The structure, functionality, isolation methods and applications of whey components, particularly the proteins and lactose, have been extensively studied. These studies have had a great impact on the food industry where whey components are increasingly being used as food ingredients. Two generations of whey protein product, namely Lactalbumin, produced by heat-induced precipitation, and Whey Protein Concentration/ Isloate, produced by ultrafiltration/ ion exchange chromatography, have been commercialised. Crystalline lactose in the food and pharmaceutical grades is also being produced. Recently, research activities in whey fractionation have shifted to the isolation of the minor components. This thesis is aimed at developing a Total Whey Utilization strategy by which the several components of the whey stream would be completely recovered by fractionation, resulting in little or no residue to be disposed of in the wastewater stream. Therefore, this study was initially dedicated to the development of novel separation methods which would be suitable for the Total Whey Utilization process. The development of those techniques revealed some previously unknown feature of whey components. The mechanisms of the separation methods have been also investigated. Although crystallization is an efficient method for fractionation or purification, its disadvantage is that the mother liquor is a wastewater containing high salt and BOD (Biological Oxygen Demand). The chromatographic method has been investigated in this work to separate the mother liquor or permeate into lactose and mineral fractions such that a goal of this thesis, namely a 'clean' water stream after processing whey, can be finally achieved. These studies have focused on the effect of resin type, salt form of the resin and the operating conditions on the separation of the lactose and mineral fraction. / Doctor of Philosophy (PhD)

Master of Science in Biomedical Sciences / Department of Anatomy and Physiology / Mark L. Weiss / Mesenchymal stromal cells (MSCs) derived from the umbilical cord (UC-MSCs) have therapeutic applications and are studied to understand their potential uses and immunomodulatory properties. Research must identify good manufacturing process (GMP) compliant methods to isolate and expand UC-MSCs. In addition, MSCs metabolism characteristics in culture are unknown, warranting further investigation. Viability of MSCs decreases after cryopreservation, which is detrimental to clinical translation. Previously published methods used to isolate MSCs from the umbilical cord included open dissection steps and xenogeneic components. Here, I developed improved methods by eliminating dissection which reduces contamination risks. Instead, I used the whole umbilical cord and Miltenyi dissociator tubes to mechanically and enzymatically dissociate cells in a closed system. Xenogeneic components were decreased by using medium containing pooled human platelet lysate instead of fetal bovine serum. The cell numbers isolated from umbilical cord averaged 2.68 x 10⁵ per cm, which represents greater than 20 fold improvement over the previous method. Moreover, expansion cell numbers were increased using 10% pooled human platelet lysate supplemented media. The UC-MSCs generated here met the International Society of Cell Therapy (ISCT) definition of MSCs. Metabolism characteristics of MSCs indicated that glucose was the critical metabolite, maintaining cells longer in culture than glutamine. Cell death followed depletion of glucose, too. Finally, the average viability after thawing cryopreserved MSCs was more than 95%, higher than previous methods. The improvements I introduced to our methodology could speed clinical translation of MSCs as an allogeneic cellular therapy

Exosomes are extracellular vesicles of endosomal origin. It was thought, that exosomes are used by cells only as carriers for cellular waste, but it was found out, that exosomes serve in the cellular communication and have a role in viral infections. Exosomes are exploited by viruses for example for the transport of viral protein or viral RNA/DNA. One of the viruses, where the mechanism of exploitation is unknown (if any exists) is murine polyomavirus. Murine polyomavirus belongs to the family Polyomaviridae, to which other human viruses belong for example, JC virus or virus of Merkel cell carcinoma. Murine polyomavirus codes for small, large and middle T antigen and three capsid proteins. Middle T antigen is known to bind to cellular membranes. Exosomes are membrane derived structures, so we investigated a possible transfer of middle T antigen. To this goal the successful isolation of exosomes and their characterization was necessary. Exosomes were isolated by ultracentrifugation and further purified by the density gradient OptiPrep. Exosomes were characterized by electron microscopy, NanoSight and by protein exosomal markers. These markers are for example Alix and flotillin-1. The cells were transfected in order to produce middle T antigen. It was shown, that exosomes isolated from these cells...

A two-step protocol has been devised as a rapid and selective alternative to conventional differential extraction techniques with an increased recovery of DNA. The protocol involves pressure cycling with the Barocycler® NEP 2320 from Pressure Biosciences. Inc. in alkaline conditions for epithelial cell lysis and removal. This step is followed by alkaline lysis at 95º C for extraction of sperm cell DNA. At 1:1 or 2:1 female to male cell ratios, high selectivity and complete separation can be achieved. But at higher ratios, male allelic dropout is observed. This protocol has been modified to generate a clean male profile at a 20:1 cell ratio through optimization of NaOH concentration and inclusion of an additional pressure cycling step. Validation studies have been performed to assess the efficiency of this method under various conditions. An additional immunomagnetic cell capture pretreatment allowed for nearly complete separation at cell ratios of up to 200:1.