Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 5 uppsatser.

Diisocyanates (dNCO) such as methylene diphenyl diisocyanate (MDI) are used primarily as cross-linking agents in the production of polyurethane products such as paints, elastomers, coatings and adhesives, and are the most frequently reported cause of chemically induced immunologic sensitization and occupational asthma (OA). Immune mediated hypersensitivity reactions to dNCOs include allergic rhinitis, asthma, hypersensitivity pneumonitis and allergic contact dermatitis. There is currently no simple diagnosis for the identification of dNCO asthma due to the variability of symptoms and uncertainty regarding the underlying mechanisms. Immunological sensitization due to dNCO exposure is traditionally thought to require initial conjugation of the dNCO to endogenous proteins to generate neoantigens, which trigger production of dNCO specific T lymphocytes and ultimately dNCO specific IgE. Testing for dNCO-specific IgE, for diagnosis of dNCO asthma is however, only specific (96-98%) but not sensitive (18-27%). The low prevalence of detectable dNCO specific IgE has been attributed to both assay limitations and a potential IgE-independent dNCO asthma mechanism(s). The identity of the conjugated proteins responsible for the sensitization also remains unknown. It is also not clear whether dNCOs bind to extracellular, cell membrane, or intracellular proteins as a way of triggering non-IgE asthma. Standardization and optimization of immunoassays used to screen for dNCO specific antibodies in sera is important if its utility as a dNCO asthma diagnostic tool is to be achieved. This will potentially improve sensitivity and allow comparison of results across studies. Current studies on assays of dNCO-specific IgE and IgG lack or have limited characterization of the conjugates used. Diisocyanates bound to hemoglobin (Hb), human serum albumin (HSA), and THP-1 proteins were quantified by HPLC with fluorescence detection. Proteomic tandem mass spectrometry (MS) was used to delineate TDI and MDI specific amino acid binding sites on Hb as well as identification of proteins from MDI exposed THP-1 cells. The trinitrobenzene sulfonic acid assay (TNBS) and SDS gel electrophoresis were used to evaluate extent of intra and intermolecular cross-linking in dNCO-HSA conjugates. Binding of monoclonal antibodies (mAbs) to dNCO bound proteins in enzyme-linked immunosorbent assay (ELISA) was used to evaluate antigenicity of dNCO-protein conjugates. The amount of dNCO binding to HSA and Hb increased with the concentration of the dNCO used for conjugation. All the dNCOs reacted with HSA more than with Hb. Eight binding sites were observed with both MDI and TDI on Hb. The N-terminal valines of both the alpha and beta subunits on Hb, lysine 40 of the alpha subunit and lysine 61 of the beta subunit were common binding sites for both TDI and MDI. Lysine 7 of the alpha subunit and lysines 8, 65 and 66 of the beta subunit were unique to MDI. On the other hand, lysines 11, and 16 of the alpha subunit and lysines 17 and 144 of the beta subunit were unique to TDI. Protein bound MDI was detected in a dose-dependent manner in membrane and cytoplasm fractions of MDI exposed THP-1 cells. MDI was also detected in 11 of the 13 cytoplasmic protein bands. The extent of MDI intracellular protein binding was not affected by cytochalasin D, a chemical that binds actin filaments and inhibits active uptake into cells. The extent of cross-linking shown using the TNBS assay was found to increase with amount of dNCO used. Clear bands from both intra and intermolecular cross-linking were observed on all dNCO-Hb/HSA SDS gels. Using ELISA, both TDI-Hb and TDI-HSA conjugates were reactive to monoclonal antibodies produced against TDI conjugated HSA indicating that dNCO-Hb is also antigenic. The best characterization of dNCO-protein conjugates is achieved by the quantitative determination of conjugated dNCO per mole of protein as well as determining the extent of dNCO cross-linking. Although HSA is more reactive to dNCOs than other serum proteins such as Hb, contribution from other serum proteins to development of OA should not be overlooked as dNCO-Hb was found to be reactive to dNCO specific mAbs. dNCO-conjugated proteins identified in the soluble fraction of MDI exposed THP-1 cells were all of intracellular origin suggesting that MDI can cross the cell membrane and react with intracellular proteins. The entry of MDI into live cells is a passive process, as the extent of intracellular binding was not affected by cytochalasin D. The present study support the potential involvement of dNCO-haptenated membrane and intracellular proteins in development of non-IgE dNCO asthma.

Ferroptosis is a regulated junction between cell death, metabolism, and disease, and it hasbeen implicated in many pathologies. The assorted ferroptosis pharmacology modulators offer valuable means to modulate ferroptosis in multiple diseases, to explore disease etiology, and to develop potential therapeutics. In the first part, the work focuses on inhibiting ferroptosis in a Huntington’s disease model. Ferrostatin-1 (Fer-1) is a potent small-molecule ferroptosis inhibitor that has been adopted to investigate the role of ferroptosis in many disease models. However, its further application is limited by its low potency, poor stability, possible toxicity, and lack of brain penetration. We developed the fourth and fifth generations of ferrostatins and investigated the in vitro and in vivo pharmacokinetics of lead compounds. We identified PHB4082 preferentially accumulating in the kidney as a potential candidate for kidney disease-relevant contexts. Moreover, TH-4-55-2 displayed an excellent brain penetration, preferentially accumulating in the brain at concentrations of magnitude higher than the in vitro IC50 values. In the in vivo toxicity study, it was well-tolerated over 30 days in wild-type and R6/2 mice and exhibited a protective effect against weight loss in a Huntington’s disease model, suggesting it is a strong candidate for application in HD and more neurodegenerative disease models. The second part describes the efforts to explore the therapeutic potential of inducing ferroptosis in a tumor model. Imidazole ketone erastin (IKE) induced ferroptosis by specifically inhibiting system xc– in a subcutaneous xenograft model of Diffuse Large B Cell Lymphoma (DLBCL), suggesting the potential of IKE as a therapeutic strategy for cancer. A biodegradable polyethylene glycol-poly (lactic-co-glycolic acid) nanoparticle formulation was used to aid in delivering IKE to cancer cells in vivo, exhibiting improved tumor accumulation and therapeutic index relative to free IKE, indicating its potential for treating DLBCL. In summary, this work explored the possibility to modulate ferroptosis using small molecule modulators in multiple disease models and identified some potential drug candidates and useful chemical probes.

Climate change is already harming public health through warmer, more erratic weather and many downstream consequences. Research can support climate change adaptation by characterizing climate-related exposures, identifying vulnerable populations, and identifying effective interventions. Furthermore, the main source of greenhouse gas emissions, fossil fuel combustion, also produces air pollutants such as fine particulate matter (PM2.5) that directly harm human health. In this dissertation my colleagues and I have examined the effects of short-term temperature exposure on myocardial infarction and stroke to inform adaption (Chapters 1 to 3) and leveraged multiple exposure models to estimate annual PM2.5 concentrations and quantify uncertainty (Chapter 4). To examine the effects of short-term temperature exposure, we conducted case-crossover analyses using an administrative dataset of hospitalizations in New York State. For PM2.5 prediction and uncertainty characterization, we applied a flexible ensemble approach to leverage seven already-developed PM2.5 models. Overall, we found that warmer hourly temperatures and greater daily temperature variability increased the risk of ischemic events, and lower hourly temperatures increased the risk of hemorrhagic stroke. The ensemble model showed high predictive accuracy, demonstrating the strength of this approach, and we observed greatest uncertainty in the Pacific Northwest and southeast coast. The methods applied in this dissertation can be applied to other exposures and in different settings to further quantify the risks of climate impacts and improve air pollution assessment. Future research should examine the joint impacts of multiple weather factors, strategies to protect people in group housing from extreme weather, measurements and models to reduce uncertainty of air pollution exposures, and propagate exposure uncertainty into health models. However, the need for further research should not delay climate action today.

Traumatic brain injury (TBI) is a prevalent condition worldwide with 1.7 million incidences in the U.S. alone. A range of clinical outcomes have been reported post TBI, including dementia, memory loss, and impaired balance and coordination. The lack FDA approved treatments for TBI drives the need for improved prevention and therapeutic strategies. Finite element (FE) models of brain injury mechanics can be used to advance these efforts. These computational models require appropriate constitutive properties in order to predict accurate brain tissue response to injury loading. Suitable experimental models need to be implemented to match the resolution and computational power of FE models. The first aim of this thesis was to characterize the mechanical properties of brain tissue. Here, human, porcine, and rat brain tissue mechanical responses to multistep indentation of increasing strains up to 30% strain were recorded. We tested whether the quasilinear theory of viscoelasticity (QLV) was required to capture the mechanical behavior of brain tissue, but observed that linear viscoelasticity was sufficient under the loading condition applied. Using this fitting model, brain tissue stiffness was found to be dependent on anatomical region, loading direction, age, sex and species to varying degrees. This analysis elucidated factors that affect brain tissue injury mechanics and can be used to improve the accuracy of FE models of brain tissue deformation to predict a biofidelic response to TBI. There is growing evidence linking TBI to pathologies leading to increased risk of neurodegeneration, like tauopathies. However better understanding of these underlying mechanisms is still needed. In our study, we utilized a custom shock tube design to induce blast TBI (bTBI). To isolate the effect of bTBI-induced tau pathology, tau was extracted from sham and shockwave exposed mice 24 hours post injury, referred to as sham and blast tau respectively. We showed that bTBI increased phosphorylation of tau and its propensity to oligomerize. Treatment with blast tau resulted in impaired behavior in mice as well as reduced long term potentiation (LTP) in acute hippocampal slices. Treatment with brain isolate from shockwave exposed tau knockout mice did not exhibit altered behavior or LTP response, eliminating the possibility that any confounding factor in the blast tau preparation was responsible for the impaired outcome. Administration of de-oligomerized blast tau prevented these cognitive impairments, suggesting that toxic effect of blast tau was attributed to its oligomeric form. Here we showed that blast injury can initiate cascades in tau pathology and exposure to this progression results in worsened neurological outcome. Tau phosphorylation is mainly regulated by protein phosphatase 2A (PP2A), whose activity can be altered by leucine carboxyl methyltransferase 1 (LCMT-1) and protein phosphatase methylesterase 1 (PME-1). We sought to leverage this mechanism by infusing LCMT-1 and PME-1 transgenic mice with sham and blast tau. LCMT-1 overexpression prevented behavior and LTP deficits induced by oligomeric blast tau. Furthermore, PME-1 overexpression worsened behavior and LTP response at subthreshold doses of oligomeric blast tau. Together, this illustrated the ability of these two enzymes to regulate the response to exposure of bTBI-induced pathogenic forms of tau. This study indicates the potential of targeting PP2A activity as a viable strategy for therapeutic intervention. In conclusion, this research expands our understanding of the complexity of brain tissue injury mechanics to inform computational models of TBI, illustrates the deleterious effect of pathogenic forms of tau induced by blast injury on cognitive function, and presents a potential target mechanism for the investigation of therapeutic strategies.

Staphylococcus aureus is a pulmonary pathogen associated with substantial morbidity and mortality. It is a common complication of influenza and SARS CoV2 infection, chronic obstructive pulmonary disease, cystic fibrosis and is a major cause of ventilator associated pneumonia. The prevalence of this specific organism as a respiratory pathogen has been attributed to its many gene products that thwart innate immunity. However, vaccines targeting virulence determinants have failed to be protective in humans, suggesting that other bacterial or host factors are also critical in pathogenesis. We postulated that S. aureus that are able to persist in the lung must adapt to substrates that are especially abundant. Here we show that among the many potential carbon sources in the infected airway, S. aureus is directed by carbon catabolite repression (CCR) to utilize proline. By following transcriptomic and metabolomic changes over the initial course of infection by human clinical isolates of S. aureus, we established that CcpA and CcpE upregulate expression of the S. aureus collagenase (scpA) and proline transporter (putP). In response to infection, airway fibroblasts synthesize collagen, of which proline is a major component. Host-adapted S. aureus is thus poised to ingest and metabolize newly available proline which fuels oxidative metabolism via the TCA cycle, outcompeting strains that have not made this metabolic transition. Thus, clinical settings characterized by airway repair processes and fibrosis provide a milieu that is intrinsically supportive of S. aureus infection.

Advances in sequencing technologies have sparked the discovery of new genetic etiologies for neurological and neurodevelopmental disorders. As new disease-causing mutations are unveiled, questions into the specific mechanisms of pathogenicity and potential therapeutic approaches arise. To address these questions, in vivo and in vitro models have been generated and analyzed; but how best to utilize these models, and how well they recapitulate the human brain, is still not fully understood. Within the work discussed in this thesis, we address this problem through the transcriptomic and functional interrogation of these models in the context of neurodevelopment and disease. In Chapter 2 of this thesis, we describe the use of single-cell RNA-sequencing to examine the longitudinal transcriptomic profiles of neuronal network establishment and maturation in ex vivo mouse cortex- and hippocampus-derived cultures. Our data highlights unique developmental transcriptomic profiles for individual genes, disease gene subclasses, and biological processes, and discusses cell population-specific divergent transcriptomic profiles between genes associated with neurological diseases, focusing on epilepsy and autism spectrum disorder. We also compared the data from our ex vivo system to transcriptomic data collected from in vivo neonatal and adult mouse brains and human cortical organoids, highlighting the importance of the generation and consideration of system-specific transcriptomic datasets when looking into a gene, disease, or biological process of interest, and serves as a vital resource for researchers. In Chapter 3, we propose a high-throughput drug discovery paradigm utilizing the application of transcriptome reversal for neurodevelopmental disorder-associated genes that affect the transcriptome. This approach describes the idea that if gene dysregulation is causal for the pathogenicity of a disease, then correcting the transcriptional signature should have a therapeutic effect. We demonstrated that small-molecule induced gene expression changes vary between both cell lines and neural cell populations, and highlight both the importance of selecting the appropriate model of disease and creating cell population-specific signatures for compounds and disease. In Chapter 4, we focus on the utilization of multi-electrode arrays for the electrophysiological characterization of primary cortical networks derived from mouse models of epileptic encephalopathy. This technique allows for the analysis of numerous neuronal and network synchronization metrics for spontaneous longitudinal activity and responses to external stimuli in the form of electrical stimulation and compound addition. In particular, mouse models with mutations in the genes Grin2a, Gnb1, and Scn1a were analyzed. We discovered significant hyperexcitability, bursting, and synchrony phenotypes, and discuss how acute and chronic compound addition can be used to interrogate biological pathways and reverse disease activity signatures.

This study was designed to assess the relationship of dietary and serum beta-carotene to the development of oral lesions in smokeless tobacco users. Eighty eight smokeless tobacco users without oral lesions and 18 with lesions participated in the study. Dietary intake of beta-carotene, personal health habits and selected dietary intake were analyzed by questionnaire. Serum levels were assessed by high pressure liquid chromotography. No correlation between dietary beta-carotene intake and serum beta-carotene levels was observed. Results of t-test analysis indicated no significant difference in the mean serum or dietary intake of beta-carotene between the groups. Mean serum beta-carotene for group-I, users without oral lesions and group-2, users with oral lesions were 12.3 ug/dl(I.02 SE), and 10.6 ug/dl(1.59 SE), respectively. Stepwise regression techniques were employed to assess the influence of selected variables on serum beta-carotene. The effects of smoking, smokeless tobacco exposure, alcohol consumption, and age as well as other dietary indices were evaluated. Age (1.015,p<0.001) was the only factor found to influence serum beta-carotene levels. The small sample size and variability within groups may have decreased the likelihood of observing statistical significance for serum beta-carotene between groups. / Master of Science

O efeito anti-aterogênico da HDL foi sugerido ser devido, parcialmente, à ação da paraoxonase (PON) associada à HDL. Três SNPs em PON1 (L55M e Q192R) e PON2 (S311C) têm sido implicados como fatores de risco independentes para doença arterial coronariana (DAC) em alguns, mas não todos os estudos. O efeito destes três polimorfismos do gene PON na DAC e na concentração sérica de lipídios, apolipoproteínas A-I e B (apo A-I e apo B) foi investigado em 221 indivíduos sem vínculos genéticos. Os três polimorfismos genéticos (L55M, Q192R e S311C) do gene PON foram analisados por PCR-RFLP. Não houve diferença entre a distribuição genotípica e a freqüência relativa dos alelos dos polimorfismos L55M, Q192R e S311C do gene PON entre pacientes e controles. Não houve associação entre os polimorfismos da PON e a DAC na população estudada. As freqüências dos haplótipos dos pacientes foram similares às encontradas no grupo controle. O polimorfismo L55M do gene PON1 está associado com variações na concentração sérica de apo A-I dos pacientes. O polimorfismo S311C do gene PON2 não está associado com variações na concentração sérica de lipídios, apo A-I e apo B dos pacientes. / The anti-atherogtenic effect of the HDL has been suggested to be due, partially, to the action of the HDL-associated paroxonase(PON). Three SNPs in PON1 (L55M e Q192R) and PON2 (S311C) have been involved as independent risk factors for coronary artery disease (CAD), in some, but not all studies. The effect of these three polymorphisms of the PON gene on CAD and on the levei of lipids, apolipoproteins A-I e B (apo A1 and apo B) was investigated in 221 genetically unrelated individuais. The three gene polymorphismis (L55M, Q192R e S311C) of the PON gene were analyzed by PCR-RFLP. Is no significant difference between distribuition of genotype and allele frequencies of the L55M, Q192R and S311C polymorfisms of the PON gene between patient group and controls. Is no association between polymorphisms of the PON gene and CAD in the population studied. The frequencies of the haplotypes in the patients were similar to those found in the control group. The L55M polymorphism of the PON1 gene is associated with variations of the level of polipoprotein A-I at patients. The S311C polymorphism of the PON2 is not associated with variations of the level of lipids, apo A-I and apo B at patients.

The vertebrate eye lens functions to focus light onto the retina to produce vision. The lens is composed of an anterior monolayer of cuboidal epithelial cells that overlie a core of organelle free fiber cells. The lens develops and grows throughout life by the successive layering of lens fiber cells via their differentiation from lens epithelial cells. Lens developmental defect and damage to the lens are associated with cataract formation, an opacity of the lens that is a leading cause of visual impairment worldwide. The only treatment to date for cataract is by surgery. Elucidating those molecules and mechanisms that regulate the development and lifelong protection of the lens is critical toward the development of future therapies to prevent or treat cataract. To determine those molecules and mechanisms that may be important for these lens requirements we employed high-throughput RNA sequencing of microdissected differentiation statespecific lens cells to identify an extensive range of transcripts encoding proteins expressed by these functionally distinct cell types. Using this data, we identified differentiation state-specific molecules that regulate mitochondrial populations between lens epithelial cells that require the maintenance of a functional population of mitochondria and lens fiber cells that must eliminate their mitochondria for their maturation. In addition, we discovered a novel mechanism for how lens epithelial cells clear apoptotic cell debris that could arise from damage to the lens and found that UVlight likely compromises this system. Moreover, the data herein provide a framework to determine novel lens cell differentiation state-specific mechanisms. Future studies are required to determine the requirements of the identified molecules and mechanisms during lens development, lens defense against damage, and cataract formation. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2016. / FAU Electronic Theses and Dissertations Collection