Bacterial infection within a joint, septic arthritis, is a serious condition in horses that can lead to long-term joint disease if the infection is not resolved quickly. Equine septic arthritis is diagnosed primarily based on clinical signs and synovial fluid cytology. Septic synovial fluid is characterized by significant elevations in total protein (TP) and total nucleated cell count (TNCC). However, in some cases it can be difficult to distinguish between septic arthritis and non-septic joint inflammation (synovitis) based on clinical signs and synovial fluid cytology alone. A rapid assay to help confirm septic arthritis would be advantageous. A new assay to quantify the major equine acute phase protein, serum amyloid A (SAA) may fulfill this need. Serum amyloid A increases in the body in response to injury, infection, and inflammation and shows promise as a useful tool in confirming a diagnosis of sepsis, as inflammation causes mild increases in SAA and infection causes marked elevations. In our study, serial serum and synovial fluid samples were collected from horses with experimental models of synovitis and septic arthritis, synovial fluid cytology was performed, and serum and synovial fluid SAA were quantified. Synovial fluid TNCC and TP concentrations increased significantly following induction of both models. Serum and synovial fluid SAA concentrations remained normal in synovitis horses and increased significantly in septic arthritis horses. Any elevation in serum or synovial fluid SAA above normal values may be supportive of synovial sepsis since synovial inflammation alone did not result in SAA elevations in our model. / Master of Science

The high cell density fermentation of recombinant Pichia pastoris for human serum albumin (HSA) production is a high oxygen demand process. The oxygen demand is usually met by increased agitation rate and use of oxygen-enriched air. Microbubble fermentation however can supply adequate oxygen to the microorganisms at relatively low agitation rates because of improved mass transfer of the microbubbles used for the sparging. Conventionally sparged fermentations were conducted for the production of HSA using P. pastoris at agitation rates of 350, 500, and 750 rpm, and were compared to MBD sparged fermentation at 150, 350, and 500 rpm agitation rates. The MBD improved the volumetric oxygen transfer coefficient (kLa) and subsequently increased the cell mass and protein production compared to conventional fermentation. Cell production in MBD fermentation at 350 rpm was 4.6 times higher than that in conventional fermentation at 350 rpm, but similar to that in the conventional 750 rpm. Maximum cell mass productivity in the conventional 350 rpm was only 0.37 g / (L·h), while the maximum value in MBD 350 rpm was 2.0 g / (L·h), which was similar to 2.2 g / (L·h) in the conventional 750 rpm. Biomass yield on glycerol Ys (g cell/ g glycerol) was 0.334 g / g in the conventional 350 rpm, 0.431 g / g in MBD 350 rpm and 0.438 g / g in the conventional 750 rpm. Protein production in MBD 350 rpm was 7.3 times higher than that in the conventional 350 rpm, but similar to the conventional 750 rpm. Maximum protein productivity in the conventional 350 rpm was 0.37 mg / (L·h), 2.8 mg / (L·h) in MBD 350 rpm, and 3.3 mg / (L·h) in the conventional 750 rpm. Protein yield on methanol Yp (mg protein / g methanol) was 1.57 mg /g in the conventional 350 rpm, 5.02 in MBD 350 rpm, and 5.21 in the conventional 750 rpm. The volumetric oxygen transfer coefficient kLa was 1011.9 h-1 in MBD 350 rpm, which was 6.1 times higher than that in the conventional 350 rpm (164.9 h-1) but was similar to the conventional 750 rpm (1098 h-1). Therefore, MBD fermentation results at low agitation of 350 rpm were similar to those in the conventional fermentation at high agitation of 750 rpm. There was considerable improvement in oxygen transfer to the microorganism using MBD sparging relative to the conventional sparging. Conventional fermentations were conducted both in a Biostat Q fermenter (small) at 500 rpm, 750 rpm, and 1000 rpm, and in a Bioflo III fermenter (large) at 350 rpm, 500 rpm, and 750 rpm. At the same agitation rate of 500 rpm, cell production in the large reactor was 3.8 times higher than that in the small one, and no detectable protein was produced in the small reactor at 500 rpm. At the same agitation rate of 750 rpm, both cell production and protein production in the large reactor were 4.6 times higher than the small reactor. Thus, the Bioflo III fermenter showed higher oxygen transfer efficiency than the Biostat Q fermenter, because of the more efficient aeration design of the Bioflo III fermenter. / Master of Science

Global food production management has become a challenge with an anticipated population of 10 billion people by 2050 and the ongoing COVID-19 epidemic. Seafood is a vital food source due to its widespread consumption, excellent nutrient profile, and low feed conversion ratio, rendering its sustainable production quintessential. Cellular agriculture or cultured meat can increase seafood production; however, the conventional use of Fetal Bovine Serum (FBS) in culture media restricts its utilization at an industrial level. FBS is effective but has many limitations: unethical animal extraction, high demand and low supply, poorly defined ingredients, variable performance, and high cost that impedes the feasibility and commercial viability of cellular agriculture. Thus, employing serum-free media becomes a quintessential need for cellular agriculture. This project aims to replace or reduce the typical 10% serum usage in Zebrafish embryonic stem cell (ESC) production media with protein hydrolysates derived from low-cost natural sources with high protein content. Enzymatic hydrolysis was performed on nine sources: insects (black army fly and cricket), plants (pea), fungi (mushroom and yeast), algae, and marine invertebrates (oyster, mussel, and lugworm). The resulting hydrolysates were evaluated for serum replacement in zebrafish ESCs. All hydrolysates were used at five different concentrations (10, 1 0.1, 0.01 and 0.001 mg/mL) in serum concentrations of 10%, 5%, and 0% with four biological replicates. The best hydrolysate sources and concentrations were selected for further testing at 2.5% and 1% serum concentrations. All hydrolysates, except for cricket, could restore or significantly increase cell growth with 50% less serum at a concentration of 0.1-0.001mg/mL. Protein hydrolysate concentration of 10 and 1mg/mL was toxic for cells. Additionally, the eight hydrolysates could reduce serum concentrations up to 75–90%. However, no protein hydrolysate could completely replace serum, as cells using only protein hydrolysates exhibited morphological aberrations and decreased growth. Replacing serum with protein hydrolysates lowers cellular agriculture's overall cost, thus enabling the commercialization of cultured meat and the development of a sustainable food system. In the future, blending various protein hydrolysate sources with or without the addition of conventional growth factors could be done to create the ideal serum-free media. / Doctor of Philosophy / With a predicted population of 10 billion by 2050 and the ongoing COVID-19 outbreak, the management of global food production has become a dilemma. However, due to its widespread consumption and good nutrient profile, seafood is an essential food supply, making its sustainable production indispensable. Both capture fisheries and aquaculture are conventional ways to produce seafood. However, they are under tremendous pressure and require alternatives that can alleviate this demand and contribute to the sustainable growth of seafood. In-vitro cultured meat, also known as lab-grown meat, is a novel technique with the potential to supplement the traditional fish sector. It appears a great option, as it completely imitates meat and offers numerous environmental, financial, and health advantages. A culture medium supports the existence, survival, growth, and multiplication of meat-producing cells and tissues in cell-based meat. However, the culture medium uses a Fetal Bovine Serum (FBS) supplement, which dramatically increases the cost and raises many ethical concerns as it is derived from a cow's fetus. In this thesis, we substitute FBS with protein hydrolysates derived from nine distinct sources. Hydrolysing proteins with enzymes produce protein hydrolysates, rich in nutrients and peptides that promote cell development. Enzymes were used to hydrolyse nine unique and protein-rich sources, including insects (black army fly and cricket), plants (pea), fungi (mushroom and yeast), algae, and marine creatures (oyster, mussel, and lugworm). The resultant hydrolysates were investigated for replacement of serum in cell culture. Eight protein hydrolysates successfully replaced 90% of serum without impairing cell growth and structure but could not replace serum entirely. In the future, serum-free media could be created by combining these various protein hydrolysates with or without adding other growth-promoting components.

Human Protein C (hPC) is a vitamin K-dependent serine protease that has a critical role in the naturally-occurring anticoagulant pathway. Upon activation of the zymogen by thrombin at the endothelial cell surface, the active form of hPC has anticoagulant activity in hemostasis due to its ability to inactivate factors Va and VIIIa. For biological activity, hPC requires several post-translational modifications including proteolytic cleavage, disulfide bond formation, b-hydroxylation, g-carboxylation, and N-linked glycosylation. Plants have the eukaryotic protein modifying mechanisms required for many human proteins and may provide a safe, cost-effective system for producing hPC on a large-scale basis. Tobacco (Nicotiana tabacum L.) is particularly well suited for use as a bioreactor for high-value recombinant proteins. Tobacco is one of the easiest plants to transform, it is an excellent biomass producer and can produce up to a million seeds from a single genetically engineered plant. Previous attempts to produce hPC in tobacco were limited by expression levels. The overall goal of the research was to develop transgenic plants that express hPC at higher levels. A cDNA encoding hPC was fused to an enhanced constitutive 35S promoter (35SDE ) and introduced into a plant transformation vector. The hPC construct was introduced into tobacco leaf disks using Agrobacterium tumefaciens-mediated transformation, and 30 transgenic plants were generated. Stable integration of the hPC gene construct into the tobacco genome and transgene copy number were determined by genomic Southern hybridization and segregation analyses. The majority of transgenic plants expressed the hPC transgene based on RNA analyses by northern hybridization. Plants utilizing the enhanced 35S promoter had equivalent levels of expression to previously generated hPC-containing plants. A variety of polyclonal and monoclonal antibodies raised against hPC were tested for detection of hPC standards and tobacco-synthesized hPC by western immunoblotting. Novel proteins in the size range of hPC heavy chain cross-reacted with anti-heavy chain hPC antibodies in 35SDE:hPC plants. Thus, plants may be capable of synthesizing hPC and proteolytically processing it to light and heavy chains. Although further experiments will be required to confirm the identity of these putative hPC proteins in tobacco, these result suggest that analyses of hPC expressed in plants have been limited by effective tools for detecting the hPC gene product rather than expression levels determined by the transgene promoter. / Master of Science

The objective of this trial was to evaluate the effect of a multi-strain probiotic on serum concentrations of selected metabolites in young calves. Holstein heifers (n = 112) from a commercial dairy were enrolled within 48 h of birth and placed into individual hutches. Calves were weaned at 60 d of age and remained in their hutches until 90 d of age, when they were moved to group housing. Calves were placed into 2 treatment groups: 1) control and 2) B. subtilis, B. lichenformis, L. animalis, and P. freudenreichii probiotic (Bovamine Dairy Plus, Chr. Hansen, Milwaukee, WI). Control calves received 0.5 g of lactose in milk once daily until weaning and 0.75 g in grain thereafter. Treatment calves received 0.5 g (1.1 × 10^10 cfu/g) probiotic in milk once daily until weaning and 0.75 g (1.65 × 1010 cfu/g) probiotic in grain thereafter. Jugular blood samples (6 mL) were collected at 14, 25, 45, 63, 77, 120, and 180 d of age for the determination of serum β hydroxy-butyrate (BHB), plasma glucose, non-esterified fatty acid (NEFA), total free amino acids (TFAA), blood urea nitrogen (BUN), and tumor necrosis factor-α (TNF). Data were analyzed as a mixed linear model to determine the fixed effects of treatment, day, and their interaction and also the random effect of calf. Single degree of freedom contrasts were used to evaluate the effect of phase (pre- vs. post-weaning) and the interaction of treatment with phase. Concentrations of BHB increased throughout the 180 d of the study and were greater (P < 0.001) in the post-weaning (661 μM) vs. pre- weaning (240 μM) phase and greater for the probiotic-treated calves in the post-weaning phase only (treatment × phase, P < 0.001). Glucose concentrations were greater (P < 0.001) for the pre-weaning (88.0 mg/dL) vs. post-weaning (81.1 mg/dL) phase and greater for calves receiving probiotics in the pre-weaning phase only (Treatment × phase, P < 0.01). Non- esterified fatty acid (NEFA) concentrations were greater (P < 0.001) in the pre-weaning vs. post-weaning phase and were greater in calves not receiving probiotics (treatment × phase, P = 0.0036).Serum TFAA concentrations were greater (P < 0.001) in the pre-weaning (3.37 mM) vs. post-weaning (3.03 mM) phase and greater for probiotic-treated vs. control calves (3.22 mM vs. 3.11 mM; P < 0.001). Concentrations of BUN were greater (P ≤ 0.001) in the post-weaning vs. preweaning phase and were higher for probiotic treated calves vs. control in the post-weaning phase (P < 0.001). Tumor necrosis factor-α (TNF) concentrations were affected by treatment (P< .001). Calves that received probiotic supplementation had lower TNF concentrations on day 63 of life when compared to control calves on day 63 of life. Data suggest that nutrient metabolism and ruminal development were enhanced by feeding a multi-strain probiotic.

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Syftet med projektet var att utvärdera hållbarheten av analyterna follikelstimulerande hormon (FSH), luteiniserande hormon (LH), progesteron (PRGE) och sexualhormonbindande globulin (SHBG) i primärrör förvarade i kyl, i serum såväl som plasma, på ADVIA Centaur XPT (Siemens Healthineers). Samtliga analyter analyserades i serum respektive plasma vid tidpunkterna 0, 24, 48, 72, 96 samt 168 h efter insamling. Analyspåverkan av förvaringstid i kyl och korrelationen mellan analyser i serum och plasma utvärderades. FSH, LH och SHBG uppvisade acceptabla medelbias vid samtliga tidpunkter i båda matriserna. PRGE uppvisade låg medelbias, &lt;15 %, vid samtliga tidpunkter i serum men uppvisade medelbias &gt;15 % i plasma. Analys i plasma skiljde sig minimalt från serum för FSH, LH och SHBG. För PRGE var skillnaderna lite större. Utifrån resultatet bedömdes FSH, LH och SHBG vara hållbara i en vecka i kyl (168 h) i både serum och plasma. PRGE bedömdes vara hållbart i kyl i en vecka i serum men inte godtagbar i plasma. Laboratoriets riktlinjer kan utvidgas för FSH, LH och SHBG att också genomföras i plasma. PRGE fortsatt bör genomföras i serum. / The purpose of the project was to evaluate the stability of the analytes follicle-stimulating hormone (FSH), luteinizing hormone (LH), progesterone (PRGE) and sex hormone-binding globulin (SHBG) in primary collection tubes stored refrigerated, in serum and plasma, on ADVIA Centaur XPT (Siemens Healthineers). All analytes were analyzed in serum and plasma at 0, 24, 48, 72, 96 and 168 h after collection. The impact of storage time on the analysis results and the correlation between analyzes in serum and plasma were evaluated. FSH, LH and SHBG obtained a mean bias &lt;15 %, the threshold, at every moment in both matrices. PRGE showed a mean bias &lt;15 % at every moment in serum but showed a mean bias &gt;15 % in plasma. Analysis in plasma differed minimally from serum for FSH, LH and SHBG. For PRGE, the differences were slightly larger. This study considered FSH, LH and SHBG to be stable for one week (168 h) in both serum and plasma stored refrigerated. PRGE was considered to be stable for one week in serum stored refrigerated, and not acceptable in plasma. Analyzes of FSH, LH and SHBG can be carried out in plasma, while analyzes of PRGE should continue to be performed in serum.

Background: Mesenchymal stromal cells (MSCs) are multipotent cells that can differentiate into various cell types and possess immunomodulatory and anti-inflammatory effects, making them interesting candidates for therapeutic applications. MSCs are present in small quantities in tissues like bone marrow and therefore need to be expanded while preserving their essential characteristics. They should adhere to plastic, differentiate into osteocytes, adipocytes and chondrocytes and express specific cell surface markers. Currently, the “golden standard” culture media supplement is fetal bovine serum (FBS). However, there is a potential contamination risk of MSCs by xenogeneic and zoonotic infectious agents, which can trigger an immune response. As an alternative, xeno-free serum supplements derived from human sources, e.g., human serum (HS) can be used.  Aim: This study aimed to identify and characterize human bone marrow derived MSCs and examine the effects different supplements have on the cells.  Methods: MSCs were cultured in 10% FBS, 2% FBS and 10% HS for 20 -21 days. Differentiation was induced and the potential was detected with immunocytochemistry. Cell surface markers CD73, CD90, CD105 and CD45 were identified with flow cytometry.  Results and Conclusion: There was no significant difference in morphology, differential potential or immunophenotype between the different serum conditions. However, HS-supplemented culture media resulted in a significantly higher number of cells with 1 x 107 cells after 20 days without affecting their differentiation potential and immunophenotype in comparison to 10% FBS with 2.2 x 106 cells (p=0.0004). MSCs cultured in 2% FBS resulted in the least number of cells (9.9 x 105) after 21 days of expansion.

No description available.

No description available.