Neurabin, a cytoskeletal protein, has been shown to be required for normal dopamine signalling, and dopaminergic systems have been previously implicated in the pathophysiology of anxiety disorders, including generalized social anxiety disorder. And results from neuroimaging studies have implicated the anterior cingulate cortex (ACC) in depression and anxiety disorders. However, lesion studies have failed to produce the expected deficits. Here, we demonstrate that the injections of muscimol and midazolam into the ACC reduced anxiety- and depression-like behaviours, and that complete absence of neurabin reduced anxiety-like behaviour but increased depression-like behaviour. However, reduction of neurabin by injecting neurabin-targeted siRNA into the ACC reduced anxiety-like behaviour but did not affect depression-like behaviour. This study provides evidence that the imbalance of excitatory and inhibitory activity in the ACC alters affective disorders, and that neurabin may be critical for the modulation of these behaviours.

<p>This project is predicated on the ability of the boranophosphate modification of siRNA to increase its therapeutic applicability for gene silencing in in vitro and in vivo systems. It has been shown that the boranophosphate (BH3-PO3) can overcome many of the limitations that are traditionally found when using RNAi, namely nuclease stability. The synthesis of siRNA modified with 5'-(alpha-P-borano)-nucleoside triphosphates (NTP) analogs alone and in combination with 2'-deoxy-2'-fluoro nucleoside triphosphate analogs were performed and optimized. It was found that normal RNA transcriptions showed the highest yield with higher NTP concentrations and shorter incubation times. Boranophosphate modified RNA and 2'F/borano modified RNA transcription yield was optimal at lower NTP concentrations and extended incubations. The boranophosphate NTPs and RNA were characterized with high performance liquid chromatography, mass spectrometry, and nuclear magnetic resonance, indicating successful synthesis of NTPalphaB and 2'F NTPs. PAGE and mass spectrometry analysis were performed to ensure full-length transcription of the modified siRNA molecules. The effects of these modifications were explored with respect to the biophysical properties of the modified homoduplex and heteroduplex siRNA. The techniques used in this work included hybridization affinity assays (melting temperature), secondary structure determination (circular dichroism), nuclease stability assays, and assessment of the lipophilicity of the modified siRNA by determining partition coefficients. </p><p>Modification of siRNA with boranophosphate and 2'fluoro/borano modified NTPs appears to have caused the homoduplexes and heteroduplexes to adopt a more B form-like helix that had lower Tm compared to unmodified RNA. The stability of the siRNA transcript to enzymatic hydrolysis by Exonuclease T was on the order of 2'fluoro/borano> normal = boranophosphate. Boranophosphate modification increased the stability of the transcript to enzymatic hydrolysis by the endonuclease RNase A, compared to both normal and 2' fluoro modified siRNA. Overall, the 2' fluoro/borano modified siRNA showed the greatest biological stability. Modification of the siRNA with increasing percentages of boranophosphates resulted in increasing lipophilicity of the molecule up to 60-fold, compared to both normal and 2' fluoro RNA. </p><p>A method to site-specifically modify the boranophosphate siRNA using T4 RNA ligase was also investigated. Finally, the siRNA in this work was tested in several in vitro systems, yielding promising results for the usage of boranophosphate siRNA for use against human viruses and cancers. It was shown that in for in vitro systems for human papillomavirus gene expression (HeLa, SiHa, and W12E) and luciferase expression (B16F10 cells), boranophosphate modified siRNA can specifically downregulate gene expression, and in the case of human papillomavirus, can downregulate cell growth.</p> / Dissertation

RNA interference (RNAi) is a conservative post-transcriptional gene silencing mechanism that can be mediated by small interfering RNAs (siRNAs). Given the effectiveness and specificity of RNAi, the administration of siRNA molecules is a promising approach to cure diseases caused by abnormal gene expression. However, as siRNA is susceptible to degradation by nucleases and it can hardly penetrate cell membranes due to its polyanionic nature, a successful translation of the RNAi mechanism for therapeutic purposes is contingent on the development of safe and efficient delivery systems. This dissertation described the development of novel siRNA delivery systems on the basis of polymeric and dendrimeric materials and also demonstrated the application of one optimized delivery system to deliver therapeutic siRNAs in a cardiovascular disease model in vivo. We studied a linear peptide polymer made from cell penetrating peptide monomers and investigated the contribution of the polymeric structure, degradability, and ligand conjugation to the siRNA loading capacity, biocompatibility, and transfection efficiency of polymeric materials. With the obtained knowledge and experience, we invented a neutral crosslinked delivery system aiming to solve the inherent drawbacks of traditional cationic delivery systems that are based on electrostatic interactions. The new concept utilized buffering amines to temporarily bind siRNA and a crosslinking reaction to immobilize the formed particles, and targeting ligands modified on the neutral dendrimer surface further enhanced the interactions between the delivery vehicles and target cells. The obtained delivery system allowed stability, safety, controllability, and targeting ability for siRNA delivery, and the method developed here could be transformed to other polymeric or dendrimeric cationic materials to make them safer and more efficient. To exploit the therapeutic potential of siRNA delivery, we developed a tadpole-shaped dendrimeric material to deliver siRNA against an Angiotensin II receptor in a rat ischemia-reperfusion model. Our results showed that the nonaarginine-conjugated tadpole dendrimer was capable of delivering siRNA effectively to cardiac cells both in vitro and in vivo, and the successful down-regulation of the Angiotensin II receptor preserved the cardiac functions and reduced the infarct size post-myocardial infarction. This dissertation paves a way for transforming multifunctional non-viral siRNA delivery systems into potent therapeutic strategies for the management of cardiovascular diseases.

The rational and directed delivery of genetic material to the cell is a formidable tool to investigate the phenotypic effects of gene expression regulation and a promising therapeutic strategy for genetic defects. RNA interference constitutes a versatile approach to gene silencing. Despite the development of numerous strategies the transfection of small interfering RNA (siRNA) is highly dependent on cell type and conditions. Direct physical access to the intracellular compartment is a promising path for high efficiency delivery independently of cell type and conditions. Silicon nanowires grant such access with minimal toxic effects, and allow intracellular delivery of DNA when actuated by atomic force microscope. These findings reveal the potential for porous silicon nanostructures to serve as delivery vectors for nucleic acids due to their porous nature, elevated biocompatibility, and biodegradability. This dissertation illustrates the development a novel platform for efficient siRNA transfection based on an array of porous silicon nanoneedles. The synthesis of biodegradable and biocompatible porous nanowires was accomplished by a novel strategy for electroless etch of silicon that allows anisotropic etch simultaneously with porosification. An ordered array of cone shaped porous silicon nanoneedles with tunable tip size, array density and aspect ratio was obtained coupling this strategy with patterned metal deposition and selective reactive ion etch. This process also granted control over porosity, nanopore size and flexural modulus. The combination of these parameters was appropriately optimized to ensure cell penetration, maximize siRNA loading and minimize cytotoxic effects. Loading of the negatively charged siRNA molecules was optimized by applying an external electric field to the nanoneedles under appropriate voltage conditions to obtain a tenfold increase over open circuit loading, and efficient penetration of the siRNA within the porous volume of the needles. Alternative surface chemistry modification provided a means for effective siRNA loading and sustained release. siRNA transfection was achieved by either imprinting the nanoneedles array chip over a culture of MDA-MB-231 cells or allowing the cells to self-impale over the needles. The procedures allowed the needles to penetrate across the cell membrane without influencing cell proliferation. siRNA was successfully transfected and was effective at suppressing gene expression. / text

Polycationic nanoparticles were synthesized with an activators regenerated by electron transfer for atom transfer radical polymerization-based (ARGET ATRP-based) emulsion in water method and investigated for their utility as biomaterials for drug delivery. The polycationic nanoparticles were composed of 2-(diethylamino)ethyl methacrylate (DEAEMA) for pH-responsiveness, poly(ethylene glycol) methyl ether methacrylate (PEGMA) for improved biocompatibility, tert-butyl methacrylate (tBMA) to impart hydrophobicity, and a tetraethylene glycol dimethacrylate (TEGDMA) cross-linking agent for enhanced colloidal stability. Dynamic light scattering demonstrated pH-responsive swelling, and cell-based assays demonstrated pH-dependent membrane disruption. The polycationic nanoparticles demonstrated low toxicity to cells. The polycationic nanoparticles were evaluated for use as drug delivery biomaterials by investigating the interactions with the drug and cells. Delivery remains a major challenge for translating small interfering RNA (siRNA) to the clinic, and overcoming the delivery challenge requires effective siRNA delivery vehicles. The polycationic nanoparticles demonstrated efficient siRNA loading. Evidence of siRNA-induced knockdown in cells was observed following transfection with the polycationic nanoparticle/siRNA complexes. Imaging techniques confirmed enhanced siRNA internalization using the polycationic nanoparticle/siRNA complexes compared to naked siRNA. An array of polycationic nanoparticles synthesized using ARGET ATRP or UV-initiated polymerization methods was characterized to examine the effect of polymerization method on material properties and the connection to molecular structure. An improved understanding of molecular structure, and its connection to polymerization method and material characteristics, may aid the design of advanced materials. The ARGET ATRP polycationic nanoparticles demonstrated increased nanoscale homogeneity compared to the UV-initiated polymerization polycationic nanoparticles; increased nanoscale heterogeneity in the UV-initiated polymerization polycationic nanoparticles was associated with broader transitions. The polycationic nanoparticles promoted cellular uptake of siRNA and induced knockdown, thus demonstrating potential as siRNA delivery vehicles. The ARGET ATRP method provides an alternative route to creating polycationic nanoparticles with improved nanoscale homogeneity. / text

Die rheumatoide Arthritis (RA) ist eine Autoimmunerkrankung mit bevorzugter Manifestation an den Gelenken. Die RA ist charakterisiert durch eine chronische, systemische Entzündung, eine abnormale zelluläre und humorale Immunantwort und eine synoviale Hyperplasie. Die Ursache der synovialen Hyperplasie ist noch nicht eindeutig geklärt, aber es wird eine veränderte oder unvollständig ablaufende Apoptose der synovialen Fibroblasten vermutet. Die vorliegende Arbeit untersucht die Rolle des Apoptosemodulators death associated protein (Daxx) in rheumatoiden synovialen Fibroblasten (RA-SF). Als erstes wurde die Expression von Daxx in RA-SF gegenüber Osteoarthrosefibroblasten (OA-SF) untersucht. Dabei konnte gezeigt werden, dass die OA-SF eine höhere Expression von Daxx auf mRNA und Proteinebene gegenüber RA-SF aufweisen. Im weiteren Verlauf erfolgte die Untersuchung der subzellulären Lokalisation von Daxx in RA-SF mittels konfokaler Laserscan-Mikroskopie (Immunfluoreszenz). Dabei zeigte sich, dass Daxx vorwiegend im Zellkern und nur zu einem geringen Anteil im Zytoplasma lokalisiert ist. Weiterhin zeigte Daxx eine Kolokalisation mit dem Promyeloischen Leukämie Protein (PML), welches ausschließlich im Zellkern lokalisiert ist. Nun erfolgten die Untersuchungen zur Rolle von Daxx in der Apoptose von RA-SF mittels RNA Interferenz (RNAi). Zu diesem Zweck wurden 3 verschiedene small interfering RNA (siRNA) synthetisiert, die unterschiedliche Abschnitte auf der mRNA von Daxx umfassen. Die Überprüfung der Effektivität der siRNA erfolgte auf mRNA Ebene mittels Quantitativer Real Time PCR(TaqMan®) und auf Proteinebene im Westernblot in HeLa-Zellen und RA-SF. Dabei zeigte die siRNA(454-472) die stärkste Hemmung. In den Untersuchungen zur Apoptose in HeLa-Zellen und RA-SF, nach der Hemmung von Daxx durch die siRNA, zeigte sich, dass eine stärkere Hemmung von Daxx zu einer verminderten Empfindlichkeit der HeLa-Zellen und RA-SF auf die FasL-induzierte Apoptose führt. Die RA-SF und Makrophagen der Deckzellschicht synthetisieren Zytokine wie den Tumor Nekrose Faktor a (TNFa). Diese Erkenntnis dient als Grundlage zur Untersuchung des Einflusses von TNFa auf die Expression von Daxx auf mRNA- und Proteinebene. Dabei zeigte sich eine konzentrationsabhängige Erhöhung der Expression von Daxx. Als letztes erfolgte die Untersuchung zur Apoptose nach der TNFa Stimulation. Hierbei zeigte sich eine Reduktion der Apoptose, die von der TNFa Konzentration abhängig war, wobei sich hohe Schwankungsbreiten bei der Konzentration von 10ng/ml TNFa zeigten. In dieser Arbeit wurde durch die Hemmung von Daxx mittels siRNA eine pro-apoptotische Funktion des Moleküls in RA-SF nachgewiesen. Diese Ergebnisse sollen der weiteren Identifizierung von Signalwegen in der Apoptose bei RA-SF dienen.

Colon cancer is the third most common type of cancer, with high mortality throughout the world. During tumorigenesis, normal cells transform into tumour cells following changes in the expression of oncogenes and/or tumour suppressor genes, which are involved in many processes including the cell cycle, differentiation and apoptosis. An imbalance in the regulation of proliferation and differentiation in colon epithelial cells is usually associated with the development of colon cancers. Uncontrolled proliferation with a lack of differentiation is one of the major characteristic features of cancer cells and a remodelling of the Ca2+ signalling is linked to these pathways. Among the Ca2+ transporting proteins, P-type Ca2+-ATPases, the plasma membrane Ca2+ ATPase (PMCA) pump, has a high-affinity for Ca2+ and is involved in the efflux of Ca2+ against the electrochemical gradient from the cytosol across the extracellular space. Four PMCA isoforms have been identified. PMCA1 and 4 are expressed in most tissues. Changes in the expression of PMCA have been documented in breast cancer cells, whereas the expression profile of PMCA isoforms in colon cancer cells remains unknown. Up-regulation of another P-type Ca2+-ATPase, expressed in the endoplasmic reticulum, SERCA3, occurs during the differentiation of colon cancer cell lines and is down-regulated in colon cancers. Changes in PMCA expression have not been assessed during colon cancer cell differentiation. The first part of this thesis describes the analysis of the expression profile of PMCA during colon cancer cell differentiation. Both PMCA mRNA and protein levels were assessed in differentiated HT-29 cells by real time RT-PCR and western blotting analysis, respectively. The results showed changes in PMCA4 expression, whereas changes in the expression of PMCA1 were not associated with differentiation of HT-29 cells. PMCA mRNA levels were also reduced in some colon cancers suggesting a remodelling of PMCA-mediated Ca2+ efflux during colon carcinogenesis. The second part of this thesis involved exploring the functional role of PMCA4 in Ca2+-mediated signalling pathways such as differentiation, proliferation and apoptosis. PMCA4 expression was altered in HT-29 colon cancer cells via transient and stable over-expression of a PMCA4 expressing plasmid or siRNA-mediated silencing of PMCA4. An increase in the PMCA4 level did not alter or induce differentiation of HT-29 cells. Hence, up-regulation of PMCA4 expression may be a consequence rather than a cause of HT-29 colon cancer cell differentiation. PMCA4-mediated reduction in proliferation was observed in HT-29 colon cancer cells where PMCA4 was stably over-expressed. Stable PMCA4 over-expression was also associated with the down-regulation of the transcription of the early response gene, FOS. Despite the apparent augmentation of cytosolic Ca2+ responses to G-protein coupled receptor Ca2+ mobilizing agents, the sensitivity of cells to the apoptotic inducing agents such as TRAIL and/or CCCP was not affected following siRNA-mediated PMCA4 inhibition in HT-29 cells. Collectively this thesis describes PMCA isoform-specific changes during differentiation of HT-29 colon cancer cells and alterations in PMCA levels in some colon cancers.Evidence is also presented to suggest that alterations in PMCA expression in colon cancer cells may provide a growth advantage by promoting proliferation without increasing sensitivity to apoptotic stimuli.

Many biopharmaceuticals, already approved for sale or currently under development, are post-translationally modified proteins, such as recombinant monoclonal antibodies or recombinant hormones. These are generally expressed in continuous (stable) mammalian cell lines, which are capable of long-term, commercial-scale production of recombinant proteins of the highest complexity. Yet, the development of a stable cell line capable of expressing heterologous proteins is very costly and can take up to 9–15 months. Therefore, transient gene expression (TGE) in animal cells has become the method of choice for many researchers who wish to obtain small to moderate quantities (1-500 mg) of novel complex recombinant proteins for further functional and structural characterisation within weeks of cDNA discovery. TGE is more cost-effective than the time-consuming establishment of stable cell clones, but a key factor in ensuring that these transient systems have practical application is the availability of efficient and robust transfection agents/methods. While chemical transfection methods currently dominate transient systems, the underlying fundamentals such as the formation of DNA complexes or their mode of function are not fully understood and the characteristics of the complexes and their subsequent ability to transfect cells are variable. This often renders the development of a successful transfection protocol for a new cell line random and researchers frequently have to resort to a trial-and-error approach, testing different media and/or conditions during DNA complex formation, as well as having to fine-tune the cell culture regime pre-, during, and post-transfection. This thesis aimed to explore novel transfection agents and develop DNA complex structure/property—transfection efficiency relationships for these reagents. Two different chemical approaches to transient transfection were investigated: i) a recently suggested inorganic nanoparticle based transfection system which utilises the anion exchange capacity of nanoparticles of a particular family of anionic clays, layered double hydroxides (LDHs), and ii) a modified polyethyleneimine (PEI)-based system, which aimed to reduce the inherent cytotoxicity of high molecular weight (MW) PEI, which is a very effective transfection agent, by constructing high MW mimics from low MW building blocks that are linked to each other via biodegradable linkers such as azomethine groups. While the LDH nanoparticles failed to give satisfactory transfection results for plasmid DNA, they were able to functionally deliver smaller nucleic acids such as siRNA. A mechanism different to that currently accepted for the transfection of mammalian cells with plasmid DNA using LDH nanoparticles as carriers is proposed. The modified polymeric transfection agents were shown to result in significantly less cell death, while maintaining the ability to transfect mammalian cells with almost similar efficiency to that obtained with high MW polyethyleneimine. Generic DNA complex structure/property—transfection efficiency relationships were developed by systematically studying the influence of particle size and zeta potential on transfection results.

La mélatonine est une hormone synthétisée et sécrétée uniquement la nuit par la glande pinéale. Son rôle principal est son implication dans la synchronisation de la saison de reproduction. La mélatonine se lie aux récepteurs, MT1 et MT,, membres de la famille des récepteurs à sept domaines transmembranaires couplés aux protéines G (RCPG).Le clonage récent du récepteur ovin MT2a remis en cause toutes les données connues. La pharmacologie et les voies de signalisation du récepteur oMT2ont été étudiées et sont communes à celles des récepteurs des autres espèces. En revanche, oMT2possède une originalité de séquence avec la présence du motif DRY, fortement impliqué dans l’établissement de l’activité constitutive des RCPG. D’une part, nous avons montré que l’ensemble des récepteurs MT possèdent une activité constitutive. D’autre part, nous avons identifié deux agonistes inverses pour les récepteurs hMT2, initialement décrits comme antagonistes. Dans l’optique de discriminer les rôles respectifs des récepteurs MT in vivo, le développement d’une approche par ARN interférents a été validée dans un modèle cellulaire, la lignée CHO-KI exprimant les récepteurs ovins et de rat. / Melatonin is a hormone synthesized and secreted only during night by pineal gland. A main role of melatoninconcerns its implication in the synchronization of reproductive seasonality. Binding sites of melatonin are MT1and MT2 receptors which belong to the superfamily of seven-transmembrane-spanning G protein-coupledreceptors (GPCRs).Recent cloning of ovine MT2 receptor has challenged the knowledge about melatonin receptors. Wedemonstrated that its pharmacology and signalling pathways were similar to subtype 2 receptor of othersspecies (human and rat). Nevertheless, oMT2 receptor possesses a particularity of sequence, with the presenceof DRY motif which is known to be involved in the establishment of constitutive activity of GPCRs. In ourstudy, we demonstrated the existence of constitutive activity for ail the melatonin receptors. In addition, weidentified two inverse agonists for human MT2 receptors, previously described as antagonists. To describe therespective roles of each subtype of melatonin receptors in vivo, siRNA approach was developed in cell line,CI-10-K Iexpressing ovine and rat melatonin receptors

abstract: Gene manipulation techniques, such as RNA interference (RNAi), offer a powerful method for elucidating gene function and discovery of novel therapeutic targets in a high-throughput fashion. In addition, RNAi is rapidly being adopted for treatment of neurological disorders, such as Alzheimer's disease (AD), Parkinson's disease, etc. However, a major challenge in both of the aforementioned applications is the efficient delivery of siRNA molecules, plasmids or transcription factors to primary cells such as neurons. A majority of the current non-viral techniques, including chemical transfection, bulk electroporation and sonoporation fail to deliver with adequate efficiencies and the required spatial and temporal control. In this study, a novel optically transparent biochip is presented that can (a) transfect populations of primary and secondary cells in 2D culture (b) readily scale to realize high-throughput transfections using microscale electroporation and (c) transfect targeted cells in culture with spatial and temporal control. In this study, delivery of genetic payloads of different sizes and molecular characteristics, such as GFP plasmids and siRNA molecules, to precisely targeted locations in primary hippocampal and HeLa cell cultures is demonstrated. In addition to spatio-temporally controlled transfection, the biochip also allowed simultaneous assessment of a) electrical activity of neurons, b) specific proteins using fluorescent immunohistochemistry, and c) sub-cellular structures. Functional silencing of GAPDH in HeLa cells using siRNA demonstrated a 52% reduction in the GAPDH levels. In situ assessment of actin filaments post electroporation indicated a sustained disruption in actin filaments in electroporated cells for up to two hours. Assessment of neural spike activity pre- and post-electroporation indicated a varying response to electroporation. The microarray based nature of the biochip enables multiple independent experiments on the same culture, thereby decreasing culture-to-culture variability, increasing experimental throughput and allowing cell-cell interaction studies. Further development of this technology will provide a cost-effective platform for performing high-throughput genetic screens. / Dissertation/Thesis / Ph.D. Bioengineering 2012