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During bio-technical processing it is important to monitorbiological parameters such as cell growth, viability andproduct formation. Many of the analyses traditionally used areslow to perform and provide only average data for thepopulation. Flow cytometry is a laser-based technique, whichmeasures physical properties of a cell in a flowing stream, ata rate of several thousand cells per second. It offers theprospect of an at-line, multi-parameter analysis of individualmicroorganisms in a population. In this project several methods for at-line measurements ofbioprocesses were developed such as protocol's for measuringcell concentration, viability and product formation. Theprimary focus was on prokaryotic organisms (E. coli) but eukaryotic organisms (P. pastoris) were included. The possibility to use volumetric cell counting to measurecell concentration (cell number) was evaluated. It was shownthat the method was applicable for high cell density processesof bothE. coliandP. pastoris. The combination of Bis- (1,3-dibutylbarbituric acid)trimethine oxonol (depolarised membranes) and propidium iodide(loss of membrane integrity) as fluorescent markers was usefulto measure viability at-line of cells in high cell densityprocesses. The protocol was shown to be reproducible forE. coliandP. pastoris. The viability staining was used to study the kinetics ofweak organic acids (food preservatives). The protocol provideddata about cell functions such as membrane depolarisation andloss of membrane integrity caused by introducing weak organicacids to shake flask cultures ofE. coli. Labeling inclusion bodies with fluorescent antibodiesprovided a method, which could specifically monitor theincreased accumulation of recombinant promegapoetin proteinwith process time. This technique was further developed forintracellular staining by application of a permeabilising stepbefore labeling with antibodies. Staining of inclusion bodiesdirectly inside permeabilised cells gave information about thedistribution of protein expression in the cell population. In conclusion, flow cytometry provides an at-line, singlecell technique for measurement of several biological parametersin bioprocesses. Key words: flow cytometry, Partec PAS, propidium iodide(PI), bis- (1,3-dibutylbarbituric acid) trimethine oxonol(BOX), Alexa fluor 488, bioprocess,E. coli,P. pastoris, inclusion body, food preservatives,viability, membrane potential

We have developed a method of calculating the average local absorbance (ALA) of a nucleus from the integrated nuclear absorbance and area. One can use the ALA, along with nuclear areas measured at different point absorbance thresholds, to determine whether a nucleus is stained too lightly or too darkly for accurate absorption measurements; this allows selection of an optimal light wavelength for the performance of these measurements. The ALA can also be used for automatic and instantaneous correction of integrated absorbance values from darkly stained cells. This allows the rapid measurement of the integrated absorbances of a large number of nuclei that are heterogeneous in stain intensity. Coefficients of variation of approximately 3% are obtained for the integrated absorbances of nuclei of nontransformed G0/G1 cells. This correction method can be applied with any image densitometer that generates both integrated absorbance and area values.

Circulating monocytes have a limited life span and will undergo apoptosis in the absence of specific stimuli. Recent studies have demonstrated that monocytes can be rescued from apoptosis via lipopolysaccharide (LPS) activation or stimulation with interleukin-1 or tumor necrosis factor-α. Based on previous studies from our laboratory, we hypothesized that, in nonseptic (e.g., autoimmune) inflammation, the presence of activated T cells may enhance monocyte longevity through T cell contact-dependent signaling. Plasma membranes prepared from 6 h activated (Tm(A)) and resting (Tm(R)) purified CD4+ T cells were added to resting elutriation-purified monocytes cultured in serum-free medium. Cells were assayed for degree of apoptosis occurring over a 72-h incubation using both agarose gel electrophoresis and flow cytometry. The addition of Tm(A) (but not Tm(R)) was capable of blocking monocyte apoptosis and the ability of Tm(A) to rescue monocytes was abrogated by the addition of anti-CD40L antibodies. Rescue of monocytes from apoptosis could also be mediated by direct cross-linking of monocyte CD40. Inhibitors of tyrosine kinase activity blocked both Tm(A) and anti-CD40-mediated rescue of monocytes from apoptosis, suggesting a primary role of a tyrosine kinase signaling pathway in the events controlling monocyte longevity.

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

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Field evaluation of frozen-thawed stallion semen has been limited to tests such as post-thaw motility and morphology that are not only subjective but also evaluate only a small population of cells. Flow cytometry has provided a quick, repeatable, objective method of evaluating a large number of cells, including spermatozoa. Two experiments were designed to first validate the use of several flow cytometric tests on frozen-thawed stallion semen and then determine a model that may best explain variation in fertility. Comparing samples that were live and freeze-killed validated the flow cytometric tests. In experiment one, six ejaculates were collected from each of three stallions. The semen from each ejaculate was centrifuged and frozen in 0.5 ml polyvinyl chloride straws. Two straws from each ejaculate were thawed and evaluated. Semen was evaluated for post-thaw motility, morphology, mitochondrial activity using Rhodamine 123 (R123), plasma membrane integrity using propidium iodide (PI) and ethidium monoazide (EMA), and chromatin structure using the sperm chromatin structure assay (SCSA). Data was recorded as percentages for all but the SCSA for both experiment one and two. The extent of chromatin denaturation was calculated using the SCSA and the alpha-t population [at = red/(red +green) fluorescence]. From the alpha-t population, statistics were calculated such mean (Xat), standard deviation (SDat), percentage of cells outside (COMPat) the main alpha-t population and the mean green fluorescence (mean green) of the population. Results from experiment one demonstrated that all flow cytometric tests except EMA were able to distinguish between live and freeze-killed samples (p < 0.0001). Also the stallion accounted for most of the variation in samples when compared to ejaculate and straw within an ejaculate. Therefore two straws could be chosen at random from a stallion and evaluated in experiment two. In experiment two, twenty-nine stallions were evaluated using the same tests as experiment one excluding EMA. Fertility data was obtained from the 1998 or 1999 breeding season. Multiple linear regression was used to determine the best-fit model to predict overall pregnancy rate. SCSA and R123-PI assays accounted for the largest amount of variation in fertility (R² = 0.65, p < 0.0004). Within SCSA and the R123/PI assays Xat and PI staining had the highest contribution to this variation in fertility (R² = 0.11, R² = 0.47) respectively. The best-fit model for predicting fertility included the assay combination listed above and the interactions between SDat and mean green staining as well as R123 and mean green staining. Post-thaw motility and morphology did not account for significant variation in fertility (p = 0.22, p = 0.46) respectively. Based on this project post-thaw motility and morphology are poor predictors of fertility in frozen-thawed stallion semen. However, through the addition of SCSA and R123-PI to the routine evaluation of frozen-thawed stallion semen time and money may be saved in advance by identifying those stallions with poor post-thaw fertility. / Master of Science

Due to the lead spike and its related health concern in the DC area, Point-of-Use (POU) filters were installed at public schools to reduce lead concentrations in water. However, the installation of POU filter could possibly lead to the growth of bacteria inside the filters, which could lead to health concerns. Therefore, the potential effects of POU filters on microbial growth was investigated. To explore the cause of filter effects on microbial growth, a sampling campaign was carried out between July and December 2017 from 25 outlets within 5 elementary schools in the DC area. The applicability of flow cytometry results as a quantification method was validated and then used to quantify the biological growth. Our results revealed that the installation of POU filters may lead to nitrification and an increase in microbial growth. Along with the increase in microbial growth, the microorganism community "fingerprints" based on flow cytometry data showed that the installation of filter could also shift the community distribution of bacteria based on their morphology. This study serves as a preliminary study to investigate the mechanics of microbial colonization on POU filters. / Master of Science

Introduction: Viruses, extracellular vesicles (EVs) and endogenous retroviruses (ERVs) are types of sub-micron particles which are known to be released from a vast range of cell types, across many species. There are many medically relevant sub-micron particles which can enter healthy cells and enable the intercellular delivery of functional host-derived and foreign products, through their enclosed lipid layers. While multiple particle subsets have been identified, many of the properties, behaviors and biochemical functions have not been fully described and have yet to be characterized. Materials and Methods: CD4⁺ naïve T-cells were isolated from female C57BL6/N mice and stimulated with varying concentrations of PMA/I. In addition to concentration, the length of PMA/I activation was assessed. Supernatants and cells were harvested, filtered, and stained to be subsequently analyzed by Nanoscale Flow Cytometry, Nanoparticle Tracking Analysis and Flow Cytometry. Particle populations were quantified and sorted by size, by NTA. Labelling dye CFSE was used in conjunction with fluorescently conjugated CD81 and CD9 antibodies to separate EVs, including exosomes, from background signal. Naïve T-cell purity, viability and levels of activation were assessed by flow cytometry using CD3, CD4 and CD62L antibodies and viability staining. Results: Increasing PMA concentration led to a global increase in particles by T-cells and a specific increase in smaller particle production and were demonstrated to be significant by Welch’s T-test, when compared to non-activated and DMSO controls (p<0.0001). In addition to concentration, activation length also correlated with increases in total particle counts and a specific increase in the secretion of smaller particles in comparison to non-activated and DMSO controls (p<0.0001). Labelling techniques by NFC revealed an increased presence of CFSE-CD81 positive and CFSE-CD9 positive particles secreted by T-cells, treated for 24 hours, compared to the 0- and 12-hour timepoints. Conclusion: This work demonstrates preliminary steps and outlines methods to begin assessing discrete particle populations and subsets secreted by murine naïve T-cells. Being able to identify patterns of particle secretions by naïve T-cells, especially under immune-stimulated conditions, may be the solution to uncovering the necessary information on EV physiology, that is required to understand the roles EVs play in pathology and how these conserved pathways may lead conditions to become exacerbated. This knowledge is essential to uncovering the roles EVs play in pathophysiology, and in the development of novel rapid diagnostic tests, to screen for cancers, infections, autoimmune disorders, and numerous other pathological conditions.

The Congenital Dyserythropoietic Anemias (CDA) are rare hereditary hemolytic disorders with large bi- to multi-nucleated erythroblasts in the bone marrow. Hemolysis is negative in a direct antiglobulin test (DAT). Based on morphology and clinical picture, three major forms of CDAs, type I, II, and III have been defined. CDA III, dominantly inherited, constitutes the rarest type with a majority of cases belonging to a family in Västerbotten, Sweden. The genetic background of CDA I and CDA II has been linked to mutations in CDAN1 and SEC23B respectively. The mutation of CDA III has been linked to 15q22 in earlier studies. In this project we have defined the causative genetic lesion in two families with CDA III. The novel mutation KIF23 c.2747C&gt;G (p.P916R) was shown to segregate with CDA III in the Swedish and American CDA III families and was absent in 356 healthy controls. KIF23 encodes mitotic kinesin-like protein 1 (MKLP1), which plays a central role in the last step of cytokinesis. RNAi-based knock-down and rescue experiments demonstrated that the p.P916R mutation causes cytokinesis failure in HeLa cells, resulting in increasing number of bi-nuclear cells, consistent with appearance of large multinucleated erythroblasts in CDA III patients. We conclude that CDA III is caused by a mutation in KIF23, encoding MKLP1, a conserved mitotic kinesin crucial for cytokinesis. Flow cytometry with eosin-5´-maleimide (EMA), anti-CD55 and anti-CD59 is commonly used when investigating non-autoimmune hemolytic anemias. Reduced fluorescence of EMA, typically detected in hereditary spherocytosis, is also seen in CDA II, while reduction of CD55 and CD59 characterizes paroxysmal nocturnal hemoglobinuria (PNH). We studied the flow cytometric profile of EMA, CD55, and CD59 on erythrocytes in CDA III. We found no abnormality of the erythrocyte membrane in CDA III and concluded that standard flow cytometry cannot be used to discriminate between CDA III and normal controls. In CDA I and CDA II a majority of patients, including those who are not transfusion dependent, suffer from iron overload, which, according to earlier studies, is not the case in CDA III. We found that individuals of the Västerbotten CDA III family carry mutations in the hemochromatosis (HFE) gene. Three CDA III patients with heterozygous or compound HFE mutations need treatment with phlebotomy due to iron overload. One of them carries heterozygous H63D mutation, which is not reported to lead to iron overload by itself in otherwise healthy individuals. We propose that molecular genetic testing of the HFE gene is indicated in all patients with CDA, including CDA III.