The fundamental element of life known to man is the gene. The information contained in genes regulates all cellular functions, in health and disease. The ability to selectively alter genes or their transcript intermediates with designed molecular tools, as synthetic oligonucleotides, represents a paradigm shift in human medicine. The full potential of oligonucleotide therapeutics is however dependent on the development of efficient delivery vectors, due to their intrinsic characteristics, as size, charge and low bioavailability. Cell-penetrating peptides are short sequences of amino acids that are capable of mediating the transport of most types of oligonucleotide therapeutics to the cell interior. It is the interaction of cell-penetrating peptides with oligonucleotides and the transport of their non-covalently formed complexes across the cellular membrane, that constitutes the main subject of this thesis. In Paper I we studied the effects of different types of oligonucleotide cargo in the capacity of cationic and amphipathic peptides to interact with lipid membranes. We found that indeed the cargo sequesters some of the peptide’s capacity to interact with membranes. In Paper II we revealed the simultaneous interaction of different molecular and supramolecular peptide and peptide/oligonucleotide species in equilibrium, with the cellular membrane. In Paper III we developed a series of peptides with improved affinity for oligonucleotide cargo as well as enhanced endosomal release and consequently better delivery capacity. In Paper IV we investigated the effect of saturated fatty acid modifications to a cationic cell-penetrating peptide. The varying amphipathicity of the peptide correlated with the complex physicochemical properties and with its delivery efficiency. This thesis contributes to the field with a set of characterized mechanisms and physicochemical properties for the components of the ternary system – cell-penetrating peptide, oligonucleotide and cell membrane – that should be considered for the future development of gene therapy. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: Manuscript.</p>

Glioblastoma multiforme is the most aggressive form of malignant brain tumor with poor prognosis. The efficacy of brain cancer treatment by chemotherapeutics is limited by the blood-brain barrier (BBB) which allows less than 2% of the small molecules and blocks almost all the macromolecules to transport into the brain. Delivery of the large molecules such as proteins and nucleic acids across the BBB is a great challenge for brain-targeted drug delivery. To overcome this obstacle, cell-penetrating peptides (CPPs) were used as vectors for delivery of nucleic acids across the BBB targeting glioblastomas. The CPPs have shown such promising carriers to deliver various cargoes ranging from small molecules to large molecules into the cells. This thesis is focused on the development of glioblastoma-targeting vectors based on modifications of the CPPs and the targeting peptides. The peptide-based vectors were developed to improve the transport of the nucleic acids across the BBB and specifically target glioblastomas. In this thesis, a series of peptide-based vectors targeting glioblastomas were synthesized and modified with targeting peptides by either covalent conjugation or non-covalent complex formation. The delivery of plasmid DNA (pDNA) in the complex with the peptide-based vectors was studied in the in vitro model of the BBB. The role of receptors expressed on the BBB was investigated. Scavenger receptors class A and B were found to be expressed on the BBB, and they were involved in the delivery of the pDNA across the BBB model. Moreover, various targeting peptides were modified with hexaglutamate to form non-covalent complexes with the CPPs for small interfering RNA (siRNA) delivery to glioblastoma cells. The non-covalent complex of the CPP and the targeting peptide showed greater gene-silencing efficiency than the consecutively covalent conjugation of the CPP and the targeting peptide for siRNA delivery to glioblastoma cells. Lastly, a number of novel, amphipathic peptides were developed based on the model amphipathic peptide. The prediction of the biological effect of the designed peptides using quantitative structure-activity relationship model showed a correlation with the experimental data. Finally, the CPP-based nucleic acid delivery vectors with homing peptide strategy have a potential for the BBB shuttle and the future use as a glioblastoma-targeted drug carrier in the in vivo studies and the clinical applications. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.</p>

Cancer testis antigens (CTA) represent attractive targets for targeted radiotherapy and imaging as their expression is restricted to cancer and germ cells. NY-ESO-1, a member of the CTA family, is highly immunogenic and expressed in multiple tumor types including carcinoma of bladder, liver lung. The aim of this study was to develop radioimmunoconjugates (RIC) to target NY-ESO-1 protein in cancer cells. Anti-NY-ESO-1 antibodies were modified by addition of DTPA for 111In-labelling or, in the presence of Iodogen, were 123I-labelled. Delivery of radiolabeled immunoconjugates across the cell membrane was achieved using a protein transfection (PT) reagent (SAINT-PhD) and by chemical linkage with the cell-penetrating and nuclear-localizing peptide, TAT (YGRKKRRQRRR). Cellular internalization, distribution and efflux of 111In-DTPA-anti-NY-ESO-1-TAT-PT and 123I-anti-NY-ESO-1-TAT-PT were investigated in cell fractionation and retention assays. It was shown that protein transfection reagent has promoted the cellular uptake of RICs into SK-MEL-37 and both of 111In-DTPA-anti-NY-ESO-1-TAT-PT and 123I-anti-NY-ESO-1-TAT-PT was retained longer in SK-MEL-37 cells in comparison to their isotope control RIC. In clonogenic assays, 111In-DTPA-anti-NY-ESO-1-TAT-PT significantly reduced surviving fraction of SK-MEL-37 cells. Cytotoxicity was inversely proportional to specific activity and the concentration of cells exposed to 111In-DTPA-anti-NY-ESO-1-TAT-PT. siRNA knock down of NY-ESO-1 resulted in partial reversal of 111In-DTPA-anti-NY-ESO-1-TAT-PT associated cytotoxicity. These promising results obtained from the in vitro study has brought the probe further into in vivo study. In preliminary biodistribution studies in SK-MEL-37 xenograft-bearing mice, tumour:muscle ratio for 111In-DTPA-anti-NY-ESO-1-TAT-PT was statistically significant compared to the control RIC 48 h post injection. This clearly indicated that the probe can be delivered into tumour in in vivo model and the successful uptake of radioactivity increased the chance of causing cytotoxicity to tumour cells through DNA damage. All of these findings have suggested that intracellular cancer associated antigen NY-ESO-1 can be reached by protein transfection reagent and cell penetrating peptide and initiates DNA damage through radio-isotope mediated cytotoxicity. Therefore, it represents a novel approach to the treatment of CTA-expressing cancers.

Membrane active peptides with the ability to cross the plasma membrane represent a promising class of therapeutic compounds. However, translocation efficacy and membrane toxicity of these peptides appear correlated and a better understanding of their mechanisms of action is needed to achieve the desired effect. Here, a range of coarse grain molecular dynamics simulations have been performed to systematically investigate the interactions of such cell-penetrating peptides (CPPs) with biologically relevant membranes. Challenges associated to the development of a suitable asymmetric mammalian membrane model demonstrated the importance of lipid species distribution on the bilayer mechanical properties, as well as the effect of coarse graining on its electrostatic properties. However, simulations successfully discriminated between two CPPs, penetratin and transportan, and were consistent with the experimental data available for these. The results obtained suggest that the ability of transportan peptides to aggregate into flexible, dynamic, transmembrane bundles is responsible for their relative membrane toxicity. The stability and structure of these aggregates, as well as the extent of the bilayer perturbations they induced, were shown to depend on the membrane composition and asymmetry, thus providing a molecular basis to explain how the toxicity of CPPs is modulated by membranes. In particular, bilayer destabilisation was enhanced by the presence of anionic lipids and hampered by that of cholesterol. Transportan aggregates were also observed to trigger lipid flip-flops above a certain size and a new pathway for such events, not relying on the formation of water defects, was characterised.

This thesis presents a novel antenna structure that satisfies the challenging requirements of an air coupled high speed ground penetrating radar (GPR). The desired GPR system is to achieve high spatial resolution and accurate inspection results while scanning at relatively high speed for highway pavement and bridge deck inspection. This work utilizes the Ultra Wide Band (UWB) antenna design to achieve both physical and electrical requirements imposed. The design procedure starts with a short survey to discuss typical UWB antennas used for GPR applications, and various tradeoffs of each type specifically when used for Air Coupled GPR applications. Our structure anatomy is presented, followed by a theory introduction that mainly focuses on achieving good impedance matching throughout the proposed antenna structure. A proof-of-concept MATLAB model is created to evaluate the preliminary physical dimensions that can achieve minimum reflections at antenna's feed point. These dimensions are then used in SolidWorks to create a 3D model that is imported later in HFSS to obtain accurate electromagnetic characteristics. Furthermore, fine tunings are performed to the antenna structure to optimize both gain and impedance matching. The SolidWorks 3-D structural model is finally used for antenna fabrication. The measurements recorded from the field experiments using the prototypes manufactured are compared to the simulation results confirming our initial findings. Both measurements and simulation results demonstrated very small reflection loss across the 700 MHz ~ 6 GHz frequency band with a very high directed gain and radiation efficiency.

Phased array radar systems employ multiple antennas to create a radar beam that can be steered electronically. By manipulating the relative phase values of feeding signals among different antennas, the effective radiation pattern of the array can be synthesized to enhance the main lobe in a desired direction while suppressing the undesired side lobes in other directions. Hence the radar scanning angles can be electronically controlled without employing the bulky mechanical gimbal structure, which can significantly reduce radar system size, weight and power consumption. In recent years, phased array technologies have received great attentions and are explored in developing many new applications, such as smart communication systems, military radars, vehicular radar, etc. Most of these systems are narrow band systems, where the phase delays are realized with narrow band phase shifter circuits. For the impulse ground penetrating radar however, its operating frequency spans an ultrawide bandwidth. Therefore the traditional phase shifters are not applicable due to their narrow band nature. To resolve the issue, in this study, a true time delay approach is explored which can precisely control time delays for the feeding pulse signals among different antennas in the array. In the design, an on chip programmable delay generator is being developed using Global Foundry 0.18 µm 7 HV high voltage CMOS process. The time delay control is realized by designing a programmable phase locked loop (PLL) circuit which can generate true time delays ranging from 100 ps (picoseconds) to 500 ps with the step size of 25 ps. The PLL oscillator's frequency is programmable from 100MHz to 500MHz through two reconfigurable frequency dividers in the feedback loop. As a result, the antenna beam angle can be synthesized to change from 9.59° to 56.4° with a step of 2.75°, and the 3dB beamwidth is 10°. The power consumption of the time delay circuit is very low, where the supply voltage is 1.8V and the average current is as low as 472uA.

The Charity Hospital Cemetery in New Orleans, Louisiana, was used as a potter's field for over 150 years. When Charity Hospital considered selling a portion of the property ground penetrating radar (GPR) and thermal infrared (TIR) data were collected in the cemetery to locate unmarked graves. The TIR data could not be used because the expert died before compiling the TIR data. Therefore, the GPR data was the sole source of subsurface information. GPR anomalies were used to excavate 3 areas where bones and hospital supplies were subsequently found, unfortunately very limited analyses were possible on the analog GPR data. The study presented here involved digitizing data and conducting a more thorough analysis of map patterns to determine whether GPR data could be used reliably to locate burials in the cemetery. The study's result indicates that GPR is a reliable source for burial detection and other anomalies in the subsurface.

El texto completo de este trabajo no está disponible en el Repositorio Académico UPC por restricciones de la casa editorial donde ha sido publicado. / A split-mouth, double-blind trial evaluated the effects of a surface sealant on the clinical performance of Class I composite resin restorations. In 16 patients, 27 pairs of maxillary and mandibular molars or premolars with Class I carious lesions or unsatisfactory restorations were restored with composite resin. For each pair, 1 surface was sealed with surface sealant. Clinical evaluations of marginal integrity, marginal discoloration, anatomical form, and secondary caries were performed by 2 experienced operators using modified US Public Health Service criteria 1-2 weeks and 6 and 12 months after treatment. Data were analyzed with the McNemar test (P < 0.05). After 6 months, only 1 (4%) sealed restoration presented a Bravo rating for marginal integrity. After 12 months, the Bravo ratings for marginal integrity were 2 (7%) for sealed restorations and 1 (4%) for nonsealed restorations. Restorations received a score of Alfa for all other parameters at all time periods. There were no statistically significant differences within or between the sealed and nonsealed groups (P = 1.0). The use of a surface sealant did not improve the clinical performance of posterior composite resin Class I restorations. / Revisión por pares

Peat soils are known to be a significant source of atmospheric greenhouse gasses. However, the releases of methane and carbon dioxide gasses from peat soils are currently not well understood, particularly since the timing of the releases are poorly constrained. Furthermore, most research work performed on peatlands has been focused on temperate to sub-arctic peatlands, while recent works have suggested that gas production rates from low-latitude peat soils are higher than those from colder climates. The purpose of the work proposed here is to introduce an autonomous Ground Penetrating Radar (GPR) method for investigating the timing of gas releases from peat soils at the lab scale utilizing samples originating from Maine and the Florida Everglades, and at the field scale in a Maine peatland. Geophysical data are supported by direct gas flux measurements using the flux chamber method enhanced by timelapse photography, and terrestrial LiDAR (TLS) monitoring surface deformation. / by William J. Wright. / Thesis (M.S.)--Florida Atlantic University, 2013. / Includes bibliography. / Mode of access: World Wide Web. / System requirements: Adobe Reader.

Este estudo avaliou, in vitro, a influência da aplicação de quatro selantes de superfície na rugosidade de duas resinas compostas. Cinqüenta espécimes foram confeccionados e divididos em 10 grupos com 5 espécimes cada, em função da combinação entre resina composta e selante de superfície, sendo: G1. Controle Concept; G2. Concept + Fortify; G3. Concept + Biscover; G4. Concept + Lasting Touch; G5. Concept + Fill Glaze; G6. Controle Esthet X; G7. Esthet X + Fortify; G8. Esthet X + Biscover; G9. Esthet X + Lasting Touch; G10. Esthet X + Fill Glaze. Os espécimes foram confeccionados com uma matriz específica, em quatro incrementos, com dimensões internas de 15mmX4mmX5mm, sendo sua polimerização realizada com o aparelho de lâmpada halógena Curing Light 2500 (500mW/cm2) por 20s cada incremento. Em seguida, foram armazenados em água destilada à 37ºC e umidade absoluta de 100% por 24 horas. Após este período, os espécimes foram adaptados em uma Politriz Metalográfica e planificados com lixas de granulometria 320, 600 e a rugosidade inicial foi aferida. Para a mensuração da rugosidade, utilizou-se o rugosímetro Hommel Tester T1000 basic. Numa segunda fase, aplicaram-se os selantes de superfície, conforme recomendações do fabricante, e uma nova aferição da rugosidade superficial foi realizada. Para o teste de abrasão, os espécimes foram submetidos a 100.000 ciclos de escovação, seguido de nova mensuração da rugosidade. Em adição à rugosidade e com o objetivo de avaliar qualitativamente a superfície de alguns espécimes, a leitura em Microscopia Eletrônica de Varredura foi empregada. Os resultados foram submetidos aos testes ANOVA a 3 critérios e Tukey para comparações individuais entre os diferentes grupos (p<0,05). A aplicação do selante de superfície diminuiu a rugosidade superficial das resinas compostas testadas (G2=0,0727, G3=0,0147, G4=0,0307, G5=0,0253, G6=0,0960, G7=0,0173, G8=0,0333, G9=0,0480). A abrasão simulada aumentou a rugosidade superficial para todos os grupos, com exceção do Fortify (G2=0,0740, G7=0,0673) e Biscover (G7=0,0440), sendo os maiores valores apresentados pelo Lasting Touch (G4= 0,1253, G9=0,0980), seguido do Fill Glaze (G5=0,0933, G10= 0,0847). / This in vitro study evaluated the influence of the application of four surface sealers on the surface roughness of composite resins. Fifty specimens were divided into ten groups according to different patterns of composite resin and surface sealers, as follows: G1. Control with Concept resin; G2. Concept + Fortify; G3. Concept + Biscover; G4. Concept + Lasting Touch; G5. Concept + Fill Glaze; G6. Control with Esthet X; G7. Esthet X + Fortify; G8. Esthet X + Biscover; G9. Esthet X + Lasting Touch; G10. Esthet X + Fill Glaze. Specimens were made in four increments using a stainless steel matrix of 15x5x4mm and resin increments were polymerized with halogen lamp Curing Light 2500 (500mW/cm2) for 20 seconds each. After complete polymerization, specimens were stored in 100% humidity in distilled water at 37ºC for 24 hours. Following storage, specimens were polished using ascending grades (320, 600) of abrasive SiC paper under running water and the initial roughness was measured with a perfilometer Hommel Tester T100 basic (Hommelwerke GmbH ref. # 240851 - Schwenningem - Germany). Surface sealers were applied following the manufacturer\'s instructions and a new measurement of roughness was made. For the abrasive resistance test, specimens were submitted to 100.000 cycles of abrasive dentifrice brushing followed by surface roughness.. Qualitative analisys was made by using MEV. Results were submitted to Three-way ANOVA (p<0,05) and Tuckey\'s test to individual comparisons between different groups. Surface sealant application provided smoother surfaces for of composite resins tested (G2=0.0727, G3=0.0147, G4=0.0307, G5=0.0253, G6=0.0960, G7=0.0173, G8=0.0333, G9=0.0480). The abrasion test increased the surface roughness for all tested groups, except Fortify (G2=0,0740, G7=0,0673) and Biscover (G7=0,0440). Lasting Touch presented rougher surfaces (G4= 0,1253, G9=0,0980), followed by Fill Glaze (G5=0,0933, G10= 0,0847).