The internal architecture of a beach system can provide clues into the processes involved in its formation, including depositional processes, and/or driving mechanisms (Billy et al., 2014). Several unique events such as cold fronts or Hurricane Irma caused conditions that resulted in erosion and accretion changes in Red Reef Beach - Boca Raton, throughout the year of 2017. Since the lateral extent of these changes is difficult to evaluate using traditional methods such as coring, a Ground Penetrating Radar (GPR) was tested, which allows for a good lateral resolution (cm scale), to image the distribution and evolution of these sediments. The objectives of this study were to 1) explore the lateral variability in the internal architecture of sediments in Red Reef beach in Boca Raton (FL) using an array of ground penetrating radar (GPR) measurements constrained with coring and sediment analysis; 2) explore how dynamics of erosion and accretion induced by changes in wave activity and related to tide variation and storm events, may affect surface topography and the sedimentary internal architecture of beach deposits, using RTK GPS and GPR time-lapse measurements; 3) to explore changes in the lateral extent of the freshsaltwater interface along the beach profile in relation to tide variation and storm events. Reflectors identified in the GPR images showed some evidence of erosional and accretionary surfaces preserved in Red Reef beach. These measurements were repeated over time coinciding with certain events (such as Hurricane Irma) to explore their effects in terms of sediment erosion and accretion as reflected in changes in topography (using time-lapse GPS-RTK measurements), and changes in the internal sedimentary architecture (using time-lapse GPR measurements). The datasets collected also revealed the temporal evolution of the salt-freshwater interface, showing how the lateral extent of saltwater saturated sediment (inferred from areas of GPR signal attenuation along the profiles) evolved over time. This study shows the potential of GPR to provide information about beach sediment processes and dynamics at resolutions beyond traditional measurements (such as coring). It also shows the importance of combining methods that are complementary, such as the use of RTK GPS to explore changes in topography, and GPR that provides information on subsurface sedimentary architecture and the mechanism of change such as post-storm recovery. This study has implications for better understanding changes in coastal sedimentary deposits and processes, both at the subsurface, particularly after high-energy events, such as hurricanes, that result in rapid changes in erosion and/or accretion of sediments. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2017. / FAU Electronic Theses and Dissertations Collection

In the work presented here we have used a function-based approach to isolate 12 novel cell-penetrating peptides from a 10,000+ member peptide library of rational design. Our unique high-throughput screen differentiates non-membranolytic from membranolytic translocation of peptides across lipid bilayers, thus allowing the selection of potential cell-penetrating peptides over potential antimicrobial peptides or peptide toxins. The 12 residue framework of the peptide library, designed with translocation in mind, is a series of 9 combinatorial sites followed by a C-terminal α-1-chymotrypsin cleavage site that is integral to the screen. The resulting residue in each of the combinatorial sites is one of 2 - 4 variable amino acids, with a hydrophobic or cationic residue available in each position. The sequences of nonpore-forming translocating peptides pulled from the screen have a 3 residue motif, Leu-Leu-Arg (p=10-5), and an overall under representation of basic residues in favor of hydrophobic amino acids. Upon characterization, these novel peptides were shown to behave akin to known cell-penetrating peptides found in nature. Ex vivo studies, in mammalian tissue cultures, revealed that the peptides translocate across the cell membrane without toxicity to the cell. In addition, structural studies showed a lack of convergence regarding a structure- function relationship, a continued trend seen among membrane-active peptides. In the course of the screen and the ex vivo studies, the peptides carried small polar molecules across lipid bilayers and biological membranes respectively; suggesting that, in addition to being cell-penetrating peptides, they could be put to use as effective therapeutic agents. The discovery of these novel cell-penetrating peptides by use of our screen supports function-based screening of peptide libraries as the best way to arrive at de novo membrane-active peptides with specific functions of interest. / acase@tulane.edu

Les peptides vecteurs (CPP) présentent un grand intérêt pour l'internalisation de principes actifs dans les cellules. Les mécanismes permettant aux peptides de traverser la membrane restent sujets à discussion. Mieux comprendre leurs interactions avec la membrane pourrait permettre d'améliorer leur efficacité. L'organisation des lipides après interaction avec le peptide pénétratine a été étudiée par DSC et par fluorescence du Laurdan, sur des membranes modèles composées de lipides naturels. La pénétratine a induit de l'hétérogénéité dans la membrane, ce qui pourrait être un élément important pour déstabiliser la membrane durant son internalisation dans la cellule. En outre, le cholestérol est un régulateur parmi les plus importants des domaines membranaires. En raison de son affinité pour les lipides saturés, il peut former des domaines ordonnés. Grâce au cholestérol-pyrène, une sonde fluorescente, nous avons étudié les domaines liquides ordonnés (Lo) et désordonnés (Ld) de la membrane. Nous avons, par analyse statistique en composante principale, déterminé les longueurs d'onde d'émission caractéristiques des domaines Lo et Ld. Les peptides pénétratine, R9 et RW9 diminuent l'assemblage du cholestérol et RW9 augmente la fluidité de la membrane. RW9 a été le seul peptide capable de traverser des membranes (Ld) sur de vésicules lipidiques dans nos conditions expérimentales. Nous pouvons ainsi en déduire que la distribution des lipides dans la membrane est un factor important pour le passage des CPP. Notamment, l'interface entre les différents domaines semble jouer un rôle prépondérant pour l'internalisation. / Cell penetrating peptides are promising vectors for molecular drug delivery in eukaryotic cells. Despite of their discovery 20 years ago, the mechanisms of peptide membrane crossing are still controversial. Understanding then how they modify the membrane will allow later on a more efficient internalisation into the cell. Lipid organisation after penetratin interaction was studied by DSC and Laurdan fluorescence. Penetratin was able to induce membrane heterogeneity, which could be important for membrane destabilisation during cell internalisation. Furthermore, cholesterol is one of the most important regulators of membrane domains. Due to its strong affinity with saturated lipids, cholesterol presents the ability to form “rafts” (ordered domains). By cholesterol-pyrene, which is a probe that mimics cholesterol, we studied the liquid ordered (Lo) and liquid disordered (Ld) domains of the membrane. Firstly, we determined the wavelengths that characterise each of these domains by multivariable analysis and then, we verify the peptide effect (R9, RW9 and penetratin) in the distribution of these domains. RW9 were the only CPP able to cross the membrane (Ld). We can deduce that lipid distribution in the membrane is important for the peptide internalisation and the interfaces between these domains may play an important role during this process.

<p>Our genome operates by sending instructions, conveyed by mRNA, for the manufacture of proteins from chromosomal DNA in the nucleus of the cell to the protein synthesizing machinery in the cytoplasm. Alternative splicing is a natural process in which a single gene can encode multiple related proteins. During RNA splicing, introns are selectively removed resulting in alternatively spliced gene products. Alternatively spliced protein products can have very different biological effects, such that one protein isoform is disease-related while another isoform is desirable. Splice switching opens the door to new drug targets, and antisense oligonucleotides (asONs), designed to switch splicing, are effective drug candidates. Cellular uptake of oligonucleotides(ONs) is poor, therefore utilization of cell-penetrating peptides (CPPs), well recognized for intracellular cargo delivery, is a promising approach to overcome this essential issue. Most CPPs are internalized by endocytosis, although the mechanisms involved remain controversial.</p><p>Here, evaluation of CPP-mediated ON delivery over cellular membranes has been performed. A protocol that allows for convenient assessment of CPP-mediated cellular uptake and characterization of corresponding internalization routes is established. The protocol is based on both fluorometric uptake measurements and a functional splice-switching assay, which in itself is based on biological activity of conveyed ONs. Additionally, splice switching ONs (SSOs) have been optimized for high efficiency and specificity. Data suggest that SSO activity is improved for chimeric phosphorothioate SSOs containing locked nucleic acid (LNA) monomers. It is striking that the LNA monomers in such chimeric constructs give rise to low mismatch discrimination of target pre-mRNA, which highlight the necessity to optimize sequences to minimize risk for off-target effects.</p><p>The results are important for up-coming work aimed at developing compounds consisting of peptides and novel synthetic nucleic acids, making these entities winning allies in the competition to develop therapeutics regulating protein expression patterns.</p>

<p>Alzheimer’s disease is the most common form of dementia. It is a neurodegenerative disorder characterized by extracellular senile plaques and intracellular neurofibrillary tangles. The main constituent of the senile plaques is the neurotoxic β-amyloid peptide. Surrounding the senile plaques are activated astrocytes and microglia, believed to contribute to neurotoxicity through secretion of proinflammatory cytokines, like interleukin-1β and interleukin-6. For many inflammatory actions, including the cytokine induction in glial cells, the transcription factor NFκB plays a key role. This suggests that therapeutical strategies aimed to control the development of Alzheimer’s disease could include administration of drugs that hinder NFκB activation.</p><p>The major aim of this thesis was to examine the effects of β-amyloid together with interleukin-1β on cytokine expression as well as NFκB activation in glial cells. The possibility to block NFκB activation, and downstream effects like interleukin-6 expression, by using an NFκB decoy was investigated. The possibility to improve the cellular uptake of the decoy by linking it to a cell-penetrating peptide was also investigated.</p><p>The results obtained provide supportive evidence that inflammatory cytokines are induced by β-amyloid, and that they can indeed potentiate its effects. The results further demonstrate that by blocking NFκB activation, the induction of interleukin-6 expression can be inhibited. By using an improved cellular delivery system, the uptake of the NFκB decoy and hence the downstream cytokine inhibition could be increased. In conclusion, these results demonstrate the possibility to decrease the inflammatory reactions taken place in Alzheimer’s disease brains, which may ultimately lead to a possible way of controlling this disorder.</p>

Ground penetrating radar (GPR) is a promising technique for humanitarian demining applications as it permits providing useful information about the subsurface based on wave reflections produced by electromagnetic (EM) contrasts. Yet, landmine detection using GPR can suffer from: (1) clutter, i.e, undesirable effects from antenna coupling, system ringing and soil surface and subsurface reflections; (2) false alarms, e.g., reflections from buried mine-like objects such as stones or metallic debris; (3) effects of soil properties on the GPR performance, such as attenuation. This thesis addresses these topics in an integrated approach aiming at reducing clutter, identifying landmines from false alarms and analysing GPR performance. For subtracting undesirable reflections, a new physically-based filtering algorithm is developed, which takes into account major antenna effects and soil surface reflection. It is applied in conjunction with a change detection algorithm for enhancing landmine detection. Landmine identification is performed using discriminant characteristics extracted from the pre-filtered data by a novel feature extraction approach in the time-frequency domain. For analysing the effects of soil properties, in particular soil dielectric permittivity, an EM model is coupled to pedotransfer functions for estimating the GPR performance on a given soil. The developed algorithms are validated using data acquired by two different hand-held GPR systems. Promising results are obtained under laboratory and outdoor conditions, where different types of soil (including real mine-affected soils) and landmines (including improvised explosive devices) are considered.

Ground penetrating radar (GPR) is a promising technique for humanitarian demining applications as it permits providing useful information about the subsurface based on wave reflections produced by electromagnetic (EM) contrasts. Yet, landmine detection using GPR can suffer from: (1) clutter, i.e, undesirable effects from antenna coupling, system ringing and soil surface and subsurface reflections; (2) false alarms, e.g., reflections from buried mine-like objects such as stones or metallic debris; (3) effects of soil properties on the GPR performance, such as attenuation. This thesis addresses these topics in an integrated approach aiming at reducing clutter, identifying landmines from false alarms and analysing GPR performance. For subtracting undesirable reflections, a new physically-based filtering algorithm is developed, which takes into account major antenna effects and soil surface reflection. It is applied in conjunction with a change detection algorithm for enhancing landmine detection. Landmine identification is performed using discriminant characteristics extracted from the pre-filtered data by a novel feature extraction approach in the time-frequency domain. For analysing the effects of soil properties, in particular soil dielectric permittivity, an EM model is coupled to pedotransfer functions for estimating the GPR performance on a given soil. The developed algorithms are validated using data acquired by two different hand-held GPR systems. Promising results are obtained under laboratory and outdoor conditions, where different types of soil (including real mine-affected soils) and landmines (including improvised explosive devices) are considered.

Based on technological advances made within the past decades, ground-penetrating radar (GPR) has become a well-established, non-destructive subsurface imaging technique. Catalyzed by recent demands for high-resolution, near-surface imaging (e.g., the detection of unexploded ordnances and subsurface utilities, or hydrological investigations), the quality of today's GPR-based, near-surface images has significantly matured. At the same time, the analysis of oil and gas related reflection seismic data sets has experienced significant advances. Considering the sensitivity of attribute analysis with respect to data positioning in general, and multi-trace attributes in particular, trace positioning accuracy is of major importance for the success of attribute-based analysis flows. Therefore, to study the feasibility of GPR-based attribute analyses, I first developed and evaluated a real-time GPR surveying setup based on a modern tracking total station (TTS). The combination of current GPR systems capability of fusing global positioning system (GPS) and geophysical data in real-time, the ability of modern TTS systems to generate a GPS-like positional output and wireless data transmission using radio modems results in a flexible and robust surveying setup. To elaborate the feasibility of this setup, I studied the major limitations of such an approach: system cross-talk and data delays known as latencies. Experimental studies have shown that when a minimal distance of ~5 m between the GPR and the TTS system is considered, the signal-to-noise ratio of the acquired GPR data using radio communication equals the one without radio communication. To address the limitations imposed by system latencies, inherent to all real-time data fusion approaches, I developed a novel correction (calibration) strategy to assess the gross system latency and to correct for it. This resulted in the centimeter trace accuracy required by high-frequency and/or three-dimensional (3D) GPR surveys. Having introduced this flexible high-precision surveying setup, I successfully demonstrated the application of attribute-based processing to GPR specific problems, which may differ significantly from the geological ones typically addressed by the oil and gas industry using seismic data. In this thesis, I concentrated on archaeological and subsurface utility problems, as they represent typical near-surface geophysical targets. Enhancing 3D archaeological GPR data sets using a dip-steered filtering approach, followed by calculation of coherency and similarity, allowed me to conduct subsurface interpretations far beyond those obtained by classical time-slice analyses. I could show that the incorporation of additional data sets (magnetic and topographic) and attributes derived from these data sets can further improve the interpretation. In a case study, such an approach revealed the complementary nature of the individual data sets and, for example, allowed conclusions about the source location of magnetic anomalies by concurrently analyzing GPR time/depth slices to be made. In addition to archaeological targets, subsurface utility detection and characterization is a steadily growing field of application for GPR. I developed a novel attribute called depolarization. Incorporation of geometrical and physical feature characteristics into the depolarization attribute allowed me to display the observed polarization phenomena efficiently. Geometrical enhancement makes use of an improved symmetry extraction algorithm based on Laplacian high-boosting, followed by a phase-based symmetry calculation using a two-dimensional (2D) log-Gabor filterbank decomposition of the data volume. To extract the physical information from the dual-component data set, I employed a sliding-window principle component analysis. The combination of the geometrically derived feature angle and the physically derived polarization angle allowed me to enhance the polarization characteristics of subsurface features. Ground-truth information obtained by excavations confirmed this interpretation. In the future, inclusion of cross-polarized antennae configurations into the processing scheme may further improve the quality of the depolarization attribute. In addition to polarization phenomena, the time-dependent frequency evolution of GPR signals might hold further information on the subsurface architecture and/or material properties. High-resolution, sparsity promoting decomposition approaches have recently had a significant impact on the image and signal processing community. In this thesis, I introduced a modified tree-based matching pursuit approach. Based on different synthetic examples, I showed that the modified tree-based pursuit approach clearly outperforms other commonly used time-frequency decomposition approaches with respect to both time and frequency resolutions. Apart from the investigation of tuning effects in GPR data, I also demonstrated the potential of high-resolution sparse decompositions for advanced data processing. Frequency modulation of individual atoms themselves allows to efficiently correct frequency attenuation effects and improve resolution based on shifting the average frequency level. GPR-based attribute analysis is still in its infancy. Considering the growing widespread realization of 3D GPR studies there will certainly be an increasing demand towards improved subsurface interpretations in the future. Similar to the assessment of quantitative reservoir properties through the combination of 3D seismic attribute volumes with sparse well-log information, parameter estimation in a combined manner represents another step in emphasizing the potential of attribute-driven GPR data analyses. / Geophysikalische Erkundungsmethoden haben in den vergangenen Jahrzehnten eine weite Verbreitung bei der zerstörungsfreien beziehungsweise zerstörungsarmen Erkundung des oberflächennahen Untergrundes gefunden. Im Vergleich zur Vielzahl anderer existierender Verfahrenstypen ermöglicht das Georadar (auch als Ground Penetrating Radar bezeichnet) unter günstigen Standortbedingungen Untersuchungen mit der höchsten räumlichen Auflösung. Georadar zählt zu den elektromagnetischen (EM) Verfahren und beruht als Wellenverfahren auf der Ausbreitung von hochfrequenten EM-Wellen, das heisst deren Reflektion, Refraktion und Transmission im Untergrund. Während zweidimensionale Messstrategien bereits weit verbreitet sind, steigt gegenwärtig das Interesse an hochauflösenden, flächenhaften Messstrategien, die es erlauben, Untergrundstrukturen dreidimensional abzubilden. Ein dem Georadar prinzipiell ähnliches Verfahren ist die Reflexionsseismik, deren Hauptanwendung in der Lagerstättenerkundung liegt. Im Laufe des letzten Jahrzehnts führte der zunehmende Bedarf an neuen Öl- und Gaslagerstätten sowie die Notwendigkeit zur optimalen Nutzung existierender Reservoirs zu einer verstärkten Anwendung und Entwicklung sogenannter seismischer Attribute. Attribute repräsentieren ein Datenmaß, welches zu einer verbesserten visuellen Darstellung oder Quantifizierung von Dateneigenschaften führt die von Relevanz für die jeweilige Fragestellung sind. Trotz des Erfolgs von Attributanalysen bei reservoirbezogenen Anwendungen und der grundlegenden Ähnlichkeit von reflexionsseismischen und durch Georadar erhobenen Datensätzen haben attributbasierte Ansätze bisher nur eine geringe Verbreitung in der Georadargemeinschaft gefunden. Das Ziel dieser Arbeit ist es, das Potential von Attributanalysen zur verbesserten Interpretation von Georadardaten zu untersuchen. Dabei liegt der Schwerpunkt auf Anwendungen aus der Archäologie und dem Ingenieurwesen. Der Erfolg von Attributen im Allgemeinen und von solchen mit Berücksichtigung von Nachbarschaftsbeziehungen im Speziellen steht in engem Zusammenhang mit der Genauigkeit, mit welcher die gemessenen Daten räumlich lokalisiert werden können. Vor der eigentlichen Attributuntersuchung wurden deshalb die Möglichkeiten zur kinematischen Positionierung in Echtzeit beim Georadarverfahren untersucht. Ich konnte zeigen, dass die Kombination von modernen selbstverfolgenden Totalstationen mit Georadarinstrumenten unter Verwendung von leistungsfähigen Funkmodems eine zentimetergenaue Positionierung ermöglicht. Experimentelle Studien haben gezeigt, dass die beiden potentiell limitierenden Faktoren - systeminduzierte Signalstöreffekte und Datenverzögerung (sogenannte Latenzzeiten) - vernachlässigt beziehungsweise korrigiert werden können. In der Archäologie ist die Untersuchung oberflächennaher Strukturen und deren räumlicher Gestalt wichtig zur Optimierung geplanter Grabungen. Das Georadar hat sich hierbei zu einem der wohl am meisten genutzten zerstörungsfreien geophysikalischen Verfahren entwickelt. Archäologische Georadardatensätze zeichnen sich jedoch oft durch eine hohe Komplexität aus, was mit der wiederholten anthropogenen Nutzung des oberflächennahen Untergrundes in Verbindung gebracht werden kann. In dieser Arbeit konnte gezeigt werden, dass die Verwendung zweier unterschiedlicher Attribute zur Beschreibung der Variabilität zwischen benachbarten Datenspuren eine deutlich verbesserte Interpretation in Bezug auf die Fragestellung ermöglicht. Des Weiteren konnte ich zeigen, dass eine integrative Auswertung von mehreren Datensätzen (methodisch sowie bearbeitungstechnisch) zu einer fundierteren Interpretation führen kann, zum Beispiel bei komplementären Informationen der Datensätze. Im Ingenieurwesen stellen Beschädigungen oder Zerstörungen von Versorgungsleitungen im Untergrund eine große finanzielle Schadensquelle dar. Polarisationseffekte, das heisst Änderungen der Signalamplitude in Abhängigkeit von Akquisitions- sowie physikalischen Parametern stellen ein bekanntes Phänomen dar, welches in der Anwendung bisher jedoch kaum genutzt wird. In dieser Arbeit wurde gezeigt, wie Polarisationseffekte zu einer verbesserten Interpretation verwendet werden können. Die Überführung von geometrischen und physikalischen Attributen in ein neues, so genanntes Depolarisationsattribut hat gezeigt, wie unterschiedliche Leitungstypen extrahiert und anhand ihrer Polarisationscharakteristika klassifiziert werden können. Weitere wichtige physikalische Charakteristika des Georadarwellenfeldes können mit dem Matching Pursuit-Verfahren untersucht werden. Dieses Verfahren hatte in den letzten Jahren einen großen Einfluss auf moderne Signal- und Bildverarbeitungsansätze. Matching Pursuit wurde in der Geophysik bis jetzt hauptsächlich zur hochauflösenden Zeit-Frequenzanalyse verwendet. Anhand eines modifizierten Tree-based Matching Pursuit Algorithmus habe ich demonstriert, welche weiterführenden Möglichkeiten solche Datenzerlegungen für die Bearbeitung und Interpretation von Georadardaten eröffnen. Insgesamt zeigt diese Arbeit, wie moderne Vermessungstechniken und attributbasierte Analysestrategien genutzt werden können um dreidimensionale Daten effektiv und genau zu akquirieren beziehungsweise die resultierenden Datensätze effizient und verlässlich zu interpretieren.

Gene therapy holds the promise of revolutionizing the way we treat diseases. By using recombinant DNA and oligonucleotides (ONs), gene functions can be restored, altered or silenced according to the therapeutic need. However, gene therapy approaches require the delivery of large and charged nucleic acid-based molecules to their intracellular targets across the plasma membrane, which is inherently impermeable to such molecules. In this thesis, two chemically modified cell-penetrating peptides (CPPs) that have superior delivery properties for several nucleic acid-based therapeutics are developed. These CPPs can spontaneously form nanoparticles upon non-covalent complexation with the nucleic acid cargo, and the formed nanoparticles mediate efficient cellular transfection. In paper I, we show that an N-terminally stearic acid-modified version of transportan-10 (PF3) can efficiently transfect different cell types with plasmid DNA and mediates efficient gene delivery in-vivo when administrated intra muscularly (i.m.) or intradermaly (i.d.). In paper II, a new peptide with ornithine modification, PF14, is shown to efficiently deliver splice-switching oligonucleotides (SSOs) in different cell models including mdx mouse myotubes; a cell culture model of Duchenne’s muscular dystrophy (DMD). Additionally, we describe a method for incorporating the PF14-SSO nanoparticles into a solid formulation that is active and stable even when stored at elevated temperatures for several weeks. In paper III, we demonstrate the involvement of class-A scavenger receptor subtypes (SCARA3 &amp; SCARA5) in the uptake of PF14-SSO nanoparticles, which possess negative surface charge, and suggest for the first time that some CPP-based systems function through scavenger receptors. In paper IV, the ability of PF14 to deliver siRNA to different cell lines is shown and their stability in simulated gastric acidic conditions is highlighted. Taken together, these results demonstrate that certain chemical modifications can drastically enhance the activity and stability of CPPs for delivering nucleic acids after spontaneous nanoparticle formation upon non-covalent complexation. Moreover, we show that CPP-based nanoparticles can be formulated into convenient and stable solid formulations that can be suitable for several therapeutic applications. Importantly, the involvement of scavenger receptors in the uptake of such nanoparticles is presented, which could yield novel possibilities to understand and improve the transfection by CPPs and other gene therapy nanoparticles. / At the time of doctoral defence the following paper was unpublished and had a status as follows: Paper nr 4: Submitted

Polythermal glaciers, i.e. glaciers with a combination of ice at and below the freezing point, are widespread in arctic and subarctic environments. The polythermal structure has major implications for glacier hydrology, ice flow and glacial erosion. However, the interplay of factors governing its spatial and temporal variations such as net mass balance, ice advection and water content in the ice is poorly investigated and as yet not fully understood. This study deals with a thorough investigation of the polythermal regime on Storglaciären, northern Sweden, a small valley glacier with a cold surface layer in the ablation area. Extensive field work was performed including mapping of the cold surface layer using ground-penetrating radar, ice temperature measurements, mass balance and ice velocity measurements. Analyses of these data combined with numerical modelling were used specifically to investigate the spatial and temporal variability of the cold surface layer, the spatial distribution of the water content just below the cold surface layer transition, the effect of radar frequency on the detection of the surface layer, and the sensitivity of the cold surface layer to changes in forcing. A comparison between direct temperature measurements in boreholes and ground-penetrating surveys shows that the radar-inferred cold-temperate transition depth is within ±1 m from the melting point of ice at frequencies above ~300 MHz. At frequencies below ~155 MHz, the accuracy degrades because of reduced scattering efficiency that occurs when the scatterers become much smaller compared to the wavelength. The mapped spatial pattern of the englacial cold-temperate transition boundary is complex. This pattern reflects the observed spatial variation in net loss of ice at the surface by ablation and vertical advection of ice, which is suggested to provide the predominant forcing of the cold surface layer thickness pattern. This is further supported by thermomechanical modeling of the cold surface layer, which indicates high sensitivity of the cold surface layer thickness to changes in vertical advection rates. The water content is the least investigated quantity that is relevant for the thermal regime of glaciers, but also the most difficult to assess. Spatial variability of absolute water content in the temperate ice immediately below the cold surface layer on Storglaciären was determined by combining relative estimates of water content from ground-penetrating radar data with absolute determination from temperature measurements and the thermal boundary condition at the freezing front. These measurements indicate large-scale spatial variability in the water content, which seems to arise from variations in entrapment of water at the firn-ice transition. However, this variability cannot alone explain the spatial pattern in the thermal regime on Storglaciären. Repeated surveys of the cold surface layer show a 22% average thinning of the cold surface layer on Storglaciären between 1989 and 2001. Transient thermomechanical modeling results suggest that the cold surface layer adapts to new equilibrium conditions in only a few decades after a perturbation in the forcing is introduced. An increased winter air temperature since mid-1980s seems to be the cause of the observed thinning of the cold surface layer. Over the last decades, mass balance measurements indicate that the glacier has been close to a steady state. The quasi-steady state situation is also reflected in the vertical advection, which shows no significant changes during the last decades. Increased winter temperatures at the ice surface would result in a slow-down of the formation of cold ice at the base of the cold surface layer and lead to a larger imbalance between net loss of ice at the surface and freezing of temperate ice at the cold-temperate transition.