Sarcoptes scabiei is a disease-causing parasitic mite of humans and animals that is prevalent worldwide. The parasite lives in burrows in the epidermis of its host. These burrows are formed by a combination of mechanical destruction by the mite and secretion of various factors. The enzyme superoxide dismutase (SOD) catalyzes the dismutation of superoxide into oxygen and hydrogen peroxide. As such, it is an important antioxidant defense in nearly all cells exposed to oxygen. In this project, the enzyme was expressed in transformed Escherichia coli cells. The SOD cDNA from S. scabiei was ligated into two different expression vectors: pPU16 and pET-14b. The S. scabiei SOD open reading frame reported here is 696 nucleotides long and yields a protein with a molecular weight of  69.5 kDa. Only one of the constructs was successfully created, using pPU16. The construct was designated pPU110 and has a sequence coding for a hexahistidine tag downstream of the SOD cDNA and has a sequence coding for the maltose binding protein (MBP) upstream. The expression plasmid pPU110 was verified by DNA-sequencing and the tested in different expression experiments. Analysis using SDS-PAGE showed that recombinant fusion SOD could be readily expressed in E.coli.

Part One. X-ray crystallography has benefited from the synthetic introduction of selenium to different positions within nucleic acids by easing the solving of the phase problem. Interestingly, its addition to the 2' position of the ribose ring also significantly enhances crystal formation. This phenomenon was investigated to describe the effect of selenium-based and other 2' modifications to the ribose ring of nucleosides in solution, as well as the incorporation of the selenium-modified nucleotides into a helical structure. This work correlates the difference in conformation propensity between the selenium containing nucleosides and oligomers towards a rationale behind the enhanced crystal forming behavior. Part Two. Recombinant protein production is a critical tool in laboratories and industries, and inducing extracellular transport of these products to the culture medium shows potential for improving cases where the yields are not sufficient in quality or quantity. This review incorporates current practices and systems with future perspectives.

B virus diagnosis presents a challenge largely complicated by the asymptomatic infection of rhesus macaques, and extremely pathogenic fatal infections in humans. Humoral detection of antibodies is generally performed using whole virus antigen for which preparation requires strict biosafety measures and specialized BSL-4 facilities. As an alternative to utilizing B virus antigen, we describe the production of a truncated form of B virus envelope glycoprotein D, gD 287, in a baculovirus expression system, and evaluate its diagnostic potential as an antigen in recombinant ELISA. After purification and characterization, gD 287 was tested using 22 negative and 72 positive macaque sera samples previously classified using the traditional method. We find that sensitivity and specificity of the recombinant ELISA are dependent on antibody titer of tested serum and gD 287 shows good to excellent predictive potential for identification of positive sera with titers higher than 500.

Recombinant Bovine Somatotropin (rBST) is a biotechnology for increasing milk production in dairy cattle. The purpose of this research was to investigate and to build a better understanding of the complexities and controversies around this product in Canada. To accomplish this, I examined the Standing Senate Committee on Agriculture and Forestrys inquiry into rBST and the drug approval process. I compared and contrasted the testimony of witnesses and Senators and I uncovered emerging issues, patterns, and themes. This research was an exploratory and qualitative exercise that analyzed how the participants of this Senate inquiry conceptualized and contested the meaning of science, safety, and the states regulatory functions. <p> This research revealed several commonalities between Health Canada management, the human safety panel, and industry representatives. These witnesses argued that the drug approval process must be efficient, standard-driven, and based upon available scientific studies. These witnesses stated that they had confidence in the neutrality and competency of internal standard setting-agencies. They emphasized transparency rather than public participation in the drug approval process. Health and safety were conceptualized as static phenomena to be measured and evaluated by experts. <p>In contrast, Health Canada employees had several commonalities with the Senators, dairy representatives, and witnesses from citizen interest groups. Their testimony supports the argument that health and safety are dynamic social constructs. These actors transformed the boundaries of science to accommodate their precautionary framing of safety. They highlighted several problems with Canadas science-based regulatory framework and demanded that they have a decisive voice in the rBST decision. They challenged the hegemony of industrial capitalism by combining both scientific and lay knowledge to expose the limits and contradictions of industrialized agriculture.

In this study, it was aimed to investigate the effects of different co-carbon sources on therapeutically important glycoprotein, recombinant human erythropoietin (rHuEPO) production by Pichia pastoris by designing feeding strategies which were applied in the production phase of the bioprocess. During the experiments, the cell growth, sorbitol, mannitol, and methanol consumptions, recombinant human EPO production, alcohol oxidase activity, total protease concentrations and the by-products organic acid concentrations were analyzed. In this context, firstly, laboratory scale air filtered shake bioreactor experiments were performed by P. pastoris Mut+ strain to investigate the effects of mannitol and sorbitol. 50 gL-1 initial concentration of co-substrates was found more affordable and appropriate for cell concentration and recombinant protein production. Thereafter, six pilot scale bioreactor operations were designed and performed. In the first designed strategy (named as SSM strategy), batch-wise 50 g L-1 sorbitol was fed at t=0 h of the production phase and then sorbitol concentration was kept constant at 50 g L-1 by fed-batch feeding with a pre-determined specific growth rate of &mu / Srb0=0.025 h-1 within t=0-15 h of the production phase together with fed-batch methanol feeding with a pre-determined specific growth rate of &mu / M0=0.03 h-1. In the following bioreactor experiments co-substrate mannitol was fed to the system with different feeding strategies together with fed-batch methanol feeding with a pre-determined specific growth rate of &mu / M0=0.03 h-1. In the second strategy (MM), only 40 g L-1 mannitol was added to the system at t=0 h of the production phase. In the third strategy (MMM), after adding 50 g L-1 mannitol at t=0 h, mannitol concentration was kept constant at 50 g L-1 by fed-batch feeding with a pre-determined specific growth rate of &mu / Man0=0.11 h-1 within t=0-9 h of the production phase when the same cell concentration was attained in SSM strategy. In the fourth one (MLM), limiting amount of mannitol, 3 g L-1, was added at t=0 h and then mannitol concentration was kept constant at 3 g L-1 by fed-batch feeding with a pre-determined specific growth rate of &mu / Man0=0.005 h-1 within t=0-10 h of the production phase. After these strategies, several pulses, batch-wise, mannitol feeding strategies were performed. In the fifth strategy (MPM), besides 50 g L-1 initial mannitol feeding at t=0 h, adding second batch-wise mannitol at t=6 h, and third one at t=12 h were applied. In the last strategy (MPMG), four 50 g L-1 pulse feeding of mannitol were performed at t=0 h, 7 h, 14 h, and 24 h, containing glycerol, with an initial concentration in the fermentation medium being 8 g L-1. The highest extracellular rHuEPO production was achieved in the fifth strategy MPM as CrHuEPO=645 mg L-1 at t=9 h while the highest cell concentration was achieved in the first strategy SSM as Cx=109 gL-1 at t=48 h. The overall cell and product yields on total substrate were calculated as YX/St=0.22 g g-1 and YP/St=2.23 mg g-1 in the highest rHuEPO production case.

Downstream purification often represents the most cost-intensive step in the manufacturing of recombinant proteins. Conventional purification processes are lengthy, technically complicated, product specific and time-consuming. To address this issue, herein we develop a one step purification system that due to the nature of the non-selective secretion system and the versatility of ion-exchange membrane chromatography can be widely applied to the production of many recombinant proteins. This was achieved through the integration of the intrinsically coupled upstream, midstream and downstream processes, a connection that is rarely exploited. A bioprocess for effective production and purification of penicillin G acylase (PAC) was developed. PAC was overexpressed in a genetically engineered Escherichia coli strain, secreted into the cultivation medium, harvested, and purified in a single step by anion-exchange chromatography. The cultivation medium developed had a sufficiently low conductivity to allow direct application of the extracellular fraction to the anion-exchange chromatography medium while providing all of the required nutrients for sustaining cell growth and PAC overexpression. It was contrived with the purposes of (i) providing sufficient osmolarity and buffering capacity, (ii) minimizing ionic species to facilitate the binding of extracellular proteins to anion-exchange medium, and (iii) enhancing PAC expression level and secretion efficiency. Employing this medium recipe the specific PAC activity reached a high level of 487 U/L/OD600, with more than 90% was localized in the extracellular medium. Both, the osmotic pressure and induction conditions were found to be critical for optimal culture performance. Furthermore, formation of inclusion bodies associated with PAC overexpression tended to arrest cell growth, leading to potential cell lysis. iv At harvest, the whole non-clarified culture broth was applied directly to a tangential flow filtration anion-exchange membrane chromatography system. One-step purification of recombinant PAC was achieved based on the dual nature of membrane chromatography (i.e. microfiltration-sized pores and anion-exchange chemistry). Due to their size, cells remained in the retentate while the extracellular medium penetrated the membrane. Most contaminate proteins were captured by the anion-exchange membrane, whereas the purified PAC was collected in the filtrate. The batch time for both cultivation and purification was less than 24 h and recombinant PAC with high purity (19 U/mg), process yield (74%), and productivity (41 mg/L) was obtained.

Bacteriophages affect bacterial evolution, pathogenesis and global nutrient cycling. They are also the most numerous and diverse group of biological entities on the planet [1, 2, 3, 4, 5, 6]. Members of the Lambda phage family share a similar genetic organization and control early gene expression by suppressing transcription, a process known as antitermination. Transcription antitermination in Lambda is mediated by a phage-encoded protein whereas in lambdoid phage HK022, antitermination is mediated by a phage-encoded RNA molecules. Recent results suggest that another bacteriophage called HK639 also appears to use RNA-mediated antitermination. To characterize this newly identified phage we generated site directed mutations and identified where the phage integrates into the chromosome of its bacterial host.

Recombinant Bovine Somatotropin (rBST) is a biotechnology for increasing milk production in dairy cattle. The purpose of this research was to investigate and to build a better understanding of the complexities and controversies around this product in Canada. To accomplish this, I examined the Standing Senate Committee on Agriculture and Forestrys inquiry into rBST and the drug approval process. I compared and contrasted the testimony of witnesses and Senators and I uncovered emerging issues, patterns, and themes. This research was an exploratory and qualitative exercise that analyzed how the participants of this Senate inquiry conceptualized and contested the meaning of science, safety, and the states regulatory functions. <p> This research revealed several commonalities between Health Canada management, the human safety panel, and industry representatives. These witnesses argued that the drug approval process must be efficient, standard-driven, and based upon available scientific studies. These witnesses stated that they had confidence in the neutrality and competency of internal standard setting-agencies. They emphasized transparency rather than public participation in the drug approval process. Health and safety were conceptualized as static phenomena to be measured and evaluated by experts. <p>In contrast, Health Canada employees had several commonalities with the Senators, dairy representatives, and witnesses from citizen interest groups. Their testimony supports the argument that health and safety are dynamic social constructs. These actors transformed the boundaries of science to accommodate their precautionary framing of safety. They highlighted several problems with Canadas science-based regulatory framework and demanded that they have a decisive voice in the rBST decision. They challenged the hegemony of industrial capitalism by combining both scientific and lay knowledge to expose the limits and contradictions of industrialized agriculture.

Mantle cell lymphoma (MCL) is one of several sub-types of B-cell lymphomas. The malignancy is very aggressive and average survival time is short. The hallmark of MCL is over expression of cyclin D1, however about 15% of all MCL cases do not display this over expression and are easily misdiagnosed. Recently the transcription factor Sox11 has been shown to be specifically over expressed in the nucleus of MCL-tumour cells, and polyclonal rabbit anti-Sox11 antibodies have been used to successfully identify MCL in both cyclin D1 positive and negative cases. Howev-er, human recombinant MCL-specific antibodies as have several advantages over these polyclonal rabbit antibodies; they can easily be produced in large quantities in vitro, their specificity is constant from batch to batch and they can possibly be used for therapeutic purposes. Because of this, it is desirable to produce human recombinant antibodies against proteins over expressed in MCL. In this study human recombinant IgGs have been produced towards two pro-teins over expressed in MCL, Sox11 and KIAA0882. This was done by cloning of single chain variable fragments (scFvs), previously selected from a large scFv library through phage display selection against Sox11- and KIAA0882-protein epitope signature tag (PrEST), into vectors containing human IgG constant regions followed by expression of human IgG antibodies in human embryonic kidney (HEK) 293 cells. One IgG clone for each antigen was shown to be functional and specific. Both clones were shown to have overlapping binding epitopes with their polyclonal rabbit antibody counterpart (rabbit anti-Sox11/KIAA0882) through competitive ELISA. The anti-Sox11 IgG was able to detect two bands in cell lysate in Western blot, of which one probably is Sox11 while the other band possibly could be Sox4. However, this needs to be confirmed in future experiments. The affinity of the anti-Sox11 IgG was measured in Biacore and compared to the affinity of its original scFv. This gave a rough estimation of the affinities, but the values are unreliable and the measurements need to be redone. Although more work has to be put into evaluating the potential of the produced IgGs, they compose a promising starting point to an improved understanding and improved diagnosis of MCL.

In der Veterinärmedizin verursachen Rotaviren als Jungtiererkrankung vor allem in der Nutztierpraxis hohe ökonomische Verluste. Über die Prävalenz von Rotavirusinfektionen bei Hunden und Katzen ist sehr wenig bekannt, obwohl von den in der Literatur als wechselseitig zwischen Mensch und Tier übertragbaren Viren ein nicht zu unterschätzendes Risiko ausgehen kann. Zunächst wurden retrospektiv Prävalenzdaten über den Nachweis von Rotaviren bei Hunden und Katzen mit Durchfall im Vergleich zu Coronaviren und Parvoviren erhoben. Dazu wurden Kotproben von 2055 Hunden und 1481 Katzen quantitativ auf das Vorhandensein von Rota-, Corona- und Parvovirus untersucht. Desweiteren wurden Aspekte der geographischen Verteilung, der Altersverteilung, mögliche Rasseprädispositionen und das Auftreten saisonaler Erkrankungsgipfel untersucht und ausgewertet. Für Rotavirusinfektionen beträgt die statistische Prävalenz 7% bei Hunden und 8% bei Katzen. Bei Hunden und Katzen konnten signifikant häufiger Dreifachinfektionen nachgewiesen werden. Bei einer Infektion mit Rota- und Coronavirus liegt beim Hund zu 100% auch eine Infektion mit Parvovirus vor. Zweifachinfektionen kamen weniger häufig vor als Monoinfektionen. Alle drei Virusinfektionen kamen bei Hunden statistisch signifikant häufiger in der Altersgruppe ≤ 1 Jahr vor. Ein statistisch signifikant häufiger Rotavirusnachweis konnte bei der Katzenrasse Siam nachgewiesen werden, während keine Hunderasse besonders hervortrat. Im Postleitzahlengebiet 3 konnten im Beobachtungszeitraum von 2000 bis 2006 statistisch signifikant häufiger Rotavirusinfektionen bei Hunden nachvollzogen werden. Es konnte sowohl für Hunde, als auch für Katzen der Trend belegt werden, dass bei steigenden Lufttemperaturen, die Anzahl der Rotavirusinfektionen sinkt. Es kann somit von einer bedingten Saisonalität ausgegangen werden. Im zweiten Teil der Arbeit wurde die Empfänglichkeit von Rotaviren gegenüber kommerziell erhältlichem Typ I Interferon (rFeIFN-ω) in vitro getestet. Zunächst wurde zum Nachweis der Aktivität der Typ I Interferone (rFeIFN-ω, rBoIFN-α, rHuIFN-α) die Expression des Mx Proteins auf Zelllinien felinen, caninen, bovinen und humanen Ursprungs, sowie auf Affenzelllinien untersucht. Es konnte gezeigt werden, dass rBoIFN-α ausschließlich auf Zellen bovinen Ursprungs eine konzentrationsabhängige Expression des Mx Proteins induziert. Das rFeIFN-ω induziert auf Zellen felinen und bei höheren Konzentrationen auch auf Zellen caninen Ursprungs die Expression des Mx Proteins. Das rHuIFN-α zeigt eine konzentrationsabhängige Induktion des Mx Proteins in Zellen humanen, caninen, felinen und bovinen Ursprunges, sowie in Affenzelllinien. Somit konnte in vitro eine Kreuz-Speziesspezifität für rekombinantes humanes Interferon nachgewiesen werden. Zum Nachweis einer immunmodulatorischen Wirkung wurde die Expression der MHC I Oberflächenrezeptoren nach Behandlung mit rFeIFN-ω und rHuIFN-α untersucht. Die Behandlung mit rFeIFN-ω führte ausschließlich in felinen Zellen zu einer konzentrationsabhängigen signifikanten Erhöhung der Rezeptordichte. Die Behandlung mit rHuIFN-α führte zu einer konzentrationsabhängigen signifikanten Erhöhung der Rezeptordichte auf felinen Zellen und in der Affenzelllinie MA104. Die Empfänglichkeit von Rotaviren gegenüber rFeIFN-ω wurde auf der embryonalen felinen Fibroblastenzelllinie (KE-R) und auf der embryonalen felinen Gehirnzelllinine (KG-R) unter steigender Interferonkonzentration (101-104 Einheiten/ml) untersucht. Beide Zelllinien zeigten eine deutliche Reduktion der infizierten Zellen bei steigender Interferonkonzentration. Die antivirale Wirkung war in KE-R Zellen deutlicher ausgeprägt. Dort konnten bereits bei einer Interferonkonzentration von 103 Einheiten/ml keine sichtbar infizierten Zellen mehr nachgewiesen werden, während KG-R Zellen erst bei einer Konzentration von 104 Einheiten/ml keine sichtbar infizierten Zellen mehr nachzuweisen waren. Abschließend wird deutlich, dass Infektionen mit Rotaviren ein vielmals vernachlässigtes Problem in der Veterinärmedizin darstellt, vor allem, wenn man von einer Vergesellschaftung mit den für Hund und Katze pathogenen Viren Corona- und Parvovirus ausgeht. Mit dem rFeIFN-ω steht in vitro eine wirksame antivirale Substanz gegen Rotavirusinfektionen zur Verfügung.