Yes / The application of vacuum venting for the removal of air from mould cavity has been introduced in injection moulding with the intent to enhance micro/nano features replication and definition. The technique is adopted to remove air pockets trapped in the micro-features, which are out of reach for conventional venting technologies and can create considerable resistance to the melt filling flow. Nonetheless, several studies have revealed a negative effect on replication that could possibly arise from the application of vacuum venting. Although the incomplete filling of micro-scale features has often been attributed to poor venting, the limited research examining the application of vacuum venting has produced mixed results. In this work, the effect of air evacuation was experimentally investigated, monitoring mould and polymer temperature evolution during the micro injection moulding process by means of a high speed infrared camera and a sapphire window, which forms part of the mould wall. The results show that air evacuation removes a mould surface heating effect caused by rapid compression of the air ahead of the flow front and subsequent conduction of that heat into the mould surface. Hence, with the increase of the surface-to-volume ratio in micro-cavities, air evacuation has a detrimental effect on the cavity filling with polymers that are sensitive to changes of the mould temperature.

Yes / Long chain branches (LCB) are successfully grafted to linear poly(lactic acid) (PLA) using functional group reactions with pentaerythritol triacrylate (PETA) and tetraethylthiuram disulfide (TETDS). Results show a high branching degree of PLA (∼49.5%) can be effectively obtained with adding only 1 wt % PETA, contributing remarkably to enhancing strain hardening. The density of the nuclei formed during nonisothermal crystallization for LCB-PLA samples is markedly increased contrasted with PLA, resulting in significantly enhancing crystallinity from 13.3% to 41%, the onset crystallization temperature (∼20 °C), and the crystallization rate. Interestingly, compared with mini-injection molding, the elevated wall shear rates (and corresponding shear stresses) prove to be beneficial to the creation of special crystalline morphologies (β-crystal form) and oriented structures under microinjection molding conditions, resulting in the improvement of tensile strength by ∼45 MPa. / Chinese Scholarship Council

The introduction of bio-molecules into cells and embryos is required in the fields of drug development, genetic engineering and in-vitro fertilization. It has been applied to create transgenic mammals and to improve pest and mold resistance in plants. However, the efficient transfection of materials still poses a problem, and a variety of techniques, broadly classified as biochemical and physical means, are actively being developed. One technique that is promising is capillary microinjection as it offers low cytotoxicity, targeted injections and high transfection efficiency. However, this process suffers from low throughput and variability as it is an operator mediated process. Other problems associated with capillary microinjection are limitations on the minimum needle size and variability in transfected volumes due to the use of pressure driven flow for injections. In this thesis we propose a device that employs microfluidic principles to enable cell microinjections in a 'lab on a chip' format and eliminates the problems associated with capillary microinjection. The device is fabricated using poly dimethylsiloxane (PDMS) rapid prototyping and features two separate channel structures-one to supply the targets and the other to supply the reagent. Integrated into the device are a microinjection capillary (10 μm tip diameter) and a suction capillary (0.5mm ID/1mm OD) which is used to immobilize the targets in the channel prior to injection. The actuation of the injection needle into the targets is achieved by the compliant deformation of the flexible PDMS substrate as a result of an externally applied displacement. This is made possible by the selective reinforcement of the PDMS substrate. From testing it was found that the effective needle actuation is 83.8% of the externally applied displacement. The injections occur in a planar configuration therefore providing precise control over the location of injection. Furthermore, the mechanism requires only one degree of freedom to perform injections, and therefore greatly simplifies existing injection techniques which require orientation in a three dimensional space. The limitations of the use of pressure driven flow for injections are overcome by performing reagent injection by electroosmotic flow, which is induced by applying a potential to electrodes embedded in the target and reagent supply channels. The applied potential induces electroosmotic flow through the embedded needle and into the injection target. This provides precise electrical dosage control. The flow rates were obtained by measuring the velocity of the interface between a neutral fluorescent marker and a clear pH 10 buffer solution. The obtained flow rates follow a predictable linear trend and correspond well to theory. The use of electroosmotic flow enables the use of smaller injection needles as it scales more favorably (r^-2) than pressure driven flow (r^-4) and becomes increasingly dominant in smaller dimensions. Present pressure microinjection systems are limited to injection needles with tip diameters larger than 0.2μm due to the high pressures required to dose at smaller dimensions. All components of the device are fully scalable and enable further miniaturization, multiple parallel injections and autonomous functionality. The device requires smaller volumes of samples and expensive reagents and also reduces the time required for performing injections. Overall, it device maintains the advantages of microinjection, while eliminating problems of low throughput, dosage control and restrictions on the injection needle size. The device was successfully used and characterized for the injection of single-cell Xenopus Laevis eggs and Zebrafish embryos. / Thesis / Master of Applied Science (MASc)

The invention of pronuclear microinjection initiated the field of transgenic research. Over 30 years later microinjection remains the most straight-forward and most commonly used transgene delivery option. In this work we address the current progress of microelectromechanical systems (MEMS) used as transgenic delivery mechanisms. The nanoinjector is a specially designed MEMS device which uses electrostatic charge to manipulate transgene molecules. The process of nanoinjection was designed as an alternative to microinjection which causes less damage to developing embryos, improves embryo survival, birth rates, and overall efficiency of injections. In vivo testing of nanoinjection demonstrates it is both safe and effective. Additionally nanoinjection has the potential to make transgenesis via yeast artificial chromosomes more practical as the nanoinjector may prevent shearing of the YAC molecules. A second nanoinjection protocol termed intracellular electroporetic nanoinjcetion (IEN) was designed to allow for cytoplasmic injections. Cytoplasmic injections are faster and easier than pronuclear injection and do not require the pronuclei to be visible; yet previous attempts to develop cytoplasmic injection have met with limited success. In IEN injections the nanoinjector is used to place transgenic molecules in the cytoplasm. The transgenes are then propelled through the cytoplasm and electroporated into the pronucleus using electrical pulses. Electroporation of whole embryos has not resulted in transgenic animals, but the MEMS device allows localized electroporation to occur within the cytoplasm, giving transgene access to the pronucleus before degradation can occur. In this report we describe the principles which allow for localized electroporation of the pronuclei including: the location of the pronuclei between 21-28 hours post-hCG treatment, modeling data predicting the voltages needed for localized electroporation of pronuclei, and data on the movement of transgenic DNA based on the voltages delivered by IEN. We further report results of an IEN versus microinjection comparative study in which IEN produced transgenic pups with viability, transgene integration, and expression rates statistically comparable to microinjection. The ability to perform injections without visualizing or puncturing the pronuclei will widely benefit transgenic research, and will be particularly advantageous for the production of transgenic animals with embryos exhibiting reduced pronuclear visibility.

Transgenic techniques have rapidly evolved in recent years. However, the efficiency of these techniques to produce viable offspring is still disappointingly low. The purpose of this study was to assess in vitro development, transgene expression, and integration following pronuclear or cytoplasmic microinjection of condensed or linear green fluorescent protein DNA into murine embryos using electroporation. In experiment 1, the effect of embryo orientation (group or linear) within the electroporation chamber on development was evaluated using zygotes which received one pulse duration (10 msec), and one of two voltages (250 or 400 V). Zygotes that received 400 V had the lowest development score (Group, 2.06 ? 0.12; Linear, 1.97 ? 0.13), irrespective of orientation. Embryos that received 250 V had the highest development of the voltage treated groups (Group 3.42 ? 0.12; Linear 3.32 ? 0.12), irrespective of orientation, and development was lower than the control embryos (Control 4.28 ? 0.12; Mannitol control 4.36 ? 0.18). In experiment 2, the efficiency of utilization of the prepared enhanced green fluorescent protein (EGFP) construct as a visual marker of protein expression was evaluated using pronuclear microinjection. Embryo development and fluorescence were evaluated following pronuclear injection of EGFP at a concentration of 3 μg/ml and compared to an uninjected control. Embryos injected with the EGFP had lower development scores (3.85 ± 0.15) than uninjected control embryos (5.72 ± 0.2). Of the embryos injected, 32.4% fluoresced due to expression of EGFP. Experiment 3 evaluated the effect of combining cytoplasmic injection of EGFP (425 μg/ml) with electroporation at 250 V on EGFP expression. The non-manipulated control embryos had significantly higher (P < 0.01) 4 d development scores (5.57 ± 0.11) than manipulated control embryos (4.6 ± 0.18), where the injection needle was inserted into the cytoplasm and no DNA was injected. Combining cytoplasmic DNA injection and electroporation caused a significant (P < 0.01) decrease in development scores, irrespective of DNA construct, when compared to embryos injected with a DNA construct alone. The mechanical effects of needle insertion combined with electroporation were not significantly different (P > 0.05) from embryos injected with DNA alone, irrespective of construct injected. Cytoplasmic injection of condensed DNA (0.38%), linear DNA (0.38%), and condensed DNA combined with electroporation (0.36%) resulted in one fluorescent embryo respectively. Cytoplasmic injection of linear DNA when combined with electroporation (3.57%) resulted in 13 fluorescent embryos. Pronuclear injection of the prepared EGFP construct results in lower development than control embryos. Electrical stimulation of zygotes reduces early embryo development. However, low amounts of electrical stimulation may allow for enhancement of gene integration in transgenic embryos. / Master of Science

Pronuclear stage murine embryos received electrical stimulation in 5, 10, or 20 µs pulse lengths, and 0, 100, 200, 250, 300 or 400 voltages. Minimal embryo development occurred with 400 V. Irreversible electroporation occurred in embryos electroporated for 5 µs pulse length at 100 and 400 V, 10 µs pulse length at 400 V, and 20 µs pulse length at 100, 250, 300 and 400 V. Electroporated embryos that underwent reversible electroporation received 100 V for 5 µs, 400 V for 10 µs, and 250 for 20 µs and had similar development (P > 0.05) between the best and worst developed groups. Enhanced green fluorescent protein on a cytomegalovirus promoter (CMV-EGFP) was condensed with MgCl₂ and injected into the cytoplasm of murine zygotes at three concentrations (100, 425 and 625 µg/ml). Zygotes injected with the highest concentration had the highest percentages of fluorescing embryos (44%), fluorescing morula and blastocysts (16.7%), and the lowest percentage of mosaicism after 4 d in culture. Five PCR analyses of tail DNA gave conflicting results between 33.3% positive in two or more analyses to 2.8% positive in all five analyses. Southern Analysis detected 2.8% transgenesis. Cytoplasmic injection of linear CMV-EGFP (625 µg/ml in water) was 3.7% transgenic. Pronuclear injections produced 7.9% transgenesis. This research identified a range of reversible electroporation that could easily be verified <i>in vitro</i> with a selectable dye or marker protein and applied in transgenic as well as preclinical treatment models of research. Furthermore this research identifies the benefits and disadvantages of using Mg²⁺ in DNA condensation and injection buffers. / Master of Science

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.

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.

Reproduction de : Thèse d'exercice : Médecine. Dermatologie et vénéréologie : Paris 12 : 2008. / Titre provenant de l'écran-titre. Bibliogr. f. 39-49.

The ability to inject DNA and other foreign particles into cells, both germ cells (e.g. to produce transgenic animals) and somatic cells (e.g. for gene therapy), is a powerful tool in genetic research. Nanoinjection is a method of DNA delivery that combines mechanical and electrical methods. It has proven to have higher cell viability than traditional microinjection, resulting in higher integration per injected embryo. The nanoinjection process can be performed on thousands of cells simultaneously using an array of microneedles that is inserted into a monolayer of cells. This thesis describes the needle array design requirements and the fabrication process used to meet them. The process uses unpassivated and passivated deep reactive ion etching (DRIE) to create needles with a constant diameter shaft and a pointed tip. The needle diameter and height are about 1 µm and 8 µm, respectively. A buckling analysis and physical testing show that the needles can withstand the force required to penetrate the cells. The chip is attached to a plastic suspension with a counter electrode and electrical connections to a voltage source. The suspension's motion is defined by two compliant orthoplanar springs that have been vertically and rotationally offset for added stability. The base of the suspension is designed to exactly fit in the bottom of a cell culture dish, where the needle array can be pushed into the cell monolayer. Injection protocol was created and followed to perform tests with needle insertion only, voltage application only, and the full nanoinjection process. The average cell viability for the full injection process was 98.2% compared to an average control viability of 99.5%. Zero volt injections with a high concentration of propidium iodide, a cell impermeable dye with two positive charges, resulted in dye uptake from diffusion, proving that the needles are penetrating the cells. Tests comparing injections with and without voltage had high variability in dye uptake. Therefore, glass cover slips were placed in the culture dishes to provide more consistent injection conditions. This reduced variation in zero voltage tests. It is recommended that this procedure be followed for performing injections with voltage.