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The opportunistic pathogen Pseudomonas aeruginosa is a significant cause of infection in immunocompromised individuals, cystic fibrosis patients, and burn victims. For its survival advantage, the bacterium adapts to a motile or sessile lifestyle when infecting the host. The active type III secretion system in motile bacterial cells enables a cytotoxic effect on the host. In contrast, an operative type VI secretion in sessile bacterial cells embedded in biofilms helps survive against bacterial competitors. P. aeruginosa switches between the lifestyles through regulatory pathways, including Gac-Rsm and secondary messengers like c-di-GMP. In this thesis, different molecular techniques were applied to understand the regulatory pathways of H1-T6SS, and the newly identified modulators were characterized. The results revealed the RNA binding protein RtcB as a switch controller of a motile and sessile bacterial lifestyle. Wildtype PAO1 in in-vitro conditions, armed with an active T3SS and inactive T6SS. In contrast, PAO1(∆retS) bears an active T6SS and repressed T3SS. Deletion of rtcB led to simultaneous expression of T3SS and T6SS in both PAO1 and PAO1(∆retS). Furthermore, the deletion of rtcB increased the biofilm formation in PAO1(∆rtcB) and restored the motility of PAO1(∆rtcB∆retS). The killing assay showed that H1-T6SS was activated in PAO1(∆rtcB) and could compete against Escherichia coli. Transcriptome analysis was performed for PAO1, PAO1(∆rtcB), PAO1(∆retS), and PAO1(∆rtcB∆retS). It revealed 370 genes of PAO1(∆rtcB) and 1030 genes in PAO1(∆rtcB∆retS) were differentially expressed. That includes genes encoding various virulence factors and the four types of secretion systems. Quantification of c-di-GMP showed elevated levels in PAO1(∆rtcB), which contributed to the altered phenotype and characteristics of PAO1(∆rtcB). The long-chain-fatty-acid—CoA ligase FadD1 was also identified as a new player in H1-T6SS regulation in this thesis. It deactivated the expression of

G protein-coupled receptors (GPCRs), a group of 7 transmembrane proteins, are the most targeted molecules in drug discovery studies. These proteins consist of an extracellular amino- and an intracellular carboxy-terminus and regulate various physiological processes in humans. Quinine as a bitter taste receptor agonist can activate G protein-coupled receptor family of proteins. One of the most extensively studied small G protein is Ras p21. The Ras p21 proteins (H-ras, K-ras and N-ras) are important in cell proliferation and upon mutations can be oncogenic. There are other small G proteins in the Ras superfamily that have ~30-50% sequence similarity with Ras p21. Ral G proteins (RalA and RalB) have high homology (~50%) with Ras p21 and participate in various cellular functions. Results from a previous study by our group showed that quinine causes activation of RalA in CHRF cells and, that these cells express the bitter taste receptor, T2R4. Ral proteins can be activated directly or through an alternative pathway that requires Ras p21 activation resulting in the recruitment of RalGDS, a guanine nucleotide exchange factor for Ral. Using MCF10A (normal mammary epithelial cells) and MCF7 (non-invasive mammary epithelial cancer cells) cell lines we investigated the effect of quinine, a bitter compound, in the regulation of Ras p21 and RalA activity. Results showed that in the presence of quinine, Ras p21 is activated in both MCF10A and MCF7 cells; however, RalA was inhibited in MCF10A cells and no effect was observed in the case of MCF7 cells. The downstream effector for Ras p21, MAP kinase, was activated in both the MCF10A and MCF7 cells. Western blot analysis confirmed expression of RalGDS in MCF10A cells that was higher than that observed in MCF7 cells. Although RalGDS was detected in MCF10A and MCF7 cells, it did not result in RalA activation due to Ras p21 activation suggesting that Ras p21-RalGDS-RalA pathway is not active. The possibility exists that quinine has a

Craniofacial development is a tightly regulated process by several cell singalling pathways. It is therefore highly vulnerable to disturbances by genetic and environmental factors. For example ethanol exposure during pregnancy causes severe birth defects including Cleft Lip and or Palate. Zebrafish (Danio rerio) is a well-established model organism for studying craniofacial defects with remarkably high similarity to humans. Mexican tetra (Astyanax mexicanus) is another teleost and a promising model organism for evolutionary biology that exists as two morphs: pigmented-eyed surface fish and eyeless cavefish. As an adaptation to dark cave environment cavefish lost their eyes, subsequently these eye lost resulted some craniofacial structural changes in the cavefish. The first objective of my study was designed to understand the effect of early eye degeneration on parasphenoid bone in Mexican tetra. My second objective was deciphering the mechanism of bmp 4 gene-ethanol interactions on palate development. Alizarin red-bone staining, micro-CT imaging, Tartrate-resistant acid phosphatase and Alkaline Phosphate staining was conducted to understand bone ossification, articulation, and remodeling within surface and cavefish. The phenotypic effect of ethanol and Dorsomorphin (bmp 4 inhibitor) in the ethmoid plate was characterized by the alcian blue staining and data was statistically analyzed. Whole-mount in situ hybridization was used to identify the expression of bmp 4 among different treatment groups. The shape of the parasphenoid bone found to be varies amongst different eye morphs in Mexican tetra. The parasphenoid bone is straightened in cavefish and curved in surface fish. Bone remodeling activity in cavefish increased compared to the surface fish. Ethanol and Dorsomorphin affect the size of the ethmoid cartilage but the severity of the ethmoid cartilage defect increased with the combined effect of both treatments. Reduction of bmp 4 expression was observed with ethan

Introduction: Adiponectin and the structurally related C1Q-Tumor Necrosis Factor-related proteins (CTRP) have important roles in innate immunity and metabolism. CTRP8 is the most recently discovered and least characterized member of this family. Our lab had identified human CTRP8 as a novel ligand of the relaxin/insulin-like family peptide receptor 1 (RXFP1) in glioblastoma. We have generated high-affinity rabbit antisera to CTRP8 and identified a selective mast cell population in human prostate normal and neoplastic tissues. Mast cells are innate immune cells that are best known for their role in mediating allergic responses. Mast cell have been shown to accumulate in prostate cancer tissues and serve as potential prognostic marker in prostate cancer. Methods: Affinity-purified rabbit polyclonal antisera directed against an epitope unique to human CTRP8 were characterized by Western blot, immunofluorescence and immunohistochemistry in clinically validated patient prostate tissues and tissue microarrays (TMAs). Results: Of several Flag-tagged human and mouse CTRP family members transiently expressed in HEK293 cells, both rabbit antisera exclusively detected human CTRP8 in Western blots. The two antisera (1438 and 1439) were also successfully used for the detection of CTRP8+ cells in human tissues. Immunoreactive CTRP8 co-localized with toludiene blue positive cells in human tissues. Immunofluorescence studies in normal and neoplastic human prostate tissues revealed a subpopulation of mast cells immunoreactive for CTRP8 and Tryptase, the latter being a marker for mature mast cells. Analysis of prostate cancer tissue microarrays (TMAs) revealed a significant increase in the proportion of Tryptase+/ CTRP8+ cells versus total number of mast cells (Tryptase+/ CTRP8+ and Tryptase+/ CTRP8- cells) in the peritumoral compartment as compared to the intratumoral regions of prostate cancer samples with Gleason grades 6 and 7. Conclusions: This is the first report on CTRP8 expre

Objective: To assess the oral health status and oral-health related quality of life (OHRQoL) of young First Nations and Metis children in Manitoba. Methods: This cross-sectional study assessed the oral health status of First Nations and Metis children <72 months of age while their parents/caregivers completed a questionnaire, including the Early Childhood Oral Health Impact Scale (ECOHIS) to assess OHRQoL. Participants were recruited from three rural Indigenous communities and one urban centre participating in a scale-up of the Healthy Smile Happy Child (HSHC) initiative in Manitoba, Canada. Analysis included descriptive statistics, bivariate analyses, and multiple regression. A p-value ≤ 0.05 was considered significant. Results: Altogether, 146 children were recruited with a mean age of 40.1±21.2 (SD) months and 49% were male. Overall, 60% had early childhood caries (ECC) while 56% had severe ECC (S-ECC). The mean dmft score was 4.9±5.3 (range 0-20) and the mean dmfs score was 14.5±20.4 (range 0-80). The total mean ECOHIS score was 4.44±5.86 (range 0-25), while the mean Child Impact Section (CIS) and Family Impact section (FIS) scores were 2.65±4.05 (range 0-10) and 1.78±2.80 (range 0-8), respectively. In total, 13.9% of children frequently experienced oral pain. Multiple linear regression showed S-ECC was associated with total mean ECOHIS scores (p=0.02). However, stepwise regression revealed that children’s age and S-ECC were independently associated with total mean ECOHIS scores (p=0.04 and p<0.001). Higher total mean ECOHIS scores (which indicates poorer OHRQoL) were observed in children with ECC compared to those caries-free (5.8 vs. 2.4, p=0.0001). Statistically significant correlations were found between increased ECOHIS scores and dmft and dmfs scores (both p<0.0001). iii Conclusion: Oral health disparities such as ECC and reduced OHRQoL exist among many First Nations and Metis children in Manitoba. This is the first Canadian study exploring the OHRQoL of I

Bitter taste receptors (referred to as T2Rs) are integral cell membrane receptors that belong to G protein-coupled receptor family. In humans, 25 T2R subtypes have been discovered thus far, and are differentially expressed in various organs. Embracing the newly recognized extraoral and functional roles of T2Rs, I investigated the significance of select T2Rs in pathophysiological conditions. The function of T2Rs in breast cancer is not clearly understood. We characterized the expression and function of T2R4 and T2R14 in breast cancer clinical samples and analyzed their physiological role using highly metastatic breast cancer and non-cancerous cell lines. The results show that compared to normal breast epithelial cells, intrinsic activation of T2Rs in breast cancer cells elicited anti-proliferative, anti-migratory and pro-apoptotic responses. These findings demonstrate that the chemosensory T2R signaling network is involved in evoking beneficial physiological responses in breast cancer. The second part of my thesis involved identifying intrinsic factors regulating T2R14 signaling in airways. The pathophysiological relevance of T2R14 in airway cells is well recognized. Using human airway smooth muscle cells and human bronchial epithelial cells, I studied the role of membrane lipids, cholesterol and sphingomyelin, in airway T2R14 signaling. The results reveal that membrane cholesterol regulates T2R14 signaling, however, T2R14 signaling was not dependent on the availability of sphingomyelin. Furthermore, this work identified the site on T2R14 that potentially binds cholesterol. The final part of my thesis involved identification of a highly conserved histidine (H208) on the intracellular side of T2R14 in determining agonist selectivity. Structure-function analysis of H208 suggests its role in regulating T2R14 active conformation and consequentially agonist promoted downstream signaling. Collectively, these studies provide novel insights into various factors governing the

Platelets are anucleated cells derived from megakaryocytes and play a crucial role in circulation including, blood clotting and repair during blood vessel injury. Quinine is one of the most bitter compound known and acts as an agonist for several bitter taste receptors (T2Rs) including, T2R4. When used as an antimalarial drug, quinine is known to cause thrombocytopenia. RalA is a small GTPase that has been shown to play a role in platelet function. RalA activity is regulated by calcium and the calcium-binding protein, calmodulin. The objective of this study is to investigate if quinine regulates RalA activity in platelets independently and/or through T2R4. The immortalized megakaryocyte cell line, CHRF-288-11, was utilized in these studies. Pull-down assays using Ral-binding domain of Ral-interacting protein 1 were employed to assess RalA activation. To investigate if CHRF cells express bitter taste receptor, T2R4, RT-PCR and western blot analysis was carried out. Treatment of CHRF cells with quinine resulted in the activation of RalA. The results from PCR and western blot analysis demonstrated the presence of T2R4 in CHRF cells. It has been shown that calmodulin and calcium are required for RalA activation. First, we investigated if quinine altered the interaction between calmodulin and RalA. Incubation of CHRF cell lysate with quinine prior to incubation with CaM-Sepharose beads demonstrated that quinine did not affect the interaction between RalA and calmodulin. Next, we investigated if quinine mediated RalA activation is through a calcium dependent pathway. CHRF cells were treated with quinine and calcium chelator (BAPTA-AM) or calmodulin antagonist (W7). The results obtained demonstrated that quinine activated RalA independently of calcium. Treatment of CHRF cells with BCML, a T2R4 antagonist, inhibited quinine mediated activation of RalA suggesting that quinine action is dependent on this bitter taste receptor. The information gained from these studies may lea

Protein hydrolysates have been shown to be sources of various bioactive peptides but there is scanty information on their ability to act as bitter taste modifiers. Meat has multiple proteins and offers the possibility of generating a wide range of peptides with desirable function as bitter taste modifiers. In this study, beef protein was hydrolyzed with each of six commercial enzymes (Alcalase, Chymotrypsin, Trypsin, Pepsin, Flavourzyme, and Thermoase). Using an electronic tongue showed that Alcalase hydrolysates (AH) had significantly (P<0.05) lower bitter scores compared to other enzymatic hydrolysates. Separation of the AH and chymotrypsin hydrolysate (CH) by RP-HPLC yielded peptide fractions with significant ability to inhibit quinine activation of a human bitter taste receptor (T2R4) in HEK 293 cells based on calcium imaging assays. Eight peptides were identified from the T2R4-inhibitory AH and CH fractions, most of which were dominated by leucine and polar amino acids.

Humans have five basic taste sensations which are bitter, sweet, umami, salt and sour. Salt and sour are mediated by ion channel-linked receptors, while bitter, sweet and umami are sensed by cell surface G protein-coupled receptors (GPCRs). Among the five basic taste modalities, the bitter taste signaling mechanism is the most complex and least understood. This is in part due to the large number of bitter taste receptors (T2Rs) in humans (n=25), and the wide range of bitter compounds they detect. The T2Rs are expressed in the oral cavity as well as in many extraoral tissues. The activation of T2Rs in the oral cavity elicits bitter taste sensation, while the activation of the extraoral T2Rs causes various physiological and pathophysiological responses. Recent findings showed that T2R activation caused muscle relaxation and bronchodilation of pre-contracted airway smooth muscle. Moreover, bacterial quorum sensing molecules were shown to activate T2Rs in extraoral tissues. In view of the importance of T2R functions in extraoral tissues, it is of great value to study the structure-function relationship of T2Rs and identify efficient ligands. Hitherto, there have been few studies published involving human bitter taste receptor 7 (T2R7). Only few agonists have been identified for the T2R7 receptor, and no structure-function studies on the T2R7 receptor have been reported. In this study, different compounds including known T2R7 agonists, common bitter compounds, antibiotics and quorum sensing molecules were tested for their ability to activate or inhibit T2R7 heterologously expressed in HEK293T cells. Results suggest that T2R7 is activated by novel ligands including dextromethorphan (DXM), diphenhydramine (DPH), thiamine, tobramycin and erythromycin. In contrast, beef protein hydrolysates purified by RP-HPLC quenched quinine activated T2R7 signal. This suggests that beef hydrolysates may contain potent T2R7 blockers. To understand the structure of the ligand binding pocket

Oral health is closely connected with the overall systemic health. Oral biofilms are dynamic and present a very specific bacterial community which affects oral health. Oral biofilms are directly associated with the major oral infections including caries and periodontal disease. Understanding the oral microbial community is important for preventing oral disease and maintaining oral and overall health. In the first part of this study, I have explored the pathogenic load for three microorganisms Porphyromonas gingivalis, Tannerella forsythia and Aggregatibacter actinomycetemcomitans in companion cats and dogs. Results showed detectable levels of P. gingivalis and T. forsythia in both healthy and diseased cats and surprisingly the number was minimal in healthy dogs. A. actinomycetemcomitans was not detected in any healthy or diseased samples as this bacterium is found only in aggressive stages of Periodontitis. Quorum sensing (QS) system is a cell-cell signaling system that enables bacteria to interact with their neighboring cells and facilitates coordinated bacterial behavior in response to environmental changes. Gram-negative bacteria contain several quorum sensing systems in which different molecules are used as signals, including acyl-homoserine lactones (AHLs), cyclic dipeptides, autoinducers 2 and 3. The LuxI/LuxR-type quorum sensing system consists of a LuxI-like autoinducer synthase that produces AHLs as signals which are detected by a LuxR-type receptor upon exceeding a threshold concentration. The AHLs are detected through binding or activating cytoplasmic receptor proteins, which then dimerize to regulate transcription of downstream genes. P. gingivalis which demonstrated the strongest association with periodontal disease in our study of cats and dogs. P. gingivalis does not produce AHL but possesses many LuxR like homologs on its chromosome. One of these LuxR homologs is PGN_1373 which has been shown to affect the interaction between Streptococcus gordonii a

Pseudomonas aeruginosa is an opportunistic bacterial pathogen, capable of causing both acute and chronic infections in humans. The establishment of infection is a result of the broad pathogen-host interactions. P. aeruginosa possesses a variety of virulence factors to gain advantage over the host during such interactions, and among them are the complex type three (T3SS) and type six (T6SS) secretion systems, which help the pathogen to invade, colonize and survive in the host environment. Both T3SS and T6SS play important roles in cell-cell interaction and pathogenesis. Expression and function of these systems relies on various extracellular signals, such as calcium levels, phosphate concentrations and the contact with host cell. Much is still unknown about the complex regulation of the two secretion systems. A transposon insertion library was previously constructed in our lab to identify genes involved in T3SS regulation. In this project, the gene fimV and a gene cluster PA3284-81 identified from the library were selected for further study. Their effect on T3SS expression was verified and the potential mechanism investigated. The results showed that fimV is important for P. aeruginosa virulence and persistence, and the knockout fimV mutant downregulated the expression of exoS, a representative of T3SS system. PA3284-81 also influenced T3SS expression, and a significant increase of ExoS protein secretion was observed when PA3284-81 was over-expressed on a plasmid. Finally, T3SS expression was found to be regulated by zinc ions; an upregulation of exoS was observed under conditions of zinc depletion. In addition, PA3284-81 was shown to respond to other environmental signals, such as calcium, iron and peracetic acid, possibly influencing virulence. To investigate the regulation of the recently discovered T6SS systems, a transposon mutagenesis library was constructed. Potential regulators of the second T6SS system H2-T6SS were screened for. A Total of eight putative reg

Pseudomonas aeruginosa is the leading cause of morbidity and mortality in patients with chronic diseases, neutropenia and those with immunocompromise. Pulmonary infection with P. aeruginosa in patients with cystic fibrosis can result in respiratory failure and death. The pathogenesis of P. aeruginosa involves a lifestyle change in the bacterium and depends on coordination between two-component regulatory systems, quorum sensing systems and signaling via small molecules such as bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP). Acute form of P. aeruginosa infection is characterized by motility and host-directed toxicity by Type Three Secretion System (T3SS). On the other hand, chronic infection is characterized by formation of biofilms. These biofilms are highly resistant to external stressors and play a role in bacterial persistence. RetS-GacS/A-PA1611-RsmA/Y/Z is a key regulatory pathway in P. aeruginosa that determines the bacterium’s lifestyle choice. Previously, PA1611 a hybrid sensor kinase was identified as a new player in this pathway that functions independent of phosphorelay and influences biofilm formation and T3SS. However, the exact nature of its interaction, the regulation and the environmental signals that activate PA1611 are still unexplored. In this study, using advanced molecular methods, the structural and mechanistic basis of the interaction between PA1611 and RetS is examined in P. aeruginosa PAO1. The histidine kinase A and histidine kinase-like ATPase domains of PA1611 are found to play crucial roles in the PA1611–RetS interaction and have been shown to exert profound effect on bacterial phenotypes. cmpX (PA1775) was identified as the regulator of PA1611. A cmpX knockout demonstrated higher biofilm formation, downregulation of the T3SS and elevated intracellular c-di-GMP levels. cmpX was also identified to exert its effects via both the RetS-GacS/A-PA1611-RsmA/Y/Z as well as via activated c-di-GMP signaling. Finally, mucin and anoxi