Streptococcus pneumoniae é a maior causa de doenças infecciosas invasivas. As vacinas conjugadas, ao contrário das vacinas polissacarídicas, se mostram eficazes na indução de uma resposta protetora em crianças menores de 2 anos de idade. Entretanto, apesar do sucesso na redução dos casos de doenças pneumocócicas, publicações recentes têm mostrado um aumento nos casos de doença por sorotipos de S. pneumoniae não inclusos nas vacinas conjugadas atualmente licenciadas. Com o objetivo de evitar a substituição dos sorotipos prevalentes e aumentar a cobertura vacinal, nosso projeto prevê a introdução de uma proteína de superfície do pneumococo como carreador em uma vacina conjugada. A Proteína de Superfície Pneumocócica A (PspA), por ser conservada entre diversos sorotipos e por sua alta imunogenicidade, foi escolhida para constituir o conjugado, juntamente com o Polissacarídeo Capsular de S. pneumoniae sorotipo 6B (PS6B), um sorotipo de elevada prevalência no Brasil. Também foi avaliada a influência de diferentes espaçadores entre a proteína carreadora e o PS sobre a resposta imune induzida. Estudos envolvendo diferentes condições reacionais tiveram como objetivo o estabelecimento de um protocolo de conjugação com elevado rendimento. A substituição do ativador EDAC (hidrocloreto de 1-[3-(dimetilamino)propil]-3-etilcarbodiimida), comumente empregado na ativação do grupo carboxila da proteína, pelo DMT-MM (cloreto de 4-(4,6-dimetoxi-1,3,5-triazin-2-il)-4-metilmorfolino) foi capaz de aumentar significativamente o rendimento reacional, o qual passou de menos de 10% para aproximadamente 50%. A proteção com formaldeído dos grupos lisinas da PspA aumentou a especificidade da reação de conjugação, com consequente aumento no rendimento reacional. A PspA modificada manteve a mesma estrutura secundária apresentada pela PspA nativa (análise por dicroísmo circular - CD) bem como sua imunogenicidade (título de anticorpos, capacidade de deposição de complemento e atividade opsonofagocítica). Apesar das análises por CD mostrarem que a conjugação alterou a estrutura secundária da PspA, ensaios imunológicos mostraram que essa desestruturação não afetou a sua capacidade de induzir uma resposta imune protetora. A resposta anti-PspA e anti-PS6B induzida pelo conjugado mostrou-se dose dependente, muito embora o PS6B tenha se comportado como um antígeno pouco imunogênico. / Streptococcus pneumoniae is the main cause of invasive infectious diseases. Unlike polysaccharide (PS) based vaccines, conjugate vaccines have shown to protect children less than 2 years old. However, regardless of the success in reducing pneumococcal disease, recent reports have shown an increase in the rate of disease caused by serotypes not included in licensed conjugate vaccines. In order to avoid serotype replacement, our project proposes to test a surface exposed pneumococcal protein as carrier. Pneumococcal Surface Protein A (PspA), a conserved protein among various serotypes, and highly immunogenic was chosen to take part in the conjugate together with the Polysaccharide Serotype B of Streptococcus pneumoniae (PS6B), a serotype of high incidence in Brazil. In this sense, studies involving different reactional conditions were aimed at stablishing a high yield conjugation protocol. Also, the influence of different spacer between carrier protein and polysaccharide were evaluated on immune response. The replacement of EDAC (1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride), usually used in the activation of protein carboxyl groups, to DMT-MM (4-[4,6-Dimethoxy-1,3,5-triazin-2-yl]-4-methylmorpholinium chloride), was able to significantly increase the reactional yield from less than 10% to approximately 50%. The protection of lysines groups of PspA with formaldehyde increased conjugation specificity, with subsequent increase in reactional yield. Modified PspA maintained the same secondary structure of native PspA (circular dichroism analyses - CD) as well as its immunogenicity (antibody titers, deposition of complement and opsonophaghocytic activity). Despite circular dichroism analyses have shown changes in PspA secondary structure, immunological tests proved that this alteration did not affect its ability in inducing a protective immune response. Anti-PspA and anti-PS6B responses proved to be dose dependent, although PS6B acted as a poor immunogenic antigen in mice.

Epidemiologic systems failure (ESF) is a major hurdle in minimizing the spread of infectious diseases during outbreaks. The reasons for ESF include the technical limitation of personnel handling epidemic crises, strictly defined health policies that limit the actions of epidemiologists, and personal perspective's reservations towards the intentions of health agencies. The purpose of this triumvirate mixed-methods case study was to examine factors of infectious disease control mechanisms useful for determining ESF. Three juxtaposed pre-emptive factors (technical [T], organizational [O], and personal [P] perspectives were used to determine how the multiple perspectives inquiring systems and fuzzy logic revealed factors causing ESF so that remedial tools may be constructed. The juxtaposed ESF-TOP model formed the research theoretical framework and allowed for clustering the ESF factors. Data sources were direct quotations from TOP based secondary data of 4 well-publicized participants; who had Ebola, HIV-AIDS, Tuberculosis, or Typhoid disease; and randomized quantitative TOP hypothetical data sets were created with Microsoft Excel software and used to model an Ebola outbreak of 10 theoretical subjects. Data were analyzed using TOP guidelines from which T, O, and P perspective themes emerged. The findings indicated that a disjointed TOP perspective specifies a serious ESF, a strictly overlapped TOP indicates an effective containment of ESF, and the overall fuzzy set with T given O and P indicates the actual ESF. The findings may result in positive social change by helping epidemiologists identify critical outbreak control factors which may minimize the outbreak impact.

The brain and spinal cord make up the central nervous system (CNS), and as an immune-privileged site, it requires special immune surveillance and regulation. The complement system is a component of innate immunity produced locally in the CNS, since size restrictions from the blood brain barrier prevent complement proteins from easily passing through from the rest of the body. The complement pathway contributes to inflammatory cell recruitment, cell lysis, and opsonization, and thus requires regulation to avoid inappropriate activation. Despite its important role in innate immunity, very little is known about complement production, regulation, and function in the CNS of healthy or diseased individuals. For this dissertation, the central goal was to investigate and characterize the regulation of complement factor H (CFH), a regulator of the alternative pathway of complement activation. CFH polymorphisms have been associated with a number of diseases including atypical hemolytic syndrome, age-related macular degeneration, and Alzheimer's disease, but the regulation of CFH is not well understood, especially in the CNS. To investigate the role of CFH in the CNS, mRNA and protein production in glial cells was first established. The murine CFH (mCFH) promoter was cloned and the transcription start site was identified in astrocytes, microglia, and liver tissue. The mCFH promoter was truncated and different regions were investigated for enhancer or silencer activity. Database mining identified potential transcription factor binding sites, and mutagenesis studies and binding assays identified transcription factor binding candidates. Specifically, the activating protein-1 (AP-1) transcription factors c-Jun and c-Fos bound to a region of the mCFH promoter between – 416 base pairs (bp) and – 175 bp in an electrophoretic mobility supershift assay. Cytokine stimulation increased mCFH mRNA and protein production, as well as the mRNA production of c-Jun and c-Fos and the protein production of c-Jun. These results suggest a relationship between cell cycle and complement regulation, and the investigation of how these transcription factors and CFH affect disease will be a valuable area of research for CNS immune regulation.

The B cell population is one of the key components of the adaptive immune system, which protects the host from a tremendous variety of pathogens by producing antibodies. B cells develop from hematopoietic stem cells through a pathway known as B lymphopoiesis. This is a process accompanied by intensive gene expression reprogramming. By the end, genes appropriate for the B lineage are activated and those that are not are continuously repressed. The regulation of lineage gene expression is conferred by a network of transcriptional regulators. Although some key components have been defined, more factors, especially those orchestrating the repression of non-B lineage genes, remain to be identified. Chemically induced mutagenesis is a potent way of identifying genes with critical biological functions. Injection of n-ethyl-n-nitrosourea, a mutagen, has generated a unique point mutation in the mouse Gon4-like (Gon4l) gene that specifically causes a loss of peripheral B cells while maintaining the T cell population. The mutation is therefore named Justy for Just T cells. The goal of this thesis project is to analyze the Justy mice and provide insights into the mechanisms underlying the regulation of B lymphopoiesis. The work presented here demonstrates that the protein encoded by Gon4l is essential for early B lymphopoiesis, which is likely through the repression of non-B lineage genes. Gon4l protein contains conserved domains implicated in transcriptional repression and associates in a complex with the transcriptional repression mediators Yin Yang 1 and Sin3a/HDAC1, after these proteins are transiently expressed in cell lines. When bound to DNA, Gon4l is capable of repressing a nearby promoter and this function correlates with its ability to form a complex. Therefore, these results suggest that Gon4l may function as a transcriptional regulator by employing its associated co-factors in the identified complex. Lastly, a wide spectrum of tumors developed in Justy mice, indicating that Gon4l can also act as a tumor suppressor.

CD40 signals are required for productive immune responses but also play a role in autoimmune disease pathogenesis. The major goal of this research was to investigate the contribution of two receptors to the development of autoimmune disease: (1) LMP1, an oncogenic EBV-encoded mimic of CD40 and (2) a naturally-occurring polymorphism in CD40, P227A, which appears to confer LMP1-like properties to the CD40 receptor. Interestingly, hCD40-P227A is overrepresented in individuals of Mexican and South American descent. Although this allele is not directly associated with SLE incidence in Hispanic populations, patients of Hispanic ethnicity have a tendency toward increased severity of SLE symptoms, particularly lupus nephritis. This work reports the initial genetic description of CD40-P227A and characterizes its gain-of-function signaling properties in mouse and human B cells. In comparison to Wt-CD40 signaling, CD40-P227A signaling results in increased binding of TRAF3, TRAF5, and Act1, as well as enhanced secretion of IL-6, TNF-α, and Ig due to a selective hyperactivation of the JNK pathway. Whereas TRAF3 is normally a negative regulator of Wt-CD40 signaling, TRAF3 is a required positive regulator of CD40-P227A signaling as demonstrated in TRAF3-deficient B cells. LMP1 is an EBV-encoded CD40 mimic which signals in an amplified and sustained manner. Although EBV is latent in >90% of humans, EBV infection is associated with autoimmunity, particularly SLE. Upon flares of autoimmunity, EBV reactivates and LMP1 is expressed, yet the contribution of LMP1 to exacerbation of disease is unknown. LMP1 transgenic mice generated in our lab have an autoreactive phenotype but do not die early due to autoimmune disease. To test the hypothesis that LMP1 cooperates with other genes in the host to exacerbate autoimmunity, mCD40-LMP1 transgenic mice were bred to two lupus-prone strains of mice. LMP1 signaling was able to enhance the autoimmune phenotype of the B6.Sle1, but not the B6.Sle3 strain. These data suggest that LMP1 is redundant with genes within the Sle3 interval, but acts cooperatively with genes within the Sle1 interval to exacerbate autoimmunity. Together, the research foci of this dissertation examine how the CD40 pathways of B cell activation can be amplified and dysregulated, by either a viral mimic of CD40, a polymorphism in its signaling domain, or its cooperation with additional gene products. Differential usage of TRAF3 as a positive, rather than a negative, regulator of signaling appears to be one common mechanism by which this occurs. In conditions where enhanced CD40 signaling may be desirable such as during chronic infections, manipulation of TRAF3-CD40 signaling may serve to enhance immune responses. It is hoped that these studies can additionally reveal important information about the normal regulation of this powerful activation pathway.

Embryonic stem (ES) cells possess the remarkable ability to form cells and tissues from all three germ layers, a characteristic known as pluripotency. In particular, the generation of ES cell-derived hematopoietic cells could serve as an alternate source of hematopoietic stem cells for transplantation in place of bone marrow cells, which are limited by donor availability and high immunogenicity. The advantages of ES cell-derived hematopoietic cells over bone marrow cells include a greater proliferative capacity, which alleviates the problems of donor shortage, and low level expression of MHC antigens, which suggests immune privilege. However, it is unclear whether the immune system is capable of recognizing and rejecting ES cell-derived hematopoietic cells following transplantation. The observation that ES cell-derivatives express low levels of MHC class I, the predominant inhibitory ligand for NK cells, led us to hypothesize that ES cell-derived hematopoietic progenitor cells (HPC) are susceptible to NK cell-mediated killing. To test this hypothesis, we first generated HPCs from murine ES cells ectopically expressing HOXB4, a homeobox transcription factor that confers hematopoietic self-renewal, and confirmed that HPCs expressed low levels of MHC class I antigens. To specifically investigate the role of NK cells in regulating the in vivo engraftment of HPCs, we transplanted NK-replete Rag2-/- or NK-deficient Rag2-/-γc-/- mice with HPCs. We observed permanent HPC engraftment in Rag2-/-γc-/- mice; however, HPC engraftment was significantly reduced in Rag2-/- mice and was eventually eliminated over time. Bone marrow harvested from these animals showed that HPC-derived Lin-c-kit+ and Lin-Sca-1+ progenitor cells, critical progenitor cells for long-term hematopoietic engraftment, were deleted in Rag2-/- but not in Rag2-/-γc-/- mice. Next, we focused on the mechanism of NK cell activation by HPCs. Increased expression of the cytotoxic proteins Granzyme B and Perforin in the NK cells of HPC-transplanted Rag2-/- mice confirmed in vivo NK cell activation. Phenotypic analysis of HPCs revealed high level expression of H60, a ligand of the NK activating receptor NKG2D, and neutralization of H60 rescued HPCs from NK cell-mediated killing. Altogether, our results demonstrate that NK cells are a major barrier to the successful engraftment of ES cell-derived hematopoietic cells, underlining an important role of the innate immune system in regulating the long-term engraftment of ES cell derivatives.

Antigen-specific CD8 T cells provide an important protective role in response to infection by viruses, intracellular bacteria, and parasites. Pathogen-specific CD8 T cells render this protection by undergoing robust expansion in numbers while gaining the ability to produce cytokines and cytolytic machinery. Following expansion and effector differentiation, pathogen-specific CD8 T cells will contract in number while further differentiating into a highly functional population of memory CD8 T cells. These antigen-experienced cells persist in secondary lymphoid organs and the periphery in order to rapidly respond to repeated infection. Creating optimal CD8 T cell responses to infection can be critical for raising sufficient armament to provide protection against invading intracellular pathogens. Although CD8 T cells have protective value, many vaccine strategies tend to focus on creating productive B cell antibody responses to promote immunological protection. Even though antibody responses can be highly protective, coupling optimal CD8 T cell responses with B cell responses could provide higher orders of protection than either one on their own. Therefore, a deeper understanding of the pathways that ultimately guide the magnitude of CD8 T cell responses is required to achieve this potential therapeutic benefit. My studies evaluate the role of receptor signaling events in guiding the expansion of activated CD8 T cells during primary and secondary responses. Specifically, the first portion of my studies dissect the mechanism by which direct IL-12 and Type I IFN stimulation can substantially bolster primary CD8 T cell responses in vivo. Within this context, I demonstrate that direct IL-12 and Type I IFN signaling increases CD8 T cell accumulation during primary expansion by prolonging division without altering survival. IL-12/Type I IFN signaling promoted prolonged division of activated CD8 T cells by maintaining high-affinity IL-2 receptor subunit (CD25) expression and IL-2 signaling. The other portion of my work was dedicated to understanding the expression and role of the inhibitory FcgR (FcgRIIB) during primary and secondary CD8 T cell responses. FcgRIIB expression could be detected as early as the peak of the CD8 T cell response and marked activated CD8 T cells that were highly sensitive to antigen stimulation. Although FcgRIIB did not appear to play a substantial role in regulating the magnitude of primary CD8 T cell responses, it played an important role in inhibiting the expansion and cytotoxicity of memory CD8 T cells during homologous challenge. Collectively, these data highlight potential avenues that could be exploited by future therapies that aim to achieve appropriately sized CD8 T cell responses.

BACKGROUND: Over the past 5 years, 634 cases of Shigatoxin E. coli (STEC) infection were reported to Tennessee Health Department 1. At our local children’s hospital, 4-5 children are hospitalized with STEC infection each year. Some of these children had no history of ingesting food items that could have placed them at risk to develop STEC infection; however, there are other ways that humans could get infected, such as exposure to contaminated water from cattle farms 2. GOALS: To determine if bodies of water in the city are contaminated with STEC. METHODS: Fifty (50) ml of water samples were collected from selected areas of Johnson City, TN. Samples were inoculated to Sorbitol McConkey Agar (SMA) plates under sterile techniques & incubated at 36C for 18 hours under aerobic conditions. RESULTS: Table 1 E. coli Strains Isolated from Water Samples Colony Types Founders Park Sinking Creek Carroll Creek Cherokee Creek Colorless (STEC) 14 (3.5) 24 (6) 32 (8) 35 (8.75) Pink (Non-STEC ) 8 (2) 3 (0.75) 7 (1.75) 4(1) DISCUSSION/ CONCLUSION: All sampled sites were positive for STEC. STEC is a normal flora of the gastrointestinal tract of cattle. Around city neighborhoods are pastures, as cattle farming is a major livelihood in Northeastern, TN. It is highly possible that water runoff from these pastures contaminates the waters around the city. Public health measures should be undertaken to inform the community that these waters are contaminated with STEC to prevent STEC infection. References: Reportable Conditions. TN Epi-news, TN Health Dept Issue 3, Volume 9, 2016 Escherichia coli O157:H7 Infections in Children Associated with Raw Milk & Raw Colostrum From Cows—California, 2006. MMWR Weekly, 57(23); 625-628, June 23, 2008.

This report describes the case of a 56-year-old man who presented with blurry vision, increased intraocular pressure, and conjunctival injection after posterior chamber intraocular lens implantation. Initially post-operative endophalmitis and foreign body inflammation were considered as differential diagnoses, but after further examination pan-uveitis was diagnosed. Uveitis is an ocular finding that may indicate several diseases, one of which is Behçet’s Disease. During the interview, the patient mentioned a history of apthous ulcers and genital ulcers which then lead to the clinical diagnosis of Behçet’s Disease. This report emphasizes that Behçet’s Disease is rare in Caucasians. Therefore, is frequently misdiagnosed in North America due to variable presentations and by not exploring the option when analyzing differential diagnoses. Early diagnosis and intervention will prevent the development of blindness and fatality due to complications of the disease.

abstract: Hepatitis C is an infectious disease that affects 71 million people worldwide and causes liver failure and death if untreated. In 2013, a direct acting antiviral drug, sofosbuvir, revolutionized treatment of the disease. Sofosbuvir showed immense promise, but the high price point at which it was launched created access barriers that prevented it from reaching its full public health potential. By 2016, fewer than 1% of Hepatitis C patients worldwide had received treatment. In the United States (US), concerns about the cost of the drug led public and private payers to implement rationing and treatment restrictions that prevented some of the most vulnerable populations from accessing Hepatitis C treatment at all. Through interviews with researchers, patients and providers, and a literature review of grants, patents, papers, court documents, and news articles, I examine the history of sofosbuvir with attention to the ways in which federal funding practices and intellectual property law encouraged the high initial pricing of the drug. I then examine the impact of this drug on healthcare systems in the United States and abroad, and discuss how the fragmented nature of the United States healthcare system has exacerbated price-based barriers to access. Finally, I discuss intellectual property laws as potential mechanisms to increase access. My study underscores how the political reluctance to use well-established federal funding and intellectual property laws has resulted in a drug development system that delivers medications that are so highly priced that the fragmented US healthcare system cannot compensate for the expense. This leads to low access and poor public health outcomes, and a continued failure to contain or control diseases for which effective therapies exist. / Dissertation/Thesis / Doctoral Dissertation Biology 2020