and smart materials has increased considerably. For instances, it has been possible to achieve intelligent drug nano-carriers, biomolecular sensors, platforms to promote cell growth and differentiation among many other striking applications. Two mentionable factors that helped such development are the incorporation of biological moieties onto this interfaces to gain specificity and the combination of more than one material in order to get a synergistic effect between the different components (i.e. conducting polymers suffer from poor mechanical strength, therefore its combination with polyesters can reduce their fragility). This thesis has been devoted to the design and development of high performance polymeric materials for multiple functions related to the biomedical field, such as passive ion transport membranes, drug delivery systems and the addition of selectivity in different surfaces. The work gives special emphasis to the characterization of these platforms, like its surface chemistry, topology, biocompatibility or its mechanical strength. Besides, these systems have been synthetized in a large variety of shapes, from free-standing nanomembranes to polymeric nanoparticles. The Thesis is divided in three blocs: Bloc A encloses all the studies realized for the generation of hybrid nanoperforated membranes in order to achieve controlled ion diffusion. Specifically, an outer membrane protein, Omp2a, was considered for these studies. Primarily, the protein was purified, folded and characterized in an ambient resembling to the one encountered in nature, its mechanical forces and conductivity were analysed. The project was followed by the immobilization of Omp2a into silicon microcantillevers to acquire greater knowledge of its folding and unfolding processes upon thermal stress. Next, artificial polymeric membranes containing nanofeatures were developed with the final purpose to immobilize Omp2a via protein confinement. Then, the conductivity of the membrane with different electrolyte media solutions was tested. Bloc B describes the state-of-the art of drug delivery systems prepared with intrinsically conducting polymers to achieve controlled drug release upon electrical stimuli. Furthermore, two systems based on poly(3,4-ethylenedioxythiophene) (PEDOT) nanoparticles are described. Particularly, curcumin was employed as a model neutral drug and incorporated within the PEDOT nanoparticles. The oxidation state of the PEDOT chains regulated the drug release. Later on, a similar system was generated with polyester microfibers loaded with curcumin and nanoparticles. The driving force for the later drug release was the actuation of the PEDOT nanoparticles. Lastly, Bloc C reports the immobilization of a pentapeptide called CREKA and its analog CR(NMe)EKA onto PEDOT and silicon surfaces. The addition of CREKA favoured the selectivity of those interfaces towards clotted plasma proteins such as fibrin and fibrinogen. PEDOT-peptide material allowed the electrochemical detection of the proteins by an increase in membrane resistance and these interactions were evaluated with microcantilevers by measuring the difference on weight when they were incubated with different protein concentrations. Overall, the compilation of the studies presented in this Thesis offer a comprehensive view on how modifying and generating hybrid materials is possible to optimize and exploit their capabilities for a wide range of applications.Durant les últimes dècades, la recerca centrada en la preparació de materials altament selectius i intel·ligents ha augmentat considerablement. Ha estat possible aconseguir nano-contenidors de fàrmacs, sensors de biomolècules, plataformes per promoure el creixement i la diferenciació cel·lular, entre moltes altres aplicacions interessants. Dos factors destacables que han ajudat aquest desenvolupament són la incorporació de cues biològiques en aquets materials per obtenir especificitat i la combinació de més d'un element per obtenir un efecte sinèrgic entre els diferents components (per exemple els polímers conductors pateixen d’una baixa resistència mecànica, per tant, la seva combinació amb polièsters pot reduir la seva fragilitat però seguir mantenint les seves propietats elèctriques). En resum, aquesta tesi s'ha centrat en el disseny i desenvolupament de materials polimèrics d'alt rendiment per a múltiples funcions relacionades amb el camp biomèdic, com ara membranes passives de transport iònic, sistemes de lliurament de fàrmacs i l'addició de selectivitat envers proteïnes del plasma en diferents superfícies. El treball fa especial èmfasi en la caracterització d'aquestes plataformes, com la seva química superficial, topologia, biocompatibilitat o resistència mecànica. A més, aquests sistemes s'han sintetitzat en una gran varietat de formes, des de films fins a nanopartícules polimèriques. La tesi es divideix en tres blocs: El bloc A inclou tots els estudis realitzats per a la generació de membranes híbrides nanoperforades amb la finalitat d’aconseguir una difusió controlada de ions. Concretament en aquests estudis es va emprar una proteïna transmembrana anomenada Omp2a. La primera etapa del treball es centra en la purificació, plegament i caracterització de la proteïna en un ambient similar al que es troba originàriament. A més a més, es van analitzar les seves forces mecàniques i de conductivitat. Seguidament, es va procedir a la immobilització d'Omp2a en microcantillevers de silici per adquirir un major coneixement sobre els seus processos de plegament i desplegament depenent de l'estrès tèrmic. Finalment, es van desenvolupar membranes polimèriques artificials amb nanoperforacions amb l'objectiu d'immobilitzar Omp2a a través del confinament de la proteïna en aquests porus. El Bloc B descriu l'estat de l’art dels sistemes d’alliberament controlat de fàrmacs, preparats amb polímers intrínsecament conductors, depenent d’estímuls elèctrics. En aquest apartat, es descriuen dos sistemes basats en nanopartícules de poli(3,4-etilendioxitiofé) (PEDOT). En el primer cas, l'estat d'oxidació de les cadenes PEDOT és el responsable de regular l'alliberament del medicament. En canvi, en el segon, on es va generar un sistema similar amb microfibres de polièster carregades de droga i nanopartícules per separat, la força motriu de l'alliberament del fàrmac és el moviment d’expansió i contracció de les nanopartícules PEDOT. Finalment, el Bloc C informa de la immobilització d'un pentapèptid anomenat CREKA i el seu anàleg CR(NMe)EKA en films de PEDOT i superfícies de silici. La incorporació de CREKA afavoreix la selectivitat d'aquestes interfícies cap a les proteïnes de coagulació del plasma com la fibrina i el fibrinogen. El material pèptid-PEDOT va permetre la detecció electroquímica de les proteïnes mitjançant un augment de la resistència a la membrana i aquestes interaccions van ser avaluades amb microcantilevers, concretament, mesurant la diferència de pes quan es van incubar amb diferents concentracions de proteïnes. En general, la recopilació d’aquets estudis ofereix una visió completa sobre com modificant i generant materials híbrids és possible optimitzar i explotar les seves capacitats particulars per a una àmplia gamma d'aplicacions.Award-winningPostprint (published version

The Regulation Of Egfr Signaling And Kras Tumorigenesis By Receptor Palmitoylation

Non-Small Cell Lung Cancer (NSCLC) is often characterized by mutually exclusive mutations in epidermal growth factor receptor (EGFR) or KRAS. The mutual exclusivity of these mutations is due to synthetic lethality, revealing a potential therapeutic vulnerability if possible to selectively activate EGFR in KRAS mutant cells. This thesis work demonstrates a previously unidentified mechanism of EGFR signal regulation through palmitoylation, the addition of the 16-carbon palmitate. The palmitoyltransferase, DHHC20, catalyzes this palmitoylation to Cys1025, Cys1122 and Cys1034 on the C-terminal tail of EGFR. Loss of EGFR palmitoylation leads to hyperactivation of the receptor, but decreased cell growth of KRAS mutant cancer cells. While KRAS is still an elusive therapeutic target, here we report that disrupting EGFR palmitoylation by ablation of DHHC20 or expression of a palmitoylation-resistant EGFR mutant blocks tumorigenesis in a KRAS-driven mouse model of lung adenocarcinoma. Mechanistically, we show that in the presence of oncogenic KRAS, unpalmitoylated, active EGFR increases signaling through the MAP Kinase pathway while simultaneously reducing PI3K/AKT signaling leading to a severe decrease in expression of the central proliferation-associated transcription factor, Myc, similarly as impossible to therapeutically target as KRAS. We find that the dysregulation of EGFR palmitoylation from DHHC20 loss disrupts the delicate balance of MAPK and PI3K signaling leading to detrimental loss of Myc expression and subsequent loss of cell growth. Initially, we discovered that inhibiting EGFR palmitoylation increases sensitivity to the EGFR tyrosine kinase inhibitor, gefitinib, in cell lines specifically harboring mutant KRAS and interestingly, in cells harboring the drug-resistant EGFR gatekeeper mutation through a mechanism that is still unclear. We have now determined that inhibition of DHHC20 induces sensitivity of KRAS mutant cells to a clinically available pan-PI3K inhibitor, Buparlisib, more effective than gefitinib in inducing cell death by directly blocking the residual, necessary PI3K signaling. Thus, this previously unappreciated mechanism of receptor signaling modulation driven by the palmitoyltransferase, DHHC20, can be exploited to treat the currently incurable mutant KRAS NSCLCs