Development and characterization of self-assembling nanoparticles using a bio-inspired amphipathic peptide for gene delivery

The design of a non-viral gene delivery vehicle capable of delivering and releasing a functional nucleic acid cargo intracellularly remains a formidable challenge. For systemic gene therapy to be successful a delivery vehicle is required that protects the nucleic acid cargo from enzymatic degradation, extravasates from the vasculature, traverses the cell membrane, disrupts the endosomal vesicles and unloads the cargo at its destination site, namely the nucleus for the purposes of gene delivery. This manuscript reports the extensive investigation of a novel amphipathic peptide composed of repeating RALA units capable of overcoming the biological barriers to gene delivery both in vitro and in vivo. Our data demonstrates the spontaneous self-assembly of cationic DNA-loaded nanoparticles when the peptide is complexed with pDNA. Nanoparticles were < 100 nm, were stable in the presence of serum and were fusogenic in nature, with increased peptide α-helicity at a lower pH. Nanoparticles proved to be non-cytotoxic, readily traversed the plasma membrane of both cancer and fibroblast cell lines and elicited reporter-gene expression following intravenous delivery in vivo. The results of this study indicate that RALA presents an exciting delivery platform for the systemic delivery of nucleic acid therapeutics.

Dynamics and structure of self-generated magnetics fields on solids following high contrast, high intensity laser irradiation

The dynamics of self-generated magnetic B-fields produced following the interaction of a high contrast, high intensity (I > 10¹⁹W cm^-2) laser beam with thin (3 μm thick) solid (Al or Au) targets is investigated experimentally and numerically. Two main sources drive the growth of B-fields on the target surfaces. B-fields are first driven by laser-generated hot electron currents that relax over ∼10-20 ps. Over longer timescales, the hydrodynamic expansion of the bulk of the target into vacuum also generates B-field induced by non-collinear gradients of density and temperature. The laser irradiation of the target front side strongly localizes the energy deposition at the target front, in contrast to the target rear side, which is heated by fast electrons over a much larger area. This induces an asymmetry in the hydrodynamic expansion between the front and rear target surfaces, and consequently the associated B-fields are found strongly asymmetric. The sole long-lasting (>30 ps) B-fields are the ones growing on the target front surface, where they remain of extremely high strength (∼8-10 MG). These B-fields have been recently put by us in practical use for focusing laser-accelerated protons [B. Albertazzi et al., Rev. Sci. Instrum. 86, 043502 (2015)]; here we analyze in detail their dynamics and structure.

Self-directedness, personality type and success in self- employment

The new economy has spirited a transformation ofwork organizations from big business structures into smaller, more flexible enterprises, many of which are launched as self-employment initiatives. The growing trend towards increasing selfemployment in Canada demands aeritical review of how educational programs support and encourage entrepreneurship and self-employment opportunities for students ofpost-secondary and adult training programs. The focus ofthis study was threefold. First, the study examined whether a relationship exists between self-directedness and success in self-employment. Secondly, the purpose of this research was to determine whether a relationship exists between psychological type as defined by Jung and success in selfemployment. Finally, this research effort attempted to develop a model for identifying individual potential for self-employment based on combined factors of self-directedness and psychological type. Success was measured in three stages: 1) Did the subject start a selfemployment initiative? 2) Did the business survive six months? 3) Did the business survive one year? The research went beyond classroom training activities to determine whether individuals actually started a business enterprise while participating ina self-employment program designed for individuals who were unemployed. Given that many people initiate a self-employment venture.without actually operating the business beyond the initial start-up, this research effort measured success based on a commitment of at least one year to the selfemployment initiative. Results ofthe study revealed that individuals with a high level of selfdirected learning readiness tended to be more likely to succeed in business in terms ofbusiness starts, survival for six months, and survival for one year. In addition, it was discovered that individuals who were extraverted intuitive types succeeded more often in business at all three levels than any other type. These findings supported a model using the SDLRS and the PET Type Check as predictors for success in entrepreneurial ventures.

The Effect of Trainer Muscularity and Expertise on Self-Presentational Concerns, Body Image, and Performance in College Men during One-Repetition Maximum Testing

This study attempted to manipulate self-presentational efficacy to examine the effect on social anxiety, social physique anxiety, drive for muscularity, and maximal strength performance during a one-repetition maximum (1-RM) chest press and leg press test. Ninety-nine college men with a minimum of six months of previous weight training experience were randomly assigned to complete a 1-RM protocol with either a muscular male trainer described as an expert or a lean male trainer described as a novice. Participants completed measures of self-presentation and body image prior to meeting their respective trainer, and following the completion of the 1-RM tests. Although the self-presentational efficacy manipulation was not successful, the trainers were perceived significantly differently on musculature and expertise. The group with the muscular, expert trainer reported higher social anxiety and attained higher 1-RM scores for the chest and leg press. Thus, trainer characteristics can affect strength performance and self-presentational concerns in this population.

Humour and Learning: A Self-Study of My Practice as an Adult Educator

This study examined my lived experiences as a frontline practitioner and adult educator in a local nonprofit community organization. Using self-study research methodology, I explored my professional practice as a facilitator of job search skills training with newcomers to Canada and the impact of humour on their learning, a topic for which I have a particular passion. To better inform my practice, I designed and delivered job search skills workshops on resume writing and cold-calling/networking. I used a variety of data sources including a literature review, personal observations, and reflections as well as secondary data sources from workshop evaluations and unsolicited feedback emails from participants. Findings from the self-study indicated that adult learners who experience entertaining and fun-filled lessons that use appropriate humour have better learning results, are more confident, and are better prepared to apply skills required for achieving career goals. Further, I learned in my practice that my challenge as an adult educator is to ensure humour is appropriately used and adds value to the learning rather than being the focus of the teaching.

Electrospinning and characterization of self-assembled inclusion complexies

L’électrofilage est une technique permettant de fabriquer des fibres polymériques dont le diamètre varie entre quelques nanomètres et quelques microns. Ces fibres ont donc un rapport surface/volume très élevé. Les fibres électrofilées pourraient trouver des applications dans le relargage de médicaments et le génie tissulaire, comme membranes et capteurs chimiques, ou dans les nanocomposites et dispositifs électroniques. L’électrofilage était initialement utilisé pour préparer des toiles de fibres désordonnées, mais il est maintenant possible d’aligner les fibres par l’usage de collecteurs spéciaux. Cependant, il est important de contrôler non seulement l’alignement macroscopique des fibres mais aussi leur orientation au niveau moléculaire puisque l’orientation influence les propriétés mécaniques, optiques et électriques des polymères. Les complexes moléculaires apparaissent comme une cible de choix pour produire des nanofibres fortement orientées. Dans les complexes d’inclusion d’urée, les chaînes polymères sont empilées dans des canaux unidimensionnels construits à partir d’un réseau tridimensionnel de molécules d’urée liées par des ponts hydrogène. Ainsi, les chaînes polymère sonts très allongées à l’échelle moléculaire. Des nanofibres du complexe PEO-urée ont été préparées pour la première fois par électrofilage de suspensions et de solutions. Tel qu’attendu, une orientation moléculaire inhabituellement élevée a été observée dans ces fibres. De tels complexes orientés pourraient être utilisés à la fois dans des études fondamentales et dans la préparation de matériaux hiérarchiquement structurés. La méthode d’électrofilage peut parfois aussi être utilisée pour préparer des matériaux polymériques métastables qui ne peuvent pas être préparés par des méthodes conventionnelles. Ici, l’électrofilage a été utilisé pour préparer des fibres des complexes stables (α) et "métastables" (β) entre le PEO et l’urée. La caractérisation du complexe β, qui était mal connu, révèle un rapport PEO:urée de 12:8 appartenant au système orthorhombique avec a = 1.907 nm, b = 0.862 nm et c = 0.773 nm. Les chaînes de PEO sont orientées selon l’axe de la fibre. Leur conformation est significativement affectée par les ponts hydrogène. Une structure en couches a été suggérée pour la forme β, plutôt que la structure conventionnelle en canaux adoptée par la forme α. Nos résultats indiquent que le complexe β est thermodynamiquement stable avant sa fonte et peut se transformer en forme α et en PEO liquide par un processus de fonte et recristallisation à 89 ºC. Ceci va dans le sens contraire aux observations faites avec le complexe β obtenu par trempe du complexe α fondu. En effet, le complexe β ainsi obtenu est métastable et contient des cristaux d’urée. Il peut subir une transition de phases cinétique solide-solide pour produire du complexe α dans une vaste gamme de températures. Cette transition est induite par un changement de conformation du PEO et par la formation de ponts hydrogène intermoléculaires entre l’urée et le PEO. Le diagramme de phases du système PEO-urée a été tracé sur toute la gamme de compositions, ce qui a permis d’interpréter la formation de plusieurs mélanges qui ne sont pas à l’équilibre mais qui sont été observés expérimentalement. La structure et le diagramme de phases du complexe PEO-thiourée, qui est aussi un complexe très mal connu, ont été étudiés en détail. Un rapport molaire PEO :thiourée de 3:2 a été déduit pour le complexe, et une cellule monoclinique avec a = 0.915 nm, b = 1.888 nm, c = 0.825 nm et β = 92.35º a été déterminée. Comme pour le complexe PEO-urée de forme β, une structure en couches a été suggérée pour le complexe PEO-thiourée, dans laquelle les molécules de thiourée seraient disposées en rubans intercalés entre deux couches de PEO. Cette structure en couches pourrait expliquer la température de fusion beaucoup plus faible des complexes PEO-thiourée (110 ºC) et PEO-urée de forme β (89 ºC) en comparaison aux structures en canaux du complexe PEO-urée de forme α (143 ºC).

Design and study of self-assembled functional organic and hybrid systems for biological applications

The focus of self-assembly as a strategy for the synthesis has been confined largely to molecules, because of the importance of manipulating the structure of matter at the molecular scale. We have investigated the influence of temperature and pH, in addition to the concentration of the capping agent used for the formation of the nano-bio conjugates. For example, the formation of the narrower size distribution of the nanoparticles was observed with the increase in the concentration of the protein, which supports the fact that γ-globulin acts both as a controller of nucleation as well as stabiliser. As analyzed through various photophysical, biophysical and microscopic techniques such as TEM, AFM, C-AFM, SEM, DLS, OPM, CD and FTIR, we observed that the initial photoactivation of γ-globulin at pH 12 for 3 h resulted in small protein fibres of ca. Further irradiation for 24 h, led to the formation of selfassembled long fibres of the protein of ca. 5-6 nm and observation of surface plasmon resonance band at around 520 nm with the concomitant quenching of luminescence intensity at 680 nm. The observation of light triggered self-assembly of the protein and its effect on controlling the fate of the anchored nanoparticles can be compared with the naturally occurring process such as photomorphogenesis.Furthermore,our approach offers a way to understand the role played by the self-assembly of the protein in ordering and knock out of the metal nanoparticles and also in the design of nano-biohybrid materials for medicinal and optoelectronic applications. Investigation of the potential applications of NIR absorbing and water soluble squaraine dyes 1-3 for protein labeling and anti-amyloid agents forms the subject matter of the third chapter of the thesis. The study of their interactions with various proteins revealed that 1-3 showed unique interactions towards serum albumins as well as lysozyme. 69%, 71% and 49% in the absorption spectra as well as significant quenching in the fluorescence intensity of the dyes 1-3, respectively. Half-reciprocal analysis of the absorption data and isothermal titration calorimetric (ITC) analysis of the titration experiments gave a 1:1 stoichiometry for the complexes formed between the lysozyme and squaraine dyes with association constants (Kass) in the range 104-105 M-1. We have determined the changes in the free energy (ΔG) for the complex formation and the values are found to be -30.78, -32.31 and -28.58 kJmol-1, respectively for the dyes 1, 2 and 3. Furthermore, we have observed a strong induced CD (ICD) signal corresponding to the squaraine chromophore in the case of the halogenated squaraine dyes 2 and 3 at 636 and 637 nm confirming the complex formation in these cases. To understand the nature of interaction of the squaraine dyes 1-3 with lysozyme, we have investigated the interaction of dyes 1-3 with different amino acids. These results indicated that the dyes 1-3 showed significant interactions with cysteine and glutamic acid which are present in the side chains of lysozyme. In addition the temperature dependent studies have revealed that the interaction of the dye and the lysozyme are irreversible. Furthermore, we have investigated the interactions of these NIR dyes 1-3 with β- amyloid fibres derived from lysozyme to evaluate their potential as inhibitors of this biologically important protein aggregation. These β-amyloid fibrils were insoluble protein aggregates that have been associated with a range of neurodegenerative diseases, including Huntington, Alzheimer’s, Parkinson’s, and Creutzfeldt-Jakob diseases. We have synthesized amyloid fibres from lysozyme through its incubation in acidic solution below pH 4 and by allowing to form amyloid fibres at elevated temperature. To quantify the binding affinities of the squaraine dyes 1-3 with β-amyloids, we have carried out the isothermal titration calorimetric (ITC) measurements. The association constants were determined and are found to be 1.2 × 105, 3.6× 105 and 3.2 × 105 M-1 for the dyes, 1-3, respectively. To gain more insights into the amyloid inhibiting nature of the squaraine dyes under investigations, we have carried out thioflavin assay, CD, isothermal titration calorimetry and microscopic analysis. The addition of the dyes 1-3 (5μM) led to the complete quenching in the apparent thioflavin fluorescence, thereby indicating the destabilization of β-amyloid fibres in the presence of the squaraine dyes. Further, the inhibition of the amyloid fibres by the squaraine dyes 1-3, has been evidenced though the DLS, TEM AFM and SAED, wherein we observed the complete destabilization of the amyloid fibre and transformation of the fibre into spherical particles of ca. These results demonstrate the fact that the squaraine dyes 1-3 can act as protein labeling agents as well as the inhibitors of the protein amyloidogenesis. The last chapter of the thesis describes the synthesis and investigation of selfassembly as well as bio-imaging aspects of a few novel tetraphenylethene conjugates 4-6.Expectedly, these conjugates showed significant solvatochromism and exhibited a hypsochromic shift (negative solvatochromism) as the solvent polarity increased, and these observations were justified though theoretical studies employing the B3LYP/6-31g method. We have investigated the self-assembly properties of these D-A conjugates though variation in the percentage of water in acetonitrile solution due to the formation of nanoaggregates. Further the contour map of the observed fluorescence intensity as a function of the fluorescence excitation and emission wavelength confirmed the formation of J-type aggregates in these cases. To have a better understanding of the type of self-assemblies formed from the TPE conjugates 4-6, we have carried out the morphological analysis through various microscopic techniques such as DLS, SEM and TEM. 70%, we observed rod shape architectures having ~ 780 nm in diameter and ~ 12 μM in length as evidenced through TEM and SEM analysis. We have made similar observations with the dodecyl conjugate 5 at ca. 70% and 50% water/acetonitrile mixtures, the aggregates formed from 4 and 5 were found to be highly crystalline and such structures were transformed to amorphous nature as the water fraction was increased to 99%. To evaluate the potential of the conjugate as bio-imaging agents, we have carried out their in vitro cytotoxicity and cellular uptake studies though MTT assay, flow cytometric and confocal laser scanning microscopic techniques. Thus nanoparticle of these conjugates which exhibited efficient emission, large stoke shift, good stability, biocompatibility and excellent cellular imaging properties can have potential applications for tracking cells as well as in cell-based therapies. In summary we have synthesized novel functional organic chromophores and have studied systematic investigation of self-assembly of these synthetic and biological building blocks under a variety of conditions. The investigation of interaction of water soluble NIR squaraine dyes with lysozyme indicates that these dyes can act as the protein labeling agents and the efficiency of inhibition of β-amyloid indicate, thereby their potential as anti-amyloid agents.

The phenology and potential for self-pollination of two Australian monoecious fig species

In this preliminary study, the reproductive phenology of two monoecious fig species, Ficus racemosa and F. rubiginosa, was examined in tropical Australia. Syconia (inflorescences) occurred on both species all year round, but pre-floral and interfloral syconia were much commoner than the wasp-receptive and wasp-emitting phases in both species. The temporal overlap of the wasp-receptive and wasp-emitting phases on a single tree indicated that self-pollination was possible in both species and that pollinators may sometimes persist through multiple generations on one tree. This sexual phase overlap was commoner in F. rubiginosa than in F racemosa. The two species also differed in their general within-tree asynchrony, with a higher diversity of phases on F. rubiginosa than on F. racemosa. The time from syconium initiation to ripening was very similar in F. rubiginosa (mean = 48.51 days) and F. racemosa (mean = 43.53 days). However, there was much more variation within and between trees for F. rubiginosa. In addition, the wasp-receptive phase was found to last up to 5 days (rnean = 4.38) in F. rubiginosa. Such longevity should contribute substantially to local pollinator population persistence. Future work should use genetic studies to determine whether self-pollination is common in these fig species.