Introduction: Rates of childhood-onset type 1 diabetes (T1D) are steadily increasing among American youth, yet Black Americans are more likely to suffer from serious T1D-related complications caused by poor glycemic control. The aim of this thesis is to determine the external factors that are causing discrepancies in the development, diagnosis, treatment, and long-term management of T1D in Black youth. Methods: Epidemiological studies were compiled from the American Diabetes Association, Center for Disease Control (CDC), International Diabetes Foundation (IDF), Kaiser Family Foundation (KFF), and the Claremont Colleges Library network to identify the sociocultural aspects that negatively affect long-term glycemic control in Black youth. Results: Studies indicate that Black youth with T1D are more likely to face disadvantages in treatment regimen which are attributed to insurance coverage, socioeconomic status, education level, and implicit bias. Most studies demonstrate that these factors result in poor glycemic control, which subsequently leads to severe dysglycemia-related complications later in life. Conclusion and Discussion: Many Black youth who suffer from T1D receive insufficient healthcare, which is often exacerbated by a lack of social and economic resources. As a result, they may not have the means to maintain consistent, healthy glycemic levels. System-level changes are necessary to change the morbidity and mortality of T1D in Black youth. Future research should include the analysis of other racial minority groups in order to uncover additional institutional disparities.

Made available in DSpace on 2014-10-09T12:42:37Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T13:59:44Z (GMT). No. of bitstreams: 0 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Tese (Doutorado em Tecnologia Nuclear) / IPEN/T / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP / FAPESP:10/52170-9

Made available in DSpace on 2014-10-09T12:42:37Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T13:59:44Z (GMT). No. of bitstreams: 0 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Imunocomprometidos são pessoas susceptíveis a adquirir doenças graves a partir de alimentos contaminados, por possuírem um baixo número de células de defesa. Em consequência a este fato, apresentam alimentação extremamente restrita, evitando qualquer alimento que represente um risco microbiológico. A irradiação é uma das poucas tecnologias que permite garantir a segurança e a qualidade do alimento, controlando microrganismos patogênicos, sem afetar significativamente qualquer atributo organoléptico. Tomando-se por base a restrição alimentar em relação a produtos frescos, este trabalho destinou-se a estudar o efeito da radiação ionizante (raios gama) em saladas de frutas para pessoas imunocomprometidas. Para tanto, frutas minimamente processadas e saladas de frutas foram submetidas às doses de 0,5; 1,0; 1,5; 2,0 e 3,0 kGy e analisadas juntamente com as amostras controle (não irradiadas). Análises físico-químicas (cor, textura, acidez titulável total, pH, atividade de água, sólidos solúveis totais e açúcares solúveis), microbiológicas (estafilococos coagulase positiva, contagem de bolores e leveduras, contagem de bactérias aeróbias, coliformes totais, E. Coli e Salmonella) e sensoriais (testes de aceitação e degustação) foram realizadas, além da aplicação de questionários com chefes de nutrição de hospitais para o entendimento de práticas acerca da dieta hospitalar de imunocomprometidos. Os resultados demonstraram que a irradiação na dose de 3,0 kGy foi eficaz na eliminação de microrganismos da salada de frutas, garantindo a segurança microbiológica dentro dos níveis exigidos pela legislação. Em relação aos resultados físico-químicos, foram observadas alterações de cor e de textura com o aumento da dose de radiação. Resultados de pH, acidez, sólidos solúveis totais e atividade de água apresentaram flutuações de valores principalmente em função dos fatores intrínsecos da fruta, ao passo que saladas de frutas irradiadas com doses de 3,0 kGy, apresentaram uma tendência de ficar mais doces que as amostras controle, obtendo boa aceitação sensorial. Além disso, os resultados dos questionários realizados em hospitais reportaram uma necessidade de incorporar alimentos frescos à dieta nutricional dos imunocomprometidos, bem como a criação ou o estabelecimento de protocolos nutricionais no país para essa área. Sendo assim, com base nos resultados microbiológicos, físico-químicos e sensoriais pode-se concluir que saladas de frutas podem ser indicadas para o consumo de imunocomprometidos, desde que tratadas com a dose de 3,0 kGy e produzidas com Boas Práticas de Fabricação, respeitando os critérios de produção desde a matéria-prima até o produto final. / Tese (Doutorado em Tecnologia Nuclear) / IPEN/T / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP / FAPESP:10/52170-9

Background Monoclonal Gammopathy of Undetermined Significance (MGUS) is an understudied precursor of multiple myeloma (MM), the second most prevalent hematologic malignancy in the United States. This dissertation was designed to: (1) Describe the trajectories of serum biomarkers over time in patients with an MGUS diagnosis, (2) Determine if an MGUS diagnosis is associated with changes in healthcare service utilization, and (3) explore the patient- and provider-level drivers of healthcare utilization in patients with MGUS. Methods Data sources include health claims and electronic health records from a community-based population of patients seeking care in central Massachusetts and primary qualitative data collected from providers and patients’ interviews. The analyses included descriptive statistics, group-based trajectory modeling, conditional Poisson regression, and qualitative data analyses. Results (1) Three distinct multi-trajectory groups of creatinine and hemoglobin were identified. (2) The rates of emergency room, hospital, and outpatient visits were higher for patients with MGUS than patients without MGUS. (3) Patients have a basic understanding of MGUS; however, some patients feel anxiety, which may affect other aspects of their lives. Patients primarily see hematologists for follow-up care; other providers have less knowledge about MGUS. Conclusions Biomarker trajectories characterize specific subpopulations of patients with MGUS over time. We found that an MGUS diagnosis is associated with higher healthcare utilization, especially during the months surrounding the diagnosis date. Finally, our study suggests that some patients with MGUS may need psychosocial support services and identifies a gap in knowledge around caring for MGUS patients among primary care providers.

Signal Transducers and Activators of Transcription (STATs) are latent transcription factors that mediate several cellular responses. This protein family consists of seven members, STAT1 – 6 including two closely related molecules, STAT5a and STAT5b, that show 96% amino acid sequence homology and are critical for lymphoid, myeloid and erythroid cell development and function. Activated STAT proteins dimerize and translocate to the nucleus, where they bind to high-affinity DNA motifs to modulate gene expression. We recently identified STAT5b as the critical regulator of IgE-mediated cytokine production in mast cells. STAT5b knockout (KO) cells show decreased sensitivity to IgE-mediated passive systemic anaphylaxis accompanied with decreased production of IL-6 and IL-13 compared to wild type counterparts. Interestingly, STAT5b KO mice demonstrated elevated levels of serum IgE but a normal response to histamine-mediated passive systemic anaphylaxis. The current work demonstrates that STAT5b regulates mast cell function both in vivo and in vitro. Additionally, activated STAT proteins can also form tetramers through an N-terminal domain-mediated oligomerization process when bound to low-affinity tandem motifs. Dr. Warren Leonard’s laboratory generated STAT5a-STAT5b double knock-in (DKI) mice in which STAT5 proteins are phosphorylated and can form dimers but not tetramers. We have now found that bone marrow-derived mast cells from STAT5 DKI mice are defective in IgE-induced cytokine and chemokine production and exhibit defective stem cell factor (SCF)-induced migration and survival responses in vitro. Similarly, IgE-mediated passive systemic anaphylaxis is decreased in STAT5 DKI mice. These data indicate that Stat5 tetramers are critical for some aspects of mast cell function in allergic and inflammatory disease.

HIV-1 protease is a very important drug target for AIDS therapy. Nine protease inhibitors have been proved by FDA and used in AIDS treatment. Due to the high replication rate and the lack of fidelity of the HIV-1 reverse transcriptase, HIV-1 virus developed various drug-resistant variants. Although experimental methods such as crystallography and isothermal titration calorimetry provide structural and thermodynamic data on drug-resistant variants, they are unable to discern the mechanism by which the mutations confer resistance to inhibitors. Understanding the drug-resistance mechanism is crucial for developing new inhibitors more tolerant to the drug-resistant mutations. Computational methods such as free energy calculations and molecular dynamic simulations can provide insights to the drug resistance mechanism at an atomic level. In this thesis, I have focused on the elucidation of the energetic and dynamics of key drug-resistant variants of HIV-1 protease. Two multi-drug resistant variants, in comparison with wild-type HIV-1 protease were used for the comparisons: Flap+ (L10I, G48V, I54V, and V82A) which contains a combination of flap and active site mutations and ACT (V82T, I84V) that only contains active site mutations. In Chapter II, I applied free energy simulations and decomposition methods to study the differential mechanism of resistance to the two variants, Flap+ and ACT, to the recently FDA-approved protease inhibitor darunavir (DRV). In this study, the absolute and relative binding free energies of DRV with wild-type protease and the two protease variants were calculated with MM-PB/GBSA and thermodynamic integration methods, respectively. And the predicted results are in good agreement with the ITC experimental results. Free energy decomposition elucidates the mutations alter not only its own interaction with DRV but also other residues by changing the geometry of binding pocket. And the VdW interactions between the bis-THF group of DRV is predominant even in the drug-resistant variants. At the end of this chapter, I offer suggestions on developing new inhibitors that are based on DRV but might be less susceptible to drug-resistant mutations. In Chapter III, 20-ns MD simulations of the apo wildtype protease and the apo drug-resistant protease variant Flap+ are analyzed and compared. In these studies, these mutations have been found to decrease the protease flexibility in the apo form but increase the mobility when the protease is binding with inhibitor. In Chapter IV, more details of the free energy simulation and decomposition are discussed. NMR relaxation experiments were set up as a control for the MD simulation study of the dynamics of the Flap+ variant. The difficulty of finishing the NMR experiment is discussed and the solution and some preliminary results are shown. In summary, the scope of this thesis was to use computational methods to study drug-resistant protease variants’ thermodynamic and dynamic properties to illuminate the mechanism of protease drug resistance. This knowledge will contribute to rational design of new protease inhibitors which bind more tightly to the protease and hinder the development of drug-resistant mutations.

Gain of function NOTCH1 mutations are common in both patients with T-ALL and in mouse models of the disease. Inhibiting the Notch pathway in T-ALL cell lines results in growth arrest and/or apoptosis in vitro, suggesting a requirement for Notch signaling in T-ALL. Therefore, we sought to examine the role of Notch1 signaling in both premalignancy and in the maintenance of leukemic growth. Using a murine model of T-ALL, in which expression of the Tal1 and Lmo2 oncogenes arrests thymocyte development, our preleukemic studies reveal that Notch1 mutations are early events that contribute to the clonal expansion of DN3 and DN4 progenitors. We also demonstrate that progenitors are maintained within the tumor and are enriched in leukemia-initiating cell (L-IC) activity, suggesting Notch1 may contribute to L-IC self-renewal. By studying the effects of Notch signaling in murine T-ALL cell lines, we also demonstrate that Notch1 promotes the proliferation and survival of leukemic blasts through regulation of Lef1 and the Akt/mTOR pathways. Given that T-ALL cell lines are dependent on Notch signaling in vitro, we investigated the effects of Notch inhibition in vivo. We provide evidence that Notch1 can be successfully targeted in vivo and that Notch inhibition, with γ-secretase inhibitors (GSIs), significantly extends the survival of leukemic mice. We also demonstrate that administration of GSIs in combination with rapamycin inhibits human T-ALL growth and extends survival in a mouse xenograft model. Given that NOTCH1 may be required to maintain both L-IC and bulk leukemic growth, targeting NOTCH1 may prove to be an efficacious targeted therapy for T-ALL patients with aberrant NOTCH1 activation.

Celiac Disease (CD) is an autoimmune disorder that emerges due to the ingestion of gluten, a protein found in a variety of common grains such as wheat, rye, and barley. Approximately 1 in 100 individuals in the US suffer from CD, making it the most commonly diagnosed gastrointestinal disorder (Ciclitira et. al., 2005). These proline-rich gluten peptides are resistant to proteolysis and accumulate in the duodenum of the small intestine. Once in the duodenum, these peptides illicit an autoimmune response resulting in villous atrophy. Current treatment for CD requires a rigorous adherence to a gluten-free diet. Nevertheless, gluten-containing grains are ubiquitous in the western diet, so accidental exposure to gluten remains as a persistent threat. The approach of this project centers on genetically engineering a strain Lactobacillus reuteri to secrete a Myxococcus xanthus prolyl endopeptidase (PEP), an enzyme that hydrolyzes a peptide bond adjacent to an internal proline residue. The data from this study revealed that recombinant M. xanthus PEP purified from E. coli was effective in degrading Suc-Ala-Pro-pNA, a chromogenic substrate containing an internal proline residue. When introduced into a L. reuteri expression vector, mutations accumulated in the vector over the course of 5 days. These data suggested that toxicity was possibly associated with M. xanthus PEP and the amyl signal peptide.

Celiac disease (CD), an autoimmune disorder affecting millions of Americans, poses significant obstacles leading to a normal life. With no known cure, adherence to a strict glutenfree diet (GFD) is essential. However, the cost and limited availability of gluten-free alternatives can burden individuals with CD. Additionally, factors such as socioeconomic status, nutrient deficiencies, and the nature of the disease may contribute to mental health issues. This study aimed to investigate the influence of adherence to a GFD on depression in CD patients. The prevalence of depression among individuals with CD from diverse backgrounds was examined, along with the analysis of macro- and micronutrient distribution and the impact of the GFD. Data from the National Health and Nutrition Examination Survey spanning three cycles (2009-2014) were extracted, including 70 CD patients and 271 participants reporting general gluten issues. The dataset was analyzed using SAS v9.4 (SAS Institute, Inc., Cary, NC) with the three cycles merged using a unique identifier sequential number. Sample weights were applied to mitigate bias in national estimates due to unequal probability of selection, while oversampling was utilized to enhance the study’s reliability when examining subgroups or minorities. Survey weight and sampling design considerations were incorporated into the SAS syntax to safeguard participants’ privacy, as managed by the National Center for Health Statistics. Multiple linear regression analysis revealed no significant association between depression and adherence to the GFD or CD; however, ethnicity showed significance. Celiac disease exhibited a prevalence of 0.12% among White individuals, 3-6 times higher than other ethnic groups, and was twice as prevalent in females compared to males. Notably, deficiencies in macro- and micronutrients among CD and GFD cases were observed. Carbohydrate intake exhibited a negative association with GFD consumers and those with CD, while individuals adhering to a GFD showed an association with decreased polyunsaturated fat consumption, yet within adequate intakes. Deficiencies in micronutrients such as thiamin, vitamins B12, D, and E, and calcium were also observed within GFD group, while a low sodium intake was observed among CD group. This study provides insights into the complex interplay between diet, mental health, and CD management.

Equine protozoal myeloencephalitis (EPM), caused by the protozoan parasite Sarcocystis neurona, is one of the most important neurological diseases of horses in the Americas. While seroprevalence of S. neurona in horses is high, clinical manifestation of EPM occurs in less than 1% of infected horses. Factors governing the occurrence and severity of EPM are largely unknown, although horse immunity might play an important role in clinical outcome. We hypothesize that EPM occurs due to an aberrant immune response, which will be discernable in the equine IgG subisotypes a, b, and (T) that recognize S. neurona in infected diseased horses versus infected but clinically healthy horses. Based on previously-established serum antibody concentrations for IgG subisotypes in healthy horses, standard curves were generated and served to establish the concentration of antigen-specific IgG subisotypes in equine serum and CSF in infected diseased and infected normal horses. The subisotype concentrations and ratios between subisotypes were analyzed to assess whether neurological disease is associated with detectable differences in the antibody response elicited by infection. Results indicate a type I biased immune response in infected diseased horses, implicating the role of immunity in the development of EPM.