<p>Bilirubin is a degradation product of haem, which is constantly formed in all</p><p>mammals. Increased levels of bilirubin in humans lead to jaundice, a condition</p><p>that is very common during the first days after birth. This neonatal</p><p>jaundice can routinely be treated by phototherapy without any serious side</p><p>effects. During this treatment, bilirubin undergoes a photoreaction to isomers</p><p>that can be excreted. The most efficient photoreaction is the isomerisation</p><p>around a double bond (Z-E-isomerisation), which results in more soluble</p><p>photoproducts.</p><p>The work presented in this thesis shows results of a femtosecond optical</p><p>spectroscopy study, combined with quantum-mechanical investigations, of</p><p>the mechanism of isomerisation of bilirubin. The spectroscopic research was</p><p>conducted with bilirubin in organic solvents, and in buffer complexed by</p><p>human serum albumin. This albumin complex is present in the blood, and</p><p>has thus medical importance. Quantum-chemical calculations (CASSCF) on</p><p>a bilirubin model were used to explain experimental results.</p><p>The fluorescence decay observed with femtosecond spectroscopy shows an</p><p>ultrafast component (~120 fs), which is explained by exciton localisation,</p><p>followed by processes with a lifetime of about 1-3 ps. These are interpreted</p><p>as the formation of a twisted intermediate, which decays with a lifetime of</p><p>10-15 ps back to the ground state, as observed by absorption spectroscopy.</p><p>CASSCF calculations, in combination with the experimental results, suggest</p><p>the ca. 1-3 ps components to be relaxation to the twisted S1 minimum, followed</p><p>by the crossing of a barrier, from where further relaxation takes place</p><p>through a conical intersection back to the ground state.</p><p>Time-dependent DFT calculations were utilised to analyse the absorption</p><p>spectrum of bilirubin. Good agreement with the measured spectrum was</p><p>achieved, and low-lying states were observed, that need further investigation.</p><p>The theoretically obtained CD spectrum provides direct evidence that</p><p>bilirubin preferentially binds to human serum albumin in the enantiomeric</p><p>P-form at neutral pH.</p> / <p>Bilirubin är en nedbrytningsprodukt av hem som ständigt bildas hos alla</p><p>däggdjur. En förhöjd bilirubinkoncentration i den mänskliga kroppen kan</p><p>leda till gulsot, något som är mycket vanligt under de första dagarna efter</p><p>födelsen (neonatal gulsot). Fototerapi används rutinmässigt som säker behandlingsmetod,</p><p>under vilken bilirubin genomgår en fotoreaktion till en</p><p>isomer som kan utsöndras. Den mest effektiva fotoreaktionen är en Z-Eisomerisation,</p><p>vilken leder till lösligare fotoprodukter.</p><p>Arbetet som presenteras i denna avhandling visar resultaten av en kombinerad</p><p>femtosekund optisk-spektroskopisk och kvantmekanisk undersökning</p><p>av mekanismen bakom bilirubins isomerisation. Den spektroskopiska</p><p>studien genomfördes med bilirubin, löst i organiska lösningsmedel och i</p><p>buffert i komplex med humant serumalbumin. Detta albuminkomplex finns i</p><p>blodet, och är därför av medicinskt intresse. Kvantmekanistiska CASSCFberäkningar</p><p>på en bilirubinmodell användes för att förklara de experimentella</p><p>resultaten.</p><p>Det uppmätta fluorescence sönderfallet visar ultrasnabba komponenter</p><p>(~120 fs). Dessa tolkas som excitonlokalisering, som följs av bildandet av</p><p>ett vridet intermediat med en hastighetskonstant på ca. 1 ps-1(beroende på</p><p>lösningsmedlet). Absorptionsmätningar visar att detta intermediat sönderfaller</p><p>tillbaka till grundtillståndet med en livstid på 10-15 ps.</p><p>CASSCF beräkningar, i kombination med de experimentella resultaten, tyder</p><p>på att sönderfallet med livslängden på ca. 1 ps är en relaxation till det</p><p>vridna S1-tillståndet. Reaktionsvägen därifrån antas passera en barriär till en</p><p>konisk genomskärning, som möjliggör snabb relaxation till grundtillståndet.</p><p>Tidsberoende DFT-beräkningar användes för att analysera bilirubins absorptionsspektrum,</p><p>vilket gav bra överensstämmelse med uppmätta data. Dessutom</p><p>hittades ett tillstånd med låg excitationsenergi, som kräver ytterligare</p><p>studier. Med hjälp av det beräknade CD-spectret kunde det visas att bilirubin</p><p>binder till albumin i P-formen vid neutralt pH.</p>

Caribbean spiny lobster (Panulirus argus) is a popular and heavily exploited seafood throughout its range. This species supports the primary fishery in many Caribbean countries, especially in the Bahamas, which reports the highest catches and where spiny lobster serves as the number one food export. P. argus possesses one of the longest pelagic larval durations of any marine species, ranging from 6-12 months. This allows for the possibility of long-range dispersal, which would make it difficult to determine if local adult populations originate from areas close-by or within the same countries/jurisdictions, thus presenting implications for conservation and management of the species. This project seeks to explore the policy implications of lobster larval dispersal in the Bahamas by examining the larval connectivity of locally spawned P. argus in order to determine the mean dispersal kernel and to identify hotspots of settlement within the area. A coupled biophysical model was used to simulate larval transport from scaled egg production of 47 release locations within the Bahamas. The model was initialized bi-weekly from April through May, the highest months of larvae production in the Bahamas, with each model run occurring for a maximum of 180 days. The dispersal kernel for the Bahamas was calculated to be an average of 100-300 km, indicating that the larvae released within its boundaries typically settled there as well. Due to the long pelagic larval duration, larval particles were able to travel extensive distances, averaging trajectories covering distances of 4000 km and greater from the source locations. Yet, those same larval particles still settled in locations within the Bahamas, suggesting local retention, which varies from the common perception that lobster in the Bahamas originate elsewhere. This knowledge can be used to assess and perhaps reevaluate conservation and management strategies related to the Bahamian P. argus fishery, including the implementation of MPAs and/or MPA networks, input and output management controls, and other management tools.

Bilirubin is a degradation product of haem, which is constantly formed in all mammals. Increased levels of bilirubin in humans lead to jaundice, a condition that is very common during the first days after birth. This neonatal jaundice can routinely be treated by phototherapy without any serious side effects. During this treatment, bilirubin undergoes a photoreaction to isomers that can be excreted. The most efficient photoreaction is the isomerisation around a double bond (Z-E-isomerisation), which results in more soluble photoproducts. The work presented in this thesis shows results of a femtosecond optical spectroscopy study, combined with quantum-mechanical investigations, of the mechanism of isomerisation of bilirubin. The spectroscopic research was conducted with bilirubin in organic solvents, and in buffer complexed by human serum albumin. This albumin complex is present in the blood, and has thus medical importance. Quantum-chemical calculations (CASSCF) on a bilirubin model were used to explain experimental results. The fluorescence decay observed with femtosecond spectroscopy shows an ultrafast component (~120 fs), which is explained by exciton localisation, followed by processes with a lifetime of about 1-3 ps. These are interpreted as the formation of a twisted intermediate, which decays with a lifetime of 10-15 ps back to the ground state, as observed by absorption spectroscopy. CASSCF calculations, in combination with the experimental results, suggest the ca. 1-3 ps components to be relaxation to the twisted S1 minimum, followed by the crossing of a barrier, from where further relaxation takes place through a conical intersection back to the ground state. Time-dependent DFT calculations were utilised to analyse the absorption spectrum of bilirubin. Good agreement with the measured spectrum was achieved, and low-lying states were observed, that need further investigation. The theoretically obtained CD spectrum provides direct evidence that bilirubin preferentially binds to human serum albumin in the enantiomeric P-form at neutral pH. / Bilirubin är en nedbrytningsprodukt av hem som ständigt bildas hos alla däggdjur. En förhöjd bilirubinkoncentration i den mänskliga kroppen kan leda till gulsot, något som är mycket vanligt under de första dagarna efter födelsen (neonatal gulsot). Fototerapi används rutinmässigt som säker behandlingsmetod, under vilken bilirubin genomgår en fotoreaktion till en isomer som kan utsöndras. Den mest effektiva fotoreaktionen är en Z-Eisomerisation, vilken leder till lösligare fotoprodukter. Arbetet som presenteras i denna avhandling visar resultaten av en kombinerad femtosekund optisk-spektroskopisk och kvantmekanisk undersökning av mekanismen bakom bilirubins isomerisation. Den spektroskopiska studien genomfördes med bilirubin, löst i organiska lösningsmedel och i buffert i komplex med humant serumalbumin. Detta albuminkomplex finns i blodet, och är därför av medicinskt intresse. Kvantmekanistiska CASSCFberäkningar på en bilirubinmodell användes för att förklara de experimentella resultaten. Det uppmätta fluorescence sönderfallet visar ultrasnabba komponenter (~120 fs). Dessa tolkas som excitonlokalisering, som följs av bildandet av ett vridet intermediat med en hastighetskonstant på ca. 1 ps-1(beroende på lösningsmedlet). Absorptionsmätningar visar att detta intermediat sönderfaller tillbaka till grundtillståndet med en livstid på 10-15 ps. CASSCF beräkningar, i kombination med de experimentella resultaten, tyder på att sönderfallet med livslängden på ca. 1 ps är en relaxation till det vridna S1-tillståndet. Reaktionsvägen därifrån antas passera en barriär till en konisk genomskärning, som möjliggör snabb relaxation till grundtillståndet. Tidsberoende DFT-beräkningar användes för att analysera bilirubins absorptionsspektrum, vilket gav bra överensstämmelse med uppmätta data. Dessutom hittades ett tillstånd med låg excitationsenergi, som kräver ytterligare studier. Med hjälp av det beräknade CD-spectret kunde det visas att bilirubin binder till albumin i P-formen vid neutralt pH.

The pathogenesis of Alzheimer’s disease (AD), the most common senile dementia, is a complex process. A crucial event in AD is the aggregation of amyloid-β protein (Aβ), a cleavage product from the Amyloid Precursor Protein (APP). Aβ40, a common component in amyloid plaques found in patients, aggregates in vitro at concentrations, much higher than the one found in vivo. But in the presence of charged lipid membranes, aggregations occurs at much lower concentration in vitro compared to the membrane-free case. This can be understood due to the ability of Aβ to get electrostatically attracted to target membranes with a pronounced surface potential. This electrostatically driven process accumulates peptide at the membrane surface at concentrations high enough for aggregation while the bulk concentration still remains below threshold. Here, we elucidated the molecular nature of this Aβ-membrane process and its consequences for Aβ misfolding by Circular Dichroism Spectroscopy, Differential Scanning Calorimetry and Nuclear Magnetic Resonance Spectroscopy. First, we revealed by NMR that Aβ40 peptide does indeed interact electrostatically with membranes of negative and positive surface potential. Surprisingly, it even binds to nominal neutral membranes if these contain lipids of opposite charge. Combined NMR and CD studies also revealed that the peptide might be shielded from aggregation when incorporated into the membrane. Moreover, CD studies of Aβ40 added to charged membranes showed that both positively and negatively membranes induce aggregation albeit at different kinetics and finally that macromolecular crowding can both speed up and slow down aggregation of Aβ.

It is well known that folded proteins in water are destabilized by the addition of urea. When a protein loses its ability to perform its biological activity due to a change in its structure, it is said to denaturate. The mechanism by which urea denatures proteins has been thoroughly studied in the past but no proposed mechanism has yet been widely accepted. The topic of this thesis is the study of the mechanism of urea-induced protein denaturation, by means of Molecular Dynamics (MD) computer simulations and Nuclear Magnetic Resonance (NMR) spectroscopy. Paper I takes a thermodynamic approach to the analysis of protein – urea solution MD simulations. It is shown that the protein – solvent interaction energies decrease significantly upon the addition of urea. This is the result of a decrease in the Lennard-Jones energies, which is the MD simulation equivalent to van der Waals interactions. This effect will favor the unfolded protein state due to its higher number of protein - solvent contacts. In Paper II, we show that a combination of NMR spin relaxation experiments and MD simulations can successfully be used to study urea in the protein solvation shell. The urea molecule was found to be dynamic, which indicates that no specific binding sites exist. In contrast to the thermodynamic approach in Paper I, in Paper III we utilize MD simulations to analyze the affect of urea on the kinetics of local processes in proteins. Urea is found to passively unfold proteins by decreasing the refolding rate of local parts of the protein that have unfolded by thermal fluctuations. Based upon the results of Paper I – III and previous studies in the field, I propose a mechanism in which urea denatures proteins mainly by an enthalpic driving force due to attractive van der Waals interactions. Urea interacts favorably with all the different parts of the protein. The greater solvent accessibility of the unfolded protein is ultimately the factor that causes unfolded protein structures to be favored in concentrated urea solutions.

Enzymes are extraordinary molecules that can accelerate chemical reactions by several orders of magnitude. With recent advancements in structural biology together with classical enzymology the mechanism of many enzymes has become understood at the molecular level. During the last ten years significant efforts have been invested to understand the structure and dynamics of the actual catalyst (i. e. the enzyme). There has been a tremendous development in NMR spectroscopy (both hardware and pulse programs) that have enabled detailed studies of protein dynamics. In many cases there exists a strong coupling between enzyme dynamics and function. Here I have studied the conformational dynamics and thermodynamics of three model systems: adenylate kinase (Adk), Peroxiredoxin Q (PrxQ) and the structural protein S16. By developing a novel chemical shift-based method we show that Adk binds its two substrates AMP and ATP with an extraordinarily dynamic mechanism. For both substrate-saturated states the nucleotide-binding subdomains exchange between open and closed states, with the populations of these states being approximately equal. This finding contrasts with the traditional view of enzyme-substrate complexes as static low entropy states. We are also able to show that the individual subdomains in Adk fold and unfold in a non-cooperative manner. This finding is relevant from a functional perspective, since it allows a change in hydrogen bonding pattern upon substrate-binding without provoking global unfolding of the entire enzyme (as would be expected from a two-state folding mechanism). We also studied the structure and dynamics of the plant enzyme PrxQ in both reduced and oxidized states. Experimentally validated structural models were generated for both oxidation states. The reduced state displays unprecedented μs-ms conformational dynamics and we propose that this dynamics reflects local and functional unfolding of an α-helix in the active site. Finally, we solved the structure of S16 from Aquifex aeolicus and propose a model suggesting a link between thermostability and structure for a mesophilic and hyperthermophilic protein pair. A connection between the increased thermostability in the thermophilic S16 and residual structure in its unfolded state was discovered, persistent at high denaturant concentrations, thereby affecting the difference in heat capacity difference between the folded and unfolded state. In summary, we have contributed to the understanding of protein dynamics and to the coupling between dynamics and catalytic activity in enzymes.

Installed photovoltaic nameplate power have been growing rapidly around the worldin the last few years. But how much energy is returned to society (i.e. net energy) by this technology, and which factors contribute the most to the amount of energy returned? The objective of this thesis was to examine the importance of certain inputs and outputs along the solar panel production chain and their effect on the energy return on (energy) investment (EROI) for crystalline wafer-based photovoltaics. A process-chain model was built using publicly available life-cycle inventory (LCI) datasets. This model has been kept simple in order to ensure transparency. Univariate sensitivity analysis for processes and multivariate case studies was then applied to the model. The results show that photovoltaic EROI values are very sensitive to assumptions regarding location and efficiency. The ability of solar panels to deliver net energy in northern regions of the earth is questionable. Solar cell wafer thickness have a large impact on EROI, with thinner wafers requiring less silicon material. Finding an alternative route for production of solar-grade silicon is also found to be of great importance, as is introduction of kerf loss recycling. Equal system sizes have been found to yield an primary EROI between approximately 5.5-19 depending on location and assumptions. This indicates that a generalized absolute EROI for photovoltaics may be of little use for decision-makers. Using the net energy cliff concept in relation to primary EROI found in this thesis shows that primary EROI rarely decreases to less than the threshold of 8:1 in univariate cases. Crystalline photovoltaics under similar system boundaries as those in the thesis model does not necessarily constrain economic growth on an energetic basis.

<p>Oceanographic features, such as fronts, eddies, and upwellings, provide important foraging areas for marine predators. These areas serve as important "hotspots" of marine life, by aggregating weakly swimming lower and mid-trophic level species which, in turn, attract foraging predators. Despite the importance of these dynamic features, we lack a comprehensive understanding of how they create foraging habitat for seabirds and other marine predators. In the first part of this dissertation, I review current knowledge of how seabirds use oceanographic features with an emphasis on developing a more mechanistic understanding of these features, and identify important considerations for future studies. I use the findings of this review to inform two field research projects in the Bay of Fundy, Canada and Onslow Bay, North Carolina. In these two projects, I examined seabird abundance and distribution in relation to oceanographic features that occur at different spatial and temporal scales. In the first project, I examined foraging habitat of red-necked phalaropes (<italic>Phalaropus lobatus</italic>) in relation fine-scale tidal forcing near the Brier Island ledges in the Bay of Fundy. This research demonstrated the importance of biophysical interactions in creating phalarope habitat, and characterized red-necked phalarope habitat in both space and time. In Onslow Bay, I investigated the effects of Gulf Stream fronts and eddies on the abundance and distribution of seabirds using both remotely sensed and in situ data. I used fisheries acoustics surveys to investigate prey distribution within Gulf Stream frontal eddies. I then developed habitat models for the six most commonly sighted species or species groups (Cory's shearwaters, <italic>Calonectris diomedea</italic>; greater shearwaters (<italic>Puffinus gravis</italic>; Wilson's storm petrel, <italic>Oceanites oceanicus</italic>; Audubon's shearwaters, <italic>Puffinus lherminieri</italic>; black-capped petrels, <italic>Pterodrama hasitata</italic>; and red and red-necked phalaropes, grouped together as <italic>Phalaropus</italic> spp.) using multivariate modeling techniques. Gulf Stream frontal eddies influenced the abundance and distribution of seabirds in Onslow Bay, although frontal features were not as important in predicting seabird habitat as demonstrated in previous studies in the South Atlantic Bight. Prey availability in Gulf Stream frontal eddies was highest in eddy cold core regions, particularly in those regions close to the Gulf Stream. Taken together, the results of my dissertation: underscore the importance of conducting standardized surveys to assess dynamic environmental variables; demonstrate the use of multivariate methods to examine seabird foraging in relation to oceanographic features; emphasize the need to evaluate both prey distributions and physical regimes within oceanographic features at depth; and highlight the importance of temporal aspects of oceanographic features, such as the persistence and age of the features, when assessing the role that these features play in creating seabird foraging habitat.</p> / Dissertation

2'-5'-oligoadenylate synthetases (OAS) are interferon-stimulated proteins that act in the innate immune response to viral infection. Upon binding to viral double-stranded RNAs, OAS enzymes produce 2'-5'-linked oligoadenylates that stimulate RNase L and ultimately slow viral propagation. Studies have linked mutations in the OAS1 gene to increased susceptibility to West Nile virus (WNV) infection, highlighting the importance of the OAS1 enzyme. Here I report that the 5'-terminal region (5'-TR) of the WNV genome, comprising both the 5'-untranslated region (5'-UTR) and initial coding region, is capable of OAS1 activation in vitro. This region contains three RNA stem loops (SLI, SLII, and SLIII), whose relative contribution to OAS1 binding affinity and activation were investigated using electrophoretic mobility shift assays and enzyme kinetics experiments. Stem loop I (SLI) is dispensable for maximum OAS1 activation, as a construct containing only SLII and SLIII was capable of enzymatic activation. Mutations to the RNA binding site of OAS1 confirmed the specificity of the interaction. Solution conformations of both the 5'-TR RNA of WNV and OAS1 were then elucidated using small-angle x-ray scattering. I also report that the 3' terminal region (3'-TR) is able to mediate specific interaction with and activation of OAS1. Binding and kinetic experiments identified a specific stem loop within the 3'-TR that is sufficient for activation of the enzyme. The solution confirmation of the 3'-terminal region was determined by small angle X-ray scattering, and computational models suggest a conformationally restrained structure comprised of a helix and short stem loop. Structural investigation of the 3'-TR in complex with OAS1 is also presented. Finally, we show that genome cyclization by base pairing between the 5'- and 3'-TRs, a required step for replication, is not sufficient to protect WNV from OAS1 recognition. The purity, monodispersity and homogeneity of all samples subjected to SAXS analysis were evaluated using dynamic light scattering and/or analytical ultra-centrifuge. These data provide a framework for understanding recognition of the highly structured terminal regions of a flaviviral genome by an innate immune enzyme. / October 2015

‘Critical Habitat’ is the habitat required to close the life history of an endangered species and is a fundamental requirement for species recovery for two reasons; the role of habitat in population limitation and viability must be determined, and the habitat must be protected. The North Atlantic right whale is an endangered species that annually migrates to the Grand Manan Basin and Roseway Basin Critical Habitats to feed on diapausing calanoid copepods that are typically aggregated at depths of 100 to 150 m. In this thesis I quantify spatial and temporal variation in the copepod prey field and occupancy of right whales in Roseway Basin, and use this information to identify the location and extent of right whale Critical Habitat. To accomplish this, I measured copepod abundance and energy density (kJ m-3) using optical, acoustic and net collection methods during 2007 to 2009. Oceanographic processes that affect variation in the copepod prey field include slope water intrusions, water mass density, gyre-like circulation and frontal features. Aggregations of diapausing copepods are maintained on the southern slope of Roseway Basin by cross-isobath tidal advection, and are advected along-isobath by the residual flow. Tidal advection at a front, coupled with along-isobath advection and shear in the horizontal currents serve to accumulate copepods along the slope where aggregations are maintained for at least 7 days. The abundance, stage-structure, species composition and aggregation locations of copepods, as well as the hydrography and circulation, were variable among the three years of the study. A 20 year time series of right whales, copepods and hydrography revealed that interannual whale occupancy in the Critical Habitats is variable and can be explained by prey field variation only in Roseway Basin. Factors other than the local prey field affect the number of whales that occupy Grand Manan Basin. Variation in the right whale prey field could not be explained by temperature and phytoplankton-dependent growth in the Scotia - Fundy -Gulf of Maine region. The results of this thesis assisted in establishing the Roseway Basin right whale Critical Habitat in 2008, and the cross-disciplinary nature of the study also provides new insights into the relationships between biology and physics in Scotian Shelf - Gulf of Maine basins.