Staphylococcus aureus (SA), an opportunistic pathogen colonizing the anterior nares in approximately 30% of the human population, causes severe hospital-associated and community-acquired infections. SA nasal carriage plays a critical role in the pathogenesis of staphylococcal infections and SA eradication from the nares has proven to be effective in reducing endogenous infections. To understand SA nasal colonization and its relation with consequent disease, assessment of nasal carriage dynamics among a diverse population and determining factors responsible for SA nasal carriage have become major imperatives. Here, we report on an extensive longitudinal monitoring of SA nasal carriage in 109 healthy individuals over a period of up to three years to assess nasal carriage dynamics. Phylogenetic analyses of SA housekeeping genes and hypervariable virulence genes revealed that not only were SA strains colonizing intermittent and persistent nasal carriers genetically similar, but no preferential colonization of specific SA strains in these carriers was observed over time. These results indicated that other non-SA factors could be involved in determining specific carriage states. Therefore, to elucidate host responses during SA nasal carriage, we performed human SA nasal recolonization in a subset of SA nasal carriers within our cohort. In these studies, SA colonization levels were determined, and nasal secretions were collected and analyzed for host immune factors responsible for SA nasal carriage. Interestingly, we observed that stimulation of host immune responses lead to clearance of SA while sustained SA colonization was observed in hosts that did not mount a response during carriage. Further, analysis of nasal secretions from hosts revealed that proinflammatory cytokines and chemokines were significantly induced during SA nasal clearance suggesting that innate immune effectors influence carriage. SA utilizes a repertoire of surface and secreted proteins to evade host immune response and successfully colonize the nose. Analysis of the most abundant immunoevasive proteins in the exoproteome of SA nasal carrier strains revealed that expression levels of Staphylococcal protein A (SPA) produced by SA nasal carrier strains in vitro corresponded to the level of persistence of SA in the human nose. To determine if SPA is involved in modulating the host's response to SA colonization, a subset of participants in our cohort was nasally recolonized with equal concentrations of both wild-type (WT) and spa-disrupted (?spa) autologous strains of SA. Interestingly, ?spa strains were eliminated from the nares significantly faster than WT when the host mounted an immune response, suggesting that the immunoevasive role of SPA is a determinant of carriage persistence. Collectively, this report augments our understanding of SA nasal carriage dynamics, in addition to identifying important host and microbial determinants that influence SA nasal colonization in humans. Better understanding of this phenomenon can lead to improved preventative strategies to thwart carriage-associated SA infections.

Over the past decades, information has accumulated concerning the mechanism how an exterior signal induced by ligand on neighboring cells is transmitted to the nucleus through the Notch receptor and the cellular effects of Notch signaling on the regulation of differentiation, proliferation and apoptosis in many cell types. However, the function and the mechanism of Notch signaling in peripheral T cells still remains to be addressed. Therefore, we asked whether Notch1 is involved in CD8+ cytolytic effector T cell (CTLs) maturation and effector functions and how Notch1 exerts its cellular function in the nucleus and in the cytoplasm. The maturation of naïve CD8+ T cells into CTLs is a critical feature of a functional adaptive immune system. Development of CTLs depends, in part, upon the expression of the transcriptional regulator, Eomesodermin (EOMES), which is thought to regulate the expression of two key effector molecules, perforin and granzyme B. In addition, the data from previous studies in our lab showed that Notch signaling results in the activation of NF-κB, IFN-γ secretion and cell proliferation both in CD4+ and CD8+ T cells. Therefore, we hypothesized that Notch1 may be involved in CD8+ T cell maturation and effector function. We observed that Notch1 regulates the expression of EOMES, perforin and granzyme B through direct binding to the promoters of these crucial effector molecules. By abrogating Notch signaling, both biochemically as well as genetically, we conclude that Notch activity mediates CTL development through direct regulation of EOMES, perforin and granzyme B. We further investigated the molecular steps leading to the formation of intracellular Notch1 (N1ICD)/CSL (also known as CBF1/RBP-Jκ in mammals; Suppressor of Hairless in Drosophila; and Lag-1 in C. elegans) with other co-factors in target promoters of Notch1 signaling. We proposed that the association of two nuclear complexes with N1ICD controls the transcription of genes, allowing the development of effector CTL in the immune system. Recent studies proposed a model where Notch1 colocalizes with CD4, a component of the immune synapse, upon T cell stimulation and directly associates with p56Lck and CD28, as well as PI3K. However, the link between Notch and the TCR signalosome needed further investigation. We found that Notch1 functions as a scaffold, associated with the cytosolic components, Carma1, Bcl10, PKCθ and the IKK complex upon TCR stimulation, leading to the activation of NF-κB and IL-2 production. We further showed that the N-terminal region of N1ICD is essential for interaction with Carma1 and that deficiency of Notch1 abolishes the nuclear binding of NF-κB on the il- 2 promoter, leading to reduced IL-2 production.

The objectives of this study were to determine (1) the prevalence of adult ticks of Dermacentor andersoni in national forest recreational sites of Utah, and (2) the incidence of spotted fever rickettsia, Rickettsia rickettsii, in the ticks of these areas. With the use of a white flannel cloth, 358 adult D. andersoni (135 males and 223 females) were collected from 48 recreational sites during the spring and summer of 1964. Ticks from each collection were put in pools, preserved in non-fat skim milk at -30° C, and subsequently tested for the presence of spotted fever rickettsia by guinea pig inoculations. The average collection rate (population density) for all collections was 6.8 per hour, but populations varied between sites. Populations were greater in the middle and southern parts of the state than in the northern. The greatest populations were at elevations between 6,000 and 8,000 feet with the upper limit just under 9,000 feet. The elevational distribution varied with the latitude--greater populations were found at higher elevations in southern than in northern Utah. The season of peak abundance was between the last week of May and the last of June. Populations were greater at lower elevations early in the season and at higher elevations later. Male ticks were more abundant early in the spring whereas females predominated later. The preferred habitat was open, unshaded areas of short, scanty, young grass. Ticks were collected in greater numbers in the afternoon than in the morning. Temperatures between 12° and 38° C apparently had little effect on tick activity. Activity was slightly greater on partly cloudy and cloudy days than on clear days, and increased proportionately relative to an increase in wind velocity. Spotted fever rickettsia were found in 3.6% of the ticks collected. These were from 13 different recreational sites, over half of which are in the northern half of the state near human population centers. Ticks positive for spotted fever were probably infected with avirulent type U or type T strain of R. rickettsii.

Although nucleic acid tests (NATs) for human immunodeficiency virus (HIV) exhibit many advantages, such as early detection and viral load quantification, over immunological assays, their widespread use is limited by their demand for high-level infrastructure, sophisticated equipment, and advanced staff competence. Furthermore, when quantifying viral loads of patients, it has been reported that these assays can underestimate viral quantities by 22- to 100-fold due to primer-template mismatches in more divergent HIV subtypes. Therefore, we have developed a cost-effective and sequence-independent assay for the detection and quantification of HIV utilizing a modified nucleic acid sequence-based amplification (NASBA) protocol coupled to an electrochemical DNA biosensor. The modified NASBA reaction involves the addition of a 22-nucleotide tag between the T7 RNA Polymerase Promoter and the hybridizing region of the reverse primer. As a result, this tag will be placed at the 5' end of each amplicon regardless of the target sequence. We then designed the DNA sensor to hybridize to this segment of the amplicon specifically. Therefore, hybridization, and subsequently, detection, is dependent only on the presence of this tag and not the viral RNA sequence itself. As a result, the issue of underestimating viral loads is eliminated as multiple primers can be used in one reaction without having to use multiple sensors. The use of an isothermal NASBA technique and a reusable gold-disc electrode for the sensor helps drive down the cost of the assay by eliminating the need for thermocyclers and fluorometers used by conventional NATs.

Viruses in the Flavivirus genus (e.g., Zika, yellow fever, dengue, West Nile, and Japanese encephalitis viruses) are arthropod-borne, globally distributed, and can cause a range of neurological or hemorrhagic diseases. The ongoing epidemics of flaviviral disease consistently demonstrate the need for new vaccines capable of outbreak control. However, safe, effective, and easy-to-produce vaccines remain relatively elusive due to limitations of conventional vaccine development that make it difficult to balance safety and efficacy. Insect-specific flaviviruses (ISFVs) are emerging as a novel method to overcome this challenge. Herein, we develop a new flavivirus vaccine platform based on the novel insect-specific flavivirus called Aripo virus, which we used to create a preclinical Zika virus (ZIKV) vaccine named Aripo/Zika virus (ARPV/ZIKV). ARPV/ZIKV is a live recombinant virus consisting of the ZIKV pre-membrane and envelope protein genes expressed on an Aripo virus backbone. In this work, we quantify the safety and efficacy of ARPV/ZIKV in multiple murine models, and begin to elucidate the mechanisms of humoral and cell-mediated immune induction for this new platform. Overall, the vaccine showed no evidence of pathogenicity in immunocompromised or suckling mice, and demonstrated a complete inability to replicate in various vertebrate cell lines. Despite this lack of replication, a single dose of live, unadjuvanted ARPV/ZIKV completely prevented ZIKV disease in mice and prevented in utero ZIKV transmission in gravid mice. Direct protection post-ZIKV challenge appears to be primarily mediated by neutralizing antibodies based on passive transfer, adoptive transfer, and T-cell depletion studies. However, vaccination studies in Rag1 KO, Tcra KO, and muMt- mice demonstrate the critical role of T-cell responses in developing immunity post-vaccination. In summary, ARPV/ZIKV is a promising vaccine candidate that induces robust adaptive immune responses, and this success is a positive indication of ARPV's potential as a new resource for flavivirus vaccine development. This body of work contributes to the rapidly expanding field of insect-specific virus-based vaccines and generates new insights into their optimization. Ultimately, this work may help protect the health of millions of people worldwide that are currently at risk of flavivirus infection. / MPH / Arthropod-borne viruses (especially flaviviruses such as Zika virus (ZIKV), yellow fever virus, West Nile virus) represent a major global health threat and a significant burden on human life. Vaccination is a critical tool for controlling the often unpredictable outbreaks of flavivirus diseases. However, licensed flavivirus vaccines remain relatively elusive. This is, in part, because the same characteristics of traditional live-attenuated vaccines that make them highly effective can also reduce their safety. Insect-specific flaviviruses (ISFVs) are emerging as a novel method to overcome this challenge. ISFVs only replicate in insects and thus are safe in humans. They do not cause disease in vertebrates, eliminating the need for the chemical or physical inactivation methods required by traditional whole inactivated vaccines and which can result in reduced efficacy. Herein, we develop a new flavivirus vaccine platform based on a novel ISFV called Aripo virus (ARPV). As proof of concept, we used ARPV to create a preclinical ZIKV vaccine named Aripo/Zika virus (ARPV/ZIKV). ARPV/ZIKV expresses immune system-stimulating ZIKV structural proteins on its virus particle. However, it remains highly safe because the genetic material from ARPV makes it incompatible for replication in human cells. Here, we demonstrate the safety and protective ability of ARPV/ZIKV, and begin to elucidate its mechanisms of protection. Overall, ARPV/ZIKV shows promise as a ZIKV vaccine candidate, which supports the potential of ARPV as a platform for new flavivirus vaccines and the potential to protect the health of the millions of people currently at risk of flavivirus infection.

Plasmodium falciparum, the organism that causes the most prevalent and most virulent cases of malaria in humans, poses a major health burden on the developing world, especially in the tropical regions of Sub-Saharan Africa, Southeast Asia, and Latin America. The burden of the disease is intensified by the fact that the parasite has developed widespread resistance to all current antimalarial therapies, such as chloroquine. This drug resistance underscores the need to develop novel therapeutics that target the parasite, but show low toxicity in the human host. Protein kinases, because of their integral roles in cell signaling networks, are considered to be attractive drug targets. Cyclin dependent kinases, or CDKs, and Mitogen-Activated Protein kinases, or MAPKs, are common to eukaryotes and regulate cellular processes of growth and proliferation. Plasmodium falciparum Protein Kinase 6, or PfPK6, is an atypical protein kinase that shows similarities to both MAPKs and CDKs. PfPK6 is expected to have an important role in the intraerythrocytic cell cycle progression and growth in the malaria organism, as it has been found to be essential in the parasite. In order to better understand the function of PfPK6 within Plasmodium, we have identified serveral potential substrates and interactors of the kinase using co-immunoprecipitation with an HA epitope-tagged cell line of PfPK6, as well as phosphoproteomic analysis. These methods resulted identification of 15 novel protein interactors, with 4 being studied for further investigation, and 45 putative substrates after strict peptide filtering, five of which are used in this study. In order to verify putative substrates and interactors, both in vitro and in vivo methods were used. In vitro kinase assays using GST-PfPK6 with 5 recombinant substrates confirmed direct phosphorylation of two novel substrates: MAL7P1.38, a regulator of chromosome condensation, and PF10_0047, a putative RNA binding protein. After attempts to generate bacterial constructs of several putative interactors and a global failure of a usable amount of protein to express under IPTG induction conditions, an alternative form of expression using a cell free Transcription and Translation reaction (TNT) with Wheat Germ Extract was used to generate radiolabeled PF11_0154, PFF0625w, and PF11_0305. Pull down analysis using GST-PfPK6 showed the kinase's ability to "pull" the interactors out of solution, confirming the interactions defined by the initial epitope tagged Co-Immunoprecipitation. Additionally, for in vivo analysis, parasites were transfected with RFP- PFF_0695w, an uncharacterized Plasmodium protein, in order to cellular localization of this interactors. Immunofluorescence assays of transfected lines showed punctate forms of PFF_0695w in the host erythrocyte in the late trophozoite and schizont stages of the parasite development, suggesting this interactor is a previously undiscovered protein in the Plasmodium secretome. The research presented here is an initial step to defining the interactome of PfPK6.

Phenylethanolamine-N-methyltransferase (Pnmt) is the enzyme that catalyzes the conversion of noradrenaline to adrenaline. These catecholamines are synthesized in the medulla of the adrenal gland and by some neurons of the central nervous system. The precise location of Pnmt action in the brain and its physiological significance are unknown. Prior studies led by Aaron Owji, a graduate student in Dr. Ebert’s laboratory, showed that mice with selectively ablated Pnmt cells show signs of neurological defects such as abnormal gait, weakened grip strength, lack of balance, reduced movement, and defective reflexes during tail suspension tests. The cerebellum is a small section of the brain that is responsible for fine-tuning motor commands. Since the Purkinje cells of the cerebellum act as the sole source of output from the cerebellar cortex, impairment of these cells could possibly account for the motor deficits seen in the mice models. The purpose of this project is to determine if there is indeed a change in Purkinje cells between wild type mice and Pnmt-ablated mice. The first aim is to identify quantitative differences in cell count between both genotypes. The second aim is to determine any morphological changes in the Purkinje cells. The main technique used in this project is immunohistochemistry in which cerebellum tissue from mice models are stained with Calbindin (a cellular marker for Purkinje neurons) and imaged with a confocal microscope. Results showed a slight reduction in the Purkinje cells of the ablated mice compared to the control genotype, accompanied with observable differences in cell structure. Understanding catecholamine pathway mechanisms in the nervous system is imperative for elucidating and targeting key players in neurodegenerative disorders.

The prevalence of herpes simplex virus (HSV) infection globally is high, and although there is no cure for it, the antiviral drug acyclovir is used to alleviate symptoms. There are two types of HSV: HSV-1, which typically infects the oral area, and HSV-2, which is associated with genital infections. A mother who carries the infection may transmit it to a neonate in different ways, most commonly via vaginal delivery in the presence of active lesions. There are three types of HSV disease that affect newborns: skin, eyes or mouth (SEM) disease, central nervous system (CNS) disease, or disseminated disease. The purpose of this study was to examine the long-term effects of the infection and the treatment used in neonates infected with HSV. Data collection consisted of original case reports published in Medline, CINHAL, and Google Scholar. Two case reports were found, and this narrative review compares the cases, which report recurrences and outcomes of HSV infection identified in the three databases. Both cases were consistent with recurrence of CNS disease, and one showed signs of a slight developmental delay that may have been related to the CNS insult.

T cells play an important role in adaptive immune system against viral infections, while premature aging and dysfunction of T cells induced by unrepaired DNA damages are always non-negligible snags during the long-term of fighting with chronic viral infections, such as Hepatitis B virus (HBV), Hepatitis C virus (HCV) or Human Immunodeficiency Virus (HIV) infection. In this dissertation, we investigated the role of topological DNA damage in reprogramming telomeric DNA damage responses (DDR), mitochondrial metabolisms, and T cell functions using CD4+ T cells derived from individuals with chronic viral infections or healthy subjects treated with topoisomerase inhibitors. The healthy human T cells were treated with camptothecin (CPT) for mitochondrial topoisomerases I (Top1mt) or ICRF-193 or etoposide (ETP) for topoisomerases IIα (Top2α) as models. We found a significant suppression of Top2α and Top1mt protein levels and enzymatic activity in CD4+ T cells in chronically HCV/HIV-infected patients compared to age and gender-matched healthy subjects, along with an accumulation of the topoisomerase cleavage complex (Topcc) in genomic DNA as well as mitochondrial DNA (mtDNA). Mechanistically, topoisomerase inhibition in healthy CD4+ T cells caused topological DNA damage, telomere attrition, mitochondrial metabolic disorder and T cell apoptosis or dysfunction via inducing Topcc accumulation, PARP1 cleavage and failure in DNA repair, thus recapitulating T cell dysregulation in the setting of chronic viral infections. In addition, T cells from virally infected subjects with lower topoisomerase levels were vulnerable to the inhibitor-induced cell apoptosis, indicating an important role for Top2α and Top1mt in preventing DNA topological disruption and cell death. These results demonstrate that accumulation of Topcc and topoisomerase deficiency lead to unrepaired DNA damage and render virally infected patients’ T cells prone to senescence and apoptosis, thus contributing to mitochondrial metabolic disturbance or dysfunction in CD4+ T cell during chronic HCV or HIV infection. This study reveals a novel mechanism by which topoisomerase deficiency promotes telomeric DNA or mtDNA damage and premature T cell aging, and provides a new therapeutic target for restoring the DNA topologic machinery protecting T cells from unwanted DNA damage and to maintain immune competence.

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