Aluminum is a neurotoxin and has been recognized as a causative agent for dialysis encephalopathy and renal osteodystrophy, as well as possibly being related to Alzheimer's disease. General public exposures to aluminum have increased in the modern, industrial age stimulating scientists to inquire into the degree of risk associated with such widespread use of aluminum. Aluminum is thought to be stored in bone, therefore, development of an in vivo method for the determination of aluminum a in human bone, suitable for routine monitoring of patients and population is the goal of this study. Using neutron activation analysis, low-energy neutrons are produced on the KN accelerator inducing the 27Al(n,y)28AI reaction in an irradiated site. Two different shapes (cylindrical and flat) of aluminum doped tissue equivalent phantoms, simulating both bone and soft tissue, have been built. Calibration lines, detection limits and doses delivered with the different shapes of phantom have been discussed, and compared to the previously published results. Two detection systems. an assembly of two large Nal{Tl) detectors and a hyperpure germani Jm detector, have been compared as well. The results achieved suggest that this technique may provide an alternative choice to painful bone biopsy for the in vivo monitoring of aluminum intoxication from long-term exposure. / Thesis / Master of Science (MS)

Thesis advisor: Jia Niu / In the last ten years, programmable CRISPR-Cas systems have been widely-used as genome editing tools for gene manipulation, epigenetic functionalization, and transcriptional regulation. Among them, fusing effector proteins directly to the Cas protein allows the resulting CRISPR machinery to direct these effector proteins to multiple sites of the same gene or multiple genes at once. Although they can be used to target multiple genetic loci simultaneously, these methods are often limited to applying one regulatory function (e.g., activation or repression) at a time. On the other hand, recruiting effector proteins via RNA aptamer-RNA-binding protein (RBP) recognition enabled multiplexed and multi-modular gene manipulations simultaneously. However, there are only a limited set of aptamer-RBP pairs that can function orthogonally and intracellularly, e.g., MS2 RNA aptamer with MS2 coat protein (MCP), and PP7 RNA aptamer with PP7 coat protein (PCP). The scarcity of orthogonal intracellular aptamer-RBP pairs imposes severe constraints on the CRISPR-mediated multifunctional manipulations of the genome and the epigenome. We established an intracellular selection platform for RNA aptamers, named CRISPR-Hybrid, and expanded the scope of aptamer-RBP toolkit for CRISPR transcription regulators. Using CRISPR-Hybrid, we successfully identified a highly active and specific aptamer for bacteriophage Qβ coat protein (QCP) in vivo, and characterized its binding affinity and specificity in vitro. We further validated the orthogonality of selected aptamer with QCP to other available intracellularly functional aptamer-RBP pairs including MS2-MCP and PP7-PCP in mammalian cells. Finally, we demonstrated the utility of this orthogonal pair in multiplexed and multi-modular regulations of endogenous genes. / Thesis (PhD) — Boston College, 2024. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Thesis (Ph.D.)--Boston University / PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / The hamster model of experimental chronic bronchitis comprises a persistent increase in the proportion of bronchial granulated secretory cells after a single intratracheal instillation of elastase. This granulated secretory cell increase, which does not occur in the trachea, has been termed secretory cell metaplasia (SCM). Susceptibility of the bronchial epithelium may be due to a large population of elastase-responsive cells specific to this region. Three dimensional reconstruction of the major form of bronchial secretory cells revealed very little or no rough endoplasmic reticulum (RER), thus demonstrating significant regional heterogeneity since all epithelial secretory cells in the trachea have abundant RER. Animals with bronchial SCM were stimulated with pilocarpine to determine whether the cells subsequent to discharge would re-accumulate granules, thus indicating a permanent phenotypic change. However, bronchial secretory cells failed to discharge at doses equal to and greater than those claimed to be effective in rats. Elastase instilled intratracheally was immuno-localized in the hamster airways to assess the possibility of regional differences in cellular uptake of the enzyme. Elastase was not seen intracellularly in trachea or bronchus suggesting that initiation of bronchial SCM results from a cell surface effect, possibly because of elastase-specific sites on bronchial but not tracheal cells. Tracheal resistance was tested by challenging the epithelial cells in vivo and in vitro with very high doses of elastase. Light and electron microscopy revealed no evidence of a stimulation of the mucus synthetic apparatus, suggesting that tracheal epithelial cells are inherently resistant to proteolytic up-regulation. / 2999-01-01

Marine mammals are known for their low susceptibility to mercury toxicity, and it was hypothesized that selenium may play a role in protection against mercury toxicity. To gain insight into the mechanisms of the low susceptibility of cetaceans, we investigated the in vitro effects (1) of mercuric chloride (HgCl₂) on the ultrastructure and cell death of Atlantic spotted dolphin renal cells (Sp1K cells), (2) of HgCl₂ on the cell proliferation and cell cycle status of Sp1K and Rhesus monkey renal cells (MK2), and (3) of sodium selenite (Na₂SeO₃) on cell proliferation and cell death of control and HgCl₂-treated Sp1K cells. HgCl₂ affected multiple organelles and nuclei in Sp1K cells, and induced apoptosis in a time-and dose-dependent manner. Both ultrastructural changes and induction of apoptosis were milder than seen in other cell types in previous publications. In addition, Sp1K cells were able to proliferate at 25 µM HgCl₂ while MK2 cells were killed at 15 µM HgCl₂. An increase in percentage of cells in the G0/G1 phase in the cell cycle and a decrease in S, and G2/M phase cells were seen in Sp1K cells exposed to more than 10 uM HgCl₂ more than 72 hours. MK2 cells showed cell cycle changes only at 24 hours exposure, and may be due to a sensitive subgroup. These data suggested that Sp1K cells were less susceptible than other cell types in a cell-specific way, which was independent of selenium protection. Concurrent exposure to Na₂SeO₃ provided protection against the HgCl₂-induced decrease in cell proliferation of Sp1K. The protective effects were greater if Na₂SeO₃ and HgCl₂ were premixed, but disappeared if exposures did not overlap. Although pretreatments with Na₂SeO₃ alone did not provide protection, they increased the protection of selenium administered later. Furthermore, Na₂SeO₃ decreased HgCl₂-induced apoptosis. These data demonstrated the Na₂SeO₃ protection against HgCl₂ toxicity in Sp1K cells in terms of cell proliferation and apoptosis. This study is the first report that reveals the existence of mercury-selenium antagonism in cultured cetacean cells. The data supported the hypothesis that selenium protection against mercury toxicity is, at least partially, through competition of binding sites and formation of mercury-selenium complex. / Master of Science

The purpose of this research was to further test a stable isotope based approach as a more reliable in vivo method to determine amino acid bioavailability from a variety of ingredients. The method was used to assess feather meal (FM), blood meal (BM), soybean meal (SBM), and a rumen protected amino acid (RPAA). An abomasal infusion of raw EAAs (isoleucine, leucine and methionine) and an abomasal infusion of sodium caseinate were used as control treatments to test the accuracy of the technique. The isotope-based results were then compared to in situ, in vitro and in vivo test results. The isotope-based technique provided AA bioavailability values for five non-essential AA and seven essential AA. The raw EAA infusion had the greatest AA recovery in plasma with an estimated absorbed RUP value of 93.4± 7.35% followed by the casein infusion (86.7 ± 4.81%), SBM (54.8 ± 5.19%), FM (52.7 ± 4.81%) and BM (47.5 ± 4.81%). The RPAA treatment had the lowest bioavailability at 9.9 ± 12.73%. Numerically, SBM supplied the most absorbable EAA of the 4 feed ingredients, but was not significantly different from that of BM and FM. Simply based on the control treatments in this research (raw EAA and casein), this isotope method was a more accurate method in determining AA bioavailability values with relatively low standard errors. Ingredients are exposed to all aspects of natural digestive processes and the method is able to determine AA appearance in the blood with no use of in situ or in vitro measurements. / Master of Science / Balancing rations for essential amino acids has beneficial effects on milk production and milk protein synthesis. However, to have predictable results, accurate knowledge of essential amino acid supply deriving from ingredient rumen undegradable protein and microbial crude protein flows is required. Methods used to assess essential amino acid supply include in vivo, in vitro and in situ methods; however these methods often generate conflicting results and have significant deficiencies that have hampered development of robust, accurate predictions of essential amino acid supply to the animal. This research tested a non-steady state, stable isotope based approach as a more reliable in vivo method to determine amino acid bioavailability for feed ingredients. Two control treatments (abomasally infused casein and raw essential amino acid) and four ingredients (feather meal, blood meal, soybean meal, and a rumen protected amino acid) were tested. Based on the control treatments, the method provided a reliable assessment of amino acid bioavailability values with relatively low standard errors. This method has the advantage of assessing essential amino acid bioavailabilities in a natural state where the ingredients of interest are components of a relatively normal diet exposed to all of the natural digestive processes. Thus values derived from this approach can be expected to be representative of most normal industry diets. With some further refinement, this method can help to create a library of true values for a variety of feed ingredients, leading to more accurately balanced diets and increased milk production. More accurate values of amino acid digestibility and rumen undegradable protein measurements for ingredients will also help to better determine their fair market value.

Thesis (Ph.D.)--Boston University / PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / The in vitro relationship of sialic acids to the A, B, M, N, C, D, and E antigens of the human erythrocyte has been studied by means of the Landsteiner hapten inhibition test with the idea that substances that strongly inhibit anti-D might find practical application in the prevention and treatment of erythroblastosis fetalis. Our results suggest that N-acetyl neuraminic acid (NANA) is a major constituent of the D (Rh0), M, and N agglutinogens, a minor constituent of the A antigen, but is probably not a functional portion of the B, C, and E antigenic structures. [TRUNCATED] / 2999-01-01

No / Purpose: The hollow fiber assay is used successfully as a routine in vivo screening model to quantitatively define anticancer activity by the National Cancer Institute. This study investigates whether the hollow fiber assay can be used as a short-term in vivo model to demonstrate specific pharmacodynamic end points, namely microtubule and cell cycle disruption. Experimental Design: The growth of A549 cells was characterized within hollow fibers over 5 days in vivo at both subcutaneous (s.c.) and intraperitoneal (i.p.) sites. Drugs were administered on day 4 (i.p.). Results: At 24 hours, cells were retrieved from fibers at both i.p. and s.c. sites of paclitaxel-treated (20 mg/kg) and combretastatin A1 phosphate¿treated (150 mg/kg) mice. Cell cycle analysis after paclitaxel treatment revealed a mean G2-M phase population of 48.04% (i.p.) and 25.76% (s.c.) compared with vehicle group mice (6.78 and 5.56%, respectively; P = <0.001 and 0.005, respectively). Tumor cells retrieved from combretastatin A1 phosphate¿treated mice had a mean G2-M phase population of 36.3% (i.p.) and 29.36% (s.c.) compared with cells retrieved from vehicle group mice (5.58 and 5.49%, respectively; P = <0.001). Using fluorescence and laser-confocal microscopy, paclitaxel was revealed to induce the formation of spindle asters and tubulin polymerization. Combretastatin A1 phosphate was shown to hold cells in mitosis. Changes in nuclear morphology were also observed. Conclusion: These data demonstrate that the hollow fiber assay can be used as a short-term in vivo model for studying the pharmacodynamic effects of both standard and novel compounds on microtubules. Evidence has also been provided to support the routine use of the in vivo hollow fiber assay for demonstrating the mechanism of action of a drug.

No / Background: In previous experiments, we showed that heparin oligosaccharides inhibit the angiogenic cytokine fibroblast growth factor-2. Here, we present the first in vivo study of size-fractionated heparin oligosaccharides in four models of angiogenesis that are progressively less dependent on fibroblast growth factor-2. Experimental Design: Heparin oligosaccharides were prepared using size-exclusion gel filtration chromatography and characterized through depolymerization and strong anion exchange high-performance liquid chromatography. Size-defined oligosaccharides (20 mg/kg/d) were given to mice bearing s.c. sponges that were injected with fibroblast growth factor-2 (100 ng/d). After 14 days, octasaccharides and decasaccharides reduced the microvessel density to levels below control. In a second experiment, HEC-FGF2 human endometrial cancer cells that overexpress fibroblast growth factor-2 were implanted in a hollow fiber placed s.c. in vivo. Oligosaccharides were given at 20 mg/kg/d for 2 weeks and the data again showed that octasaccharides significantly reduced microvessel density around the fiber (P = 0.03). In a more complex model, where angiogenesis was induced by a broad spectrum of growth factors, including vascular endothelial growth factor, we implanted H460 lung carcinoma cells in hollow fibers and treated the animals with oligosaccharides at 20 mg/kg/d over 3 weeks. Octasaccharides reduced the microvessel density to that of control. Preliminary investigation of 6-O-desulfated heparins showed that these also had antiangiogenic activity. Results: Finally, we examined the inhibitory potential of hexasaccharides and octasaccharides given at 20 mg/kg/d and these inhibited the growth of H460 lung carcinoma in vivo. At clinically attainable concentrations, significant anticoagulation (activated partial thromboplastin time, anti-factor Xa, and anti-factor IIa) was not observed in vitro unless species containing 16 saccharide residues were investigated. Conclusions: Thus, our preclinical data show that heparin octasaccharides represent novel antiangiogenic compounds that can be given without the anticoagulant effects of low molecular weight heparin.

Many medical therapies, such as thermal tumor detection and hypothermia cancer treatments, utilize heat transfer mechanisms of the body. The focus of this work is the development and experimental validation of a method to simultaneously estimate thermal conductivity, volumetric specific heat, and perfusion of in-vivo tissue. The heat transfer through the tissue was modeled using a modified Pennes' equation. Using a least-squares parameter estimation method with regularization, the thermal properties could be estimated from the temperature response to the known applied heat flux. The method was tested experimentally using a new agar-water tissue phantom designed for this purpose. A total of 40 tests were performed. The results of the experiments show that conductivity can be successfully estimated for perfused tissue phantoms. The values returned for volumetric specific heat are lower than expected, while the estimated values of perfusion are far greater than expected. It is believed that the mathematical model is incorrectly accounting between these two terms. Both terms were treated as heat sinks, so it is conceivable that it is not discriminating between them correctly. Although the method can estimate all three parameters simultaneously, but it seems that the mathematical model is not accurately describing the system. In the future, improvements to the model could be made to allow the method to function accurately. / Master of Science

Die Interleukin-6-Zytokinfamilie vermittelt pleiotrope physiologische und pathologische Wirkungen. Einerseits sind die Zytokine bei inflammatorischen Prozessen involviert, an-dererseits spielen sie auch eine Rolle in der Entwicklung und Differenzierung verschie-dener Zelltypen. Zusätzlich wirken einige von ihnen, vor allem das Interleukin-6 selbst, durch Induktion der Akute-Phase-Reaktion auf die Leber. Gemeinsames Kennzeichen der Familie der Interleukin-6-Zytokine ist die Wirkung über Rezeptorkomplexe, die als signaltranszudierende Untereinheit das Glykoprotein 130 enthalten. Ein Schwerpunkt dieser Arbeit liegt darauf, wie verschiedene toxische und inflammatorische Schädigun-gen von Zellen die Endozytose des Glykoproteins 130 beeinflussen. Es sind wichtige Bereiche in der Aminosäuresequenz des Proteins beschrieben, die eine zentrale Rolle in den Abläufen der Internalisierung via Clathrin-vermittelter Endozytose einnehmen. Re-sultate dieser Arbeit legen nahe, dass die Aktivierung der p38-Kinase durch schädigende Stimuli eine mögliche Reduzierung der Zelloberflächenexpression des Glykoproteins 130 induzieren kann. Durch Versuche mit gp130-mutanten Zellen kann zudem die es-sentielle Bedeutung eines im zytoplasmatischen Bereich von gp130 lokalisierten Dileu-cin-Motivs für die Zelloberflächenexpression weiter bestätigt werden. Auch der Einfluss des Zytoskeletts auf eine der Situation angepasste Endozytose wird durch den Einsatz verschiedener Zytoskelettinhibitoren nahegelegt. Der zweite Schwerpunkt dieser Arbeit ist die Untersuchung der Prozesse während der hepatischen Ischemia-Reperfusion Injury. Die hepatische Ischemia-Reperfusion Injury ist ein hochkomplexer Prozess aus dem Zusammenspielen sowohl physio- als auch pa-thologischer Reaktionen des Organismus. Im Rahmen dieses Prozesses ist eine leber-schützende Wirkung des Interleukin-6 über Vermittlung des JAK/STAT-Signalwegs und der Induktion von Akute-Phase-Proteinen in der Literatur vorbeschreiben. In Versuchen dieser Arbeit mit murinen Leberproben, kann gezeigt werden, dass durch die Ischämie und die anschließende Reperfusion eine gesteigerte RNA-Expression des Interleukin-6, des gp130s sowie der Akutphase-Proteine induziert wird. Jedoch ist dieser Anstieg auch für scheinoperierte Tiere nachweisbar, sodass die hier gezeigten Ergebnisse klar zeigen, dass für weiterführende Versuche zur IRI in der Zukunft spätere Zeitpunkte nach dem Abklingen der durch die OP-bedingten Akutphase gewählt werden müssen. / The interleukin-6 family cytokines mediate pleiotropic physiological and pathological effects. On the one hand, the cytokines are involved in inflammatory processes, on the other hand, they also play a role in the development and differentiation of various cell types. In addition, some of them, especially interleukin-6 itself, act on the liver by inducing the acute phase reaction. A main feature of these cytokines is their effect on the orga-nism via receptor complexes that contain glycoprotein 130 as a signal-transducing sub-unit. One focus of this work is on how various toxic and inflammatory damages of cells influence the endocytosis of glycoprotein 130. There are several important regions in the amino acid sequence of the protein described that are involved in the processes of inter-nalization via clathrin-mediated endocytosis. Results of this work suggest that activation of p38 kinase by damaging stimuli can induce a possible reduction in cell surface ex-pression of glycoprotein 130. In addition, experiments with gp130 mutant cells further confirm the importance of a dileucine motif localized in the cytoplasmic part of gp130 for the expression on the cell surface. The influence of the cytoskeleton on endocytosis adapted to the situation is also suggested by the use of various cytoskeletal inhibitors. The second focus of this work is the investigation of the processes during hepatic ische-mia-reperfusion injury. Hepatic ischemia-reperfusion injury is a highly complex process resulting from the interplay of both physiological and pathological reactions of the orga-nism. Within this process, a hepatoprotective effect of interleukin-6 via mediation of the JAK/STAT signaling pathway and induction of acute phase proteins has been described in the literature. In experiments of this work with murine liver samples, it can be shown that an increased RNA expression of interleukin-6, gp130 and acute phase proteins is induced by ischemia and subsequent reperfusion. However, this increase is also de-tectable in sham-operated animals so that the results shown here clearly indicate that for further IRI experiments in the future, later time points after the acute phase caused by the surgery has subsided must be selected.