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Craniofacial development requires orchestrated and complex interactions between multiple tissues of different origins. Cranial neural crest stem cells migrate from the dorsal neural tube into the frontonasal process and branchial arches where they ultimately form most of the skeletal structures and connective tissue of the craniofacial complex, as well as contributing neurons and glia to cranial ganglia. The timing and mechanism by which cranial neural crest cells progressively differentiate from multipotent stem cells into lineage restricted and terminally differentiating cell types has previously not been investigated. In addition, there are many deficits in our knowledge of the molecular controls regulating early development of neural crest cells within the branchial arches. Spatial and temporal changes in migratory and lineage potential in neural crest populations contributing to the developing first branchial arch and trigeminal ganglia were examined by back-transplanting cells from quail into chick embryos. Neural crest cells that had barely entered the first branchial arch had largely lost both the ability to localise to the trigeminal ganglia and neurogenic differentiation capacity but were still capable of long-distance migration. However, after a further 12 hours residence in the branchial arch, neural crest cells had lost long-distance migratory ability.

Thesis (Ph.D.)--University of California, San Francisco, 2004. / Bibliography: leaves 110-137. Also available online.

The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file. Title from PDF of title page (University of Missouri--Columbia, viewed on September 22, 2009). Thesis advisor: Dr. Naz E. Islam. Includes bibliographical references.

Thesis (M.S.)--Virginia Polytechnic Institute and State University, 1992. / Vita. Abstract. Includes bibliographical references (leaves 36-40). Also available via the Internet.

La crête neurale céphalique (CNC) est une structure pluripotente à l'origine de la totalité du squelette de la face et de la voûte crânienne.
L'absence de la CNC est associée à des malformations du cerveau antérieur et des défauts oculaires qui reproduisent les malformations congénitales humaines: microcéphalie, holoprosencéphalie, aniridie, chambre antérieure manquante, colobome congénital, désorganisation du cristallin, agénésie des
paupières,
strabisme précoce (Alward 2000, Creuzet 2009, Williams and Bohnsack 2015). Dans notre étude, l'augmentation hétérochronique de la signalisation Bmp7 au niveau de la plaque préchordale (PPC) a engendré une perturbation de la migration de la CNC associée à une fusion des deux champs oculaires en un seul œil cyclope. Cette cyclopie, contrairement aux études antérieures (Chiang et al. 1996, Golden et al. 1999) est précédée par une perturbation de la constriction apicale au niveau des vésicules optiques (VO) au stade Hamburger-Hamilton (HH) 12 et qui est responsable de l'invagination dorsale des VO et leur séparation du reste du tube neural. Au niveau moléculaire, nous avons enregistré une augmentation de la signalisation Wnt1 très tot après le traitement (2h) dans la CNC. Cette augmentation s'étend rostralement ce qui suggère que Wnt1 pourrait etre impliqué dans la perturbation du développement oculaire. Pour tester cette hypothèse, nous avons tenté des expériences de sauvetage par l'inactivation de la signalisation Wnt1 par ARN interférence. Nous avons observé une amélioration du phénotype mais les yeux sont restés morphologiquement en hypotélorisme, c'est-à-dire trop proches. Des coupes coronales au stade E8 (32-33HH) ont montré que les yeux sont synophtalmiques, deux yeux dans un seul orbite, et sont restés fusionnés au télencéphale. Au niveau moléculaire, nous avons observé aussi une diminution de l'expression de Wnt2b au niveau des VO chez l'embryon cyclope et que cette diminution persiste malgré le sauvetage par Wnt1. Pour comprendre cette signalisation, nous avons tenté une deuxième expérience de sauvetage avec ARN interférence contre Wnt1 et un vecteur plasmidique exprimant Wnt2b dans le tube neural et les VO respectivement. Vers E8 l'embryon ressemble au contrôle mais avec persistance de fusion entre les VO et le télencéphale. L'hybridation in situ a montré une diminution du niveau d'expression de Foxg1 à 12HH dans la partie dorsale des VO au niveau de la frontière entre la partie rostrale du diencéphale et le télencéphale (RD/T) dans les deux tentatives de sauvetage ce qui suggère que ce gène pourrait être impliqué dans la séparation rostrale des VO du télencéphale. / Cephalic neural crest (CNC) is a pluripotent structure giving rise to entire skeleton of the face and skull. Absence of CNC is associated with forebrain and eye defects that mimic human congénital malformations: microcephaly, holoprosencephaly, aniridia, missing anterior chamber, coloboma, congenital dislocation of the lens, agenesis of eyelid and early strabismus. (Alward 2000 Creuzet 2009, Williams and Bohnsack 2015). In our study, heterochronic increased Bmp7 signaling in the prechordal plate (Pcp), caused disruption of CNC migration associated with fusion of two optical fields into one cyclopic eye. This cyclopia, unlike previous studies (Chiang et al. 1996, Golden et al. 1999), is preceded by a disturbance of the apical constriction in optic vesicles at Hamburger-Hamilton (HH)12 stage witch is responsible for dorsal invagination of OVs and their separation from the rest of the neural tube. At the molecular level, we recorded an increase in Wnt1 expression very early after treatment (2h) in CNC and this increase extends rostrally suggesting that Wnt1 could be involved in the disturbance of eye development. To test this hypothesis, we attempted rescue experiments by inactivating Wnt1 signaling by RNA interference and we observed an improvement of the phenotype but the eyes remained morphologically in hypotelorism, too close,. At E8 (32-33HH) stage the eyes are remained fused to the forebrain and are synophtalmic, two eyes in a single orbit,. We also observed a decrease in Wnt2b expression in OV in the cyclopic embryo and that this decline persists despite Wnt1 rescue. To understand Wnt2b signaling, we attempted a second rescue experience with RNA interference against Wnt1 and a plasmid vector expressing Wnt2b in the CNC and OV respectively. Coronal sections at E8 stage resemble the control but with persistence of dorsal fusion between OVs and telencephalon. In situ hybridization showed a decrease in Foxg1 expression at 12HH in the dorsal part of OVs in the RD/T boundry in the two rescue attempts suggesting that this gene may be involved in the separation of OVs at 12HH stage rostrally from the telencephalon.

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

No / This work investigated the effect of electroporation on human epidermal penetration of a model neutral lipophilic compound (estradiol) from saturated aqueous solution and when encapsulated in ultradeformable liposomes. Total amount penetrated and skin deposition were compared with values obtained from passive diffusion. The effect of electrical pulsing on liposome size was investigated. The action of phosphatidylcholine on skin that was structurally altered by such pulses was determined. Electroporation did not affect liposome size. Skin pulsing considerably increased estradiol penetration and skin deposition from solution, relative to passive delivery, with subsequent partial recovery of skin resistance to molecular penetration. Surprisingly, with liposomes, electroporation did not markedly affect estradiol skin penetration. Importantly, liposomal phosphatidylcholine applied during or after pulsing accelerated skin barrier repair, i.e. provided an anti-enhancer or retardant effect.

Irreversible electroporation (IRE) is a local ablation technique that has been shown to be both safe and effective in the treatment of solid tumors. The treatment typically consists of inserting needle electrodes directly into the treatment zone and applying high-voltage pulses with widths on the order of hundreds of microseconds. These pulses permeabilize tissue leading to loss of homeostasis among the cells in the treatment zone. Predicting these treatments is challenging as the electric field (EF) induced through the electrode configuration is heterogeneous and is affected by several adjustable parameters. Computational treatment planning models aim to provide a visualization of the treatment zone, and they rely on two critical pieces of information: the electric field distribution (EFD) within the tissue, and the lethal EF threshold for the target tissue type. This work primarily aims to quantify tissue properties necessary for computing the EFD for any electrode configuration, for both traditional IRE as well as next-generation high-frequency IRE treatments. Also included is the determination of pancreatic tumor lethal EF threshold using collagen tissue mimics. Additionally, this work builds on previous reports of an optimal resistance reached during IRE by examining the changes in patients' immune cell populations following treatment, and proposing a method of optimizing these populations by monitoring real-time current achieved during IRE. / Doctor of Philosophy / We are in dire need of new options in cancer therapy, especially in the treatment of tumors that are unresectable, particularly aggressive, or resistant to drugs. Irreversible electroporation (IRE) is a local tumor treatment that has been shown to safely and effectively destroy tumor tissue while leaving behind important structures like blood vessels. As IRE treatments depend on the electric field (EF) generated within the target tissue, it is difficult for clinicians to predict the amount of tissue that will be treated ahead of time. This work aims to collect and examine the information about tumors and the surrounding healthy tissue that is critical to models that can help visualize the treatment and ensure the tumor is exposed to enough lethal energy. Additionally, a new and improved, high-frequency version of IRE (H-FIRE) is explored in terms of its impact on how tissue behaves during the delivery of these types of pulses. In addition to informing models of these therapies, we also explore strategies that clinicians can employ during treatment in order to know when to stop in order to avoid over-treating the area.