Numerical Study on Critical Wedge Angle of Cellular Detonation Reflections

The critical wedge angle (CWA) for the transition from regular reflection (RR) to Mach reflection (MR) of a cellular detonation wave is studied numerically by an improved space-time conservation element and solution element method together with a two-step chemical reaction model. The accuracy of that numerical way is verified by simulating cellular detonation reflections at a 19.3∘ wedge. The planar and cellular detonation reflections over 45∘–55∘ wedges are also simulated. When the cellular detonation wave is over a 50∘ wedge, numerical results show a new phenomenon that RR and MR occur alternately. The transition process between RR and MR is investigated with the local pressure contours. Numerical analysis shows that the cellular structure is the essential reason for the new phenomenon and the CWA of detonation reflection is not a certain angle but an angle range.

Biotechnologies for cancer diagnostics : cell sorting, protein analysis and imaging of cellular metabolism

This thesis presents the development of chip-based technology for informative in vitro cancer diagnostics. In the first part of this thesis, I will present my contribution in the development of a technology called “Nucleic Acid Cell Sorting (NACS)”, based on microarrays composed of nucleic acid encoded peptide major histocompatibility complexes (p/MHC), and the experimental and theoretical methods to detect and analyze secreted proteins from single or few cells.

Secondly, a novel portable platform for imaging of cellular metabolism with radio probes is presented. A microfluidic chip, so called “Radiopharmaceutical Imaging Chip” (RIMChip), combined with a beta-particle imaging camera, is developed to visualize the uptake of radio probes in a small number of cells. Due to its sophisticated design, RIMChip allows robust and user-friendly execution of sensitive and quantitative radio assays. The performance of this platform is validated with adherent and suspension cancer cell lines. This platform is then applied to study the metabolic response of cancer cells under the treatment of drugs. Both cases of mouse lymphoma and human glioblastoma cell lines, the metabolic responses to the drug exposures are observed within a short time (~ 1 hour), and are correlated with the arrest of cell-cycle, or with changes in receptor tyrosine kinase signaling.

The last parts of this thesis present summaries of ongoing projects: development of a new agent as an in vivo imaging probe for c-MET, and quantitative monitoring of glycolytic metabolism of primary glioblastoma cells. To develop a new agent for c-MET imaging, the one-bead-one-compound combinatorial library method is used, coupled with iterative screening. The performance of the agent is quantitatively validated with cell-based fluorescent assays. In the case of monitoring the metabolism of primary glioblastoma cell, by RIMChip, cells were sorting according to their expression levels of oncoprotein, or were treated with different kinds of drugs to study the metabolic heterogeneity of cancer cells or metabolic response of glioblastoma cells to drug treatments, respectively.

Fluorescence microscopy of nicotinic acetylcholine receptors

Neuronal nicotinic acetylcholine receptors (nAChRs) are pentameric ligand gated ion channels abundantly expressed in the central nervous system. Changes in the assembly and trafficking of nAChRs are pertinent to disease states including nicotine dependence, autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE), and Parkinson’s disease (PD). Here we investigate the application of high resolution fluorescence techniques for the study of nAChR assembly and trafficking. We also describe the construction and validation of a fluorescent α5 subunit and subsequent experiments to elucidate the cellular mechanisms through which α5 subunits are expressed, assembled into mature receptors, and trafficked to the cell surface. The effects of a known single nucleotide polymorphism (D398N) in the intracellular loop of α5 are also examined.

Additionally, this report describes the development of a combined total internal reflection fluorescence (TIRF) and lifetime imaging (FLIM) technique and the first application of this methodology for elucidation of stochiometric composition of nAChRs. Many distinct subunit combinations can form functional receptors. Receptor composition and stoichiometry confers unique biophysical and pharmacological properties to each receptor sub-type. Understanding the nature of assembly and expression of each receptor subtype yields important information about the molecular processes that may underlie the mechanisms through which nAChR contribute to disease and addiction states.

Part I. Regulation of development in the cellular slime mold Dictyostelium discoideum. Part II. Polysomes and RNA synthesis during early development of the surf clam Spisula solidissima

Part I. The cellular slime mold Dictyostelium discoideum is a simple eukaryote which undergoes a multi-cellular developmental process. Single cell myxamoebae divide vegetatively in the presence of a food source. When the food is depleted or removed, the cells aggregate, forming a migrating pseudoplasmodium which differentiates into a fruiting body containing stalk and spore cells. I have shown that during the developmental cycle glycogen phosphorylase, aminopeptidase, and alanine transaminase are developmentally regulated, that is their specific activities increased at a specific time in the developmental cycle. Phosphorylase activity is undetectable in developing cells until mid-aggregation whereupon it increases and reaches a maximum at mid-culmination. Thereafter the enzyme disappears. Actinomycin D and cycloheximide studies as well as studies with morphologically aberrant and temporally deranged mutants indicate that prior RNA and concomitant protein synthesis are necessary for the rise and decrease in activity and support the view that the appearance of the enzyme is regulated at the transcriptional level. Aminopeptidase and alanine transaminase increase 3 fold starting at starvation and reach maximum activity at 18 and 5 hours respectively.

The cellular DNA s of D. discoideum were characterized by CsC1 buoyant density gradient centrifugation and by renaturation kinetics. Whole cell DNA exhibits three bands in CsCl: ρ = 1.676 g/cc (nuclear main band), 1.687 (nuclear satellite), and 1.682 (mitochondrial). Reassociation kinetics at a criterion of Tm -23°C indicates that the nuclear reiterated sequences make up 30% of the genome (Cot_1/2 (pure) 0.28) and the single-copy DNA 70% (Cot_1/2(pure) 70). The complexity of the nuclear genome is 30 x 10⁹ daltons and that of the mitochondrial DNA is 35-40 x 10⁶ daltons (Cot_1/2 0.15). rRNA cistrons constitute 2.2% of nuclear DNA and have a ρ = 1.682.

RNA extracted from 4 stages during developmental cycle of Dictyostelium was hybridized with purified single-copy nuclear DNA. The hybrids had properties indicative of single-copy DNA-RNA hybrids. These studies indicate that there are, during development, qualitative and quantitative changes in the portion of the single-copy of the genome transcribed. Overall, 56% of the genome is represented by transcripts between the amoeba and mid-culmination stages. Some 19% are sequences which are represented at all stages while 37% of the genome consists of stage specific sequences.

Part II. RNA and protein synthesis and polysome formation were studied during early development of the surf clam Spisula solidissima embryos. The oocyte has a small number of polysomes and a low but measurable rate of protein synthesis (leucine-³H incorporation). After fertilization, there is a continual increase in the percentage of ribosomes sedimenting in the polysome region. Newly synthesized RNA (uridine-5-³H incorporation) was found in polysomes as early as the 2-cell stage. During cleavage, the newly formed RNA is associated mainly with the light polysomes.

RNA extracted from polysomes labeled at the 4-cell stage is polydisperse, nonribosomal, and non-4 S. Actinomycin D causes a reduction of about 30% of the polysomes formed between fertilization and the 16-cell stage.

In the early cleavage stages the light polysomes are mostly affected by actinomycin.

Metallothionein as an indicator of water quality: assessment of the bioavailability of cadmium, copper, mercury and zinc in aquatic animals at the cellular level

The study of metallothioneins (MTs) has greatly improved our understanding of body burdens, metal storage and detoxification in aquatic organisms subjected to contamination by the toxic heavy metals, Cd, Cu, Hg and Zn. These studies have shown that in certain organisms MT status can be used to assess impact of these metals at the cellular level and, whilst validation is currently limited to a few examples, this stress response may be linked to higher levels of organisation, thus indicating its potential for environmental quality assessment. Molluscs, such as Mytilus spp., and several commonly occurring teleost species, are the most promising of the indicator species tested. Natural variability of MT levels caused by the organism's size, condition, age, position in the sexual cycle, temperature and various stressors, can lead to difficulties in interpretation of field data as a definitive response-indicator of metal contamination unless a critical appraisal of these variables is available. From laboratory and field studies these data are almost complete for teleost fish. Whilst for molluscs much of this information is lacking, when suitable controls are utilised and MT measurements are combined with observations of metal partitioning, current studies indicate that they are nevertheless a powerful tool in the interpretation of impact, and may prove useful in water quality assessment.

HIV-1 Capture and Transmission by Dendritic Cells: The Role of Viral Glycolipids and the Cellular Receptor Siglec-1

Dendritic cells (DCs) are essential in order to combat invading viruses and trigger antiviral responses. Paradoxically, in the case of HIV-1, DCs might contribute to viral pathogenesis through trans-infection, a mechanism that promotes viral capture and transmission to target cells, especially after DC maturation. In this review, we highlight recent evidence identifying sialyllactose-containing gangliosides in the viral membrane and the cellular lectin Siglec-1 as critical determinants for HIV-1 capture and storage by mature DCs and for DC-mediated trans-infection of T cells. In contrast, DC-SIGN, long considered to be the main receptor for DC capture of HIV-1, plays a minor role in mature DC-mediated HIV-1 capture and trans-infection.

Low Molecular Weight Chitosan (LMWC)-based Polyplexes for pDNA Delivery: From Bench to Bedside

Non-viral gene delivery vectors are emerging as a safer alternative to viral vectors. Among natural polymers, chitosan (Ch) is the most studied one, and low molecular weight Ch, specifically, presents a wide range of advantages for non-viral pDNA delivery. It is crucial to determine the best process for the formation of Low Molecular Weight Chitosan (LMWC)-pDNA complexes and to characterize their physicochemical properties to better understand their behavior once the polyplexes are administered. The transfection efficiency of Ch based polyplexes is relatively low. Therefore, it is essential to understand all the transfection process, including the cellular uptake, endosomal escape and nuclear import, together with the parameters involved in the process to improve the design and development of the non-viral vectors. The aim of this review is to describe the formation and characterization of LMWC based polyplexes, the in vitro transfection process and finally, the in vivo applications of LMWC based polyplexes for gene therapy purposes.