Browsing by Author "Kim, Yonghyun"
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Item Bioengineering of Heterogenous Glioblastoma Multiforme Microenvironment(University of Alabama Libraries, 2021) Park, Seungjo; Kim, Yonghyun; University of Alabama TuscaloosaGlioblastoma (GBM) is the most aggressive brain tumor that originates from glioblastoma stem cells (GSCs). In the brain, GSCs are supported by a tumor microenvironment (TME) wherein the perivascular niche and hypoxic region are present. The glioblastoma microenvironment (GBME) exhibits high heterogeneity, vast cell-to-cell interactions, and stiff mechanical properties. To produce in vitro models mimicking the GBME features, GBM organoid (GBO) models have been developed. Conventional organoid studies rely on growing them in serum-free media, resulting in sphere formation. However, this conventional method is not scalable and often fails in recapitulating inter- and intratumor heterogeneity. Also, the conventional method is not ideal to produce adequate quantities of GBOs to screen drugs for personalized medicine. Therefore, development of a reproducible and scalable GBO culture method can provide a better platform to simulate novel treatments.First, the bioreactor design was optimized by using different diameters of impellers and bioreactor vessels. Even with similar shear stresses, cell proliferation was inhibited or promoted depending on the ratio of the impeller diameters to the vessel diameters. With the optimized vessel geometry, shear stress and media supplements were optimized for GBO production. The bioreactor GBOs (bGBOs) were produced in uniform size, not by clonal aggregations, but by cell proliferation. With the optimal agitation rate, bGBOs displayed upregulation of genes involved in stemness, hypoxia, angiogenesis, proliferation, and migration. The statistical analysis revealed the synergetic effects of the high agitation rate and the size of the bGBOs. Next, bGBO models were characterized by their morphologies and transcriptional and translational profiles. The bGBOs exhibited high and strong cell-to-cell contact. Multivariate gene analysis found a significant correlation between gene expression and the size of the bGBOs. GBME was established and spatially organized in bGBOs greater than 800 µm in diameter. Hypoxic TME was developed in bGBOs greater than 400 µm in diameter. Inside the bGBOs, spatially separated features of the hypoxic niche and the perivascular niche were demonstrated. Also, the large bGBOs displayed angiogenesis features. Self-established GBME was organized by transdifferentiated GBM into endothelial cells, pericytes, and astrocytes. GBME containing necrotic regions displayed more spatially distinctive and hierarchically organized GSC niches. The GSCs in the niche were regulated by transcription factors involved in dedifferentiation. Hydrogels have been employed to further understand underlying mechanisms of the transformation of GBM in the bGBO model. Mechanical properties of GBME was engineered using hyaluronic acid (HA)-based hydrogels. Cell behavior in response to the hydrogel stiffness was examined. Transcription factors dedifferentiating GBM into GSCs were translocated to the nucleus in response to stiffer substrates. Collectively, these studies provided a small-scale model for a high-throughput production of GBOs that recapitulate in vivo GBM features, including high heterogeneity and high cell-to-cell interactions. Hydrogel model and the bioreactor culture conditions described here suggest that GBOs can be biomanufactured by modulating mechanical stress.Item Biophysical analysis of fluid shear stress induced cellular deformation in a microfluidic device(American Institute of Physics, 2018) Landwehr, Grant M.; Kristof, Andrew J.; Rahman, Sharif M.; Pettigrew, Jacob H.; Coates, Rachael; Balhoff, Joseph B.; Triantafillu, Ursula L.; Kim, Yonghyun; Melvin, Adam T.; Louisiana State University; North Carolina State University; University of Alabama TuscaloosaEven though the majority of breast cancers respond well to primary therapy, a large percentage of patients relapse with metastatic disease, for which there is no treatment. In metastasis, a tumor sheds a small number of cancerous cells, termed circulating tumor cells (CTCs), into the local vasculature, from where they spread throughout the body to form new tumors. As CTCs move through the circulatory system, they experience physiological forces not present in the initial tumor environment, namely, fluid shear stress (FSS). Evidence suggests that CTCs respond to FSS by adopting a more aggressive phenotype; however, to date single-cell morphological changes have not been quantified to support this observation. Furthermore, the methodology of previous studies involves inducing FSS by flowing cells through the tubing, which lacks a precise and tunable control of FSS. Here, a microfluidic approach is used for isolating and characterizing the biophysical response of single breast cancer cells to conditions experienced in the circulatory system during metastasis. To evaluate the single-cell response of multiple breast cancer types, two model circulating tumor cell lines, MDA-MB-231 and MCF7, were challenged with FSS at precise magnitudes and durations. As expected, both MDA-MB-231 and MCF7 cells exhibited greater deformability due to increasing duration and magnitudes of FSS. However, wide variations in single-cell responses were observed. MCF7 cells were found to rapidly deform but reach a threshold value after 5 min of FSS, while MDA-MB-231 cells were observed to deform at a slower rate but with a larger threshold of deformation. This behavioral diversity suggests the presence of distinct cell subpopulations with different phenotypes. Published by AIP Publishing.Item CD200R/Foxp3-mediated signalling regulates microglial activation(Nature Portfolio, 2016) Yi, Min-Hee; Zhang, Enji; Kim, Jwa-Jin; Baek, Hyunjung; Shin, Nara; Kim, Sena; Kim, Sang Ryong; Kim, Hang-Rae; Lee, Sung Joong; Park, Jin Bong; Kim, Yonghyun; Kwon, O-Yu; Lee, Young Ho; Oh, Sang-Ha; Kim, Dong Woon; Chungnam National University; University of Texas Medical Branch Galveston; Yanbian University; Chungnam National University Hospital; Kyungpook National University; Seoul National University (SNU); University of Alabama TuscaloosaThe heterogeneity of microglial functions have either beneficial or detrimental roles in specific physiological or pathological environments. However, the details of what transcriptional mechanisms induce microglia to take beneficial phenotypes remain unknown. Here, we report that Foxp3 is essential for beneficial outcome of the microglial response and depends upon signalling by the immunoglobulin CD200 through its receptor (CD200R). Foxp3 expression was up-regulated in microglia activated by excitotoxicity-induced hippocampal neuroinflammation. Suppression of CD200R prevented anti-inflammatory phenotype of microglia, but over-expression of Foxp3 enhanced it. Phosphorylation of STAT6, a downstream effector of CD200R, modulated transcription of Foxp3. Finally, CD200R/Foxp3-mediated signalling enhanced hippocampal neuronal viability and conferred a degree of neuroprotection, presumably by counteracting inducible nitric oxide synthase. We conclude that enhancement of Foxp3 through CD200R could be neuroprotective by targeting the microglia.Item Cellular Mechanisms of Circulating Tumor Cells During Breast Cancer Metastasis(MDPI, 2020) Park, Han-A; Brown, Spenser R.; Kim, Yonghyun; University of Alabama TuscaloosaCirculating tumor cells (CTCs) are cancer cells that detach from the primary site and travel in the blood stream. A higher number of CTCs increases the risk of breast cancer metastasis, and it is inversely associated with the survival rates of patients with breast cancer. Although the numbers of CTCs are generally low and the majority of CTCs die in circulation, the survival of a few CTCs can seed the development of a tumor at a secondary location. An increasing number of studies demonstrate that CTCs undergo modification in response to the dynamic biophysical environment in the blood due in part to fluid shear stress. Fluid shear stress generates reactive oxygen species (ROS), triggers redox-sensitive cell signaling, and alters the function of intracellular organelles. In particular, the mitochondrion is an important target organelle in determining the metastatic phenotype of CTCs. In healthy cells, mitochondria produce adenosine triphosphate (ATP) via oxidative phosphorylation in the electron transport chain, and during oxidative phosphorylation, they produce physiological levels of ROS. Mitochondria also govern death mechanisms such as apoptosis and mitochondrial permeability transition pore opening to, in order eliminate unwanted or damaged cells. However, in cancer cells, mitochondria are dysregulated, causing aberrant energy metabolism, redox homeostasis, and cell death pathways that may favor cancer invasiveness. In this review, we discuss the influence of fluid shear stress on CTCs with an emphasis on breast cancer pathology, then discuss alterations of cellular mechanisms that may increase the metastatic potentials of CTCs.Item Cellular uptake of protic ruthenium complexes is influenced by pH dependent passive diffusion and energy dependent efflux(Elsevier, 2020) Park, Seungjo; Gray, Jessica L.; Altman, Sarah D.; Hairston, Angela R.; Beswick, Brianna T.; Kim, Yonghyun; Papish, Elizabeth T.; University of Alabama TuscaloosaThe lipophilic vs. hydrophilic properties of three protic ruthenium compounds were studied as a function of pH. Specifically, we measured Log(D-o/w) values for [(N,N)(2)Ru(6,6'-dhbp)](2+) complexes (where N,N = 2,2'-bipyridine (1(A)), 1,10-phenanthroline (2(A)), 2,3-dihydro-[1,4]dioxino[2,3-f][1,10]phenanthroline (3(A)) and 6,6'-dhbp is the diprotic 6,6'-dihydroxy-2,2'-bipyridine ligand) from pH 4.0 to 8.0. This study allowed us to demonstrate that as the ligand is deprotonated at higher pH values the resulting neutral charge on the complex improves its lipophilic properties. Thus, improved uptake by passive diffusion is expected with protic ligands on Ru(II). Furthermore, cellular studies have demonstrated that passive diffusion is the dominant pathway for cellular uptake. However, metabolic inhibition has also shown that energy dependent efflux reduces the amount of the ruthenium complex (as measured by mean fluorescence intensity) in the cells. These compounds have been shown by fluorescence microscopy to accumulate in the nuclei of cancer cells (MCF7, MDA-MB-231, and HeLa). Taken together, this data shows that uptake is required for toxicity but uptake alone is not sufficient. The greatest light activated toxicity appears to occur in breast cancer cell lines with relatively moderate uptake (MCF7 and MDA-MB-231) rather than the cell line with the greatest uptake of complex 3(A) (normal breast cell line MCF-10A).Item The Deprotonation and Dissociation of Amino Acids and Peptides by Negative Ion Mode Mass Spectrometry(University of Alabama Libraries, 2021) Cui, Can; Cassady, Carolyn J.; University of Alabama TuscaloosaInvestigation of negative ion mass spectrometry (MS) is important to proteomics due to its superior ability to analyze acidic peptides and provide complementary information to positive ion mode. This dissertation focuses on the deprotonation and dissociation of amino acids and peptides, which provides fundamental knowledge and assists in development of negative ion MS. Gas-phase acidities (GA) of acidic peptides were determined experimentally and compared to computational values. In electrospray ionization (ESI), either one major structure or multiple structures with very similar GAs were formed. Peptides with acidic residues at the C-terminus are more acidic than those at the N-terminus. Replacing glutamic acid residues (E) with aspartic acid (D) can increase the acidities when E is at the C-terminus but has no effect if E is at the N-terminus. Bond dissociation energies of deprotonated amino acids, dipeptides, and their amides were investigated by combining MS and computations. The loss of H2O occurs in collision-induced dissociation (CID) from all amides except glycine amide. Loss of the C-terminus is only seen in CID of deprotonated dipeptides. In addition, an intense y1 ion forms from dipeptides and their corresponding amides. In the comparison among radical-based dissociation techniques, negative ion in-source decay (nISD) has the best performance for peptides. nISD gives the highest sequence coverage and consistently generated singly charged c- and z-ions. Besides, nISD produced the least neutral loss products and is the fastest technique. The disadvantage of nISD is its inability to select precursor ions. For the investigation of nISD with oxidizing matrices on acidic peptides, 4-nitro-1-naphthylamine (4N1NA) was used as a MALDI matrix for the first time and was found to be the most useful matrix. The overall sequence coverage obtained with 4N1NA is higher than with three other matrices. The formation of c- and a-ions indicates that 4N1NA has both reducing and oxidizing characteristics.Item Developing 3D Engineered in Vitro Models to Study the Impact of Brain Microenvironment Derived Cues and Chemotherapy Drugs on Dormancy in Brain Metastatic Breast Cancer(University of Alabama Libraries, 2023) Kondapaneni, Raghu Vamsi; Rao, Shreyas SBreast cancer cells are known to disseminate to distant organs (brain, liver, lungs, bone, and lymphnodes) to develop secondary (metastatic) tumors. Upon arriving in the secondary organ, disseminated tumor cells (DTCs) exhibit dormancy to evade cell death and gain therapeutic resistance via tumor microenvironment-derived biomechanical, cellular and/or bio-chemical cues. Metastatic breast cancers are considered to be incurable with a dismal 5-year survival rate of only 27%. Among breast cancer metastasis, breast cancer brain metastasis (BCBM) is very aggressive with a median survival rate of 15 months. A mechanistic understanding of tumor cell-brain microenvironment interactions involved in attaining a dormant state in BCBM cells is crucial inidentifying new therapeutic targets for BCBM. This dissertation focuses on studying the impact of extracellular matrix derived cues, cellular cues and chemotherapy drugs on BCBM dormancy. Specifically, hyaluronic acid (HA) hydrogels with matrix stiffness comparable to both native brain and brain metastatic niche were utilized to study the impact of biomechanical cues on tumor dormancy in BCBM cell clusters (spheroids). The impact of spheroid size on tumor dormancy was also studied. In addition, this hydrogel system was employed to study regulation of tumor mass dormancy in BCBM spheroids. Further, impact of a chemotherapeutic drug (Paclitaxel) on BCBM dormancy was studied. Finally, HA hydrogel based dormancy model was utilized to study the impact of cellular cues on theregulation of BCBM dormancy, by co-culturing dormant BCBM cells with astrocytes. Taken together, the development of such models is poised to provide key scientific insights into the mechanisms involved in BCBM dormancy.Item Effect of increased intracranial pressure on blood flow through cerebral arteries and aneurysms -a fluid-structure interaction study(University of Alabama Libraries, 2016) Syed, Hasson Basha Quadri; Unnikrishnan, Vinu U.; University of Alabama TuscaloosaThe pathological changes due to many cerebral diseases lead to increase in intracranial pressure (ICP), which is a life threatening condition especially in severe head injuries such as traumatic brain injury, hydrocephalus, sub arachnoid hemorrhage etc. Elevated intracranial pressure (ICP) is a major contributor to morbidity and mortality in severe head injuries. Maintaining the ICP within acceptable range is important to contain the failure of auto regulation which maintains and regulates adequate cerebral blood flow inside the brain. These increased intracranial pressures are found to significantly affect the Wall Shear Stresses (WSS) distribution in the artery, which is an important hemodynamic parameter and may lead to the formation, progression and rupture of cerebral aneurysms (pathological dilatations in cerebral arteries) which go unnoticed until a stage when they are severe. Earlier research on cerebral arteries and aneurysms involves using constant mean ICP values. Recent advancements in ICP monitoring techniques have led to measurement of the ICP waveform and by incorporating time varying ICP waveform in the analysis of cerebral arteries helps in better understanding their effects on wall deformation and shear stresses. To date, such a robust computational study on the effect of increasing intracranial pressures on the cerebral arterial walls and aneurysms has not been attempted to the best of our knowledge. In this work, fully coupled fluid structural interaction (FSI) simulations are carried out to investigate the effect of variation of intracranial pressure (ICP) waveforms on the cerebral arterial walls and aneurysms. Three time varying ICP waveforms and three constant ICP profiles acting on the cerebral arterial wall are analyzed in this work. It has been found that the arterial and aneurysmal walls experiences significant deformation depending on the time varying ICP waveforms, while the WSS changes at peak systole for all the ICP profiles. Also, the maximum wall shear stresses decreased with increase in ICP inside the aneurysm dome and the minimum area of WSS distribution increased.Item The effects of physiological fluid shear stress on circulating tumor cells(University of Alabama Libraries, 2018) Triantafillu, Ursula Lea; Kim, Yonghyun; University of Alabama TuscaloosaThe focus of this dissertation is on the effects of physiological fluid shear stress (FSS) on circulating tumor cells (CTCs). FSS occurs on cells both in vitro and in vivo. FSS is typically considered as a major variable in large scale bioprocessing while FSS is assumed to have a negligible role in bench scale culture. In physiological settings, FSS impacts cells in cancer where local, regional, and distant cancers experience FSS through interstitial, lymphatic, and hematological flow, respectively. CTCs in hematological flow experience the highest FSS and are involved in the transit stage of metastasis. One challenge of metastatic cancer is the lack of secondary tumor detection. Detection of CTCs largely relies on the epithelial cell adhesion molecule (EpCAM). CTC phenotype also includes expression of cancer stem cell (CSC) and epithelial to mesenchymal transition (EMT), which are correlated to increased resistance to chemotherapy. The study of FSS using an in vitro model can provide a better understanding on CTC phenotype expression and drug resistance. FSS was first examined using a baseline study with in vitro cell spheroid culture. Spheroids provide better representation of the stem and tumor cell environment than 2D culture. These cells experience FSS during cell dissociation. Since FSS can detrimentally affect cells, a gentler mechanical platform was developed for dissociation. Furthermore, this method, as well as traditional dissociation methods, was tested to study how FSS affects cell viability and expression. This platform was further used to model breast CTCs as suspension cells under FSS. This metastatic model allowed for testing the effects of FSS on CTC phenotype, and it was found that FSS increased CSC and CTC expression. Since an increase in CSC expression is correlated to increased drug resistance, drug resistance on CTCs under FSS was tested with chemotherapy drugs. It was found that the combination of FSS and drug resistance synergistically increases drug resistance expression in the model CTCs, corroborating clinical reports of CTC drug resistance. Finally, the effects of FSS and drug resistance was tested on estrogen receptor positive (ER+) molecular subtype. Collectively, these studies provide a better understanding on CTC behavior during metastasis.Item Electron transfer dissociation mass spectromerty studies of peptides(University of Alabama Libraries, 2014) Feng, Changgeng; Cassady, Carolyn J.; University of Alabama TuscaloosaElectron transfer dissociation (ETD) is an important tandem mass spectrometry technique in peptide and protein sequencing. In the past, ETD experiments have primarily involved basic peptides. A limitation of ETD is the requirement that analytes be at least doubly cationized by electrospray ionization (ESI). In this research, a method has been developed for enhancing protonation of acidic and neutral peptides. This has allowed doubly protonated ions, [M+2H]2+, to be produced from peptides without basic residues and has enabled their study by ETD. This dissertation includes the first extensive study of non-basic peptides by ETD. The effects of a basic residue on ETD were investigated using a series of heptapeptides with one lysine, histidine, or arginine residue. The spectra contain primarily c"- and z'-ions, which result from cleavage of N-C_α bonds along the backbone. Almost all of product ions include the basic residue. Enhanced fragmentation occurs on the C-terminal side of the basic residue. Also, cn-1 formation is enhanced, where n is the number of residues in the peptide. Addition of Cr(III) nitrate to a solution of the neutral peptide heptaalanine yields abundant [M+2H]2+ formation by ESI. Eleven metal ions were tested and Cr(III) gave by far the most intense supercharging of peptides. In contrast, Cr(III) does not increase protonation of proteins. Experiments were performed to explore the supercharging mechanism. Addition of Cr(III) to the sample solution was used to produce [M+2H]2+ in the remainder of this research. Neutral peptides with alkyl side chains were studied by ETD and found to produce b- and c-ions. Two mechanisms are proposed for b-ion formation, which involves cleavage of backbone amide (O=C)-N bonds. The length of peptide chain affects ETD fragmentation, but the identity of the alkyl residue has minimal effect. Acidic peptides with one or two aspartic or glutamic acid residues produce b-, c- and zOe-ions. The mechanism of b-ion formation is probably the same as that for neutral peptides, while c- and zOe-ions result from a radical mechanism involving oxygen atoms on the acidic side chains. For highly acidic heptapeptides, c- and zOe-ions are the major products, which supports a radical mechanism.Item Endoplasmic reticulum stress impairment in the spinal dorsal horn of a neuropathic pain model(Nature Portfolio, 2015) Zhang, Enji; Yi, Min-Hee; Shin, Nara; Baek, Hyunjung; Kim, Sena; Kim, Eunjee; Kwon, Kisang; Lee, Sunyeul; Kim, Hyun-Woo; Bae, Yong Chul; Kim, Yonghyun; Kwon, O. -Yu; Lee, Won Hyung; Kim, Dong Woon; Chungnam National University; Chungnam National University Hospital; Kyungpook National University; Yanbian University; University of Alabama TuscaloosaEndoplasmic reticulum (ER) stress has been implicated in neurodegenerative diseases, but its role in neuropathic pain remains unclear. In this study, we examined the ER stress and the unfolded protein response (UPR) activation in a L5 spinal nerve ligation (SNL)-induced rat neuropathic pain model. SNL-induced neuropathic pain was assessed behaviorally using the CatWalk system, and histologically with microglial activation in the dorsal spinal horn. L5 SNL induced BIP upregulation in the neuron of superficial laminae of dorsal spinal horn. It also increased the level of ATF6 and intracellular localization into the nuclei in the neurons. Moreover, spliced XBP1 was also markedly elevated in the ipsilateral spinal dorsal horn. The PERK-elF2 pathway was activated in astrocytes of the spinal dorsal horn in the SNL model. In addition, electron microscopy revealed the presence of swollen cisternae in the dorsal spinal cord after SNL. Additionally, inhibition of the ATF6 pathway by intrathecal treatment with ATF6 siRNA reduced pain behaviors and BIP expression in the dorsal horn. The results suggest that ER stress might be involved in the induction and maintenance of neuropathic pain. Furthermore, a disturbance in UPR signaling may render the spinal neurons vulnerable to peripheral nerve injury or neuropathic pain stimuli.Item Expression of PGC1 alpha in glioblastoma multiforme patients(Spandidos, 2017) Cho, Sang Yeon; Kim, Seon-Hwan; Yi, Min-Hee; Zhang, Enji; Kim, Eunjee; Park, Jisoo; Jo, Eun-Kyeong; Lee, Young Ho; Park, Min Soo; Kim, Yonghyun; Park, Jongsun; Kim, Dong Woon; Chungnam National University; Chungnam National University Hospital; Yanbian University; University of Alabama TuscaloosaPeroxisome proliferator-activated receptor. coactivator 1 alpha (PGC1 alpha) is a key modulator of mitochondrial biogenesis. It is a coactivator of multiple transcription factors and regulates metabolic processes. However, little is known about the expression and function of PGC1 alpha in glioblastoma multiforme (GBM), the most prevalent and invasive type of brain tumor. The purpose of the present study was to investigate the biological function, localization and expression of PGC1 alpha in GBM. It was observed that PGC1 alpha expression is increased in the tumor cells, and a higher level of expression was observed in the mitochondria. Bioinformatics analyses identified that metabolic and mitochondrial genes were highly expressed in GBM cells, with a high PGC1 alpha mRNA expression. Notably, mitochondrial function-associated genes were highly expressed in cells alongside high PGC1 alpha expression. Collectively, the results of the present study indicate that PGC1 alpha is associated with mitochondrial dysfunction in GBM and may have a role in tumor pathogenesis and progression.Item The Impact of Astrocytes and Endothelial Cells on Glioblastoma Stemness Marker Expression in Multicellular Spheroids(Springer, 2021) Nakod, Pinaki S.; Kim, Yonghyun; Rao, Shreyas S.; University of Alabama TuscaloosaIntroduction Glioblastoma multiforme (GBM), the most common primary brain tumor in adults, is extremely malignant and lethal. GBM tumors are highly heterogenous, being comprised of cellular and matrix components, which contribute to tumor cell invasion, cancer stem cell maintenance, and drug resistance. Here, we developed a heterotypic 3D spheroid model integrating GBM cells with astrocytes and endothelial cells (ECs) to better simulate the cellular components of the tumor microenvironment and investigate their impact on the stemness marker expression of GBM cells, which has not been previously investigated. Methods We used U87 GBM cells, C8-D1A mouse astrocytes, and human umbilical vein ECs to construct co- and tri-culture spheroid models in low-attachment U-well plates. We characterized the expression of known stemness markers NESTIN, SOX2, CD133, NANOG, and OCT4 in these models and compared it to respective mixed monoculture spheroids (control) using qRT-PCR and immunostaining. Results We incorporated GBM cells and astrocytes/ECs in 1:1, 1:2, 1:4, and 1:9 ratio and observed spontaneous self-assembled spheroids in all coculture conditions. We observed changing spheroid size dynamics over 7 days and an increased expression in stemness markers in GBM-astrocyte and GBM-EC coculture spheroids in 1:4 and 1:9 coculture conditions, respectively. In a triculture model employing GBM cells, astrocytes, and ECs in a 1:4:9 ratio, we found an increased expression of all the stemness markers. Conclusions We elucidated the impact of astrocytes and ECs on GBM stemness marker expression. This multicellular spheroid model may provide an important tool for investigating the crosstalk between cell types in GBM.Item Light Activated Protic Ruthenium(II) Compounds: Structure Function Relationships and Determining Which Factors Influence Cytotoxicity(University of Alabama Libraries, 2024) Oladipupo, Olaitan; Papish, Elizabeth TCancer is a significant health challenge that has been difficult to treat. There is a continuous search for treatments with fewer side effects. The study of metallodrugs with antitumor properties has increased since the discovery and clinical approval of the Pt(II) drug, cisplatin, to treat many cancerous tumors. Photodynamic therapy (PDT) is a clinically approved targeted cancer treatment. Ru(II) polypyridyl complexes have gained attention over the years for their photochemical and photophysical properties, which can be harnessed for PDT. Ru(II) polypyridyl complexes have electronic properties that make their excited state properties tunable by ligand and charge modification. Many Ru(II) compounds have been studied as PDT agents, but only a few have been protic. The research in this dissertation is focused on the synthesis of diprotic Ru(II) polypyridyl compounds and fundamental studies of how ligand modifications influence lipophilicity, singlet oxygen generation, emission energies, luminescence quantum yields, luminescence lifetimes, and cytotoxicity of light-activated protic Ru(II) compounds. The diprotic ligands allow investigation into the influence of Ru-N strain and the electronic effects of protonation and deprotonation of the diprotic ligands on the photoexcited state properties and the photoexcited state reaction of Ru(II) polypyridyl compounds. For this dissertation, diprotic Ru(II) polypyridyl compounds have been synthesized and studied using three protic polypyridyl ligands with varying π-conjugation and hydroxyl group substitution. To investigate the impact of lipophilicity and ligand substitution on diprotic Ru(II) compounds, two new lipophilic bathophenanthroline compounds with 4,4′-dhbp and 6,6′-dhbp ligands were synthesized and investigated. The compound with the 6,6′-dhbp has a Ru-N strain and photodissociates because of the thermal accessibility of the antibonding triplet excited state. The compound with 4,4′-dhbp ligand has no Ru-N strain, does not photodissociate, and was the most photocytotoxic of the two compounds against the breast cancer cell line, MCF7. To probe the effect of (de)protonation events on the excited state properties and excited state reactions of diprotic Ru(II) compounds, eight compounds were synthesized using the 6,6′-dhbp and the 4,4′-dhbp, from which eight doubly deprotonated Ru(II) compounds were isolated. Studies showed that deprotonation quenches excited state properties, and only diprotic Ru(II) compounds of the 4,4′-dhbp have long-lived luminescence (ƮEm), high photoluminescence quantum yield (ɸPL), and high singlet oxygen quantum yield (ɸΔ). Going forward, the use of 6,6′-dhbp ligand was discontinued, and a new ligand, 4,7-dhphen, which is similar to but more conjugated than the 4,4′-dhbp ligand, was introduced.To study the impact of π-expansion of diprotic ligands on the photocytotoxicity of Ru(II) compounds, five new compounds were synthesized using the 4,4′-dhbp and 4,7-dhphen ligands. Photoactivation studies of these compounds against human skin melanoma cells (SK-MEL-28), breast cancer cells (MCF7), and triple-negative breast cancer cells (MDA-MB-231) show that, in most cases, the Ru(II) compounds of the 4,7-dhphen ligand are not as photocytotoxic as the Ru(II) compounds of 4,4′-dhbp. These studies show that extensive conjugation does not necessarily enhance the photocytotoxicity of the diprotic Ru(II) compounds in this dissertation.Item Light-activated protic ruthenium anticancer compounds: structure function relationships and determining which factors influence toxicity(University of Alabama Libraries, 2020-12) Gray, Jessica Lee; Papish, Elizabeth T.; University of Alabama TuscaloosaWhile research in the field of metallo-based chemotherapy drugs is extensive, understanding the effects of pH responsive ligands within these systems is limited. In 2017, the Papish group reported a new class of pH sensitive light-activated metallo drugs that are activated by light-triggered ligand dissociation also known as PACT or photoactivated chemotherapy. Three ruthenium complexes of the type [(N,N’)2Ru(6,6’-dhbp)]Cl2 (the photolabile ligand 6,6’-dhbp = 6,6’-dihydrohybipyridine; 1A: N,N’ = 2,2′-bipyridine (bpy) ; 2A: N,N = 1,10-phenanthroline (phen); 3A: N,N = 2,3-dihydro-[1,4]dioxino[2,3-f ][1,10]phenanthroline (dop)) were synthesized and found to be toxic against various breast cancer cell lines upon irradiation (λ = 450 nm) with compound 3 eliciting EC50 values as low as 4 µM. Phototoxicity indices with 3 were as high as 120, which shows that dark toxicity is limited. The complexes exhibited low overall photodissociation (ΦPD) despite good toxicity suggesting the mode of toxicity is not through a PACT driven pathway. Discussed herin, are the efforts to study the mode of action, physical properties, and which characteristics have the largest impact on light driven toxicity for compounds 1-3 and further investigation into newly developed compounds. The hydrophobicity (Log(Do/w)) and uptake properties of 1-3 are reported. Due to the presence of the protic ligand, 6,6’-dhbp, all of the complexes studied increase in hydrophobicity with pH with 3 being the most hydrophobic (3>2>1). Cellular studies have demonstrated that passive diffusion is the dominant pathway for cellular uptake and compound 3 accumulates in the nuclei of cancer cells (MCF7, MDA-MB-231, and HeLa); however it competes with active transport out of the cell (efflux). Subsequent research has shown that an increase in photodissociation does not result in an increase in toxicity and the primary mode of toxicity is likely via the production of singlet oxygen (1O2); a process known as photodynamic therapy (PDT). Singlet oxygen quantum yields (ΦΔ) were higher for 1-3 upon deprotenation with values as high as 0.87(9) for complex 2. New complexes are also reported which demonstrate improved ΦΔ’s, toxicity, and light selectivity against breast cancer cells demonstrating the importance of studying protic anticancer mettalo drugs.Item Light-responsive and Protic Ruthenium Compounds Bearing Bathophenanthroline and Dihydroxybipyridine Ligands Achieve Nanomolar Toxicity towards Breast Cancer Cells(dagger)(Wiley, 2022) Oladipupo, Olaitan E.; Brown, Spenser R.; Lamb, Robert W.; Gray, Jessica L.; Cameron, Colin G.; DeRegnaucourt, Alexa R.; Ward, Nicholas A.; Hall, James Fletcher; Xu, Yifei; Petersen, Courtney M.; Qu, Fengrui; Shrestha, Ambar B.; Thompson, Matthew K.; Bonizzoni, Marco; Webster, Charles Edwin; McFarland, Sherri A.; Kim, Yonghyun; Papish, Elizabeth T.; University of Alabama Tuscaloosa; Mississippi State University; Oak Ridge Associated Universities; United States Department of Energy (DOE); Oak Ridge Institute for Science & Education; University of Texas ArlingtonWe report new ruthenium complexes bearing the lipophilic bathophenanthroline (BPhen) ligand and dihydroxybipyridine (dhbp) ligands which differ in the placement of the OH groups ([(BPhen)(2)Ru(n,n '-dhbp)]Cl-2 with n = 6 and 4 in 1(A) and 2(A), respectively). Full characterization data are reported for 1(A) and 2(A) and single crystal X-ray diffraction for 1(A). Both 1(A) and 2(A) are diprotic acids. We have studied 1(A), 1(B), 2(A), and 2(B) (B = deprotonated forms) by UV-vis spectroscopy and 1 photodissociates, but 2 is light stable. Luminescence studies reveal that the basic forms have lower energy (MLCT)-M-3 states relative to the acidic forms. Complexes 1(A) and 2(A) produce singlet oxygen with quantum yields of 0.05 and 0.68, respectively, in acetonitrile. Complexes 1 and 2 are both photocytotoxic toward breast cancer cells, with complex 2 showing EC50 light values as low as 0.50 mu M with PI values as high as >200 vs. MCF7. Computational studies were used to predict the energies of the (MLCT)-M-3 and (MC)-M-3 states. An inaccessible (MC)-M-3 state for 2(B) suggests a rationale for why photodissociation does not occur with the 4,4 '-dhbp ligand. Low dark toxicity combined with an accessible (MLCT)-M-3 state for O-1(2) generation explains the excellent photocytotoxicity of 2.Item Metabolic engineering and process development in butanol production with clostridium tyrobutyricum(University of Alabama Libraries, 2016) Ma, Chao; Liu, X. Margaret; University of Alabama TuscaloosaAs a sustainable and environmentally friendly biofuel, biobutanol is a potential substitute for gasoline without any engine modification. The multiple Omics studies were applied to evaluate the change of the expression of host protein and intracellular metabolism in Clostridium tyrobutyricum in response to butanol production. The key enzymes related to carbon balance (i.e. acid and solvent end products and carbohydrates in central pathway), redox balance, energy balance, and cell growth has been studied. It was found that rebalancing both carbon and redox was critical to improve butanol production. These findings were used to achieve high production of biobutanol via integrated metabolic cell-process engineering (MCPE). In a comparative genomics study, the wild type C. tyrobutyricum, the metabolically engineered mutant with down-regulated acetate kinase and evolutionarily engineered strain showing fast cell growth were used to evaluated in butyrate fermentation at pH 6.0 and 37 oC. It was found that the cell growth rate was increased by 61-100% and butyrate productivity was improved by 44-102% by the evolutionarily engineered strain. To understand the mechanism of butyric acid production and cell growth regulation in engineered C. tyrobutyricum mutant, a comparative genomics study was performed. It was concluded that the genome mutations in transcription, translation, amino acid and phosphate transportation and cofactor binding might play important role in regulating cell growth and butyric acid production. Comparative proteomics, which covered 78.1% of open reading frames and 95% of core enzymes, was performed using wild type, mutant producing 37.30 g/L of butyrate and mutant producing 16.68 g/L of butanol. Carbon regulation enzymes in the central metabolic pathway that correlated with butanol production were identified, including thiolase (thl), acetyl-CoA acetyltransferase (ato), 3-hydroxybutyryl-CoA dehydrogenase (hbd) and crotonase (crt). The apparent imbalance of energy and redox was also observed due to the downregulation of acids production and the addition of butanol synthesis pathway. The understanding of the mechanism of carbon redistribution enabled the rational design of metabolic cell and process engineering strategies were revealed to achieve high butanol production in C. tyrobutyricum. With the fundamental understanding, the C. tyrobutyricum was metabolically engineered by rebalancing carbon and redox simultaneously. The overexpression of aldehyde/alcohol dehydrogenase (adhE2) and formate dehydrogenase (fdh) improved butanol titer by 2.15 fold in serum bottle and 2.72 fold in bioreactor. In addition, the proteomics study and metabolite analysis showed that more than 90% of the amino acid in the medium was consumed before the cell entered the stationary phase and some enzymes involved in amino acid metabolism had low expression in butanol producing mutant. Extra yeast extract or casamino acids was fed to the free-cell fermentation the mid-log phase, improving the butanol titer to more than 18 g/L compared to 14 g/L without extra nitrogen supplement. The rational metabolic cell-process engineering facilitated with systems biology understanding was demonstrated a powerful approach in butanol production. Finally, the C. tyrobutyricum was further rationally engineered by integrating multiple regualtors, including 1) heterologous NAD+-fdh that provides extra reducing power, 2) the thiolase (thl) that redirects metabolic flux from C2 to C4, and 3) AdhE2. Two novel mutatns, ACKKO-adhE2-fdh and ACKKO-thl-adhE2-fdh, were constructed and produced 18.37 g/L and 19.41 g/L, respectively. This study demonstrated that systems biology-based metabolic cell-process engineering of C. tyrobutyricum enabled a high production of butanol.Item Optimizing and characterizing fundamentals of magnetic heating in iron oxide nanoparticles for use in cancer hyperthermia therapy(University of Alabama Libraries, 2016) Shah, Rhythm; Brazel, Christopher S.; University of Alabama TuscaloosaIron oxide magnetic nanoparticles generate heat upon application of high frequency magnetic field that can be harnessed to treat cancer by achieving a temperature rise of > 43 °C. This study investigates and optimizes parameters such as magnetic field strength and frequency, solution viscosity, nanoparticle size, composition, and magnetic properties to maximize the heat generation in iron oxide magnetic nanoparticles (MNPs). Data were normalized to calculate SAR (specific absorption rate, W/g Fe) to determine the most effective parameters for magnetic heating, resulting in values as high as 1001 W/g Fe for the MNPs. Calculations based on the linear response theory and the Stoner-Wohlfarth theory were used to estimate nanoparticle SAR, and yielded results in agreement with the experiments. Efforts were made to determine nano-scale temperatures in the core of polymeric micelles, which are imbedded with a temperature sensitive fluorescent dye and magnetic nanoparticles. Experimental and computational macro-scale tumor models were investigated to observe the bulk temperature rise (>43 °C), and determine the applicability of the MNPs investigated for magnetic hyperthermia. It was determined that although there was minimal local temperature rise in the core of magnetic micelles, a temperature rise sufficient to reach hyperthermia conditions was observed in bulk tumor models measuring at least 0.8 mm in diameter. Additionally, bulk heating of Chinese Hamster Ovary (CHO) cells using MNPs demonstrated that hyperthermia led to cell death in the temperature range of 43 °C to 46 °C, while causing minimal toxicity concerns. A separate investigation (Chapter 9) was undertaken to validate single-use cell culture bags by developing a methodology to measure the toxicity caused by a leachate found to release from these bags, which are finding greater acceptance in the biopharmaceutical industry. CHO cells were used to investigate the dependence of leachate toxicity on cell culture type, mixing, culture volume, and the duration of cell exposure to the leachate. It was determined that the leachates posed a higher toxicity to suspended CHO-K1 cells compared to adherent cells, while use of constant mixing and long term exposure of cells to leachates led to considerable cell death.Item P22 virus templated synthesis of plasmonic photocatalytic nanostructures(University of Alabama Libraries, 2015) Zhou, Ziyou; Gupta, Arunava; University of Alabama TuscaloosaThe objective of the work presented in this dissertation is to examine the fabrication of novel viral-templated gold-cadmium sulfide (Au-CdS) plasmonic photocatalytic nanostructures and to investigate their photocatalytic behavior via photodegradation of methylene blue. Bacteriophage P22 shells, assembled from 420 copies of coat protein, have been demonstrated to act as selective biotemplates for the growth of ordered gold nanostructures in two different routes: 1) the incubation of gold precursor with P22 biotemplates before the addition of reducing agent; 2) and the direct reduction of gold precursor in the solution of P22 biotemplates with a pre-existing reducing agent. In both routes, gold nanocrystals could find their selective binding sites to form an ordered nanostructure over P22 shells. However, in our current study, the incubation of inorganic precursor with biotemplates, which is a common practice in the biotemplated synthesis of inorganic nanoparticles, did not produce better outcome compared to direct reduction without protein/gold precursor interaction. Genetically engineered scaffolding proteins evenly positioned inside P22 virus-like particles (VLP) can act as selective biotemplates for the constrained growth of CdS nanocrystals. The formation of CdS confined inside the VLP involves initial uniform nucleation and growth at the genetically engineered sites of the scaffolding proteins, followed by a more stochastic growth for longer reaction periods. The presence of the biotemplates does not affect the reaction order for the formation of CdS, but significantly influences the rate constant for the hydrolysis of thioacetamide (TA) and subsequent reaction of released S ions with Cd2+. Combining the coat protein templated synthesis of gold and the scaffolding protein templated synthesis of CdS, we have been able to utilize genetically engineered P22 VLP as a robust nanoplatform for fabricating gold/CdS plasmonic photocatalytic nanostructures. The controlled formation of gold nanoparticles on the outer shell of VLP-CdS dramatically enhances the photoactivity of CdS confined inside the VLP. However, the gold nanoparticles by themselves exhibit no significant effect on the photodegradation of MB. These findings are relevant for the synthesis of a wide range of alternative plasmonic photocatalytic materials with desired components, architectures, and performance.Item Primary cilia modulate TLR4-mediated inflammatory responses in hippocampal neurons(Biomed Central, 2017) Baek, Hyunjung; Shin, Hyo Jung; Kim, Jwa-Jin; Shin, Nara; Kim, Sena; Yi, Min-Hee; Zhang, Enji; Hong, Jinpyo; Kang, Joon Won; Kim, Yonghyun; Kim, Cuk-Seong; Kim, Dong Woon; Chungnam National University; Chungnam National University Hospital; University of Texas Medical Branch Galveston; Yanbian University; University of Alabama TuscaloosaBackground: The primary cilium is an organelle that can act as a master regulator of cellular signaling. Despite the presence of primary cilia in hippocampal neurons, their function is not fully understood. Recent studies have demonstrated that the primary cilium influences interleukin (IL)-1 beta-induced NF-kappa B signaling, ultimately mediating the inflammatory response. We, therefore, investigated ciliary function and NF-kappa B signaling in lipopolysaccharide (LPS)-induced neuroinflammation in conjunction with ciliary length analysis. Methods: Since TLR4/NF-kappa B signaling is a well-known inflammatory pathway, we measured ciliary length and inflammatory mediators in wild type (WT) and TLR4(-/-) mice injected with LPS. Next, to exclude the effects of microglial TLR4, we examined the ciliary length, ciliary components, inflammatory cytokine, and mediators in HT22 hippocampal neuronal cells. Results: Primary ciliary length decreased in hippocampal pyramidal neurons after intracerebroventricular injection of LPS in WT mice, whereas it increased in TLR4(-/-) mice. LPS treatment decreased primary ciliary length, activated NF-kappa B signaling, and increased Cox2 and iNOS levels in HT22 hippocampal neurons. In contrast, silencing Kif3a, a key protein component of cilia, increased ARL13B ciliary protein levels and suppressed NF-kappa B signaling and expression of inflammatory mediators. Conclusions: These data suggest that LPS-induced NF-kappa B signaling and inflammatory mediator expression are modulated by cilia and that the blockade of primary cilium formation by Kif3a siRNA regulates TLR4-induced NF-kappa B signaling. We propose that primary cilia are critical for regulating NF-kappa B signaling events in neuroinflammation and in the innate immune response.