<p>Polymeric nanoparticles are potential candidates as carriers for pharmaceutical agents. Development of such nanoparticles generally requires molecules immobilized on the particle surfaces to ensure biocompatibility and/or targeting abilities. Following particle preparation and surface modification, excess reagents must be removed. Ultracentrifugation, which is the most widely used purification technique as per today, is not feasible in industrial applications. In this diploma work, tangential flow filtration is studied as an alternative purification method which is better suited for implementation in a large-scale process.</p><p>Comparison of ultracentrifugation and tangential flow filtration in diafiltration mode for purification of nanoparticles, indicate that they are comparable with respect to particle stability and the removal of the surfactant SDS from methacrylic anhydride nanoparticles. The purification efficiency of tangential flow filtration is superior to that of ultracentrifugation. Conductivity measurements of filtrates and supernatant liquids show that a stable conductivity value can be reached 6 times faster in filtration than in centrifugation with equipment and settings used. This conductivity arises from several types of molecules, and the contribution from surfactant molecules alone is not known. However, protein adsorption on the particles indicates successful removal of surfactant. Conductivity and tensiometry were evaluated as potential methods to quantify surfactant in solutions, but both proved unsatisfactory.</p><p>Using bovine serum albumin as a model protein, the extent of immobilization to nanoparticles is evaluated at different pH. A maximum amount of 6,8 mg/m2 is immobilized, whereof an unknown part is covalently bound. This coverage is achieved at pH 4,0 and is probably partly due to low electrostatic repulsion between particle and protein. An estimation of 2,0 µmol covalently bound BSA per gram of nanoparticles corresponds to 5,3 mg/m2 and a surface coverage of 76%. Removal of excess reagents after surface modification is done with ultracentrifugation instead of filtration, as particle aggregates present after the immobilization reaction might foul the membrane.</p>

In recent years, biocatalytical steps in chemical synthesis are becoming increasingly important for economical and environmental-friendly production. In order to evaluate the use of enzymes in a process at Cambrex Karlskoga AB, an expression system was developed for a dehydrogenase. A synthetic gene was cloned into Escherichia coli DH5a cells, using the pTZ19R expression vector, as previously described in the literature. Protein expression was carried out at 25°C, 30°C and 37°C and results were measured using SDS-PAGE and activity assays. To improve expression, the gene was modified in three ways using PCR, yielding eight clones: It was inserted into the pSE420 expression vector, shortened to avoid inclusion body formation and a missing nucleotide was inserted into the sequence. A protocol for inclusion body screening was also developed. Finally, an assay for determining the kinetic constants of dehydrogenase was designed. It is concluded that further experiments must be done to obtain expression of the dehydrogenase and recommendations for additional work are given. / Biokatalytiska processteg har de senaste åren blivit ett allt viktigare inslag i kemisk syntes för att åstadkomma ekonomisk och miljövänlig produktion. För att utvärdera användandet av enzymer i en process hos Cambrex Karlskoga AB utvecklades ett expressionssystem för ett dehydrogenas. En syntetisk gen klonades in i Escherichia coli DH5a och uttrycktes med hjälp av expressionsvektorn pTZ19R, som tidigare finns beskrivet i litteraturen. Proteinuttrycket utfördes vid 25°C, 30°C och 37°C och resultatet mättes med hjälp av SDS-PAGE och aktivitetsmätningar. Genen för dehydrogenaset modifierades på tre sätt, vilket gav upphov till åtta varianter. Genen fördes över till expressionsvektorn pSE420, kortades för att undvika bildning av inklusionskroppar och en nukleotid som fattades från gensekvensen återinfördes. Ett protokoll utarbetades även för undersökning av inklusionskroppar. Till sist sammanställdes en metod för att undersöka de kinetiska konstanterna hos dehydrogenaset. Slutsatsen av arbetet är att fortsatta studier måste utföras för att erhålla uttryck av dehydrogenaset och rekommendationer ges för framtida undersökningar.

Fotosyntesen kallas den process som omvandlar ljusenergi till kemisk energi. Fotosyntesen sker i tylakoidmembranet och drivs av två stora proteinkomplex, fotosystem II (PSII) och fotosystem I (PSI) då de tillförs energi i form av fotoner. PSI-N är ett mindre protein på ca 10 kDa som ingår i PSI. På något sätt, som ännu inte är klarlagt, samverkar PSI-N med PSI-F och plastocyanin när det dockar till PSI. Det är därför av viktigt att rena fram större mängder av PSI-N för att få djupare kunskaper om proteinet samt dess struktur och funktioner. Tidigare undersökningar har utförts i ämnet och ett fusionsprotein innehållande PSI-N har uttryckts i Escherichia coli (E.coli). Problem har dock uppstått efter lysis av cellerna då det har visat sig att fusionsproteinet har degraderats. Vårt examensarbete strävar efter att rena fram intakt fusionsprotein med hjälp av, framför allt, mekanisk lysis och proteasinhibitorer. / The process where light is converted into chemical energy is called photosyntesis. The reaction takes place in the thylakoid membrane and is driven by two major protein complexes, photosystem II (PSII) and photosystem I (PSI) when energy in form of photons are received. PSI-N, a subunit in PSI, is a smaller protein with a mass of approximately 10 kDa. In some way, which is not yet clarified, PSI-N collaborates with PSI-F and plastocyanin when plastocyanin is docking to PSI. It is therefore important to purify larger amounts of the protein to acquire deeper knowledge of its structure and function. In earlier research the PSI-N protein has been expressed in Escherichia coli (E.coli). The problem has been degradation of the fusion protein after lysis. Our goal with this project is to obtain the purified protein intact using mechanic lysis and protease inhibitors.

In this study, Tetrahydrofuran (THF) hydrates were formed in-situ in the Zetasizer Nano ZS90. With various concentrations of SDS, we attempted to characterize the SDS adsorption on the surface of the hydrate particles. In doing so, we tried to correlate the adsorption of SDS to THF hydrate induction times with respect to SDS concentration (0 – 3.47 mM), to determine whether the fast nucleation of THF hydrates is due to the adsorption of SDS. The measured ζ-potential for pure THF hydrates was -100 ± 10 mV, indicating anion adsorption. An adsorption curve was observed where there is saturation leveling. Correlating this data to the hydrate induction times, we see that when the saturation level is reached, a significant reduction in induction time can be seen.

In this study, Tetrahydrofuran (THF) hydrates were formed in-situ in the Zetasizer Nano ZS90. With various concentrations of SDS, we attempted to characterize the SDS adsorption on the surface of the hydrate particles. In doing so, we tried to correlate the adsorption of SDS to THF hydrate induction times with respect to SDS concentration (0 – 3.47 mM), to determine whether the fast nucleation of THF hydrates is due to the adsorption of SDS. The measured ζ-potential for pure THF hydrates was -100 ± 10 mV, indicating anion adsorption. An adsorption curve was observed where there is saturation leveling. Correlating this data to the hydrate induction times, we see that when the saturation level is reached, a significant reduction in induction time can be seen.

Introduction: While the influence of parental behavior and economic status on children’s weight status is well-known, little is known about the impact of specific family-related aspects such as parental and grandparental social support. This study investigates the importance of parental and grandparental social support and economic capital for children’s weight status in a clinical sample of preschoolers with obesity. Methods: Baseline data from an obesity intervention study for preschoolers, 4-6 years of age (n = 39, 56 % girls) was used. Among parents, 73% were overweight/obese, 60% had a 3-year high school education and 50% were of non-Swedish origin. Linear regression analyses, simple and multiple, were performed separately for mothers and fathers on indicators of economic capital and social support with child BMI SDS as the dependent variable. Additionally, combined analyses were conducted in which parental income was stratified by emotional support. Results: Low levels of income for both parents and low emotional support from grandparents for fathers were significantly associated with a higher child BMI. Moreover, the association between parental income and the child’s BMI SDS was stronger among those parents who had low emotional support versus those who had high emotional support. Conclusion: The study indicate that both economic capital and social support of parents may influence the level of obesity in their children and that emotional support of grandparents is especially important when parental income is low.

The development of metal-tolerance and antibiotic resistance in bacteria may be caused by metals polluting a particular environment. During mining and mineral processing activities, large quantities of metals are deposited into the soil. These high concentrations of metals are evolutionary pressures selecting for microorganisms tolerant to these metals. Metaltolerance maybe conferred to these organisms by mobile genetic elements such as plasmids. This study describes the characteristics of plasmids isolated from various bacteria that displayed an ability to withstand high metal concentrations. The isolated plasmids were individually transformed into Escherichia coli JM109. Transformants were then evaluated for metal-tolerant capabilities using a microdilution approach. Plasmids were then isolated from the transformants and the concentration of the plasmid DNA ranged between 11.75 – 118.06 ng/μl. These plasmids were of the same size as the original ones. This demonstrated that successful transformations with plasmid DNA were conducted. In order to determine the compatibility group, plasmids were subjected to PCR amplification using IncQ, IncP-9 and IncW specific primers. Only the IncW provided positive results. To demonstrate that the plasmids were free of genomic DNA, a 16S rDNA PCR test was included. The plasmids that were positive for IncW PCRs were all negative for the rDNA PCRs. Plasmids were stably inherited and at least three, isolated from three different Gram positive species, belonged to the Inc W group of plasmids. These were originally isolated from Paenibacillus ginsingari, Paenibacillus lautus and Bacillus cereus. Minimum inhibition concentrations (MICs) were carried out to determine the ability of transformed E. coli JM109 to tolerate metals at varying concentrations. Results indicated that transformed E. coli JM109 developed ability to grow in the presence of several heavy metals. Some strains were resistant to high concentrations (+10 mM) of Ni2+/Al3+, Pb2+ and Ba2+. The order of metal resistance was Ni/Al=Pb>Ba>Mn>Cr>Cu>Co=Hg. All the x transformants were sensitive to 1 mM of Co2+ and Hg2+. Moreover, protein profiling was used to determine the impact of plasmids on E. coli JM109. Proteins were extracted from both transformed and un-transformed E. coli JM109 using acetone-SDS protocol and subjected to one-dimensional (1D) and two-dimensional (2D) Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS- PAGE). Transformed E. coli JM109 were grown under the metal stress. One dimension SDS-PAGE illustrated general similarity of the profiles except for two banding positions in the 30 to 35 kDa region where bands were present in the transformants that were grown in the Ni/Al alloy containing media. Twodimensional electrophoresis PAGE analysis showed that some of the proteins were upregulated while others were down-regulated. The largest numbers of proteins were from 15 – 75 kDa. The majority of these proteins had isoelectric points (pI) between 5 and 6. It was concluded that plasmids isolated from various heavy metal-tolerant bacterial species were successfully transformed into E. coli JM109 rendering various new metal-tolerant E. coli JM109 strains. Furthermore, the study showed that metal resistance was due to the presence of the plasmids. Two-dimensional SDS-PAGE resolved more differences in the protein expression profiles. Since the plasmids rendered the E. coli JM109 tolerant to metals tested, it also can be concluded that the change in the protein profiles was due to the effects of the plasmids. Furthermore, plasmids were also re-isolated from the transformants and these plasmids were of the same size as the original ones.. All the plasmids in this study were also stably inherited, a feature associated with IncW plasmids. More detailed genetic characterization of these plasmids is required. Plasmids isolated and characterized in this study may hold biotechnology potential. Such features should be exploited in follow-up experiments. / Thesis (Master of Environmental Sciences)--North-West University, Potchefstroom Campus, 2013.

The development of metal-tolerance and antibiotic resistance in bacteria may be caused by metals polluting a particular environment. During mining and mineral processing activities, large quantities of metals are deposited into the soil. These high concentrations of metals are evolutionary pressures selecting for microorganisms tolerant to these metals. Metaltolerance maybe conferred to these organisms by mobile genetic elements such as plasmids. This study describes the characteristics of plasmids isolated from various bacteria that displayed an ability to withstand high metal concentrations. The isolated plasmids were individually transformed into Escherichia coli JM109. Transformants were then evaluated for metal-tolerant capabilities using a microdilution approach. Plasmids were then isolated from the transformants and the concentration of the plasmid DNA ranged between 11.75 – 118.06 ng/μl. These plasmids were of the same size as the original ones. This demonstrated that successful transformations with plasmid DNA were conducted. In order to determine the compatibility group, plasmids were subjected to PCR amplification using IncQ, IncP-9 and IncW specific primers. Only the IncW provided positive results. To demonstrate that the plasmids were free of genomic DNA, a 16S rDNA PCR test was included. The plasmids that were positive for IncW PCRs were all negative for the rDNA PCRs. Plasmids were stably inherited and at least three, isolated from three different Gram positive species, belonged to the Inc W group of plasmids. These were originally isolated from Paenibacillus ginsingari, Paenibacillus lautus and Bacillus cereus. Minimum inhibition concentrations (MICs) were carried out to determine the ability of transformed E. coli JM109 to tolerate metals at varying concentrations. Results indicated that transformed E. coli JM109 developed ability to grow in the presence of several heavy metals. Some strains were resistant to high concentrations (+10 mM) of Ni2+/Al3+, Pb2+ and Ba2+. The order of metal resistance was Ni/Al=Pb>Ba>Mn>Cr>Cu>Co=Hg. All the x transformants were sensitive to 1 mM of Co2+ and Hg2+. Moreover, protein profiling was used to determine the impact of plasmids on E. coli JM109. Proteins were extracted from both transformed and un-transformed E. coli JM109 using acetone-SDS protocol and subjected to one-dimensional (1D) and two-dimensional (2D) Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS- PAGE). Transformed E. coli JM109 were grown under the metal stress. One dimension SDS-PAGE illustrated general similarity of the profiles except for two banding positions in the 30 to 35 kDa region where bands were present in the transformants that were grown in the Ni/Al alloy containing media. Twodimensional electrophoresis PAGE analysis showed that some of the proteins were upregulated while others were down-regulated. The largest numbers of proteins were from 15 – 75 kDa. The majority of these proteins had isoelectric points (pI) between 5 and 6. It was concluded that plasmids isolated from various heavy metal-tolerant bacterial species were successfully transformed into E. coli JM109 rendering various new metal-tolerant E. coli JM109 strains. Furthermore, the study showed that metal resistance was due to the presence of the plasmids. Two-dimensional SDS-PAGE resolved more differences in the protein expression profiles. Since the plasmids rendered the E. coli JM109 tolerant to metals tested, it also can be concluded that the change in the protein profiles was due to the effects of the plasmids. Furthermore, plasmids were also re-isolated from the transformants and these plasmids were of the same size as the original ones.. All the plasmids in this study were also stably inherited, a feature associated with IncW plasmids. More detailed genetic characterization of these plasmids is required. Plasmids isolated and characterized in this study may hold biotechnology potential. Such features should be exploited in follow-up experiments. / Thesis (Master of Environmental Sciences)--North-West University, Potchefstroom Campus, 2013.

<p>&nbsp / </p> <p align="left">One of the most common HIV-associated opportunistic infections is candidiasis, caused by <i><font face="TimesNewRoman,Italic">Candida albicans </font></i><font lang="KO" face="TimesNewRoman">or other </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">species. In immune suppressed subjects, this commensal organism can cause an increase in patient morbidity and mortality due to oropharyngeal or systemic dissemination. Limited information exists on the prevalence and antifungal susceptibility of </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">species in the African continent, the most HIV-affected region globally and home to new and emerging drug resistant </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">species. The mechanisms of </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">drug resistance in the African continent have also not been described. In this study, 255 </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">species isolated from the oral mucosa of HIV-positive South African and Cameroonian patients were identified using differential and chromogenic media and their drug susceptibility profiles tested using the disk diffusion method and the TREK Sensititre system, an automated broth microdilution method. </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">cell wall fractions were run on SDSPAGE and HPLC-MS with the aim of identifying peptides specifically expressed by antifungal drug resistant isolates. Comparisons between the two groups of isolates revealed differences in </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">species prevalence and drug susceptibility with interesting associations observed between specific drug resistance and duration of ARV therapy. This study showed that fluconazole, the drug of choice for the treatment of candidiasis in the African continent, is not an effective therapy for most cases of </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">infection, and suggests that regional surveillance be implemented in the continent. A multiple-drug resistant </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">strain was identified in this study, a finding that has not previously been documented. The use of proteomics tools allowed for the identification of peptides involved in drug resistance and the elucidation of </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">colonization mechanisms in HIV-infected African patients.</font></i></i></i></i></i></i></i></i></i></i></p>

<p>&nbsp / </p> <p align="left">One of the most common HIV-associated opportunistic infections is candidiasis, caused by <i><font face="TimesNewRoman,Italic">Candida albicans </font></i><font lang="KO" face="TimesNewRoman">or other </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">species. In immune suppressed subjects, this commensal organism can cause an increase in patient morbidity and mortality due to oropharyngeal or systemic dissemination. Limited information exists on the prevalence and antifungal susceptibility of </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">species in the African continent, the most HIV-affected region globally and home to new and emerging drug resistant </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">species. The mechanisms of </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">drug resistance in the African continent have also not been described. In this study, 255 </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">species isolated from the oral mucosa of HIV-positive South African and Cameroonian patients were identified using differential and chromogenic media and their drug susceptibility profiles tested using the disk diffusion method and the TREK Sensititre system, an automated broth microdilution method. </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">cell wall fractions were run on SDSPAGE and HPLC-MS with the aim of identifying peptides specifically expressed by antifungal drug resistant isolates. Comparisons between the two groups of isolates revealed differences in </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">species prevalence and drug susceptibility with interesting associations observed between specific drug resistance and duration of ARV therapy. This study showed that fluconazole, the drug of choice for the treatment of candidiasis in the African continent, is not an effective therapy for most cases of </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">infection, and suggests that regional surveillance be implemented in the continent. A multiple-drug resistant </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">strain was identified in this study, a finding that has not previously been documented. The use of proteomics tools allowed for the identification of peptides involved in drug resistance and the elucidation of </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">colonization mechanisms in HIV-infected African patients.</font></i></i></i></i></i></i></i></i></i></i></p>