I characterize the behavior of microcantilever arrays which utilize the in-plane photonic transduction that I've previously developed and evaluate the performance of the microcantilever arrays in simple sensing scenarios with integrated microfluidics. First the thermal responses of microcantilevers with a variety of patterns of deposited gold films are compared. Using a scanning electron microscope, I observe the deflection thermal sensitivities of 300 µm long microcantilevers to be -170.82 nm/K for a full gold coating and -1.93 nm/K for no gold coating. Using the photonic transduction method I measure a thermal sensitivity of -1.46 nm/K for a microcantilever array with no gold. A microcantilever array integrated with microfluidics is exposed to a solution of bovine serum albumin (BSA) followed by solutions of various pH's. In all cases I observe a previously unreported transient deflection response. We find that the transient response is due to temporary nonuniform concentration distributions. In response to nonspecific binding of BSA, I observe a transient surface stress of -0.23 mN/m that agrees well with the -0.225 mN/m predicted by simulations. We hypothesize that the deflection response to pH changes is due to stress generated by conformational changes of bound BSA.The deflection response of an integrated microcantilever array to different types of flow and different flow rates is observed. Simulations of the deflection response match well with experimental results but disagree at higher flow rates. For flow rates greater than 200 µL/min, the limitation of the differential signal's dynamic range becomes apparent. We then investigate flow driven by an on-chip reciprocating reservoir pump. We demonstrate that it is possible to use the reciprocating pump to achieve high flow rates while making deflection measurements in-between reservoir actuations. Investigations of the microcantilever array noise show that flicker noise dominates below 10 Hz, while above 10 Hz, readout noise dominates. A minimum deflection noise density of 15 pW/√Hz is achieved. To improve the signal-to-noise ratio I develop algorithms for a digital lock-in amplifier with a digital phase-lock loop. In simulation the lock-in amplifier is able to improve the SNR by up to a factor of 6000, and self-lock to a noisy carrier signal without an external reference signal.

Discovery of Novel Serum Biomarkers for Diagnosing and Staging Alzheimer’s DiseaseDipti Jigar ShahDepartment of Chemistry and Biochemistry, BYUDoctor of PhilosophyAlzheimer’s disease (AD) is an untreatable neurologic disease affecting more than 5 million Americans, most over 60 years of age. Protein plaques and neurofibrillary tangles typify AD brain pathology and are thought to cause the progressive dementia and brain shrinkage observed in AD. Currently there are no methods to diagnose the disease at a time before damage becomes irreversible.Biochemical tests for AD using cerebrospinal fluid analysis or neuroimaging are not yet sufficiently sensitive and specific, and they are invasive. This points to a need for a more easily applied and more sensitive diagnostic test. Although the gross anatomical changes are localized to the brain, AD is likely to involve changes throughout the body. As a result of this, changes in the abundance of certain biomolecules present in the circulation system are likely to occur. Consequently, a serum proteomics approach able to measure such changes, when applied to AD, would likely find quantitative changes in relevant molecules that can help diagnose the disease correctly, ideally early in the disease process. The goal of this work was to discover and validate novel diagnostic serum biomarkers for AD. For biomarker discovery and validation, we used a novel serum proteomics approach involving reversed phase capillary-liquid chromatography-electrospray ionization-quadrupole-time of flight mass spectrometry. Our samples were protein depleted, which helped us survey low molecular weight species in the serum without ion suppression from larger proteins like albumin. We were able to observe more than 8000 molecular species in a single run. The overall project was comprised of four studies: (i) discovery of novel potential serum AD markers, (ii) blinded validation of diagnostically promising biomarkers found in the initial study, with their further chemical identification, (iii) exploring gender-based serum AD biomarkers, and (v) discovery of biomarkers that distinguish early versus moderate stage AD. In the first study, the approach found 38 significant (p < 0.05) biomarkers and 21 near significant (p = 0.05 to 0.099) biomarkers. On using the forward selection approach, we built multi-marker panels with specificities and sensitivities higher than 80%.The second study reports on a blinded validation study that was performed on a new set of serum samples. We focused on the 13 most promising AD biomarkers found as part of the initial study. We successfully validated 4 of these biomarkers that showed highly significant statistical p-values. As part of this study, research was conducted to identify these 4 biomarkers, which was accomplished using tandem mass spectrometry with fragmentation experiments. The third study used data from the initial study but looked at gender specific biomarkers. We found 31 significant and near significant serum AD biomarkers for women, 16 for men, and 25 that were gender independent. Multi-marker panels of AD biomarkers for women or men had sensitivities of >60% and specificities >85%.In the fourth study, cases with moderate AD were compared to cases with very mild or mild AD to find novel biomarkers that could be used for staging. We found 44 significant and near significant biomarkers that were quantitatively different between mild and severe AD. In conclusion, we were successful in accomplishing the goal of this work of finding, validating and identifying novel serum biomarkers that diagnose AD.

<p> The distribution of manganese in blood serum and erythrocytes has been investigated using a combination of radioactive tracer method with both gel chromatography and disc gel electrophoresis. </p> <p> In serum, there are two manganese-binding proteins. The first is a(beta)1 globulin with a molecular weight of 70,000. This forms a relatively labile manganese complex both in vitro and in vivo, and is remarkably similar in both its chromatographic and electrophoretic behaviour to the iron-binding protein, transferrin. The second protein is a higher molecular weight (beta) globulin. It is found to incorporate radiomanganese in vivo only, thereupon forming a very stable entity. </p> <p> In erythrocytes, manganese occurs predominantly in a porphyrin bound to apoglobin, giving rise to a species similar to hemoglobin . </p> / Thesis / Doctor of Philosophy (PhD)

Les déterminants de l’exposition aux substances per- et polyfluoroalkyliques (PFAS) chez les enfants sont mal connus. Cette étude visait à analyser les concentrations sériques de 9 PFAS chez 204 enfants participant à l’étude MIREC-Endo ; évaluer les associations entre ces concentrations et celles dans le sérum maternel (grossesse) et le lait maternel ; évaluer les déterminants des concentrations. Nous avons effectué des statistiques descriptives des concentrations et évalué leurs associations à l’aide de corrélations de Pearson et de tests de comparaisons de moyennes. Des analyses de régression ont été faites pour quantifier l’influence de l’allaitement sur les concentrations sériques. Les moyennes géométriques de PFOS, PFOA, PFHxS, PFNA et PFDA (détectés dans >67% des échantillons) étaient de 1,37 ; 1,21 ; 0,55 ; 0,42 et 0,13 µg/L, respectivement. Les concentrations sériques de certains PFAS étaient corrélées avec les concentrations sériques maternelles (r=0,315 [PFOS], 0,314 [PFOA], 0,328 [PFHxS]) et les concentrations dans le lait (r=0,273 [PFOA], 0,509 [PFHxS], 0,237 [PFNA]). Les concentrations sériques de certains PFAS chez les enfants étaient négativement associées avec l’âge maternel à l’accouchement (PFOS, PFHxS, PFDA), le tabagisme durant la grossesse (PFNA, PFDA), l’emploi de la mère (PFOS), alors qu’elles étaient positivement associées avec le niveau d’éducation maternel (PFNA) et le revenu familial (PFOA). Nous avons observé une augmentation de 2,8% (PFOS) et 1,85% (PFOA) dans les concentrations sériques par mois d’allaitement exclusif. En conclusion, les concentrations sériques de certains PFAS chez les enfants étaient associées aux expositions périnatales, à l’âge de la mère, au revenu familial et à la durée d’allaitement. / The determinants of exposure to per- and polyfluoroalkyl substances (PFAS) in children are unclear. The objectives of this study were to analyze serum concentrations of 9 PFAS in 204 children aged 7.5 to 11.8 years participating in the MIREC-Endo study, to assess associations between these concentrations and those in maternal serum (during pregnancy) and breast milk; and to evaluate the determinants of these concentrations. We performed descriptive statistics of the concentrations and assessed their associations using Pearson correlations and comparison tests. Regression analyses were performed to quantify the influence of breastfeeding on serum concentrations. The geometric means of PFOS, PFOA, PFHxS, PFNA, and PFDA (detected in >67% of samples) were 1.37, 1.21, 0.55, 0.42, and 0.13 µg/L, respectively. Serum concentrations of selected PFASs were correlated with maternal serum concentrations (r=0.315 [PFOS], 0.314 [PFOA], 0.328 [PFHxS]) and milk concentrations (r=0.273 [PFOA], 0.509 [PFHxS], 0.237 [PFNA]). Serum concentrations of children PFAS were negatively associated with maternal age at delivery (PFOS, PFHxS, PFDA), smoking during pregnancy (PFNA, PFDA), maternal employment (PFOS), whereas they were positively associated with maternal education level (PFNA) and family income (PFOA). We observed an increase of 2.8% (PFOS) and 1.85% (PFOA) in serum concentrations per month of exclusive breastfeeding. In conclusion, serum concentrations of selected PFAS in children were associated with perinatal exposures, maternal age, family income, and duration of breastfeeding.

Natural Killer (NK) cells can recognize and lyse a large variety of tumor cells and have been of interest as a potential cancer treatment option. Our group has developed a particle-based NK cell expansion method that utilizes plasma membrane particles (PM-particles) derived from K562 cells genetically engineered to express membrane bound IL21 and 41BBL(K562-mbIL21-41BBL), two proteins that stimulate growth and activity of NK cells. This method selectively expands highly cytotoxic NK cells > 400-fold in 14 days of culture. Currently NK cells are expanded in vitro using Fetal Bovine Serum (FBS) as a serum-supplement to promote cell growth. While effective, the use of animal products is not preferred in cell cultures grown for clinical purposes. This project tested Human Platelet Lysates (HPL) as a potential replacement for FBS in NK cell culture. NK cells were expanded using PM21-particle based expansion method with either FBS or HPL as supplements. Their growth characteristics, phenotype and functionality were assessed and compared. Results of this study determined that HPL is a viable option to replace FBS in NK cell culture for clinical applications, as there was no significant difference between the two serum supplements.

Translation of multipotent mesenchymal stromal cell (MSC)-based therapies is advancing in human and veterinary medicine. One critical issue is the in vitro culture of MSC before clinical use. Using fetal bovine serum (FBS) as supplement to the basal medium is still the gold standard for cultivation of many cell types including equine MSC. Alternatives are being explored, with substantial success using platelet lysate-supplemented media for human MSC. However, progress lags behind in the veterinary field. The aim of this study was to establish a scalable protocol for equine platelet lysate (ePL) production and to test the ePL in equine MSC culture. Whole blood was harvested into blood collection bags from 20 healthy horses. After checking sample materials for pathogen contamination, samples from 19 animals were included. Platelet concentrates were prepared using a buffy coat method. Platelets, platelet-derived growth factor BB, and transforming growth factor b1 concentrations were increased in the concentrates compared with whole blood or serum (p < 0.05), while white blood cells were reduced (p < 0.05). The concentrates were lysed using freeze/thaw cycles, which eliminated the cells while growth factor concentrations were maintained. Donor age negatively correlated with platelet and growth factor concentrations after processing (p < 0.05). Finally, all lysates were pooled and the ePL was evaluated as culture medium supplement in comparison with FBS, using adipose-derived MSC from four unrelated donor horses. MSC proliferated well in 10% FBS as well as in 10% ePL. However, using 5 or 2.5% ePL entailed highly inconsistent proliferation or loss of proliferation, with significant differences in generation times and confluencies (p < 0.05). MSC expressed the surface antigens CD90, CD44, and CD29, but CD73 and CD105 detection was low in all culture media. Adipogenic and osteogenic differentiation led to similar results in MSC from different culture media. The buffy coat method is useful to produce equine platelet concentrate with increased platelet and reduced white blood cell content in large scales. The ePL obtained supports MSC expansion similar as FBS when used at the same concentration (10%). Further investigations into equine MSC functionality in culture with ePL should follow.

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<p>Recent evidence suggests that vitamin D may be important for immune function. Canadian studies have reported varying prevalences of low levels of vitamin D. Whether these low vitamin D levels are associated with susceptibility to respiratory tract infections (RTIs) and infection severity remains unclear given the inconsistent association in recent studies. Influenza virus as a cause of RTI is of particular interest given its prevalence, morbidity and economic burden. Vaccination is a key strategy in prevention, but little is known about the effect of vitamin D on influenza vaccine response.</p> <p>A prospective cohort study of children 3 to 15 years old living in Hutterite communities in Alberta, Saskatchewan and Manitoba was conducted to assess the prevalence and predictors of low vitamin D levels and evaluate the association between vitamin D and the incidence and severity of laboratory proven respiratory tract infections. In those who received influenza vaccination, the relationship between vitamin D and influenza vaccine immunogenicity was examined.</p> <p>A total of 743 children were included in the study. The median serum 25-hydroxyvitamin D level (25[OH]D) was 62.0 nmol/L (interquartile range 51.0, 74.0). Levels lower than 50 nmol/L were present in 152 children (20.5%) and lower than 75 nmol/L in 565 children (76%). Lower serum 25(OH)D levels were associated with increased risk of RTI. No association was found between serum 25(OH)D level and disease severity. There was also no relationship found between serum 25(OH)D level and seroprotection or seroconversion from inactivated influenza vaccine.</p> <p>In conclusion, low serum 25(OH)D levels are a significant problem in Canadian Hutterite communities. Furthermore, low serum 25(OH)D levels were associated with increased risk of proven upper RTIs. Studies evaluating the role of vitamin D supplementation to reduce the burden of disease are warranted, and strategies to improve vitamin D status in rural communities in Canada are needed.</p> / Master of Science (MSc)