Master of Science / Department of Diagnostic Medicine/Pathobiology / Roman Reddy Ganta / Ehrlichia chaffeensis a tick transmitted Anaplasmataceae family pathogen responsible for human monocytic ehrlichiosis. Differential gene expression appears to be an important pathogen adaptation mechanism for its survival in dual hosts. One of the ways to test this hypothesis is by performing mutational analysis that aids in altering the expression of genes. Mutagenesis is also a useful tool to study the effects of a gene function in an organism. Focus of my research has been to prepare several modified Himar transposon mutagenesis constructs for their value in introducing mutations in E. chaffeensis genome. While the work is in progress, research team from our group used existing Himar transposon mutagenesis plasmids and was able to create mutations in E. chaffeensis. Multiple mutations were identified by Southern blot analysis. I redirected my research efforts towards mapping the genomic insertion sites by performing the semi-random two step PCR (ST-PCR) method, followed by DNA sequence analysis. In this method, the first PCR is performed with genomic DNA as the template with a primer specific to the insertion segment and the second primer containing an anchored degenerate sequence segment. The product from the first PCR is used in the second PCR with nested transposon insertion primer and a primer designed to bind to the known sequence portion of degenerate primer segment. This method aided in identifying the genomic locations of four E. chaffeensis mutants and also was valuable in confirming four other sites mapped previously by the rescue cloning method. This is the first mutational analysis study in the genome of an Ehrlichia species. Mapping the genomic transposon insertion sites is the first critical step needed for the continued research to define the importance of the mutations in understanding the pathogenesis caused by the organism.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Thu Annelise Nguyen / Colorectal cancer is one of the most common cancers in the United States with an early detection rate of only 39%. Colorectal cancer cells along with other cancer cells exhibit many deficiencies in cell-to-cell communication, particularly gap junctional intercellular communication (GJIC). GJIC has been reported to diminish as cancer cells progress. Gap junctions are intercellular channels composed of connexin proteins, which mediate the direct passage of small molecules from one cell to the next. They are involved in the regulation of the cell cycle, cell differentiation, and cell signaling. Since the regulation of gap junctions is lost in colorectal cancer cells, the goal of this study is to determine the effect of GJIC restoration in colorectal cancer cells. Overexpression of connexin 43 (Cx43) in SW480 colorectal cancer cells causes a 6-fold increase of gap junction activity compared to control un-transfected cells. This suggests that overexpressing Cx43 can restore GJIC. Furthermore, small molecule directly targeting gap junction channel was used to increase GJIC. Gap junction enhancers, PQs, at 200 nM showed a 4-fold increase of gap junction activity in SW480 cells. Using Western blot analysis, Cx43 isoform expression was seen to shift from P0 to P1 and P2 isoforms after treatment with PQ1 200 nM for 1 hour. Overall, the results show that overexpression of connexin and small molecules such as gap junction enhancers, PQs, can directly increase gap junction activity. The findings provide an important implication in which restoration of gap junction activity can be targeted for drug development.

Doctor of Philosophy / Department of Division of Biology / Rollie J. Clem / Mosquitoes are carriers of a variety of harmful human pathogens, including viruses. In order to be successfully transmitted, a virus must evade mosquito immune responses. In this work, the innate immune role of apoptosis in mosquito-virus interactions was examined utilizing the disease vector Aedes aegypti and Sindbis virus. Ae. aegypti is the main vector for yellow fever and dengue virus, which result in over 100 million infections per year. Sindbis virus (Togaviridae) can be transmitted to vertebrates by Ae. aegypti in the laboratory. Sindbis is also well characterized molecularly, making it a good model system for understanding virus-vector interactions. Sindbis MRE-16 recombinant virus clones were utilized to express either an antiapoptotic or pro-apoptotic gene during virus replication. Mosquitoes were infected with recombinant virus clones during a blood meal or by intrathoracic injection. Midgut tissue and whole body samples were analyzed for virus infection and dissemination. Virus was also quantified in saliva and mosquito survival was assayed. Decreased infection in the midgut and delayed virus replication were observed in mosquitoes that were infected with virus expressing a pro-apoptotic gene. Infection with this virus clone also resulted in less virus in the saliva and reduced survival of infected mosquitoes. In addition, negative selection against pro-apoptotic gene expression during virus replication was observed. Collectively, these data suggest that apoptosis can serve as an antiviral defense in Ae. aegypti and may potentially be exploited to control virus transmission. An additional study included in this dissertation focused on zebrafish development and migration of somitic precursors from the tailbud. The tailbud consists of a population of stem cells at the posterior tip of the embryonic tail. The exit of these stem cells from the tailbud is required for the formation of tail somites. A novel double mutant was identified that lacked the t-box transcription factor spadetail and the BMP inhibitor chordin. Double mutants completely lacked somites and had an enlarged tailbud due to accumulation of stem cells that were unable to exit the tailbud. This study indicates the importance of BMP inhibition and spadetail expression in the proper exit of muscle precursors from the tailbud.

Master of Science / Department of Entomology / Kun Yan Zhu / Cytochrome P450 monooxygenases (P450s) are important enzymes involved in the metabolism of a variety of xenobiotics, including insecticides and plant allelochemicals, and endogenous compounds, including juvenile hormones, ecdysteroids and fatty acids, in insects. Despite rapid advances in revealing various P450 genes in insects, our knowledge on the role of these genes in detoxification of insecticides is very limited. This research was to perform a genome-wide analysis of P450 genes and evaluate the role of selected P450 genes in detoxification of three commonly used pyrethroid insecticides in the yellow fever mosquito (Aedes aegypti). Our genome-wide analysis of revealed 159 P450 genes that can be classified into 18 families and 63 subfamilies. These genes are distributed in four clans, including 11 genes in the CYP2 clan, 80 in the CYP3 clan, 58 in the CYP4 clan and 10 in the mitochondrial CYP clan. The largest families are CYP6, CYP9, CYP4 and CYP325. The intron-exon organization of the genes is very diverse among the gene families, and the highest conservation of gene structures was observed in the CYP6 and CYP9 families predominantly containing single-intron genes. The phylogenetic analysis suggested that the CYP6 and CYP9 families might be derived from a common ancestor. The expression patterns of five transcripts including three individual genes (CYP6AA5, CYP6AL1 and CYP9J32) and two alternative splicing variants (CYP4J16A and CYP4J16B) of CYP4J16 were investigated in various tissues and at different developmental stages of the mosquito. Our results indicated differential expressions of these transcripts in different tissues and at different developmental stages examined. Furthermore, the exposure of the mosquitoes (larvae and adults) to each of three pyrethroid insecticides (permethrin, cypermethrin and deltamethrin) resulted in either down or up-regulation of these transcripts. Functional analyses of the selected P450 transcripts were conducted by using RNA interference (RNAi) followed by insecticide bioassay. RNAi was achieved by feeding mosquito larvae with chitosan/double stranded RNA (dsRNA) nanoparticles or injecting dsRNA to the adults. For the larvae, we obtained relatively low repressions of the P450 transcripts but the repressions were sufficient for carrying out our functional studies. Our study showed increased mortalities by 41.2% to cypermethrin when CYP6AA5 was silenced and 46.0% to permethrin when CYP9J32 was silenced. Similarly, the injection of dsRNAs in adults resulted in significant repressions of the P450 transcripts, and subsequent insecticide exposures led to a 29.3% increase in the adult mortality to cypermethrin when CYP6AA5 was silenced. Our further analysis of the nuclear receptor HR96 in the up-regulation of the P450 genes showed that when HR96 was silenced by RNAi, the up-regulation of CYP4J16B by cypermethrin was reduced by 10.1-fold but silencing HR96 did not affect the up-regulation of other P450 genes examined. These results suggest that HR96 is likely involved in regulating the expression of CYP4J16B in Ae. aegypti. However, different regulatory mechanism (s) may be involved in the up-regulation of other P450 genes examined. Model structure of CYP6AA5 was created by homology modeling and insecticides substrates were docked into the active site of this protein. Our results indicate that all three insecticides can fit into the catalytic pocket. The interaction distances between the heme iron and the putative aromatic hydroxylation site were 9.2, 9.4 and 7.2 Å for permethrin, cypermethrin and deltamethrin, respectively, whereas for aliphatic hydroxylation site these distances were 5.3, 2.8 and 2.9 Å. These results showed that CYP6AA5 may be able to metabolize cypermethrin and deltamethrin preferentially by aliphatic hydroxylation as indicated by the close interaction with the heme iron.

Doctor of Philosophy / Department of Biology / Michael A. Herman / All multicellular organisms consist of a variety of cell types. One of the mechanisms to generate this cellular diversity is the asymmetric cell division, which requires the establishment of cell polarity. In Caenorhabditis elegans hermaphrodites, 807 of 949 somatic cell divisions are asymmetric. The centrosome and the Wnt signaling pathway both have been shown to regulate cell polarity and subsequently asymmetric divisions in many model organisms. However, it is not clear whether the Wnt signaling pathway manipulates the cell polarity through specific cellular organelles, such as the centrosome. To address this question, we examined a centrosomal component, SPD-5, to see whether it cooperates with the Wnt signaling pathway to regulate certain asymmetric cell divisions. We showed that SPD-5, which was originally found to be critical for the embryonic development, also played a role during certain post-embryonic cell divisions in C. elegans. Specifically the asymmetric divisions of seam cells that required SPD-5 function were also known to be regulated by the Wnt signaling pathway. Thus the stem-cell like seam cell divisions could be an intriguing system to study the interaction of centrosomes and the Wnt pathway. We found that SPD-5 was required for a successful cell division, similar to other centrosomal components. This suggests that SPD-5 still functions as a centrosomal component during C. elegans post-embryonic development. It has been shown that establishment of seam cell polarity relies on the asymmetric localization of certain Wnt pathway components. Interestingly, we found that SPD-5 was required for the proper localization of several Wnt components in a way that was independent of a key MTOC (microtubule-organizing center) member γ-tubulin. In addition, SPD-5 genetically interacted with the Wnt pathway components APR-1/APC and POP-1/Tcf to regulate asymmetric divisions of seam cells. These data suggest that SPD-5 interacts with the Wnt signaling pathway in controlling the polarity of seam cells. Overall, our results suggest a novel role of SPD-5 in cooperating with the Wnt signaling pathway to regulate cell polarity and asymmetric cell division, in addition to its function as a centrosomal component.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Sanjeev K. Narayanan / Acute diarrhea induced by Escherichia coli is an important illness in humans, especially in children under age of two in developing countries. Citrobacter rodentium is used as murine model for E. coli infection in humans because it causes ultrastructural changes in murine colonic epithelium comparable to lesions produced by enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC). Adult mice of many strains develop self-limiting epithelial hyperplasia when infected, whereas adult C3H and FVB mice are highly susceptible to infection and demonstrate mortality rates between 60 and 100% two weeks after infection. These susceptible strains of mice also have higher bacterial translocation to mesenteric lymph nodes. In mice, the cause of death could be hypovolemia due to dehydration that may occur due to an increase in paracellular permeability as well as dysregulation of apical and basolateral ion transporting proteins. C. rodentium virulence factors resemble those of E. coli and are believed to primarily alter tight junctions of colonic epithelial cells. Effectors delivered via the type III secretory system have been associated with actin condensation and pedestal formation. The exact mechanisms of C. rodentium infection, as well as changes that occur in vitro as well as in the intestine of various strains of mice are not completely understood. This study introduced a new in vitro Ptk6 cell line for C. rodentium infection, which can also serve as a model for EPEC in humans. Effect of C. rodentium on colonic epithelial cells of susceptible and resistant mice was determined in in vivo study. C. rodentium attached to Ptk6 colonic epithelial cells, inducing attaching and effacing (A/E) lesions and loss of monolayer integrity, which charachterizes this cell line as a relevant in vitro model of C. rodentium and EPEC infections. Murine studies revealed that C. rodentium induced more severe disease and 100% mortality in juvenile C3H mice whereas Swiss Webster (SW) mice expressed only moderate morbidity. The colonic lesions and changes in barrier function of colonic epithelium were more prominent in C3H mice. This study determined potential targets in the murine colon that play role the establishment and the outcome of the infection, indicating multifactorial nature of C. rodentium-induced diarrhea. This study identified host factors involved in the initiation of C. rodentium-associated diarrhea and the outcome of infection, which can be useful in developing of novel strategies for preventing and treatment of infectious colitis.

Master of Science / Division of Biology / Kristin Michel / To date malaria is the most important tropical disease, which is caused by Plasmodium sp. and vectored by anopheline mosquitoes. The mosquito’s immune system is one of the limiting factors of malaria transmission. Immune reactions, such as the prophenoloxidase (PPO) pathway result in the melanization of pathogens, and are effective at limiting parasite numbers. Novel strategies for malaria control aim to exploit the immune system to interrupt parasite transmission by boosting the immune responses in the mosquito vector. Serpins play a crucial role in regulating protease cascades involved in immunity of arthropods. In Anopheles gambiae, the major malaria vector in Sub-Saharan Africa, 18 SRPN genes encoding 23 distinct proteins have been identified. So far, two are identified as active inhibitors, and both affect parasite survival. This research aims to identify additional inhibitory serpins in An. gambiae and elucidate their potential function. Identification of such serpins will enhance our understanding of the immune system of this important vector species and may identify immunoregulators to be used in malaria control. SRPN7, 9, and 18 were tested for their ability to inhibit commercial proteases in vitro. Recombinant SRPN18 had no inhibitory activity, while SRPN7 and 9 inhibited several serine proteases. SRPN7, 9 and 18 were tested against two recombinant An. gambiae clip serine proteases (CLIPBs) that are required for activation of phenoloxidase and thus regulate melanization. Only SRPN9 strongly inhibited CLIPB9 in vitro, suggesting that this serpin is a potential negative regulator of melanization. This hypothesis is further supported by the finding that SRPN9 can inhibit PO activity in insect hemolymph, ex vivo. Taken together, this research identifies SRPN18 as the first non-inhibitory serpin described in mosquitoes. Additionally, this study describes the larval-specific SRPN7 as a functional inhibitor. Future studies on these proteins will elucidate their precise physiological functions. Finally, this thesis provides strong evidence that SRPN9 is a negative regulator of melanization in An. gambiae and may therefore affect pathogen survival within this important vector species.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Sanjeev K. Narayanan / Fusobacterium necrophorum is a Gram-negative, anaerobic, and rod-shaped to pleomorphic bacterium. It is frequently associated with necrotic infections of animals and humans. It is a major bovine pathogen and causes hepatic abscesses, foot rot, and necrotic laryngitis (calf-diphtheria). Liver abscesses in feedlot cattle and foot rot in beef and dairy cattle are of significant economic importance to the cattle industry. Fusobacterium necrophorum is classified into two subspecies, subsp. necrophorum and subsp. funduliforme. The subsp. necrophorum is more virulent and isolated more frequently from bovine hepatic abscesses than subsp. funduliforme. Outer membrane proteins (OMPs) of Gram-negative bacteria play an important role in their adhesion to host eukaryotic cells and hence, help in the establishment of infection and disease. Our objectives were to characterize OMPs of the two subspecies of F. necrophorum and assess their role in adhesion to bovine cells. Electrophoretic separation of extracted OMPs of subsp. necrophorum showed a total of 19 bands. Four bands of 38, 40, 60 and 74 kDa were more prominent than others. The OMPs of subsp. funduliforme showed a total of 20 proteins bands, of which, five were prominent (37.5, 58, 70, 140 and 150 kDa). The 40 kDa band was prominent in subsp. necrophorum while 37.5 kDa band was prominent in subsp. funduliforme. The human strains of F. necrophorum subsp. funduliforme had more heterogeneous banding patterns than the bovine strains of subsp. funduliforme. The role of OMPs in adhesion was studied using bovine endothelial cell line (EJG cells). A significant decrease in the attachment of subsp. necrophorum and subsp. funduliforme to bovine endothelial cell line (EJG cells) was observed when the cell line was preincubated with the OMPs of each subspecies. Treatment of the bacterial cells with trypsin also decreased their binding. In addition, when each subspecies was incubated with the polyclonal antibody produced against their OMPs before adding them to endothelial cells, there was a significant reduction in the bacterial attachment and the inhibition was subspecies specific. A 40 kDa OMP of subsp. necrophorum was identified that binds to the bovine endothelial cells with high affinity. The protein when preincubated with the endothelial cells, lead to a significant decrease in the bacterial binding to the endothelial cells. The N-terminal sequencing of the protein indicated similarity to FomA, an outer membrane protein of Fusobacterium nucleatum, an oral pathogen of humans. In summary, OMPs of F. necrophorum subsp. necrophorum and subsp. funduliforme differ from each other and they play a significant role in binding to bovine endothelial cells. We identified a 40 kDa OMP in subsp. necrophorum that binds to the bovine endothelial cells with high affinity and have a potential role as adhesin.

Doctor of Philosophy / Department of Biology / Susan J. Brown / How animals polarize and establish the main axis during embryogenesis has been one of the most attractive questions in Biology. Increasing body of work in various model organisms implicates that most metazoans utilize the canonical Wnt signaling pathway to pattern the anterior-posterior (AP) axis, despite the limited evidence from arthropods. In Drosophila, a highly derived insect, canonical Wnt activity is not required for global AP patterning, but in typical insects including Tribolium castaneum, loss of canonical Wnt activity results in posterior truncation. To determine the eff ects of increased canonical Wnt levels, I analyzed the function of axin, encoding a highly conserved negative regulator of the pathway. Tc-axin transcripts are maternally localized to the anterior pole in freshly laid eggs. Parental RNAi for Tc-axin produced progeny phenotypes that ranged from mildly a ffected embryos with cuticles displaying a graded loss of anterior structures, to severely a ffected embryos lacking cuticles and condensing to the posterior pole of the egg without any de finable structures. Altered expression patterns of several blastodermal markers indicated anterior expansion of posterior fates. Epistasis analysis of other canonical Wnt pathway components and the expansion of Tc-caudal expression, a Wnt target, suggest that the eff ects of Tc-axin depletion are mediated through this pathway and that canonical Wnt activity must be repressed for proper anterior development in Tribolium. These studies provide unique evidence that canonical Wnt activity must be carefully regulated along the AP axis in an arthropod, and support an ancestral role for Wnt signaling in de fining AP polarity and patterning in metazoan development. Additionally, as an anterior structure, the extraembryonic serosa is reduced in Tc-axin RNAi progeny. However, in Tc-pangolin (Tc-pan, a homolog of Wnt downstream component) RNAi progeny, an interesting phenotype was produced that serosa was not only reduced but also separated into distinct anterior and dorsal domains. I carefully recorded this phenomenon with live imaging using a Tribolium transgenic line that expresses GFP in each nucleus. Through careful examination with embryonic fate-map markers, I found that the tissue between separated serosa domains is dorsally extended head lobe. And I also found that in severe phenotype, dorsal serosa was completely gone while anterior serosa not, suggesting independent regulation mechanisms for anterior and dorsal serosa formation. This descriptive data will complement future study in the genetic mechanism underlying serosa formation by providing more details in morphogenesis.

Master of Science / Department of Entomology / Marcelo Ramalho-Ortigao / Sand flies (Diptera:Psychodidae) are vectors of parasites of the genus Leishmania transmitted to suitable vertebrate host during blood feeding. For blood feeding arthropods, including sand flies, blood meal digestion requires the secretion of inhibitory molecules, such as Kazal-type serine proteinase inhibitors that are involved in preventing the blood from coagulating within the mouthparts and the midgut. Previous studies have identified such molecules in mosquitoes, ticks, and triatomine bugs. Following studies of the midgut transcriptome of Phlebotomus papatasi, the principal vector of Leishmania major, two non-classical Kazal-type serine proteinase inhibitors were identified (PpKzl1 and PpKzl2). We are interested in the role of these proteins as inhibitors of coagulation cascades, in addition to their potential effects on blood digestion in P. papatasi. Ppkzl1 is similar to thrombin and trypsin inhibitors in triatomines and mosquitoes and Ppkzl2 is similar to Kazal-type inhibitors in mosquitoes with unknown function. Analyses of expression profiles indicated that although both transcripts are expressed prior to blood feeding in the midgut of P. papatasi they are tightly regulated by the blood meal. Reverse genetics studies using RNAi-targeted knockdown of PpKzl1 and PpKzl2 by dsRNA injection did not result in a detectable effect on mRNA expression levels. Thus, we expressed a recombinant PpKzl2 in a mammalian expression system (CHO-S free style cells) that was applied to in vitro studies to assess activity against various serine proteinases. Recombinant PpKzl2 inhibited chymotrypsin at nanomolar levels and also inhibited thrombin and trypsin at micromolar levels, suggesting that native PpKzl2 is an active serine proteinase inhibitor and may regulate digestive enzymes and thrombin in the midgut. Leishmania development within the sand fly midgut is faced with several barriers that can severely impact the parasites. For transmission to occur, parasites must be able to overcome these barriers including digestive proteinases, escape from the peritrophic matrix, and midgut attachment. Early stages of Leishmania are susceptible to killing by digestive proteinases in the sand fly midgut. Thus, targeting serine proteinase inhibitors may provide a new strategy to prevent transmission of Leishmania.