T cells are central regulators of adaptive immunity in infectious and pathophysiological diseases. The activation of T cells is regulated by the T cell antigen receptor (TCR) and co-stimulatory receptors like toll-like receptor 2 (TLR2). These receptors activate distinct and overlapping intracellular signaling pathways that ultimately shape T cell responses. Therefore, studies that elucidate the molecular mechanisms of signal transduction downstream of receptors like the TCR and TLR2 will highlight key pathways required for T cell activation. These pathways could then be clinically targeted to alter dysfunctional T cell responses that promote many human diseases. Focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (Pyk2) are two tyrosine kinases activated by multiple surface receptors expressed on T cells. FAK and Pyk2 signaling regulate cell morphology, migration, adhesion, proliferation, and survival in other cell types. However, their functions in T cells are not well-described. Because FAK and Pyk2 functions are dysregulated in many disease states, it is important to understand their function in human T cells so that clinicians can safely target these kinases to treat various disorders. The studies described in this dissertation aim to more fully elucidate how FAK and Pyk2 control T cell activation mediated by the TCR. We used recombinant microRNAs or kinase inhibitors to transiently suppress FAK and Pyk2's expression or enzymatic function in human T cells. In doing so, several novel functions of FAK and Pyk2 were uncovered. In Chapter III, we revealed that FAK is a negative regulator of TCR signal transduction and function. Interestingly, in contrast to its function in other immune cell lineages, the work described in Chapters III and IV demonstrates that FAK is not required to regulate actin cytoskeletal responses downstream of the TCR. The data presented in Chapter IV demonstrate that Pyk2 regulates TCR-mediated actin cytoskeleton reorganization. This function appears to be linked to Pyk2's scaffolding function and not its enzymatic activity. In Chapter V, we demonstrated that the catalytic function of Pyk2 controls phosphatidylinositol-3-kinase (PI3K) activation in human T cells. Together, these data revealed that FAK and Pyk2 serve distinct functions in TCR signal transduction, actin cytoskeletal rearrangement, and effector responses. TCR-driven cytokine production and proliferation are enhanced when T cells are concurrently activated via TLR2 ligands. In Chapter VI, we describe the signaling pathways that TLR2 activates in human T cells, and we characterize how TCR and TLR2 signals converge to augment T cell responses. In contrast to studies performed using isolated murine T cells, we demonstrated that TLR2 does not activate nuclear-factor kappa B in human T cells. Instead, we found that TCR and TLR2 co-ligation selectively augments extracellular signal-related kinase 1 (Erk1)/Erk2 and Akt activation in human T cells. Thus, TLR2 co-stimulates murine and human T cells via distinct signaling mechanisms.

Neonatal sepsis is an important cause of neonatal morbidity and mortality. There are two distinct types of sepsis- early and late onset. Group B streptococcus and Listeria are the most common causes of early onset neonatal sepsis historically. Physicians select antibiotics for neonates with fever based on historically common bacterial pathogens such as GBS, Ecoli, Listeria, and Staphylococcal aureus. However, the landscape of bacterial pathogens causing sepsis and fever in neonates seems to be changing. This could potentially change the first choice of antibiotics for this susceptible population. In this case study, we will present early-onset sepsis in a late preterm infant due to Streptococcus pneumoniae as confirmed by blood culture. The only maternal risk factors present in this case for septicemia were delivery less than 37 weeks. Patient initially had respiratory distress at delivery and required CPAP for 3 days. On day 2 of life, cultures were taken due to acute deterioration. Ampicillin and Gentamycin were given to the patient for empiric coverage initially. On day 2 of antibiotics, cultures were reported positive. Patient’s antibiotics had to be altered at that time to cover the isolated organism. The patient was inadequately treated up until cultures were positive. This case raises the question if Ampicillin and Gentamycin remain the best choice for broad antibiotic coverage in neonates with possible sepsis.

Introduction: Toxoplasmosis is caused by infection with the protozoan Toxoplasma gondii (T. gondii), an obligate intracellular parasite. T. gondii infects a large portion of the world’s population, but uncommonly causes clinically significant disease. Those that are at greatest risk for more severe disease with toxoplasmosis are the immunologically impaired, fetuses, and newborns. T. gondii infection in immunocompetent patients can present as a self-limiting acute infection, or as an acute systemic disease. There are three main T. gondii genotypes, I, II, and III, with varying geographical prevalence. T. gondii is most commonly acquired via ingestion of infectious oocysts, from the environment, tissue cysts from contaminated food items, vertical transmission, or via organ transplantation from an infected donor. Diagnosis can be made via histological and serologic testing in suspected patients. Seropositive testing should be considered within the clinical context, as IgM antibodies may persist for months to years. IgG antibody avidity patterns further help delineate acute versus chronic infections. Histopathology from tissue biopsy of lymphadenopathy is more commonly pursued to establish diagnosis in immunocompetent patients. Case Report: We present a 37-year-old male who presented to the clinic with persistent bilateral non-tender occipital lymphadenopathy of two months duration. Patient also endorsed an acute fluid filled blister on the penis, recurrent cold sores, and significant fatigue. Review of systems were unremarkable. Patient’s immunizations were up-to-date. Patient is an active military serviceman with history of overseas deployment. Patient reports consuming undercooked meat overseas, as well as game meat preparation while hunting. Similar symptoms were also reported by another fellow veteran. Laboratory studies revealed normal CBC, CMP, and TSH. HIV, gonorrhea, and chlamydia testing were negative. Urology referral found no abnormalities. Aspiration biopsy of the right occipital lymph node demonstrated granulomas and aggregates of histiocytes compatible with reactive hyperplasia. Findings were suggestive of toxoplasmosis and no malignancy was found. Follow up T. gondii serological testing results revealed Ab IgM: 104 AU/ML (reference range 0.0-7.9). Toxoplasma gondii Ab IgG: >400 AU/ML (reference range 0.0-7.1), which were consistent for active infection. Patient was referred to Infectious Disease and supportive therapy was recommended. A three month follow up showed improvement in symptoms. Discussion: Although acute infections with T. gondii in immunocompetent patients typically are self-limiting, more serious systemic infections may occur. A pyrimethamine-containing antibiotic regimen is recommended for treating systemic infections. We propose educating high-risk individuals with appropriate preventive measures, which may be beneficial in preventing Toxoplasmosis.

Infectious diseases from novel viruses have become a major public health concern. Rapid identification of virus–host interactions can reveal mechanistic insights into infectious diseases and shed light on potential treatments. Current computational prediction methods for novel viruses are based mainly on protein sequences. However, it is not clear to what extent other important features, such as the symptoms caused by the viruses, could contribute to a predictor. Disease phenotypes (i.e., signs and symptoms) are readily accessible from clinical diagnosis and we hypothesize that they may act as a potential proxy and an additional source of information for the underlying molecular interactions between the pathogens and hosts. We developed DeepViral, a deep learning based method that predicts protein– protein interactions (PPI) between humans and viruses. Motivated by the potential utility of infectious disease phenotypes, we first embedded human proteins and viruses in a shared space using their associated phenotypes and functions, supported by formalized background knowledge from biomedical ontologies. By jointly learning from protein sequences and phenotype features, DeepViral significantly improves over existing sequence-based methods for intra- and inter-species PPI prediction. Lastly, we propose a novel experimental setup to realistically evaluate prediction methods for novel viruses.

The major histocompatibility complex class I (MHC-I) antigen presentation pathway is necessary for the immune system to be able to detect, control, and eliminate cancers. MHC-I binds oligopeptides derived from cellular proteins and presents them on the cell surface to CD8+ T cells. Consequently, the CD8+ T cells can monitor whether any cells are making abnormal proteins and, if so, can destroy those cells. Because MHC-I presentation is not essential for cell viability, immune selection pressure often leads to cancers that are MHC-I low as they can better evade CD8+ T cell recognition. It is, therefore, important to fully understand the mechanisms of MHC-I presentation as this will identify new ways to target and exploit the pathway for cancer therapeutics. Although several components of the MHC-I pathway have already been characterized, some knowledge gaps remain. Unbiased forward genetic screens from our lab identified some novel gene candidates, such as IRF2, which positively regulate MHC-I presentation. In this dissertation, I will reveal which antigen presentation pathway genes are transcriptionally controlled by IRF2 and contribute to the MHC-I presentation deficiency observed in cells lacking IRF2 and I will also show that IRF2 negatively regulates PD-L1 expression. By influencing both MHC-I antigen presentation and PD-L1 expression in this manner, cancers lacking IRF2 (of which there are many) are both harder to see and more difficult to eliminate.

Doctor Pharmaceuticae - DPharm / Treatment of the human immunodeficiency virus (HIV) and tuberculosis (TB) infections have become very complicated due to the advent of drug resistance. Drug combinations offer an alternative approach to reducing the emergence of drug resistance. Pharmaceutical co-crystals have provided the pharmaceutical industry with the ability to optimise the physicochemical properties of active pharmaceutical ingredients (APIs) while preserving the biological activity. Pharmaceutical co-crystals are formed between APIs and suitable co-formers that are biologically safe or even a second or third API.

archives@tulane.edu / We extend the conventional models in mathematical epidemiology to account for more practical (yet complicated) situations in infectious disease transmissions, such as behavior change, risk level differentiation and infectiousness as a function of time since infection. We allow the transmission rate and recovery rate to vary as functions of time since infection. We present the derivation of the integral differential equation model and analyze the associated analytical and long-time solutions. We prove the well-posedness of an initial boundary value problem for the model. We also derive the threshold quantities for the epidemic to grow. We then extend the approach for the vector-borne infectious disease models. We compare several risk distribution functions due to geographic reasons. We construct the behavior change factor for the host population to account for different levels of infectiousness due to behavior distinction and behavior change. We establish the well-posedness of an initial boundary value problem of the new model. Sensitivity analysis shows that different risk distribution functions that are designed to adjust for spatial and geographic reasons have a large impact on the solution. / 1 / Li Guan

Dendritic cells (DCs) are antigen presenting cells (APCs), a subtype of immune cells that present cellular information to T cells in the immune system. Hypoxia inducible factor 1 alpha (HIF-1 alpha) is an important transcription factor that facilitates dendritic cell metabolism by upregulating glycolysis in activated DCs. In this project, we examined the effects of HIF-1 alpha inhibition on metabolic processes of dendritic cells. Using techniques such as flow cytometry, western blotting, and extracellular flux analyzers, we used a selective inhibitor of HIF-1 alpha to test the hypothesis that HIF-1 alpha promotes glycolytic dependent processes such as glucose production, survival, and maturation. The results revealed that HIF-1 alpha impacts oxygen consumption rates in DCs, but does not affect survival, maturation rates, and glycolytic rates under the conditions studied. Dendritic cell secretion of IL-12, a proinflammatory cytokine upregulated during metabolism, decreased in a dose dependent manner under HIF-1 alpha inhibition. Understanding the effects of HIF-1 alpha can provide insight on how dendritic cells utilize their fuel source to facilitate immunological tasks and how in the future, we can optimize these sources to improve immune system functionality.

Anemia is known to worsen treatment outcomes in malaria, but there are not many studies to identify the predictors of anemia in Nigerian children with malaria. The objective of this study is to identify some of those predictors. Socio-demographic variables are predictors of anemia among under five children in Nigeria was the hypothesis tested. This is a cross-sectional study that used the 2018 demographic health survey (DHS) data from Nigeria to explore some of the factors that determine the presence of malaria-anemia co-morbidity in Nigerian children less than five years (N= 265). The outcome variable was anemia status in children under five with malaria and the explored predictors include age, sex, residential type, region of residence, mother’s education status and family’s wealth index. The study analyzed unweighted and weighted frequencies of the variables and conducted comparison of the outcome groups based on the predictor variables using Chi-square. Univariable and multivariable logistic regression was used to explore the strength of relationship between the outcome variable and the significant predictor variables in bivariate analysis. SAS 9.4 was used for the statistical analysis. Analysis of weighted frequencies showed that 55% of the children were less than 2 years of age while the sex was almost equally distributed between males and females (50.9% vs 49.1%). Just over two-thirds lived in a rural area, 63.2% resided in the Northern part of the country, 59.1% had a rich family and majority (69.1%) had anemia. When cross-tabulated with the outcome variable of anemia status, there was a significant difference in the categories of age (P=0.0048), residential type (P=0.0031), mother’s education status (P=0.0210) and family’s wealth index (P=0.0010). Univariable logistic regression showed that children less than 2 years had over two times higher odds of developing anemia when infected with malaria compared to older children aged 3-4 years (OR:2.17, 95% CI:1.26-3.74, P=0.0052). Urban-dwelling children had 57% reduced odds of developing anemia compared to rural-dwelling children (OR:0.43, 95% CI:0.25-0.76, P=0.0034). Children of educated mothers had 50% reduced odds of developing anemia compared to children of uneducated mothers (OR:0.50, 95% CI:0.28-0.91, P=0.0222), while children in poor families had 165% increased odds of developing anemia compared to those born into rich families (OR:2.65, 95% CI 1.47-4.78, P=0.0012). Once adjusted for all significant variables in the bivariate analysis, only age remained significant as a predictor of anemia in children under five years with malaria (OR:2.29, 95% CI:1.31-4.02, P=0.0039). Younger age seems to be an important predictor of anemia in Nigerian children with malaria in real life settings given its significance on the multivariable model. This finding should inform clinicians on the need to pre-empt and treat anemia in Nigeria’s younger children with malaria for better treatment outcome.

Although pharmaceutical industries have produced a number of new antibiotics in the last three decades, resistance to these drugs by infectious microorganisms has increased. For a long period of time, plants have been a valuable source of natural products for maintaining human and animal health. The use of plant compounds for pharmaceutical purposes has gradually increased worldwide. This is because there are many bioactive constituents in plants which hinder the growth or kill microbes. Plants could be considered a potential gold mine for therapeutic compounds for the development of new drugs. In this study, sixteen South African plant species were selected based on their antibacterial activity after a wide screening of leaf extracts of tree species undertaken in the Phytomedicine Programme, University of Pretoria. Literature search excluded eleven plants because of the work already performed on their antibacterial activities, while Pavetta schumaniana was found toxic and thus not included in the screening. The remaining four plants namely; Buxis natalensis, Macaranga capensis, Dracaena mannii and Garcinia livingstonei were screened for antibacterial activity by determining the minimum inhibitory concentrations (MIC) against 4 nosocomial bacterial pathogens Staphylococcus aureus, Enterococcus faecalis, Escherichia coli and Pseudomonas aeruginosa, and also by using bioautography. The extracts of Macaranga capensis, Garcinia livingstonei, Diospyros rotundifolia and Dichrostachys cinerea had good antibacterial activity with MIC values of 0.03, 0.04, 0.06 and 0.08 mg/ml against different pathogens. The average MIC values of the plant extracts against all the tested pathogens ranged from 0.23-1.77 mg/ml. S. aureus was the most susceptible bacterial pathogen with average MIC of 0.36 . The extract of Diospyros rotundifolia was the most active with an average MIC against all the organisms of 0.23 mg/ml. The extracts of Buxus natalensis, Dracaena mannii, and Pittosporum viridiflorum, Acacia sieberiana, Erythrina lattissima, Cassine papillosa and Pavetta schumanniana had lower antibacterial activity. G. livingstonei was selected for further work on the basis of its good activity. The bulk acetone extract of Garcinia livingstonei (20g) was subjected to solvent-solvent fractionation which yielded seven fractions. Only the chloroform and ethyl acetate fractions showed good bioactivity in the microdilution assay and bioautography. Column chromatography was used to isolate two bioactive biflavonoids from the ethyl acetate fraction. The structures of the two compounds were elucidated using nuclear magnetic resonance (NMR) spectroscopy, and were identified as amentoflavone (1) and 4′ monomethoxyamentoflavone (2). These two compounds have been previously isolated from plants that belong to the Clusiaceae. The two compounds were isolated in sufficient quantity with a percentage yield of 0.45% for amentoflavone and 0.55% for 4′ monomethoxyamentoflavone from 20 g crude acetone extract. The antibacterial activity was determined against four nosocomial bacterial pathogens (Escherichia coli, Staphylococcus aureus, Enterococcus faecalis and Pseudomonas aeruginosa). The MIC values ranged from 8-100 μg/ml. Except for Staphylococcus aureus which showed resistance to amentoflavone at >100 μg/ml. All the other tested organisms were sensitive to both compounds. It has long been recognized that naturally occurring substances in higher plants have antioxidant activity. Based on this, the antioxidant activities of the two isolated compounds were tested using the Trolox assay. The two flavones had good antioxidant activity. Amentoflavone had a Trolox equivalent antioxidant capacity (TEAC) of 0.9. The second compound 4′ monomethoxyamentoflavone had a TEAC value of 2.2 which is more than double the antioxidant activity of Trolox, a vitamin E analogue. To assess the safety of the two compounds on cell systems, cytotoxicity was determined using a tetrazolium based colorimetric assay (MTT assay) using Vero monkey kidney cells. The compounds indicated little to low toxicity against the cell line with cytotoxic concentration (CC50) of 386 μg/ml and >600 μg/ml for compound 1 and 2 respectively. Berberine (used as the control toxic substance) had a CC50 of 170 μg/ml. The Ames genotoxicity assay is used to assess the mutagenic potential of drugs, extracts and phytocompounds. The compounds isolated in this study were assayed for genotoxicity using the Salmonella typhimurium TA98 strain. Amentoflavone was genotoxic at the concentration of 100 μg/plate, but 4′ monomethoxyamentoflavone was inactive at the highest concentration of 400 μg/plate tested. The results of the antibacterial, antioxidant and cytotoxicity testing were encouraging and indicated the potential usefulness of Garcinia livingstonei in traditional medicine and drug discovery. However, the genotoxicity assay revealed potential mutagenic effects of amentoflavone, a compound isolated from the plant. Therefore, it is suggested that application of Garcinia livingstonei extracts in the treatment of human and animal ailments be done with caution to avoid mutagenic effects on the treated subjects. A relatively small change in the structure of the two compounds by replacing an hydroxyl group with a methoxy group had a major effect in increasing antibacterial and antioxidant activity and in decreasing cellular and genotoxicity. This illustrates the potential value of modifying a molecule before its possible therapeutic use. Copyright / Dissertation (MSc (Veterinary Science))--University of Pretoria, 2009. / Paraclinical Sciences / unrestricted