Oligonucleotide-based drugs hold great promise for the treatment of many types of diseases, ranging from genetic disorders to viral infections and cancer. The problem is that efficient delivery across the cell membrane is required for oligonucleotides to have their desired effect. Cell-penetrating peptides (CPPs) provide a solution to this problem. CPPs are capable of transporting cargoes such as drugs or nucleic acids for gene therapy into the cell, either by covalent conjugation to the cargo or by non-covalent complex formation. This thesis is focused on the development of a class of peptides called PepFects, peptides with fatty acid modifications capable of forming nanoparticle-sized complexes with oligonucleotides. These complexes are efficiently internalized by many different cell types and are generally non-toxic and non-immunogenic. We have developed a number of novel PepFect peptides and a quantitative structure-activity model to predict the biological effect of our peptides. In addition, the involvement of scavenger receptors class A in the endocytic uptake of PepFect complexes as well as other CPPs and polymeric transfection agents was studied. Lastly, we have developed a series of PepFect peptides for delivery across the blood-brain barrier and a model system mimicking the blood-brain barrier in order to evaluate the passage of these peptides. The general aim of this thesis is to improve the understanding of intracellular delivery of oligonucleotides with PepFect peptides from both a chemical and a biological viewpoint, and further improve the efficacy of this delivery system with the long-term goal of making it useful in clinical settings.

Therapeutically restricted sites present a formidable barrier in medicine. Herein, chemical strategies to overcome two restricted sites, HIV reservoirs and intracellular bacteria, will be discussed. First, cellular and anatomical HIV reservoirs, such as those in the brain, limit HIV eradication using currently known therapeutic regimes. HIV therapies are unable to localize in the brain, in part, due to high expression of efflux transporters, such as P-glycoprotein (P-gp), at the BBB, because many of these therapies are P-gp substrates. In an effort to overcome therapeutically restricted HIV sanctuaries, a dimerized combination HIV therapy was designed to act two-fold. First, the dimeracts as a P-gp inhibitor allowing therapeutic access to restricted sites. Second, the dimeractsas a prodrug, which once in the reducing environment of the cell, may release monomeric HIV therapies. The dual conjugate, Abacavir-S2-Darunavir, was shown to potently inhibit P-gp across two separate cell lines, was able to regenerate the component monomers in a reducing environment and contained modest anti-HIV activity.<div><br><div>Further, mammalian cells create sanctuary sites for bacteria to grow and proliferate, because many common antibiotic therapies are unable to cross the mammalian cell membrane. Therefore, these pathogens are able to proliferate without therapeutic constraint. Here, a chemical strategy was developed to deliver a dual antibiotic therapy inside mammalian cells in an effort to clear these intracellular pathogens. First, a new synthetic strategy was developed for facile synthesis of dual conjugates, composedof an aminoglycoside and a cell penetrating peptide (CPP) linked with a reversible disulfide tether, using kanamycin and the known CPP Arg8as a model system. Next, this synthetic methodology was expanded for use with theaminoglycoside tobramycin and theknown broad-spectrum antibiotic and cell penetrating peptide, P14LRR, once again linked via the reversible disulfide tether (TobP14). Two distinct isomers of TobP14 were synthesized, isolated, and fully characterized by 2D NMR. The TobP14 isomers were shown to be an effective antibiotic across various Gram positive and negative pathogens such as MRSA, S. epidermidis, P. aeruginosa, and A. baumannii. Further, the isomers effectively releasedthe monomeric therapies (tobramycin and P14-SH) in a reducing environment and werenontoxic to mammalian cells up to 16 μM. Finally, the dual conjugate isomers significantly reduce two different strains of intracellular A. baumanniiwithin macrophages.<br><div><br></div><div><br></div></div></div>

De nos jours, une des stratégies majeures dans la modulation de la pharmacocinétique des composés bioactifs est leur vectorisation et l’obtention de formes prodrogues. Ce travail est centré sur la vectorisation d’antitumoraux phosphorés à l’aide de peptides favorisant le passage membranaire. Nous avons alors réalisé la conjugaison d’aminoalkyl-bisphosphonates avec des séquences peptidiques afin de modifier leur temps de rétention dans l’organisme et d’augmenter leur internalisation cellulaire. Différents espaceurs possédant un motif carbonylé insaturé ont été évalués dans le couplage par la réaction d’addition aza- et thiaMichael afin d’aboutir à l’obtention d’un conjugué peptide-alendronate. Une approche prodrogue a également été réalisée avec la synthèse de dérivés de type bisphosphinates et l’obtention d’un analogue de l’alendronate. / Nowadays, one of the main strategies for pharmacokinetic modifications of bioactive compounds is their vectorization and the synthesis of prodrug derivatives. This work is focused on the vectorization of phosphorus antitumor agents with cell-penetrating peptides. We have then conjugated aminoalkyl-bisphosphonates with peptidic sequence to modify their retention time and increase their cellular internalization. Several linkers bearing an insaturated carbonyl moiety have been evaluated in conjugation by aza- and thia-Michael addition reaction to obtain a conjugated peptide-alendronate compounds. A prodrug approach has been conducted with the synthesis of bisphosphinate derivatives and an analog of alendronate has been obtained.

Maintenance of aging buried infrastructure and reinforced concrete are critical issues in the United States. Inexpensive non-destructive techniques for mapping and imaging infrastructure and defects are an integral component of maintenance. Ground penetrating radar (GPR) is a widely-used non-destructive tool for locating buried infrastructure and for imaging rebar and other features of interest to civil engineers. Conventional acquisition and interpretation of GPR profiles is based on the arrival times of strong reflected/diffracted returns, and qualitative interpretation of return amplitudes. Features are thereby generally well located, but their material properties are only qualitatively assessed. For example, in the typical imaging of buried pipes, the average radar wave velocity through the overlying soil is estimated, but the properties of the pipe itself are not quantitatively resolved. For pipes on the order of the radar wavelength (<5-35 cm), pipe dimensions and infilling material remain ambiguous. Full waveform inversion (FWI) methods exploit the entire radar return rather than the time and peak amplitude. FWI can generate better quantitative estimates of subsurface properties. In recent decades FWI methods, developed for seismic oil exploration, have been adapted and advanced for GPR with encouraging results. To date, however, FWI methods for GPR data have not been specifically tuned and applied on surface collected common offset GPR data, which are the most common type of GPR data for engineering applications. I present an effective FWI method specifically tailored for common-offset GPR data. This method is composed of three main components, the forward modeling, wavelet estimation and inversion tools. For the forward modeling and iterative data inversion I use two open-source software packages, gprMax and PEST. The source wavelet, which is the most challenging component that guarantees the success of the method, is estimated with a novel Sparse Blind Deconvolution (SBD) algorithm that I have developed. The present dissertation indicates that with FWI, GPR can yield better quantitative estimates, for example, of both the diameters of small pipes and rebar and their electromagnetic properties (permittivity, conductivity). Also better estimates of electrical properties of the surrounding media (i.e. soil or concrete) are achieved with FWI.

Acquiring GPR data at multiple frequencies is useful because higher-frequency profiles have better spatial resolution, although they suffer from reduced depth penetration. Lower-frequencies can generally resolve to greater depths, but at the cost of spatial resolution. For concise presentation of GPR data, it would be useful to combine the best features of each profile into a composite radargram. This study explores effective ways to present GPR data acquired at multiple frequencies. An example is shown from a survey of hurricane overwash deposits from Santa Rosa Island, Florida. The methodology used to create a composite radargram is dependent on which of two goals the composite radargram is designed to achieve. These goals are broadening the spectral bandwidth of GPR data to increase the effectiveness of deconvolution and enhancing the resolution and depth of GPR data by plotting high-frequency data at early two-way travel times, low-frequency data at late two-way travel times, and using filters to smoothly transition from high-frequency to lower-frequency data. The steps towards creating a composite radargram include: 1) applying standard processing to nominal frequency data sets, 2) creating spatially coincident data sets, 3) equalizing the amplitude spectra among each nominal frequency data set, and 4) summing nominal frequency data sets together. Spectral bandwidth broadening is achieved by applying optical spectral whitening and summing nominal frequency data sets using a single ramped. Deconvolving this composite radargram did not show the same success observed by Booth et al. (2009). Enhancing the resolution and depth of GPR data can be achieved by applying amplitude envelope equalization (AEE) and summation using double ramped filters. AEE calculates the coefficients required to make equivalent average amplitude envelopes for GPR data that has been gained with automatic gain control . Double ramped filters suppress low-frequency energy for two-way travel times when a higher-frequency data set has adequate signal strength and higher frequency energy for two-way travel times when higher- frequency energy exhibits significant attenuation. A composite radargram built with AEE and double ramped filters achieves the goal enhancing resolution and depth of GPR data. Shallow reflections are interpreted as dune and hurricane overwash stratigraphy.

Ground Penetrating Radar is a tool for mapping the subsurface in a noninvasive way. The radar instrument transmits electromagnetic waves and records the resulting scattered field. Unfortunately, the data from a survey can be hard to interpret, and this holds extra true for non-experts in the field. The data are also usually in 2.5D, or pseudo 3D, meaning that the vast majority of the scanned volume is missing data. Interpolation algorithms can, however, approximate the missing data, and the result can be visualized in an application and in this way ease the interpretation. This report has focused on comparing different interpolation algorithms, with extra focus on behaviour when the data get sparse. The compared methods were: Linear, inverse distance weighting, ordinary kriging, thin plate splines and fk domain zone-pass POCS. They were all found to have some strengths and weaknesses in different aspects, although ordinary kriging was found to be the most accurate and created the least artefacts. Inverse distance weighting performed surprisingly well considering its simplicity and low computational cost. A web-based, easy-to-use visualization application was developed in order to view the results from the interpolations. Some of the tools implemented include time slice, crop of a 3D cube, and iso surface.

Runion is a protohistoric Native American village located on the floodplain of the Nolichucky River in western Washington County. Previous archaeological excavations and radiocarbon dates suggest that the village was occupied during the mid-16th to mid-17th century. The Nolichucky River, in contrast, has been flowing through the area for millennia. Geophysical surveys are used to image the subsurface non-invasively, without disturbing protected land and/or organisms. Preliminary geophysical data collected at Runion include ground penetrating radar (GPR), electromagnetic induction (EMI), and magnetometry. These data show a linear feature surrounding the protohistoric village. Given its placement around the margins of the village, the feature could be interpreted as a fortification ditch, which is often paired with a palisade wall to defend a village from attack. The feature is also consistent with typical meandering floodplain stratigraphy, where sections of channel are often abandoned to form oxbow lakes. Over time these abandoned channels fill in and are called paleochannels. Each geophysical method measures the properties and characteristics of the linear feature, a presumed paleochannel. GPR sends electromagnetic radar waves into the ground, which reflect off different subsurface layers and are recorded as radargrams. Magnetometry measures subtle changes in earth magnetism, including the magnetization of rocks, soils, and/or ferrous objects. EMI systems transmit low frequency electromagnetic waves to measure both electrical conductivity (EC) and magnetic susceptibility (MS). Each of these instruments are used to collect data in transects and then processed to produce profiles, maps and, in the case of GPR, three-dimensional datasets of the subsurface. It is anticipated that GPR will reveal details about the stratigraphy of the linear feature. Magnetic, EC, and MS measurements will further help to interpret the GPR data by distinguishing between different types of sediments. These data may show if the feature is a paleochannel or a ditch excavated into older stratigraphic layers by village inhabitants for fortification. Ultimately, the feature will be tested with soil cores to study the sediments directly. At this preliminary stage the feature is interpreted to be a paleochannel. The stratigraphic layers revealed by GPR show a broad depression with stratigraphic layers characteristic of a paleochannel. In addition, magnetic readings are anomalously low on the eastern margin (closer to the modern river channel) and high on the western margin. This could indicate paired point bar sands and paleochannel fill, respectively. This interpretation is still tentative, however, because we have not yet integrated the EMI data, extracted soil cores, or dated the feature. Radiocarbon dates might help determine the relative age of the feature if organic carbon is present. In conclusion, preliminary data currently suggests that the structure is geological rather than archaeological. In the coming months we will collect more GPR data with different frequency GPR antennas, integrate the EMI data, and test the findings by extracting soil cores and reconstructing the stratigraphy.

The variable contexts of Fremont habitation sites in Utah Valley often make identification of those sites very challenging for archaeologists. Pit houses and other structures throughout the valley are frequently in plowed fields or other disturbed contexts that obscure their more exact location and nature. The application of geophysical technologies at archaeological sites throughout the world, including in North America, has proven to be an effective means of subsurface archaeological survey. However, geophysical techniques have been underutilized in Fremont archaeology. This paper reports on the employment of two geophysical methods, ground-penetrating radar (GPR) and fluxgate gradiometer surveys, at three known Fremont habitation sites in southern Utah Valley – the Wolf Village, Wolf Mound, and Snow Farm sites. The preliminary geophysical surveys and later ground-truthing of various geophysical anomalies reveals the effectiveness of these methods in identifying where architectural or other cultural features exist below the surface.

Ground penetrating radar (GPR) is limited by depth penetration and signal-to-noise ratio (SNR), impacting the ability to resolve subsurface features. Stacking, a process of averaging multiple scans in the same location, improves SNR. Digital antennas are capable of stacking at much higher rates than analog antennas. Four sites were examined using a GSSI SIR-4000 GPR unit with a 400 MHz analog antenna and a 350 MHz digital “hyperstacking” (350 HS) antenna. Sites represent various soil conditions, with known features. Data were compared qualitatively and quantitatively for differences in antenna outputs. Visual inspection of radargrams indicate a reduction in noise in the 350 HS data compared to the 400 MHz data. Quantitative assessments identified significant differences in standard deviation of radar reflection amplitude occurring at depth with both antennas and a reduction in noise and marginal increases in depth of penetration in low-loss conditions with the 350 MHz HS antenna.

Pathogens such as <i>Listeria</i>, <i>Shigella</i>, <i>Brucella</i>, <i>Salmonella</i>, <i>Mycobacterium tuberculosis</i> and <i>methicillin-resistant Staphylococcus aureus</i> (MRSA) can traverse into mammalian cells, such as phagocytic macrophages. Once inside, these bacteria can survive and reproduce, causing chronic infections. It is of utmost importance to develop novel antibiotics with broad spectrum activity to control these deadly bacteria. Broad spectrum activity will allow for targeting of pathogens with different structures and cell membrane components.<div>This work focuses on the synthesis of a dual antibiotic agent, composed of a cationic amphiphilic polyproline helix (CAPH) possessing cell penetrating and nonmembrane lytic antimicrobial capabilities (P14LRR), and a derivative of the polymyxin B (PMX) antibacterial peptide. This dual antibiotic conjugate was created to be a tool to potentially clear intracellular pathogenic bacteria. Overall, the reduction of the disulfide bond linking the two antibiotics within the reducing environment of cells would release the individual antimicrobial agents, and could have improved cell membrane penetration and intracellular synergistic activity. Herein, the synthesis of the dual antibiotic agent, P14LRR-PMX, is discussed. </div>