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.

Proton magnetic resonance studies of ribonucleic acid complexes. I. Complexes of biological bases and oligonucleotides with RNA. II. Template recognition and the degeneracy of the genetic code

Part I. Complexes of Biological Bases and Oligonucleotides with RNA

The physical nature of complexes of several biological bases and oligonucleotides with single-stranded ribonucleic acids have been studied by high resolution proton magnetic resonance spectroscopy. The importance of various forces in the stabilization of these complexes is also discussed.

Previous work has shown that purine forms an intercalated complex with single-stranded nucleic acids. This complex formation led to severe and stereospecific broadening of the purine resonances. From the field dependence of the linewidths, T₁ measurements of the purine protons and nuclear Overhauser enhancement experiments, the mechanism for the line broadening was ascertained to be dipole-dipole interactions between the purine protons and the ribose protons of the nucleic acid.

The interactions of ethidium bromide (EB) with several RNA residues have been studied. EB forms vertically stacked aggregates with itself as well as with uridine, 3'-uridine monophosphate and 5'-uridine monophosphate and forms an intercalated complex with uridylyl (3' → 5') uridine and polyuridylic acid (poly U). The geometry of EB in the intercalated complex has also been determined.

The effect of chain length of oligo-A-nucleotides on their mode of interaction with poly U in D₂0 at neutral pD have also been studied. Below room temperatures, ApA and ApApA form a rigid triple-stranded complex involving a stoichiometry of one adenine to two uracil bases, presumably via specific adenine-uracil base pairing and cooperative base stacking of the adenine bases. While no evidence was obtained for the interaction of ApA with poly U above room temperature, ApApA exhibited complex formation of a 1:1 nature with poly U by forming Watson-Crick base pairs. The thermodynamics of these systems are discussed.

Part II. Template Recognition and the Degeneracy of the Genetic Code

The interaction of ApApG and poly U was studied as a model system for the codon-anticodon interaction of tRNA and mRNA in vivo. ApApG was shown to interact with poly U below ~20°C. The interaction was of a 1:1 nature which exhibited the Hoogsteen bonding scheme. The three bases of ApApG are in an anti conformation and the guanosine base appears to be in the lactim tautomeric form in the complex.

Due to the inadequacies of previous models for the degeneracy of the genetic code in explaining the observed interactions of ApApG with poly U, the "tautomeric doublet" model is proposed as a possible explanation of the degenerate interactions of tRNA with mRNA during protein synthesis in vivo.

TRANSFER OF ANALYTICAL METHODS FOR THE CHARACTERISATION OF NATURAL PRODUCTS TO R&D PROCESSES OF IDOKI

IDOKI SCF Technologies S.L. is a technology-based company, set up on September 2006 in Derio (Biscay) with the main scope of developing extraction and purification processes based on the use of supercritical fluid extraction technology (SFE) in food processing, extraction of natural products and the production of personal care products. IDOKI¿s researchers have been working on many different R&D projects so far, most of them using this technology. However, the optimization of a SFE method for the different matrices cannot be performed unless we have an analytical method for the characterisation of the extracts obtained in each experiment. The analytical methods are also essential for the quality control of the raw materials that are going to be used and also for the final product. This PhD thesis was born to tackle this problem and therefore, it is based on the development of different analytical methods for the characterisation of the extracts and products. The projects that we could include in this thesis were the following: the extraction propolis, the recovery of agroindustrial residues (soy and wine) and the dealcoholisation of wine.On the one hand, for the extraction of propolis, several UV-Vis spectroscopic methods were used in order to measure the antioxidant capacity and the total polyphenol and flavonoid content of the extracts. A SFC method was also developed in order to measure more specific phenolic compounds. On the other hand, for the recovery of agroindustrial residues UV-Vis spectroscopy was used to determine the total polyphenol content and two SFC methods were developed to analyse different phenolic compounds. Extraction methods such as MAE, FUSE and rotary agitation were also evaluated for the characterisation of the raw materials.Finally, for the dealcoholisation of wine, the development of a SBSE-TD-GC-MS and DHS-TD-GC-MS methods for the analysis of aromas and a NIR spectroscopic method for the determination of ethanol content with the help of chemometrics was necessary. Most of these methods are typically used in IDOKI¿s lab as routine analyses apart from others not included in this PhD thesis.

The promoter of cell growth- and RNA protection-associated SND1 gene is activated by endoplasmic reticulum stress in human hepatoma cells

Background: Staphyloccocal nuclease domain-containing protein 1 (SND1) is involved in the regulation of gene expression and RNA protection. While numerous studies have established that SND1 protein expression is modulated by cellular stresses associated with tumor growth, hypoxia, inflammation, heat- shock and oxidative conditions, little is known about the factors responsible for SND1 expression. Here, we have approached this question by analyzing the transcriptional response of human SND1 gene to pharmacological endoplasmic reticulum (ER) stress in liver cancer cells. Results: We provide first evidence that SND1 promoter activity is increased in human liver cancer cells upon exposure to thapsigargin or tunicamycin or by ectopic expression of ATF6, a crucial transcription factor in the unfolded protein response triggered by ER stress. Deletion analysis of the 5'-flanking region of SND1 promoter identified maximal activation in fragment (-934, +221), which contains most of the predicted ER stress response elements in proximal promoter. Quantitative real- time PCR revealed a near 3 fold increase in SND1 mRNA expression by either of the stress- inducers; whereas SND1 protein was maximally upregulated (3.4-fold) in cells exposed to tunicamycin, a protein glycosylation inhibitor. Conclusion: Promoter activity of the cell growth- and RNA-protection associated SND1 gene is up-regulated by ER stress in human hepatoma cells.

Enhancement of three-photon absorption cross-sections in a novel class of symmetrical charge transfer fluorene-based molecules

The three-photon absorption effect (3PA) of two novel symmetrical charge transfer fluorene-based molecules (abbreviated as BASF and BMOSF) has been determined by using a Q-switched Nd:YAG laser pumped with 38 ps pulses at 1064 nm in DMF. The measured 3PA cross-sections are 84 x 10(-78) and 114 x 10(-78) cm(6) s(2), respectively. The geometries and electronic excitations of these two molecules are systematically studied by PM3 and ZINDO/S methods. The relationships between 3PA cross-sections and intramolecular charge transfer are discussed micromechanically. The experimental and theoretical results have shown that the larger intramolecular charge transfer, which was characterized by the charge density difference between the ground state (SO) and the first excited state (S-I), the greater enhancement of the 3PA cross-sections. (c) 2005 Elsevier B.V. All rights reserved.

Large three-photon absorption and intramolecular charge transfer of the bis-donor fluorene-based molecules

Three-photon absorption (3PA) of two fluorene-based molecules with D-pi-D structural motifs (abbreviated as BPAF and BCZF) has been determined by using a Q-switched Nd: YAG laser pumped with 38 ps pulses at 1064 nm in DMF. The measured 3PA cross-sections are 222 and 140 x 10(-78) cm(6) s(2) for BPAF and BCZF, respectively. AM1 calculations show that attaching different donors changes the charge density distribution of the fluorene skeleton, and it is observed that the 3PA cross-section can be enhanced with increasing intramolecular charge transfer character, measured by the parameter Delta p(1)/Delta p(2)/Delta p(1)'. (c) 2005 Elsevier B.V. All fights reserved.

Molecular Antennas and Photoactive Nanomaterials based on Energy Transfer Processes

110 p.

Theoretical investigation on the one- and two-photon absorption properties for a series of symmetrical charge transfer fluorene-phenylene and fluorene-thiophene derivatives

One- and two-photon absorption properties of a series of fluorene derivatives with symmetrical charge transfer D-IT-D and A-IT-A structural motifs have been theoretically investigated with ZINDO/S method. The optimized structures and the characterization of frontier molecular orbitals were obtained by using AMI calculations. Two-photon absorption properties of molecules have been studied using three-state model. The calculation results have shown that fluorene-thiophene derivatives exhibit larger two-photon absorption cross-section as compared with other studied molecules. To illustrate the results, the crucial effects of thiophene ring on fluorenethiophene derivatives and the net charge changes on the pi-conjugated bridges are analyzed theoretically. (c) 2006 Elsevier B.V. All rights reserved.

Energy transfer and infrared-to-visible upconversion luminescence of Er3+/Yb3+-codoped halide modified tellurite glasses

We report on the energy transfer and frequency upconversion spectroscopic properties of Er3+-doped and Er3+/Yb3+-codoped TeO2-ZnO-Na2O-PbCl2 halide modified tellurite glasses upon excitation with 808 and 978 nm laser diode. Three intense emissions centered at around 529, 546 and 657 nm, alongwith a very weak blue emission at 4 10 nm have clearly been observed for the Er3+/Yb3+-codoped halide modified tellurite glasses upon excitation at 978 nm and the involved mechanisms are explained. The quadratic dependence of fluorescence on excitation laser power confirms the fact that the two-photon contribute to the infrared to green-red upconversion emissions. And the blue upconversion at 410 nm involved a sequential three-photon absorption process. (c) 2005 Elsevier Ltd. All rights reserved.

Energy transfer between Cr3+ and Ni2+ in transparent silicate glass ceramics containing Cr3+/Ni2+ co-doped ZnAl2O4 nanocrystals

The emission intensity of Ni2+ at 1200 nm in transparent ZnO-Al2O3-SiO2 glass ceramics containing ZnAl2O4 nanocrystals is improved approximately 8 times by Cr3+ codoping with 532 nm excitation. This enhanced emission could be attributed to an efficient energy transfer from Cr3+ to Ni2+, which is confirmed by time-resolved emission spectra. The energy transfer efficiency is estimated to be 57% and the energy transfer mechanism is also discussed. (C) 2008 Optical Society of America.