Synthetic long peptide-based vaccine formulations for induction of cell mediated immunity: A comparative study of cationic liposomes and PLGA nanoparticles.

Nanoparticulate formulations for synthetic long peptide (SLP)-cancer vaccines as alternative to clinically used Montanide ISA 51- and squalene-based emulsions are investigated in this study. SLPs were loaded into TLR ligand-adjuvanted cationic liposomes and PLGA nanoparticles (NPs) to potentially induce cell-mediated immune responses. The liposomal and PLGA NP formulations were successfully loaded with up to four different compounds and were able to enhance antigen uptake by dendritic cells (DCs) and subsequent activation of T cells in vitro. Subcutaneous vaccination of mice with the different formulations showed that the SLP-loaded cationic liposomes were the most efficient for the induction of functional antigen-T cells in vivo, followed by PLGA NPs which were as potent as or even more than the Montanide and squalene emulsions. Moreover, after transfer of antigen-specific target cells in immunized mice, liposomes induced the highest in vivo killing capacity. These findings, considering also the inadequate safety profile of the currently clinically used adjuvant Montanide ISA-51, make these two particulate, biodegradable delivery systems promising candidates as delivery platforms for SLP-based immunotherapy of cancer.

Particle detector link system modifications in RE1/1 sector

The large hadron collider constructed at the European organization for nuclear research, CERN, is the world’s largest single measuring instrument ever built, and also currently the most powerful particle accelerator that exists. The large hadron collider includes six different experiment stations, one of which is called the compact muon solenoid, or the CMS. The main purpose of the CMS is to track and study residue particles from proton-proton collisions. The primary detectors utilized in the CMS are resistive plate chambers (RPCs). To obtain data from these detectors, a link system has been designed. The main idea of the link system is to receive data from the detector front-end electronics in parallel form, and to transmit it onwards in serial form, via an optical fiber. The system is mostly ready and in place. However, a problem has occurred with innermost RPC detectors, located in sector labeled RE1/1; transmission lines for parallel data suffer from signal integrity issues over long distances. As a solution to this, a new version of the link system has been devised, a one that fits in smaller space and can be located within the CMS, closer to the detectors. This RE1/1 link system has been so far completed only partially, with just the mechanical design and casing being done. In this thesis, link system electronics for RE1/1 sector has been designed, by modifying the existing link system concept to better meet the requirements of the RE1/1 sector. In addition to completion of the prototype of the RE1/1 link system electronics, some testing for the system has also been done, to ensure functionality of the design.

Lysine-based surfactants in nanovesicle formulations: the role of cationic charge position and hydrophobicity in in vitro cytotoxicity and intracellular delivery

Understanding nanomaterial interactions within cells is of increasing importance for assessing their toxicity and cellular transport. Here, we developed nanovesicles containing bioactive cationic lysine-based amphiphiles, and assessed whether these cationic compounds increase the likelihood of intracellular delivery and modulate toxicity. We found different cytotoxic responses among the formulations, depending on surfactant, cell line and endpoint assayed. The induction of mitochondrial dysfunction, oxidative stress and apoptosis were the general mechanisms underlying cytotoxicity. Fluorescence microscopy analysis demonstrated that nanovesicles were internalized by HeLa cells, and evidenced that their ability to release endocytosed materials into cell cytoplasm depends on the structural parameters of amphiphiles. The cationic charge position and hydrophobicity of surfactants determine the nanovesicle interactions within the cell and, thus, the resulting toxicity and intracellular behavior after cell uptake of the nanomaterial. The insights into some toxicity mechanisms of these new nanomaterials contribute to reducing the uncertainty surrounding their potential health hazards.

Differential Regulation of c-FLIP Isoforms Through Post-translational Modifications

Cells are constantly responding to signals from the surrounding tissues and the environment. To dispose of infected and potentially dangerous cells, to ensure the optimal execution of developmental processes and to maintain tissue homeostasis, a multicellular organism needs to tightly control both the number and the quality of its cells. Apoptosis is a form of active cellular self-destruction that enables an organism to regulate its cell number by deleting damaged or potentially dangerous cells. Apoptosis can be induced by death ligands, which bind to death receptors on the cell surface. Ligation of the receptors leads to the formation of an intracellular death inducing signaling complex (DISC). One of the DISC components is caspase-8, a protease that triggers the caspase cascade and is thereby a key initiator of programmed cell death. The activation of caspase-8 is controlled by the cellular FLICE-inhibitory proteins (c-FLIPs). Consequently, sensitivity towards receptor-mediated apoptosis is determined by the amount of c-FLIP, and the c-FLIP levels are actively regulated for example during erythroid differentiation of K562 erythroleukemia cells and by hyperthermia in Jurkat leukemia cells. The aim of my thesis was to investigate how c-FLIP is regulated during these processes. We found that c-FLIP isoforms are short-lived proteins, although c-FLIPS had an even shorter half-life than c-FLIPL. In both experimental models, increased death receptor sensitivity correlated with induced ubiquitylation and consequent proteasomal degradation of c-FLIP. Furthermore, we elucidated how phosphorylation regulates the biological functions and the turnover of c-FLIP, thereby contributing to death receptor sensitivity. We mapped the first phosphorylation sites on c-FLIP and dissected how their phosphorylation affects c-FLIP. Moreover, we demonstrated that phosphorylation of serine 193, a phosphorylated residue common to all c-FLIPs, is primarily mediated by the classical PKC. Furthermore, we discovered a novel connection between the phosphorylation and ubiquitylation of c-FLIP: phosphorylation of S193 protects c-FLIP from ubiquitylation. Surprisingly, although all c-FLIP isoforms are phosphorylated on this conserved residue, the biological outcome is different for the long and short isoforms, since S193 specifically prolongs the half-lives of the short c-FLIP isoforms, but not c-FLIPL. To summarize, we show that c-FLIP proteins are modified by ubiquitylation and phosphorylation, and that the biological outcomes of these modifications are isoform-specifically determined.

Interaction between cationic and conventional nonionic surfactants in the mixed micelle and monolayer formed in aqueous medium

Mixed micellization and surface properties of cationic and nonionic surfactants dimethyl decyl-, tetradecyl- and hexadecyl phosphineoxide mixtures are studied using conductivity and surface tension measurements. The models of Rubingh, Rosen, and Clint, are used to obtain the interaction parameter, minimum area per molecule, mixed micelle composition, free energies of mixing and activity coefficients. The micellar mole fractions were always higher than ideal values indicating high contributions of cationics in mixed micelles. Activity coefficients were less than unity indicating synergism in micelles. The negative free energies of mixing showed the stability of the surfactants in the mixed micelles.

Chemical modifications of a natural xanthone and antimicrobial activity against multidrug resistant Staphylococcus aureus and cytotoxicity against human tumor cell lines

A series of 15 ω-aminoalkoxylxanthones containing methyl, ethyl, propyl, tert-butylamino and piperidinyl moieties were synthesized from a natural xanthone isolated from a lichen species. These compounds were tested for their in vitro antibacterial properties against Gram-positive and Gram-negative bacteria and cytotoxicity against a number of human tumor cell lines was too evaluated. The newly synthesized derivatives revealed selective activity against Staphylococcus aureus (Gram-positive), and the most promising results are for a multidrug resistant strain, for which six of these compounds showed good activity (MICs 4 µg/mL). Many derivatives inhibited tumor cells growth and most compounds were active on multiple lines.

Centromere domain organization and histone modifications

Centromere function requires the proper coordination of several subfunctions, such as kinetochore assembly, sister chromatid cohesion, binding of kinetochore microtubules, orientation of sister kinetochores to opposite spindle poles, and their movement towards the spindle poles. Centromere structure appears to be organized in different, separable domains in order to accomplish these functions. Despite the conserved nature of centromere functions, the molecular genetic definition of the DNA sequences that form a centromere in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, in the fruit fly Drosophila melanogaster, and in humans has revealed little conservation at the level of centromere DNA sequences. Also at the protein level few centromere proteins are conserved in all of these four organisms and many are unique to the different organisms. The recent analysis of the centromere structure in the yeast S. pombe by electron microscopy and detailed immunofluorescence microscopy of Drosophila centromeres have brought to light striking similarities at the overall structural level between these centromeres and the human centromere. The structural organization of the centromere is generally multilayered with a heterochromatin domain and a central core/inner plate region, which harbors the outer plate structures of the kinetochore. It is becoming increasingly clear that the key factors for assembly and function of the centromere structure are the specialized histones and modified histones which are present in the centromeric heterochromatin and in the chromatin of the central core. Thus, despite the differences in the DNA sequences and the proteins that define a centromere, there is an overall structural similarity between centromeres in evolutionarily diverse eukaryotes.

Modifications of the sympathetic skin response in workers chronically exposed to lead

The long-term effects of low-level lead intoxication are not known. The sympathetic skin response (SSR) was evaluated in a group of 60 former workers of a primary lead smelter, located in Santo Amaro, BA, Brazil. The individuals participating in the study were submitted to a clinical-epidemiological evaluation including questions related to potential risk factors for intoxication, complaints related to peripheral nervous system (PNS) involvement, neurological clinical examination, and also to electromyography and nerve conduction studies and SSR evaluation. The sample consisted of 57 men and 3 women aged 34 to 69 years (mean ± SD: 46.8 ± 6.9). The neurophysiologic evaluation showed the presence of lumbosacral radiculopathy in one of the individuals (1.7%), axonal sensorimotor polyneuropathy in 2 (3.3%), and carpal tunnel syndrome in 6 (10%). SSR was abnormal or absent in 12 cases, representing 20% of the sample. More than half of the subjects (53.3%) reported a history of acute abdominal pain requiring hospitalization during the period of work at the plant. A history of acute palsy of radial and peroneal nerves was reported by about 16.7 and 8.3% of the individuals, respectively. Mean SSR amplitude did not differ significantly between patients presenting or not the various characteristics in the current neurological situation, except for diaphoresis. The results suggest that chronic lead intoxication induces PNS damage, particularly affecting unmyelinated small fibers. Further systematic study is needed to more precisely define the role of lead in inducing PNS injury.

The cytotoxicity of methacryloxylethyl cetyl ammonium chloride, a cationic antibacterial monomer, is related to oxidative stress and the intrinsic mitochondrial apoptotic pathway

Antibacterial monomers incorporated in dentin bonding systems may have toxic effects on the pulp. Thus, the cytotoxicity of antibacterial monomers and its underlying mechanisms must be elucidated to improve the safety of antibacterial monomer application. The influence of an antibacterial monomer, methacryloxylethyl cetyl ammonium chloride (DMAE-CB), on the vitality of L929 mouse fibroblasts was tested using MTT assay. Cell cycle progression was studied using flow cytometry. Production of intracellular reactive oxygen species (ROS) after DMAE-CB treatment was measured using 2,7-dichlorodihydrofluorescein diacetate staining and flow cytometry analysis. Loss of mitochondrial membrane potential, disturbance of Bcl-2 and Bax expression, as well as release of cytochrome C were also measured using flow cytometry analysis or Western blot to explore the possible involvement of the mitochondrial-related apoptotic pathway. DMAE-CB elicited cell death in a dose-dependent manner and more than 50% of cells were killed after treatment with 30 µM of the monomer. Both necrosis and apoptosis were observed. DMAE-CB also induced G1- and G2-phase arrest. Increased levels of intracellular ROS were observed after 1 h and this overproduction was further enhanced by 6-h treatment with the monomer. DMAE-CB may cause apoptosis by disturbing the expression of Bcl-2 and Bax, reducing the mitochondrial potential and inducing release of cytochrome C. Taken together, these findings suggest that the toxicity of the antibacterial monomer DMAE-CB is associated with ROS production, mitochondrial dysfunction, cell cycle disturbance, and cell apoptosis/necrosis.

Modifications in the chemical compounds and sensorial attributes of Engraulis anchoita fillet during marinating process

Marinated fish are fish products preserved by the combined action of salt and organic acids. The objective of this work was to analyze the variations in the chemical compounds of anchovy fillets that give them sensorial characteristics during the marinating process of Engraulis anchoita. The protein content decreased slightly and the TVB-N level decreased significantly in both the brining and marinating stages. In the marinating stage an increase in the total free aminoacids was observed. The NBV level in the brining and marinating solutions increased during these stages due to the solubilization of the non-protein nitrogenous compounds and the degradation of some protein compounds.The decrease of the contents of protein and TVB-N, and the increase of the acidity and the free aminoacids content during the marinating process give the marinated fillets the characteristic texture and aroma.

Physicochemical properties, modifications and applications of starches from different botanical sources

Present trends towards technologies and processes that increase the use of residues make starchy vegetal biomass an important alternative material in various applications due to starch’s versatility, low cost and ease of use when its physicochemical properties are altered. Starch is increasingly used in many industrial applications and as a renewable energy resource. Starch can be modified to enhance its positive attributes and eliminate deficiencies in its native characteristics. In this article, the state of knowledge on conventional and unconventional starches and their properties, characteristics, modifications and applications are reviewed.

The effects of mutated cationic amino acid transporter y+LAT1 at the cellular and systemic level

y+LAT1 is a transmembrane protein that, together with the 4F2hc cell surface antigen, forms a transporter for cationic amino acids in the basolateral plasma membrane of epithelial cells. It is mainly expressed in the kidney and small intestine, and to a lesser extent in other tissues, such as the placenta and immunoactive cells. Mutations in y+LAT1 lead to a defect of the y+LAT1/4F2hc transporter, which impairs intestinal absorbance and renal reabsorbance of lysine, arginine and ornithine, causing lysinuric protein intolerance (LPI), a rare, recessively inherited aminoaciduria with severe multi-organ complications. This thesis examines the consequences of the LPI-causing mutations on two levels, the transporter structure and the Finnish patients’ gene expression profiles. Using fluorescence resonance energy transfer (FRET) confocal microscopy, optimised for this work, the subunit dimerisation was discovered to be a primary phenomenon occurring regardless of mutations in y+LAT1. In flow cytometric and confocal microscopic FRET analyses, the y+LAT1 molecules exhibit a strong tendency for homodimerisation both in the presence and absence of 4F2hc, suggesting a heterotetramer for the transporter’s functional form. Gene expression analysis of the Finnish patients, clinically variable but homogenic for the LPI-causing mutation in SLC7A7, revealed 926 differentially-expressed genes and a disturbance of the amino acid homeostasis affecting several transporters. However, despite the expression changes in individual patients, no overall compensatory effect of y+LAT2, the sister y+L transporter, was detected. The functional annotations of the altered genes included biological processes such as inflammatory response, immune system processes and apoptosis, indicating a strong immunological involvement for LPI.

The effect of cationic-anionic polyelectrolyte multilayer surface treatment on inkjet ink spreading and print quality

The focus of the work reported in this thesis was to study and to clarify the effect of polyelectrolyte multilayer surface treatment on inkjet ink spreading, absorption and print quality. Surface sizing with a size press, film press with a pilot scale coater, and spray coating, have been used to surface treat uncoated wood-free, experimental wood-free and pigmentcoated substrates. The role of the deposited cationic (polydiallydimethylammonium chloride, PDADMAC) and anionic (sodium carboxymethyl cellulose, NaCMC) polyelectrolyte layers with and without nanosilica, on liquid absorption and spreading was studied in terms of their interaction with water-based pigmented and dye-based inkjet inks. Contact angle measurements were made in attempt to explain the ink spreading and wetting behavior on the substrate. First, it was noticed that multilayer surface treatment decreased the contact angle of water, giving a hydrophilic character to the surface. The results showed that the number of cationic-anionic polyelectrolyte layers or the order of deposition of the polyelectrolytes had a significant effect on the print quality. This was seen for example as a higher print density on layers with a cationic polyelectrolyte in the outermost layer. The number of layers had an influence on the print quality; the print density increased with increasing number of layers, although the increase was strongly dependent on ink formulation and chemistry. The use of nanosilica clearly affected the rate of absorption of polar liquids, which also was seen as a higher density of the black dye-based print. Slightly unexpected, the use of nanosilica increased the tendency for lateral spreading of both the pigmented and dye-based inks. It was shown that the wetting behavior and wicking of the inks on the polyelectrolyte coatings was strongly affected by the hydrophobicity of the substrate, as well as by the composition or structure of the polyelectrolyte layers. Coating only with a cationic polyelectrolyte was not sufficient to improve dye fixation, but it was demonstrated that a cationic-anionic-complex structure led to good water fastness. A threelayered structure gave the same water fastness values as a five-layered structure. Interestingly, the water fastness values were strongly dependent not only on the formed cation-anion polyelectrolyte complexes but also on the tendency of the coating to dissolve during immersion in water. Results showed that by optimizing the chemistry of the layers, the ink-substrate interaction can be optimized.