The joining of two molecular functions or the strategy of adding functions to proteins has been tremendously important for the development of proteins as tools in research and clinic. Depending on the intended application, there are a wide variety of functions that can be added to a proteins. In clinical applications drugs are a commonly conjugated to antibodies and in research adding reporter groups such as biotin, enzymes or fluorophores is a routine procedure. The chemistries and methods most often used suffer from drawbacks such as lack of stringency, which could lead to undesired effects on the protein. Many site-specific methods of labeling of antibodies require modification or insertion of handles in the antibody recombinantly, before labeling can be performed. The core of this thesis is the development of a strategy for covalent specific labeling of antibodies by exploiting the site specific binding of the Z domain to Protein A. Photoreactive Z-domains were produced by solid phase peptide synthesis, which provides the opportunity to insert a photoreactive amino acid and a reporter biotin at specific positions in the domain. The inherited binding to the Fc-part of the antibody in combination with the incorporated photoreactive amino acid, BPA, is used for site-specific interaction, and thereafter, covalent coupling to the antibody. The exposure with the appropriate wavelength of light enables the formation a covalent linkage between the Z domain and the antibody. The biotinylated photoactivable domains were subsequently used to site-specifically label a number of different types of antibodies, polyclonal rabbit IgG, monoclonal human IgG1 and monoclonal mouse IgG2a, and thereafter the antibodies was employed in a variation of applications. The photolabeling procedure of antibodies by the use of photoactivable Z domains has proven to be successful and could serve as a valuable tool in several applications. / QC 20120507

The recent expansion in chemical and manufacturing and innovation has led to a large influx of chemicals to the market, and subsequent release into the environment. Many of these new chemicals, as well as legacy chemicals are untested for their potential developmental toxicity, especially in early embryonic stages. This creates a need for a timely and cost-effective method for screening these chemicals. Furthermore, advances in in vitro methods and human pluripotent cell culturing techniques have revealed some weaknesses in our current animal model-based paradigms. Here we tested an in vitro model for developmental toxicity screening using human embryonic stem cells (hESCs) for environmental chemicals. In this study, hESCs were exposed to three known developmental toxicants that are prevalent in the environment, bisphenol A (BPA), perfluorooctane sulfonate (PFOS), or lead chloride (PbCl₂), at environmentally relevant concentrations of 0-2500 µg/L, 0-2275 µg/L, and 0-6200 µg/L respectively, for 6-days. hESCs were evaluated for dose responses on proliferation level by assaying cell viability, mitochondrial dehydrogenase activity (MDHA), cell confluency, and cell cycle distribution. Differentiation capability was assayed by induction of differentiation into ectoderm, mesoderm, and endoderm; hESCs and differentiated cells were then sequenced for their full transcriptome. Gene expression effects were analyzed by a single cell transcriptome sequencing and analysis of global DNA methylation. Proliferation and methylation effects were tested for all 3 chemicals, while differentiation and single cell sequencing was only tested on PbCl₂. Our results show hESCs were able to identify known and novel proliferation effects of BPA, PFOS, and PbCl₂, reflect differentiation level effects of PbCl₂, and elucidate molecular level drivers of these toxic effects. We also showed that hESCs responded to developmental toxicants at lower doses than in vivo models. In conclusion, our hESC-based model could act as an effective developmental toxicity screening tool for pre- peri- and post-implantation stages of embryo development.

The facile synthesis of engineering thermoplastics enabled the development of structure-morphology-property relationships for a wide range of applications. Utilizing step-growth polymerization techniques, a myriad of reaction conditions probed various polymer families including polysulfones, polyesters, polyimides and polyureas. Copolymers ranging from random to segmented sequences provided insight into the influence of segment length on physical properties. Melting temperatures, glass transition temperatures, and mechanical properties responded systematically to segment length and morphology. Leveraging several complementary analytical techniques identified critical segment lengths required for phase separation and crystallization within these copolymers. Introduction of hydrogen bonding further complicated the interrelationships between thermal and mechanical properties, and possible co-crystallization between dissimilar segments occurred. Finally, branching out from linear copolymers to other topologies determined the influence of branch length on rheological and mechanical properties. The commercially-viable synthesis of these various thermoplastics further highlights the immediate impact on state-of-the-art materials, and the fundamental development described herein provides a road map for future development in this field. / Ph. D.

The aim of this degree project was to study the use and presence of plastic in municipal large-scale catering establishments from preschool to high school and in geriatric care. It was also to get a deeper knowledge about the substance BPA and the plastic PVC and their connection with food. The third aim was to lay forward some suggestions for Eskilstuna about continued work and substitution in their municipal large-scale kitchens. The means for data gathering was databases, websites, scientific reports, on site visits, interviews, online survey and communication by phone and email. The result from the case study was that plastic articles as containers, disposable materials, utensils and food packages for storing food were used in hot, ambient, cold and freezing temperatures. Nine types of plastic were found: PP, PE, PC, PVC, PA, PS, PET, melanin and SAN. All large-scale kitchens had disposable gloves, plastic films and plastic bags. In the first two examples, some brands were made of PVC-plastic. PC-plastic was found in some drinking glasses, plastic food pans, utensils, bowls and jugs. BPA is a common additive in production of PC-plastic and therefore these articles may contain BPA. Between all ten municipal large-scale kitchens, there are some differences and similarities. Of all the kitchens, no one had exactly the same articles or methods, and some had also special routines that differed from the others. The suggestions to Eskilstuna municipality were to remove those articles that contained BPA or chlorinated plastics (especially those products that already had an alternative on the procurement contract), contact the suppliers and wholesalers and ask which products that both are made of PC and contain BPA or other bisphenols, develop a exchange plan for products that do not fulfil the future regulations of KRAV and finally demand in procurement that the product do not contain BPA, PVC or other chlorinated plastics. / KRAV-certifiering av kommunala storkök inom Eskilstuna kommun

Degradation of steel pipes in district heating systems is often a result of corrosion processes. To maintain the power in the systems the pipes gets replaced when they have been worn-out, resulting in high cost and long service time due to excavation. A more affordable and time- effective method is known to be relining which implies renovation and facing of the already existing pipes. This thesis covers an initial review of the applicability of a polyamide curing solvent-free epoxy based relining for lifetime extension of corroded pipes in Stockholm Exergi's pipeline network. The research study is comprised of a literature search focusing on the permeability properties of epoxy coatings, an experimental part where the actual coating was tested for defined parameters similar to the prevailing ones in the system of Stockholm Exergi and finally an evaluation of the usability of the coating. Critical parameters such as the adhesion between the coating and the steel substrate, the sorption of water within the coating and the emit of Bisphenol A were carefully investigated during the analysis. Analysis methods such as pull of test, TGA, DSC and GC-MS were utilized in order to investigate the critical parameters. The adhesion between the coating and the substrate was found to be inadequate for the purpose, the TGA and DSC analysis showed a time-dependent increasing water sorption when exposure to 120 °C. At a higher temperature of 190 °C the sorption decreased. It was concluded that the coating cured at higher exposure temperatures which implied higher degree of conversion and thus a more brittle epoxy matrix. Additionally, the hydrophobicity decreased during 28 days of exposure, at both 120 °C and 190 °C, which can be supposed to affect the flow in the pipeline system during service. It can be stated that a spray coating consisting a polyamide curing solvent-free epoxy will not answer to a total solution for a lifetime extension for the pipes in Stockholm Exergi's pipeline network. An extended investigation regarding the exterior isolation of the pipes is suggested in order to circumvent the most critical breakdowns due to corrosion.

Violent crimes involving bloodshed may result in the formation of a number of blood drops that move through air and impact onto a surface producing a bloodstain pattern. Bloodstain Pattern Analysis (BPA), the analysis of the position, distribution, size and morphology of the stains within the pattern present at a crime scene, may provide information about the events that gave rise to the bloodshed. The location of blood origin, i.e. victim’s position at the moment of wounding and (or) wound location, determination is of major interest to BPA. This study investigated the dynamics of formation and flight of blood drops commonly found at a crime scene (so-called passive, cast-off, impact and gunshot drops) with the aim to facilitate blood origin determination. Features of blood drop formation at passive dripping with correlation to dripping surface characteristics were studied experimentally. A numerical scheme for accurate blood drop flight characteristics modelling, including oscillations, deformation and disintegration, was developed and validated against a number of analytical and experimental cases with special attention to the passive blood drop oscillations and ultimate deformation at terminal velocity, cast-off and impact blood drop deformation and breakup features. This provided an efficient and accurate method for typical blood drop flight reconstruction from the blood origin to impact as well as from the bloodstain location to the possible blood origin. Factors affecting blood drop trajectory and blood origin estimation were studied using the developed scheme.

Background. Endocrine disruptors (EDC) are exogenous compounds that mimic the action of natural hormones and alter the normal endocrine system. Life-long chronic exposure to Bisphenol A (BPA), a putative EDC, has been linked with risk of metabolic disorders in epidemiological studies. Objectives. The aim was to study the human health effects of exposure to BPA, using an integrated approach combining environmental epidemiology and toxicology. Methods. Urinary levels of BPA exposure were measured in participants of the InChianti longitudinal study, a representative population-based study of Italian adults, at the Baseline (1998-00) and nine years later (3rd Wave, 2007-09). Hormones levels and the gene expression of specific target genes were the end points considered. Results were validated in laboratory studies on a human leukemic T-cell line (Jurkat cells). Results. In general, urinary BPA (uBPA) concentrations were higher among men and younger respondents, and within subjects uBPA concentrations were correlated (r=0.58; p=0.013, model adjusted for age, sex, urinary creatinine). At baseline, uBPA concentration were associated with higher total testosterone concentrations in men (β = 0.05; 95% CI, 0.02–0.08). In the 3rd wave, gene expression analysis revealed positive associations between uBPA concentrations and ESR2 (estrogen receptor beta) expression (β=0.18; 95% CI: 0.04, 0.32) and ESRRA (estrogen related receptor alpha) expression (β= 0.17; 95% CI: 0.02, 0.32). In a following in vitro study, BPA exposure (0.001-1 micro molar) led to enhanced expression of ESRRA and ESR2 in Jurkat cells over a period of 72 hours. Conclusions. Results indicate that BPA is bioactive in the human body and is able to alter circulating hormone concentrations and estrogen receptor/estrogen-related receptr gene expression. In particular, given the role of ERRα as a major control point for oxidative metabolism and heart development, this research provides indications on the possible molecular mechanisms of action of BPA in metabolic diseases.

Bisphenol A (BPA), a widely used chemical in plastic, has drawn wide attention due to its presence in many consumer products and the environment. The purpose of this study was to examine the association between urinary BPA and the reporting of cardiovascular diseases (CVD), and then to examine the effect of C-reactive protein (CRP) as a moderating variable. The data used in this research were extracted from the National Health and Nutrition Examination Survey collected in 2009-2010. Guided by the advanced epidemiological triangle, analysis involved 2 stepwise binary logistic regressions. The first step suggested that the controls were significant in predicting CVD (Ï?2 (5) = 83.72, p < .001, R2 = .15). The Nagelkerke R2 coefficient of determination indicated that the controls explained approximately 15% of the variance in instances of CVD. The second step of the binary logistic regression included the controls and BPA level in the model together. The regression analysis suggested that the Nagelkerke coefficient of determination (Ï?2 (6) = 83.76, p < .001, R2 = .15) did not increase from the 15% explained by the controls, and BPA level was found to be a nonsignificant predictor of CVD (p = .853). Due to lack of association between BPA and CVD, the analysis was shifted to examine the association between urinary BPA and serum CRP. The association between urinary BPA and serum CRP was also statistically nonsignificant (Spearman correlation coefficient, rs= .06, p = .015). The results may have positive social change by contributing to the body of knowledge on BPA and by increasing scientific scrutiny for substances used in people's daily lives.

Bisphenol A (BPA), a widely used chemical in plastic, has drawn wide attention due to its presence in many consumer products and the environment. The purpose of this study was to examine the association between urinary BPA and the reporting of cardiovascular diseases (CVD), and then to examine the effect of C-reactive protein (CRP) as a moderating variable. The data used in this research were extracted from the National Health and Nutrition Examination Survey collected in 2009-2010. Guided by the advanced epidemiological triangle, analysis involved 2 stepwise binary logistic regressions. The first step suggested that the controls were significant in predicting CVD (Ï?2 (5) = 83.72, p < .001, R2 = .15). The Nagelkerke R2 coefficient of determination indicated that the controls explained approximately 15% of the variance in instances of CVD. The second step of the binary logistic regression included the controls and BPA level in the model together. The regression analysis suggested that the Nagelkerke coefficient of determination (Ï?2 (6) = 83.76, p < .001, R2 = .15) did not increase from the 15% explained by the controls, and BPA level was found to be a nonsignificant predictor of CVD (p = .853). Due to lack of association between BPA and CVD, the analysis was shifted to examine the association between urinary BPA and serum CRP. The association between urinary BPA and serum CRP was also statistically nonsignificant (Spearman correlation coefficient, rs= .06, p = .015). The results may have positive social change by contributing to the body of knowledge on BPA and by increasing scientific scrutiny for substances used in people's daily lives.

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