Type 2 diabetes mellitus (T2DM) is a heterogeneous disease affecting millions of people worldwide. Both genetic and environmental factors contribute to the pathogenesis. The disease is characterized by alterations in many genes and their products. Historically, genomic alterations have mainly been studied at the transcriptional level in diabetes research. However, transcriptional changes do not always lead to altered translation, which makes it important to measure changes at the protein level. Proteomic techniques offer the possibility of measuring multiple protein alterations simultaneously. In this thesis, the proteomic technique surface enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF MS) has been applied and evaluated in the context of T2DM research. Protocols for pancreatic islet and serum/plasma protein profiling and identification have been developed. In addition, the technique was used to analyze the influence of genetic background versus diabetic environment by determining serum protein profiles of individuals with normal glucose tolerance (NGT) and T2DM with or without family history of diabetes. In total thirteen serum proteins displayed different levels in serum from persons with NGT versus patients with T2DM. Among these proteins, apolipoprotein CIII, albumin and one yet unidentified protein could be classified as being changed because of different genetic backgrounds. On the other hand, ten proteins for instance transthyretin, differed as a result of the diabetic environment. When plasma protein patterns of NGT and T2DM individuals characterized by differences in early insulin responses (EIR) were compared, nine proteins were found to be varying between the two groups. Of these proteins five were identified, namely two forms of transthyretin, hemoglobin α-chain, hemoglobin β-chain and apolipoprotein H. However no individual protein alone could explain the differences in EIR. In conclusion, SELDI-TOF MS has been successfully used in the context of T2DM research to identify proteins associated with family history of diabetes and β-bell function.

The field of proteomics has rapidly expanded due to the completion of the human genome sequence. This thesis validates affinity-purified monospecific antibodies of polyclonal origin, for protein profiling in a broad spectrum of normal tissues and cells. Validation of antibodies is crucial for development of reliable binders for target proteins and this thesis evaluates the generation and application of large sets of msAbs in different settings. MsAbs were generated towards recombinant Protein Epitope Signature Tag (PrEST) antigens using a stringent affinity-purification strategy, presented in the first study. The specificity of msAbs was studied using reverse phase protein arrays and immunohistochemistry (IHC), and results presented over 90% success rate in the protein array analysis. In IHC, 81% of the msAbs displayed apparent specific staining in normal tissues. MsAbs were also compared with commercial analogs (cAbs) using IHC and Western blot. Results presented similar outcome between msAbs and cAbs in both applications, although interpretation suggested more extensive IHC staining patterns with msAbs than with monoclonal analogs. For antibody validation, an approach called paired antibodies was presented and involved the generation of two msAbs towards non-overlapping epitopes on the same protein. Similarities in protein detection between paired antibodies were studied using three different antibody-based methods. Similar results were observed in several applications, indicating that this strategy can be a useful tool for studying known and unknown proteins. Given the reliability of msAbs, they were also applied in a study investigating the impact of tissue fixatives on protein detection. The study showed that different fixation mechanisms appeared to affect protein recognition by indicating that aldehyde-based fixation, e.g. induced by neutral buffered formalin, was preferred for tissues used in IHC and non-aldehyde based fixation was applicable for tissues used in protein extraction analysis and Western blotting. Conclusively, validation results suggest that msAbs are reliable affinity binders that can be used as valuable tools for proteome-wide protein profiling in tissues and cells.

Offenders who target children are a negative phenomenon in our society. These offenders are often seen as the worst of the worst of criminals and are therefore a priority for investigators trying to solve these crimes as fast as possible. The purpose of this thesis is to see if there are common denominators among these offenders in their modus operandi (MO) and their spatial patterns. If similar patterns emerge amongst these offenders, that would be of investigative importance for those who work with crimes against children. In Study I, a group of child molesters and their MO were studied in order to see how they found their victims and where they committed their crimes. The results were consistent with previous studies on child molesters in that they all committed their crimes at home or close to their homes. In Study II, a geographical profiling tool was tested in order to see if such a program could be used to find an offender who made obscene phone calls (OPC) to children. The results showed that the geographical software based on spatial behaviour, was able to narrow down the search area in which the offender actually lived when he committed his crimes. In Study III, the focus was on the potential witnesses/victims and how much a child could remember correctly of a staged event simulating a potential child molester looking for new victims. The results showed that the children’s event memory were comparable with an adult control group. The combined results could be summarized as follows: offenders who target children usually commit their crimes at home or close to home (or base), they tend to lure children to go with them by using bribes or the recruitment of former victims. Girls seem to be the preferred sex over boys. Children could be used as accurate witnesses in these types of crimes.

The rheological properties of cement based grouts change with water/cement ratio and time, during the course of hydration. For this reason, it is desirable to be able to measure this change continuously, in-line, with a robust instrument during the entire grouting operation in the field. The rheological properties of commonly used cement grouts were determined using the Ultrasound Velocity Profiling combined with the Pressure Difference (UVP+PD) method. A non-model approach was used that directly provides the properties, and the results were compared with the properties obtained using the Bingham and Herschel-Bulkley rheological models. The results show that it is possible to determine the rheological properties, as well as variations with concentration and time, with this method. The UVP+PD method has been found to be an effective measuring device for velocity profile visualization, volumetric flow determination and the characteristics of the grout pump used. / <p>QS 2013</p>

Food inevitably plays a vital role in our lives and is of great importance to our health and wellbeing. With increasing age, it is equally important to achieve adequate nutrition to prevent and alleviate age-related diseases. One problem is that far too many older adults find it difficult to eat enough nutritious food which in the long term may lead to malnutrition. With an increasing life expectancy the older population is growing and the problem with malnutrition is of great concern. Malnutrition can be caused by many different factors which make it difficult to find a single unique solution to the problem. Oral nutritional supplementation is one approach which has proved to be useful for improving the nutritional intake. This paper examines the possibility of creating high fat fruit emulsion with mango puré, rapeseed oil and lecithin, using simple blending equipment.  The puré-like products were evaluated for emulsion stability by a storage test, oil droplet size by a light microscope and light scattering device, viscosity by a viscometer, sensory properties by Flavoring profiling. Furthermore the nutritional values were calculated.  Successful emulsions were created using up to 50 g/100g rapeseed oil with adequate emulsion stability without lecithin. The energy content of the highest fat emulsion was 475kcal/100g. The quantities of lecithin used in these products reduced the oil droplet size but lowered the emulsion stability perhaps by depleting the stabilizing effect of mango originated particles. The lecithin made the product more viscous, also the oily and creamy/Rich mouth-feel were perceived higher with increasing lecithin. In these products and with the quantities used the lecithin was redundant. Further development of similar products but with addition of protein and perhaps sugar, to enhance flavor, should be of high interest.

Rhomboids are unique membrane proteins that use a serine protease hydrolysis mechanism to cleave a transmembrane substrate within the lipid bilayer. This remarkable proteolytic activity is achieved by a core domain comprised of 6 transmembrane segments that form a hydrophilic cavity submerged in the membrane. In addition to this core domain, many rhomboids also possess aqueous domains of varying sizes at the N- and/or C-terminus, the sequences of which tend to be rhomboid-type specific. The functional role of these extramembranous domains is generally not well understood, although it is thought that they may be involved in regulation of rhomboid activity and specificity. While extramembranous domains may be important for rhomboid activity, they are absent in all x-ray crystal structures available. For this reason, we have focused on uncovering the structural and functional relationship between the rhomboid cytoplasmic domain and its catalytic transmembrane core. To investigate the structure and function of the bacterial rhomboid cytoplasmic domain, full-length rhomboids from Escherichia coli and Pseudomonas aeruginosa were studied using solution nuclear magnetic resonance (NMR) spectroscopy, mutation and activity assays. The P. aeruginosa rhomboid was purified in a range of membrane-mimetic media, evaluated for its functional status in vitro and investigated for its NMR spectroscopic properties. Results from this study suggested that an activity-modulating interaction might occur between the catalytic core transmembrane domain and the cytoplasmic domain. Further investigation of this hypothesis with the E. coli rhomboid revealed that protease activity relies on a short but critical sequence N-terminal to the first transmembrane segment. This sequence was found to have a direct impact on the rhomboid active site, and should be included in future structural studies of this catalytic domain. The structure of the cytoplasmic domain from the E. coli rhomboid was also determined by solution NMR. We found that it forms slowly-exchanging dimers through an exchange of secondary structure elements between subunits, commonly known as three-dimensional domain swapping. Beyond this rare example of domain swapping in a membrane protein extramembranous domain, we found that the rate of exchange between monomeric and dimeric states could be accelerated by transient interactions with large detergent micelles with a phosphocholine headgroup, but not by exposure to other weakly denaturing conditions. This novel example of micelle-catalyzed domain swapping interactions raises the possibility that domain swapping interactions might be induced by similar interactions in vivo. Overall, the results of this thesis have identified detergent conditions that preserve the highest level of activity for bacterial rhomboids, defined the minimal functional unit beyond what had been identified in available x-ray crystal structures, and characterized a novel micelle-catalyzed domain-swapping interaction by the cytoplasmic domain.

By integrating the simplicity of 18O-labeling and the low signal-to-noise of MS/MS spectra with supporting software and combining them with global shotgun protein identification, a robust quantitative pipeline has been created that avoids the disadvantages of other quantitative approaches. Test mixtures of labeled and unlabeled peptides were subjected to LC-MS/MS profiling experiments. Software programs were developed and applied to automatically determine protein ratios between two samples while applying a correction for incomplete labeling. The measurement of relative abundance at the product ion (MS/MS) level, instead of at the full scan (MS) level, is shown to provide excellent accuracy and sensitivity. Ratio distributions approached the expected means, allowing empirical derivation of confidence level cutoffs for determining statistically significant fold-changes in protein abundance. A set of stringent criteria for detecting spurious ratios based on consistency checking between unlabeled and labeled y-ion pairs was found to highlight putative false positive identifications.

Field Programmable Gate Arrays (FPGAs) are a reconfigurable hardware platform which enable the acceleration of software code through the use of custom-hardware circuits. Complex systems combining processors with programmable logic require partitioning to decide which code segments to accelerate. This thesis provides tools to help determine which software code sections would most benefit from hardware acceleration. A low-overhead profiling architecture, called LEAP, is proposed to attain real-time profiles of an FPGA-based processor. LEAP is designed to be extensible for a variety of profiling tasks, three of which are investigated and implemented to identify candidate software for acceleration. 1) Cycle profiling determines the most time-consuming functions to maximize speedup. 2) Cache stall profiling detects memory-intensive code; large memory overheads reduce the benefits of acceleration. 3) Energy consumption profiling detects energy-inefficient code through the use of an instruction-level power database to minimize the system's energy consumption.

By integrating the simplicity of 18O-labeling and the low signal-to-noise of MS/MS spectra with supporting software and combining them with global shotgun protein identification, a robust quantitative pipeline has been created that avoids the disadvantages of other quantitative approaches. Test mixtures of labeled and unlabeled peptides were subjected to LC-MS/MS profiling experiments. Software programs were developed and applied to automatically determine protein ratios between two samples while applying a correction for incomplete labeling. The measurement of relative abundance at the product ion (MS/MS) level, instead of at the full scan (MS) level, is shown to provide excellent accuracy and sensitivity. Ratio distributions approached the expected means, allowing empirical derivation of confidence level cutoffs for determining statistically significant fold-changes in protein abundance. A set of stringent criteria for detecting spurious ratios based on consistency checking between unlabeled and labeled y-ion pairs was found to highlight putative false positive identifications.

Field Programmable Gate Arrays (FPGAs) are a reconfigurable hardware platform which enable the acceleration of software code through the use of custom-hardware circuits. Complex systems combining processors with programmable logic require partitioning to decide which code segments to accelerate. This thesis provides tools to help determine which software code sections would most benefit from hardware acceleration. A low-overhead profiling architecture, called LEAP, is proposed to attain real-time profiles of an FPGA-based processor. LEAP is designed to be extensible for a variety of profiling tasks, three of which are investigated and implemented to identify candidate software for acceleration. 1) Cycle profiling determines the most time-consuming functions to maximize speedup. 2) Cache stall profiling detects memory-intensive code; large memory overheads reduce the benefits of acceleration. 3) Energy consumption profiling detects energy-inefficient code through the use of an instruction-level power database to minimize the system's energy consumption.