MS, University of Cincinnati, 2022, Medicine: Cancer and Cell Biology

Microglia is the primary brain resident macrophage and is an integral component of brain development and functions including neurogenesis, angiogenesis, and synapse formation. The microglia exhibit spatial and temporal diversity in their morphology and functions. The microglial functions depend on the region and stage of development. Despite the advancement in research on microglia, the diversity profile of the microglia has yet been fully understood. Besides microglia, immune cells in the normal brain also include macrophages like the choroid plexus, meningeal, and perivascular macrophages. The role of microglia and different brain resident macrophages in normal brain physiology, neurological diseases, and brain tumors remains to be poorly defined. As the deadliest immune-privileged brain tumor, glioblastomas are largely treatment-resistant, which is in part due to the immunosuppressive tumor microenvironment. Specifically, the tumor-associated macrophages (TAM) act as a pro- and anti-tumorigenic component in GBM and are emerging as a potential target for developing anti-tumor drugs. Here, we discuss the origin, biomarkers, regulatory factors, and physiology of disease-associated microglia/macrophages. A better understanding of disease-associated microglia/macrophages and tumor microenvironment may facilitate the development of effective treatment strategies for neurodegenerative diseases and brain cancers.

Committee: Qing Lu Ph.D. (Committee Member); Ty Troutman Ph.D. (Committee Member); Ziyuan Guo Ph.D. (Committee Member); Theresa Alenghat V.M.D. Ph.D. (Committee Member) Subjects: Cellular Biology

Doctor of Philosophy, The Ohio State University, 2014, Molecular, Cellular and Developmental Biology

Despite recently declining rates of incidence and mortality, breast cancer remains the second leading cause of cancer death in American women. Much of the difficulty in treating this disease stems from the extreme diversity of cell types that compose the mammalian breast and the complexity of communication among them. Recently, macrophages and cells of the myeloid lineage have been discovered as key mediators of tumor progression, yet the precise mechanism through which this occurs remains relatively unsolved. The answer may lie in studying the ETS family of transcription factors, known to regulate many genes in cancer-relevant cell processes such as growth, proliferation, and motility. Ets2 in particular has been an attractive target as previous research suggests it has stroma-specific oncogenic potential. Through transgenic mouse tumor models, here we show that disabling Ets2 specifically within tumor-associated macrophages results in stunted tumor growth at both the primary and metastatic sites. Gene expression profiling and chromatin immunoprecipitation experiments reveal Ets2 directly binds and represses key anti-angiogenic genes, resulting in reduced tumor vasculature, thus limiting breast tumor growth and spread. Furthermore, PU.1, an ETS family member with wide influence over the cells of the hematopoietic lineage, was found to collaborate with Ets2 to regulate expression of key oncogenes Hif1a and IL-6. These results reveal that ETS factors work together as master regulators to influence angiogenesis and inflammation from within the macrophage compartment. These findings have implications for novel breast cancer therapies and prognostic tools. Macrophage-specific Ets2 deletion resulted in dysregulation of hundreds of genes. Comparing these genes with human breast cancer patient datasets resulted in a novel gene signature capable of retroactively predicting patient survival in multiple breast cancer subtypes including the highly aggressive ER-negat (open full item for complete abstract)

Committee: Michael Ostrowski PhD (Advisor); Gustavo Leone PhD (Committee Member); Thomas Ludwig PhD (Committee Member); Qianben Wang PhD (Committee Member) Subjects: Biology; Biomedical Research; Cellular Biology; Genetics; Health; Molecular Biology; Oncology

Doctor of Philosophy, Case Western Reserve University, 2014, Molecular Medicine

Macrophage migration inhibitory factor (MIF) exhibits both cytokine and chemotactic properties and is expressed by many cell types, including hepatocytes and nonparenchymal cells. Excessive alcohol abuse can lead to alcoholic liver disease (ALD), which has multiple stages of pathogenesis including hepatic steatosis and hepatic fibrosis. During steatohepatitis, several precipitating events, including triglyceride accumulation in the liver, hepatocyte damage and leukocyte infiltration, lead to further pathogenesis of ALD. Hepatic fibrosis is dependent on interactions between hepatocytes, hepatic stellate cells (HSC) and leukocytes. Recruitment of monocytes to the liver is a key contributor to hepatic fibrosis. Due to MIF's chemotactic properties, we hypothesized that MIF would be a key contributor to ALD pathogenesis. Female C57BL/6 or MIF-/- mice were fed an ethanol-containing liquid diet or pair-fed control diet for 25 days. Expression of MIF messenger RNA was induced after 25 days of ethanol feeding. After chronic ethanol feeding, chemokine expression and monocyte recruitment was increased in wild-type mice, but not MIF-/- mice. In a model of 12 liver fibrosis, female C57BL/6 or MIF-/- mice were challenged with carbon tetrachloride (CCl4). Expression of mRNA for MIF, as well as plasma MIF content, were increased in female wild-type mice in response to an acute dose of CCl4. a Smooth muscle actin (aSMA) mRNA and protein, indicators of HSC activation, were increased in liver of wild-type after chronic CCl4 challenge. This response was dampened in MIF-/- mice. Despite lower activation of HSC, MIF deficient mice developed similar fibrosis after CCl4 exposure when compared to wild-type mice. Inflammatory scar-associated macrophages (SAMs) were recruited to the liver from the periphery in response to chronic CCl4 in wild-type and MIF-/- mice, but later converted to a restorative SAM phenotype during fibrosis resolution. Fewer inflammatory and restorative monocytes were (open full item for complete abstract)

Committee: Laura Nagy PhD (Advisor); Maria Hatzoglou PhD (Committee Chair); Pierre Gholam MD (Committee Member); Booki Min DVM, PhD (Committee Member); Xiaoxia Li PhD (Committee Member) Subjects: Biology; Biomedical Research; Health Sciences; Immunology

Doctor of Philosophy in Clinical-Bioanalytical Chemistry, Cleveland State University, 2012, College of Sciences and Health Professions

RNase L is one of the key enzymes in the 2-5A system of interferon (IFN) action against viral infection and cellular proliferation. Tissue distribution analysis has revealed that RNase L is highly expressed in the spleen, thymus, lung, testis, intestine and most of immune cells such as T, B cells and macrophages. However, the physiological role of RNase L in the immune system is largely unknown. My thesis thus focused on studying the possible physiological role of RNase L in macrophages. By using bone marrow-derived macrophages (BMMs) from RNase L+/+and -/- mice, we demonstrated that RNase L is involved in macrophage functions and migration ability. RNase L deficient BMMs showed a significant reduction of endocytic activity to FITC-Dextran 40,000 compared to wild type cells. In addition, lack of RNase L remarkably decreased the migration of BMMs under both normal condition and condition induced by M-CSF, GM-CSF or CCL2. To determine the role of RNase L in tumor growth, P53-/- RL-/- cancer cells were subcutaneously implanted on the back of RNase L null and wild type mice with C57BL/6 background, respectively. Surprisingly, the average tumor weight from RNase L+/+ mice was 3-fold heavier than that from RNase L-/- mice indicating that presence of RNase L was overtly favorite for tumor growth. Immunofluorescence staining revealed that the numbers of infiltrated macrophages were markedly higher in the tumor tissues from the wild type mice. Depletion of macrophages clearly inhibited tumor growth on RNase L+/+ mice, suggesting that RNase L may promote tumor growth through regulating the function of tumor-associated macrophages (TAMs). Taken together, our findings implicate that RNase L may play a dual role in innate immunity and tumor promotion. Additionally, in a collaborated project, we successfully identified and investigated the molecular targets of an anti-cancer drug candidate. In this study, we performed protein pull down assays to purify the anti-cancer targets o (open full item for complete abstract)

Committee: Aimin Zhou PHD (Committee Chair); Wenquan Zou PHD (Committee Member); Xuelong Sun PHD (Committee Member); David Anderson PHD (Committee Member); Michael Kalafatis PHD (Committee Member) Subjects: Biochemistry; Biomedical Research; Immunology

Doctor of Philosophy, University of Akron, 2024, Polymer Science

As the demand for implantable medical devices by the healthcare industry continues to rise, the demand for novel commercial, medically relevant polymers grows as well. However, the time and resources required for the research and development of new polymer biomaterials remain a significant barrier for the entry of these materials into the medical device field. One of the critical issues that polymer biomaterials face is the lack of standardization of polymer characteristics, especially regarding biocompatibility and immunogenicity. These parameters require greater investigation as the foreign body response (FBR) is an inevitable immunological reaction to implanted medical devices and the biomaterials that comprise them. Broadly speaking, when biomaterials are first implanted, an inflammatory phase will begin which will be followed by a tissue proliferation and remodeling phase, which are greatly mediated by the host's population of macrophages and their associated cytokines. However, instead of the classic wound healing response to tissue damage, the presence of a foreign body provokes the FBR, wherein pro-inflammatory M1 macrophages stimulate chronic inflammation due to the inability to remove the implant. M1 macrophages are then stimulated with IL-4 and IL-13 cytokines, polarizing into pro-remodeling M2 macrophages, which continue to fuse into Foreign Body Giant Cells (FBGCs). This ultimately results in the fibrotic encapsulation and degradation of the medical implant, greatly reducing functionality and stability of the implant. While there exists research that focuses on biomaterials that modulates the FBR, especially that of hydrogels, there is no significant research available on the correlation between polymer characteristics such as chemical structure or physical properties and the macrophage polarization response and the subsequent FBR, obfuscating potential predictions of biomaterial immunogenicity. In this presentation, an attempt is made to show that an i (open full item for complete abstract)

Committee: Abraham Joy (Advisor); Mesfin Tsige (Committee Chair); Ge Zhang (Committee Member); Nita Sahai (Committee Member); Richard Londraville (Committee Member) Subjects: Biomedical Engineering

Doctor of Philosophy in Regulatory Biology, Cleveland State University, 2022, College of Sciences and Health Professions

Transplantation requires that an organ be removed from the circulation of the donor and then reintroduced to the circulation of the recipient. Organs from deceased donors are often removed from the donor in one hospital and then transported on ice to another hospital for the transplant procedure. Consequently, transplanted organs are subjected to varying periods of ischemia before reperfusion in the recipient. This results in ischemia reperfusion injury (IRI) to the organ. For transplanted kidneys, IRI is frequently severe enough to cause delayed function requiring dialysis and even total failure of the graft. Identifying mechanisms that mediate this injury remains a crucial goal of transplantation. It is known that IRI causes a surge of apoptosis. During normal homeostatic turnover of cells, apoptotic bodies are cleared without inducing inflammation or immune responses by C1q, the initial component of the complement system. In patients with the rare genetic deficiency of C1q, apoptotic bodies are not effectively cleared and immune responses develop to autoantigens in the apoptotic bodies. We examined the role of C1q in modulating the early immune response to IRI in renal transplants by investigating the absence of C1q on early graft injury in an MHC mismatched murine transplant model. We found that A/J kidney grafts transplanted into C1q KO mice exhibited high levels of urinary injury marker, neutrophil gelatinase associated lipocalin (Ngal) within 1 and 2 days posttransplant and decreased survival compared to WT mice. Immunohistology demonstrated increased platelet aggregates in the renal medulla typical of unresolved ischemia induced injury in grafts undergoing early rejection 5 days after transplantation. Sources of C1q were identified during IRI in renal transplants by isolating infiltrating and resident Ly6C+ cell populations from the graft. Resident Ly6C+ cells in the donor kidney were found to express high levels of C1q even 6 days a (open full item for complete abstract)

Committee: William Baldwin PhD (Committee Chair); Keith McCrae MD (Committee Member); Barsanjit Mazumder PhD (Committee Member); Tom McIntyre PhD (Committee Member); Trine Jorgensen PhD (Other); Anna Valujskikh PhD (Other) Subjects: Biology; Cellular Biology; Immunology; Molecular Biology

Doctor of Philosophy (PhD), University of Toledo, 2023, Biomedical Sciences (Cancer Biology)

Immunotherapy at best yields moderate responses in breast cancer, despite its success in other cancer types. Such therapeutic inefficacy is instigated by the immunosuppressive nature of the breast Tumor Microenvironment (TME). Tumor-Associated Macrophages (TAMs) harbored within the TME, predominantly contribute to this hostile milieu by manifesting an immunosuppressive, pro-tumorigenic M2-TAMs while down-modulating the immunostimulatory, anti-tumorigenic M1-TAMs. Therefore, reprogramming M2-TAMs to M1 could induce an immunogenic shift in breast TME and increase the efficacy of immunotherapeutics. Switching gears from current toxicogenic cytokine-mediated reprogramming methods, we aimed to target the l-arginine metabolism in TAMs for immunogenic reprogramming. As M2-TAMs are deficient in the synthesis of tetrahydrobiopterin (BH4), the co-factor of nitric oxide synthase (NOS), we hypothesized that supplementing Sepiapterin (SEP), the precursor of BH4 would redirect arginine metabolism from polyamine (PA) to nitric oxide production (NO), reprogram to M1-TAMs and increase anti-tumorigenicity. We recently reported that SEP treatment redirected arginine metabolism in M2-TAMs towards NO pathway and induced their phenotypic conversion to M1 type. In the present study, we demonstrate that SEP treatment functionally reprograms M2-TAMs to M1-TAMs, significantly elevate their immunogenic responses including activation of cytotoxic T cells and induce immunogenic cell death (ICD), a distinctive form of apoptosis, in HER2+ breast cancer cells. Consistently, SEP treatment suppresses HER2+ mammary tumor growth in vivo and enhances immunostimulation in breast TME. This study provides new evidence that SEP-mediated immunometabolic reprogramming induces an immunogenic shift in the HER2+ breast TME and can be utilized as a novel immunotherapeutic strategy.

Committee: Ivana de la Serna (Committee Chair); Saori Furuta (Committee Member); Heather Conti (Committee Member); Kandace Williams (Committee Member); Kam Yeung (Committee Member) Subjects: Biomedical Research; Oncology

Master of Science, University of Toledo, 2023, Bioengineering

Extracellular vesicles have emerged as a promising tool for cell-free therapies in tissue regeneration and disease treatment. Although regenerative properties and therapeutic effects of extracellular vesicles have already been started to be investigated, most of the EV research is associated with stem cell lineage. As both fibroblasts and macrophages are ubiquitous in the human body and are highly active in tissue repair and remodeling, the effect of extracellular vesicles secreted by fibroblasts on macrophages towards immunomodulation is yet to be known. In this regard, the objective of this study is to first isolate extracellular vesicles successfully from fibroblasts, and then to observe whether the fibroblast-derived extracellular vesicles promote macrophage polarization from pro-inflammatory to anti-inflammatory phenotype within ECM like 3D matrix, leading that EVs are enough to create anti-inflammatory response in case of treating diseases with cell-free therapies towards tissue regeneration.

Committee: Dr. Eda Yildirim-Ayan (Committee Chair); Dr. Yuan Tang (Committee Member); Dr. Brent Cameron (Committee Member) Subjects: Biomedical Engineering; Engineering

Doctor of Philosophy, The Ohio State University, 2023, Pharmaceutical Sciences

Acute respiratory distress syndrome (ARDS) is a life-threatening condition in which the lungs suffer from an inflammatory lung injury following insults such as pneumonia, sepsis, trauma, and coronavirus disease 2019 (COVID-19). Patients with ARDS often requires life-support with mechanical ventilation (MV) which can generate injurious physical forces that exacerbate the injury known as ventilator-induced lung injury (VILI). These forces can damage the alveolar-capillary barrier and trigger the release of pro-inflammatory mediators which further damage lung cells including alveolar epithelium, capillary endothelium, and alveolar macrophages (AMs). Although clinicians have tried to minimize lung injury during mechanical ventilation (MV) by using low tidal volume ventilation, it is not possible to completely eliminate it. Clinical trials have failed to identify molecularly targeted pharmacologic therapies for ARDS patients. An incomplete understanding of the role of each lung cell type plays in the development of injury and the mechanosensitive pathways involved in VILI are contributing factors to the unsuccessful outcomes of ARDS clinical trials. Another potential reason is that there has been a relative lack of studies that utilize the pulmonary drug delivery route. In addition, drug delivery during ARDS is challenging due to the heterogeneous nature of lung injury and occlusion of lung units by edema fluid and inflammation. In this dissertation, we demonstrated that AMs respond to mechanical forces by upregulating an anti-inflammatory microRNA, miR-146a, but that this native mechanotransduction response is insufficient to mitigate VILI. Due to various challenges associated with the delivery of miRs, we developed a lipid nanoparticle (LNP) formulation that facilitates the pulmonary delivery of miR-146a. We used an in vitro barotrauma model and an in vivo VILI model to evaluate the therapeutic efficacy of miR-146a loaded LNPs. Our findings indicate that increasing (open full item for complete abstract)

Committee: Joshua Englert MD (Advisor); Mitch Phelps PhD (Committee Member); Alex Sparreboom PhD (Committee Member); Samir Ghadiali PhD (Advisor) Subjects: Pharmaceuticals

Doctor of Philosophy, The Ohio State University, 2022, Neuroscience Graduate Studies Program

The glucocorticoid receptor (GR), part of the nuclear receptor superfamily of transcription factors, is ubiquitously expressed in all cell types and regulates cellular responses to glucocorticoids (GCs). Stress, a recurring and unavoidable experience throughout life, causes the release of GCs into the bloodstream which exert actions through binding GR. While GRs mainly function to maintain homeostasis and regulate metabolism, they also act in specific cell types to diversely modulate their function. GCs induce structural plasticity in neurons and many other cells throughout the nervous system. While plasticity is essential for adaptation and learning and memory stress-induced plasticity can be maladaptive. Elevated GCs can alter neuron structure and function in the brain leading to cognitive impairment and contribute to the development of neuropsychiatric disorders. GCs also exacerbate neuropathic pain, but less is known about GR mediated structural plasticity in sensory neurons and in the context of pain. Here, we show that GR mediates growth and regeneration in sensory neurons. We found that basal GR expression in dorsal root ganglia (DRG) sensory neurons is 15-fold higher than in neurons in canonical stress-responsive brain regions, making them uniquely sensitive to GCs. In response to stress or applied GCs, adult DRG neurite growth in vitro increases through mechanisms involving GR-dependent gene transcription. We also identify a new set of regeneration associated genes (RAGs) (e.g., Gilz, Cebpa) that are increased by stress. In vivo, acute stress increases peripheral nerve regeneration, providing evidence for a structural correlate of stress-exacerbated pain. Building on these findings, we further investigate sensory neuron GRs in the context of a newly developed clinically relevant model of neuroma. We characterize this new model and find that sural nerve ligation induces allodynia in both male and female mice and develop neuromas with features consi (open full item for complete abstract)

Committee: Phillip Popovich (Advisor) Subjects: Neurosciences

Bachelor of Science (BS), Ohio University, 2022, Biological Sciences

Analyzing host cell response using RNA-seq allows us to understand what is happening within the host cell that could be missed by narrower analysis. From the analyses in this study, we can broadly state that extracellular vesicles (EVs) produced by Staphylococcus aureus at different physiological temperatures have varying effects on host cell gene expression. Infection and immune system-related genes appear to be upregulated in THP-1-derived macrophages treated with EVs produced at 37°C (core body temperature). These genes then appear to be downregulated in macrophages treated with EVs produced at 34ºC (the temperature of the anterior nares, where S. aureus colonizes). This pattern observed with EV-treated macrophages was not observed in EV-treated human nasal epithelial cells (HNECs). By utilizing RNA-seq we can see on a global level how RNA introduced to human cells, via EVs or a future vaccine, has an effect on our own gene expression.

Committee: Soichi Tanda Dr. (Other); Ronan Carroll Dr. (Advisor) Subjects: Biology; Microbiology

PhD, University of Cincinnati, 2022, Medicine: Molecular Genetics, Biochemistry, and Microbiology

Obesity is classified as a low grade chronic inflammatory disease that leads to the development of co-morbidities. Disease progression of obesity and co-morbidity have been linked to increase inflammation caused by high calorie diet. A group of genes in the low-density lipoprotein receptor (LDLR) family has been linked to inflammation in macrophages. One member of this family, apolipoprotein E receptor-2 (apoER2), is expressed in macrophages but its role in macrophage function is unknown. Another group of proteins that is linked to diet-induced obesity are enzymes secreted from the exocrine pancreas involved in the breakdown of dietary lipids for absorption. One such enzyme is group 1b phospholipase A2 (PLA2g1b), which is known to break down phospholipids to produce fatty acids and lysophosphatidylcholine (LPC). LPC is broken down further to LPA by autotaxin. Both LPC and LPA are associated with increased inflammation and metabolic disease development. The first goal is to determine the role of apoER2 on macrophage function and effects on diet-induced inflammation and obesity. The second goal is to determine the role PLA2g1b and autotaxin in inflammatory disease. ApoER2 role in inflammation was assessed in bone marrow derived macrophages generated with macrophage colony stimulating factor (MCSF). Macrophages lacking apoER2 displayed increased inflammatory cytokines and reduced resolution cytokines in response to lipopolysaccharide (LPS) and interleukin-4 (IL-4) challenge, implying that apoER2 in macrophages may be important for resolution of tissue inflammation in obesity. Two mouse models placed on western diet were used to test this possibility. The first model was bone marrow transplant model and the second model was mice with global loss of apoER2. Both models had similar fasting plasma lipid levels, but loss of apoER2 specifically in the bone marrow resulted in increased body weight due to larger fat mass after diet while global loss of apoER2 resulted in r (open full item for complete abstract)

Committee: David Hui Ph.D (Committee Chair); David Wieczorek Ph.D (Committee Member); Phillip Owens Ph.D (Committee Member); Thomas Thompson Ph.D (Committee Member); William Miller Ph.D (Committee Member) Subjects: Biochemistry

Doctor of Philosophy in Clinical-Bioanalytical Chemistry, Cleveland State University, 2021, College of Sciences and Health Professions

Gangliosides are involved in various biological functions, like modulation of LPS/TLR4 signaling pathway, but specific mechanism is still unknown. Gangliosides are desialylated from various sialidases. However, sialidase specificity toward individual ganglioside is unknown. In this study, sialidase substrate specificity was investigated and endogenous ganglioside carbohydrate profile change upon LPS stimulation in THP1 macrophages was explored. First, membrane-mimic system with liposome ganglioside conjugates containing different lipids with was developed to evaluate substrate specificity of sialidase and the lipid effect on the enzyme activity. Their desialylation profiles with Arthrobacter ureafaciens and H1N1 sialidase were determined using HPLC. Diversity of substrate preference was found for sialidase was found. In addition, the apparent Km and Vmax of different sialidases were found to be dependent on the type of lipid. Secondly, major gangliosides (GM3, GM2, GM1, GD3) in THP1 macrophages were quantified, where gangliosides were extracted from total cell, cell membrane, and lipid rafts. Ceramide chain was digested with ceramide glycanase, and carbohydrate unit of gangliosides were labeled with 2-anthranilic acid (2-AA). They were quantified using HPLC, in comparison to the 2-AA labeled glucose polymer ladder. A comparison in gangliosides was made in amount between the total cell membrane and lipid rafts. Next, the expression change of ganglioside was determined upon LPS stimulation of THP1 macrophages. With LPS stimulation, all gangliosides showed a decline with the most significant decrease of GM3, and least reduction of GD3 in cell lysate, lipid raft, and GM2 on the cell membrane. A total sialidase was found to be increased upon LPS stimulation. Moreover, endogenous ganglioside change was observed with Neu3 sialidase, which further investigated with sialidase inhibitor (DANA) on cell surface and in total. Finally, the sialidase specificity of end (open full item for complete abstract)

Committee: Xue-Long Sun Ph.D (Advisor); David Anderson Ph.D (Committee Member); Bin Su Ph.D (Committee Member); Chandra Kothapalli Ph.D (Committee Member); Aimin Zhou Ph.D (Committee Member) Subjects: Biochemistry; Molecular Biology

Master of Science, University of Toledo, 2021, Bioengineering

The wound is a disease with serious health risks as it can lead to mortality and morbidity issues which impact vastly the economy. Thus, finding the method of wound healing in the fastest and cheapest way is very beneficial for the world. M2 phenotype Macrophage is known for being responsible for healing such as wound healing. Thus, using correct stimuli to polarize macrophages is crucial. Non-thermal plasma, the 4th state of matter, is known to have many medical applications such as cancer treatment, blood coagulation, sterilization, and wound healing. Among the stimuli to polarize the macrophages, non-thermal plasma is cost-effective with an easy set-up. Thus, in this research, non-thermal plasma with a DBD electrode was applied on monocyte-derived Macrophages. Representation of cells in a 2D environment does not represent the cell environment within our body. Thus, a study in 3D environment is very important as the result might even be different to the findings in 3D matrix. Thus, cells were embedded in collagen prior to plasma treatment. First, working gas was optimized between air and Helium. Gene was expressed to observe plasma treatment effect with air and Helium. Further experiments such as detecting reactive species were performed to explain the change in genes. After selecting a working gas, the plasma dose was optimized with gene expression and cell viability. This study suggests that non-thermal plasma in air may polarize the macrophages within collagen to M2 phenotype and 120s plasma was susceptible for cells. This result is of utter significance because a study on the effect of non-thermal plasma in Macrophages within collagen matrix had been performed for the first time.

Committee: Halim Ayan PhD (Committee Chair); Eda Yildirim-Ayan PhD (Committee Member); Arunan Nadarajah PhD (Committee Member) Subjects: Biomedical Engineering

Doctor of Philosophy, Case Western Reserve University, 2022, Pathology

Methods that induce immune responses against tumors have emerged as a promising approach for cancer treatment. Recent research has shown that Cow Pea Mosaic Virus (CPMV) nanoparticles can induce anti-tumor immune responses in various animal tumors. The molecular mechanisms underlying these effects, as well as CPMV's potential to activate human cells were investigated in these studies. In vitro stimulation of CD14+ monocytes with CPMV resulted in the induction of HLA-DR, CD86, PD-L1, IL-15R, CXCL10, MIP-1a and MIP-1b. CPMV also caused activation of dendritic cells and monocyte-derived macrophages. Our findings demonstrated that CPMV activates human monocytes via Syk signaling, endosomal acidification, and recognition by Toll-like Receptor (TLR) 7/8. These findings suggest that CPMV may be a useful immune-based approach in humans. Importantly, immune cell activation is hindered in the tumor microenvironment by immunosuppressive mechanisms. These mechanisms may include an influence on macrophage maturation, ultimately generating macrophages that favor tumor growth. To better understand how tumor microenvironments influence macrophage maturation, we studied the effects of tumor cell supernatants (FaDu and SCC) on monocyte-derived macrophages. Purified monocytes incubated with FaDu or SCC9 supernatants showed improved survival, decreased surface HLA-DR, CD86, IL-15R and IP-10 expression, and increased surface PD-L1, CD14 and CD206 expression. Despite expressing TLR4 and CD14, macrophages matured in tumor supernatants did not respond to the TLR4 agonist LPS. This could be due to overexpression of WIP-1 phosphatase in tumor supernatant-exposed macrophages, resulting in inhibition of p38 phosphorylation that is implicated in many inflammatory signaling pathways. Several phenotypic changes in tumor supernatant exposed macrophages could be reversed by depleting fatty acids from tumor supernatants or by inhibition of fatty acid oxidation. The phenotype of macrophages incuba (open full item for complete abstract)

Committee: Scott Sieg PhD (Advisor); George Dubyak PhD (Committee Chair); Alex Huang MD/PhD (Committee Member); John Tilton MD (Committee Member); Clive Hamlin PhD (Committee Member) Subjects: Biomedical Research; Immunology; Oncology; Pathology

Doctor of Philosophy, Case Western Reserve University, 0, Pathology

Regulatory mechanisms of the immune system are essential for human health by maintaining homeostasis and preventing disease states such as excessive inflammation and autoimmunity. These vital functions are coordinated by a wide range of cells from multiple lineages that employ unique and inventive techniques to suppress immune overactivation. Here, we describe four instances of novel regulatory mechanisms, which all in their own capacities are rooted in host and microbial glycans. In one case, we report that FoxP3+ regulatory T cells (Tregs) respond to a dual cytokine signal discovered downstream of the microbial factor polysaccharide A (PSA) by eliciting robust immunosuppression through the production of IL-10 and the selective proliferation of IL-10-expressing Tregs. In another study, we find that the suppressive T cell phenotype downstream of PSA that has been the focus of decades of work is preceded by the ability of PSA to polarize an anti-inflammatory macrophage subtype. Another study examines the cellular compensation that occurs during genetic ablation of IL-10 in FoxP3+ Tregs, where type I regulatory T cells (Tr1) in the colon upregulate their production of IL-10 to prevent the spontaneous development of disease. In the final study, we describe a new role for CD22 on macrophages that transduces tissue sialylation status and drives the polarization of anti-inflammatory macrophages that are critical for shaping immune tone over time. Using the modalities described in this work, multiple mouse models of autoimmune and inflammatory diseases, including asthma, multiple sclerosis, and inflammatory bowel disease were therapeutically prevented or reversed, demonstrating the astounding power of glycans and the immune cells that respond to them.

Committee: Brian Cobb (Advisor); George Dubyak (Committee Chair); Clive Hamlin (Committee Member); Booki Min (Committee Member); Robert Fairchild (Committee Member) Subjects: Cellular Biology; Immunology; Pathology

Doctor of Philosophy, The Ohio State University, 2021, Biomedical Sciences

Cystic fibrosis (CF) is an autosomal recessive disease that mainly affects the Caucasian population with no definitive available cure. CF is caused by specific mutations in the Cystic fibrosis transmembrane conductance regulator (CFTR) gene that encodes an ion channel which has been mostly studied in epithelial cells. CFTR F508del mutation is the most common mutation which, in epithelial cells, prevents the CFTR protein from reaching the plasma membrane due to misfolding. People with CF (pwCF) are more prone to infections by opportunistic pathogens such as Burkholderia cenocepacia (B. cenocepacia) resulting in fatal respiratory infections. Macrophages play critical roles in the process of clearance of lung pathogens, the locations and functions of CFTR in macrophages are unclear. In CF, human and mouse macrophages expressing F508del CFTR are defective in their ability to kill bacteria. Furthermore, the autophagy process in CF macrophages is halted, and the underlying mechanism remains to be elucidated. Using 3D reconstruction and confocal microscopy, we demonstrate that CFTR protein is expressed in the macrophage and on the autophagosome. We identified that CFTR is recruited to LC3-labeled autophagosomes harboring B. cenocepacia, but not to vacuoles enclosing Escherichia coli (E. coli). Using several complementary approaches, we found that F508del CFTR macrophages display defective lysosomal acidification as well as impaired degradative function for cargos destined to autophagosomes such as B. cenocepacia, whereas non-autophagosomal cargos such as E. coli are effectively degraded. CFTR modulators are used for treating CF patients, via correction of misfolded CFTR, and improving its function in epithelial cells. Yet, their effect on macrophages is still unclear. Here, we show that treatment of CF macrophages with CFTR modulators increased F508del CFTR localization to B. cenocepacia containing vacuoles, improved the autophagy flux, and lysosomal function. Additionall (open full item for complete abstract)

Committee: Amal Amer (Advisor); Mark Wewers (Committee Member); Stephen Kirkby (Committee Member); Estelle Cormet-Boyaka (Committee Co-Chair); Stephanie Seveau (Committee Member) Subjects: Biomedical Research

Doctor of Philosophy, University of Akron, 2021, Chemistry

Cysteinyl leukotrienes (cys-LTs) - leukotriene C4 (LTC4), leukotriene D4 (LTD4) and leukotriene E4 (LTE4) are potent inflammatory lipid mediators that act via two different G-protein coupled receptors: CysLT1R and CysLT2R. These metabolites are implicated in pathophysiology of various inflammatory diseases like asthma, cancer, cardiovascular diseases and monocyte recruitment to the inflammation site. However, the molecular mechanism by which cys-LTs modulate their function is still elusive. Macrophages use receptor-mediated phagocytosis to identify foreign particles and cellular debris and maintain tissue homeostasis during inflammatory processes. It was shown in Chapter III of the dissertation that cys-LTs increases phagocytosis of zymosan bioparticles and ox-LDL in macrophages. Furthermore, the underlying mechanism revealed that cys-LT-mediated activation of scavenger receptors CD36 and OLR1, as well as an increase in the cytokine MCP-1, improves macrophage phagocytic abilities. Macrophages tend to alter their function dynamically in response to local micro environmental cues. To understand the role of cys-LT/CysLTR signaling in modulating macrophage plasticity, BMDMs were polarized into M1 and M2 phenotypes which were then validated with the literature. In chapter IV of the dissertation, murine bone marrow cells polarization into M1 phenotype was shown to be NFkB, STAT1, and p38 dependent whereas M2 polarization was shown to require KLF4 and PPARγ. Using CysLT1R and CysLT2R null mice, it was shown for the first time that BMDMs and peritoneal macrophages require CysLT1R to attain inflammatory as well as resolution phenotype. Additionally, the study revealed protection form LPS induced septic shock in absence of CysLT1R which was supported by attenuated systemic response compared to WT and CysLT2R null mice. Further, in chapter VI of the dissertation it was observed that wound healing process requires CysLT1R signaling which could be a result of impaired macr (open full item for complete abstract)

Committee: Sailaja Paruchuri (Advisor); Leah Shriver (Committee Member); Nic Leipzig (Committee Member); Yi Pang (Committee Member); Adam Smith (Committee Member) Subjects: Biochemistry; Chemistry; Immunology

Doctor of Philosophy, Case Western Reserve University, 2021, Molecular Medicine

Genome-wide analyses have uncovered important roles of microRNAs in diabetes mellitus pathogenesis, including evidence suggesting a regulatory relationship between microRNAs, glucose metabolism, and inflammation. Due to their specific expression profiles, microRNAs are interesting as targets for precision medicine or as clinical biomarkers; miR expression can be fine-tuned by different stressors, adding complexity in regulation of miRNAs and its targets. Hyperglycemia (high blood glucose levels) is often a consequence of insulin resistance and obesity. It is diagnosed with a random blood glucose sample ≥ 200 mg/dL with symptoms presenting as increased thirst, weight loss, blurry vision, or polyuria. Individuals diagnosed with diabetes or obesity have low-grade systemic inflammation and predisposition for developing certain cancers, like breast cancer. Interventional studies suggest that maintaining better blood glucose control improves overall health and reduces breast cancer risk. First, we identified a differential regulation of glucose clearance: inhibiting miR-467, using an antagonist, in chow-fed mice raised insulin to levels comparable to western diet-fed mice, increased fasting blood glucose levels, inhibited glucose clearance, and increased inflammation and macrophage content in adipose tissue. Our results suggest that miR-467 normally prevents inflammation in adipose tissue in response to hyperglycemia, and targets unknown transcripts that regulate glucose and insulin levels. Interestingly, the anti-inflammatory role of miR-467 is lost in mice fed a long-term western diet (rich in fat, sugar and cholesterol): antagonist injections in western diet mice lowered insulin levels and improved glucose clearance despite elevated fasting blood glucose levels, suggesting improved sensitivity to insulin. Second, we investigated how miR-467 regulates hyperglycemia-induced inflammation and breast cancer growth, exploring its potential as a clinical biomarker (open full item for complete abstract)

Committee: Olga Stenina-Adognravi PhD (Advisor); Suneel Apte M.B.B.S., D.Phil. (Committee Chair); Keith McCrae MD (Committee Member); Donna Driscoll PhD (Committee Member); Eugene Podrez MD, PhD (Committee Member) Subjects: Biomedical Research

Doctor of Philosophy, The Ohio State University, 2020, Molecular, Cellular and Developmental Biology

Chronic lymphocytic leukemia is the most common type of leukemia in adults in the United States. Characterized by the accumulation of mature B cells, this malignancy requires the presence of a supportive microenvironment that promotes cell proliferation and survival. Nurse-like cells (NLCs) are the CLL tumor-associated macrophages, whose role has been explored for the last 20 years. Nevertheless, there is still a long way to understand their biology and to study new strategies that modify their supportive role, for better therapeutic efficacy. Here, we studied the potential use of targeting NLCs for therapeutic approaches. In the first part, we explored the nature of NLC development and found a therapeutic approach to block their differentiation and supportive role in CLL. We found that NLC differentiation is characterized by a significant loss of methylation, especially in AP-1 transcription factor binding sites. This finding further led us to the discovery that the MEK signaling pathway may be responsible for NLC differentiation and that its inhibition results in decreased disease burden in a mouse CLL model. Thus, inhibition of MEK signaling, especially with the FDA approved trametinib, could potentially offer a therapeutic option. We finally found that during NLC differentiation, there are significant changes in gene expression, some of them related to the MEK signaling pathway; this may be of use to further explore potential therapeutic targets to inhibit NLC development. In a second study, we focused on the regulation of antibody-mediated responses by inhibitory, checkpoint receptors. NLCs, similar to macrophages, have the potential to phagocytose antibody-coated CLL cells. With the increase of therapeutic antibody use in the clinic and the critical role of the anti-CD20 rituximab in CLL therapy, understanding how the IgG antibody receptors (FcγR) are regulated by checkpoint molecules is of importance. In this context, we found that the platelet endothelia (open full item for complete abstract)

Committee: Susheela Tridandapani Ph.D. (Advisor); Jonathan Butchar Ph.D. (Committee Member); Christopher Oakes Ph.D (Committee Member); Jennifer Woyach M.D. (Committee Member); Santiago Partida-Sánchez Ph.D. (Committee Member) Subjects: Immunology; Oncology