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Objective: To investigate the association between feeding practices (exclusive breastfeeding, exclusive bottle-feeding, and mixed) with early childhood caries (ECC) and the association between the prevalence of oral Rothia, at the species and genus level, with ECC. Methods: A total of 438 children (178 caries-free, 260 with ECC) were included in this case-control study. A comprehensive questionnaire that included a section on feeding history, was completed by the parent or caregiver of each participant. Based on the feeding history responses, participants were classified as either exclusively breastfed, exclusively bottle-fed, or mixed (both bottle-fed and breastfed). A supragingival plaque sample was collected from each participant and then assessed with 16S rRNA sequencing for Rothia genus and species’ relative abundances. The association between feeding practices and Rothia prevalence with ECC, accounting for confounding variables, was analyzed using multivariable logistic regressions. Results: A low relative abundance of Rothia aeria was significantly associated with ECC (p<0.05), whereas there were no significant associations found between the relative abundance of Rothia dentocariosa or Rothia mucilaginosa with caries status (p>0.05). While the odds of association between a low relative abundance of Rothia (genus) and ECC was 1.63, this relationship failed to reach the threshold of significance (p=0.054). Feeding practices were not significantly associated with ECC after adjusting for confounding variables. However, bedtime bottle use, bedtime snacking, living in a rural/remote area, being an older age, the use of fluoridated toothpaste, and having less-educated guardians were significantly associated with ECC (p<0.05) Conclusions: Findings in this study suggest that low relative abundances of R. aeria and Rothia (genus) are associated with ECC. Feeding practices were not key risk factors for ECC in this sample, as other factors related to the social environme

The loss of effective therapeutic options due to the increase instances of antimicrobial resistant bacterial infections is of great concern to the World Health Organization (WHO). The WHO released a list to prioritize efforts in fighting this problem and placed carbapenem-resistant Acinetobacter baumannii as the top priority in need of development of new therapeutic options. A. baumannii is a Gram-negative opportunistic pathogen that can survive within clinical environments. A lot is known about the ability of this species to resist and tolerate the effects of antimicrobials. It possesses three efflux pumps of the resistance-nodulation-division (RND) efflux family, AdeAB(C), AdeFGH, and AdeIJK, that when overexpressed are responsible for reducing the susceptibility of A. baumannii to several antibiotic classes. These RND efflux pumps, and AdeFGH in particular, were the focus of my study. In this study I have investigated regulation and physiological function of AdeFGH, which is the least well studied RND efflux pump of the three. I have shown that AdeFGH expression has profound effects on the motility and biofilm formation of A. baumannii and that this is most likely due to increased efflux of acyl homoserine lactones. The effect of AdeFGH on motility and biofilm formation appears to be AdeL-independent. Interestingly, the overexpression of AdeL led to increased virulence in Galleria mellonella. I have discovered that the expression of adeFGH is induced by nitrosative stress and regulated by not only its cognate regulator, AdeL, but also by the global regulator, CsrA. I have investigated the role of CsrA in the physiology of A. baumannii via transcriptomics and phenotypic microarray. Additional investigations into AdeIJK showed it was crucial for A. baumannii virulence in G. mellonella and increased expression lead to increased tolerance to osmotic stress. All of this combined furthers our understanding of A. baumannii physiology and identifies several new potential

Background: The C1Q-tumor necrosis factor related protein (CTRP) family has functions in metabolism, immunity, and cancer. Among the CTRP family, CTRP8 remains the least well characterized in terms of function and receptors. In the brain tumor glioblastoma (GBM), CTRP8 impacts migration and treatment resistance through its receptor RXFP1. We investigate a new interaction between CTRP8 and the EGF-like protein Delta-like homolog 1 (DLK1). DLK1 is a well-known regulator of differentiation and is considered a marker of stemness in several cancers. In GBM, elevated DLK1 expression promotes migration, stemness and has been correlated with poor prognosis. There is interest in further understanding complex mechanisms of DLK1 in a cancer context. Methodology: A yeast-two hybrid (Y2H) screen and co-immunoprecipitation (co-IP) were used to characterize CTRP8-DLK1 interaction domains. The U251 cell line was used to assess stemness using tumor sphere formation and glioma stem cell (GSC) marker expression. In the same U251 model we investigate mechanisms responsive to CTRP8-DLK1 that regulate GSCs. Results: The Y2H identified DLK1 as a CTRP8 interaction partner. In co-IP CTRP8 binds full-size DLK1 and a truncated, soluble form of DLK1. DLK1 enhanced sphere formation and GSC marker expression in U251. The pro-stemness effect of DLK1 was counteracted by CTRP8, resulting in an overall reduction in stemness. Reduced stemness in cells expressing a cleavage-resistant DLK1 form revealed potential functions of DLK1 cleavage products as factors regulating the GSC population. In U251 cells expressing DLK1, CTRP8 treatment increased DLK1 intracellular domain (ICD) release and nuclear translocation. DLK1 ICD may act as a transcriptional co-factor revealing a new function for DLK1 ICD. Conclusions: This is the first study describing CTRP8 as a DLK1 ligand. Our findings reveal DLK1 as a promoter of glioma stemness, and CTRP8 as an antagonist. The CTRP8-DLK1 interaction may be part of a novel

Objective: To investigate the oral health-related quality of life (OHRQoL) of young children in a randomized clinical trial of silver diamine fluoride (SDF) with 5% fluoride varnish (FV) to manage early childhood caries (ECC). Methods: Children < 72 months of age with active dentinal caries lesions (ICDAS 5 or 6) in primary teeth received two applications of 38% SDF and 5% FV as part of a randomized clinical trial testing three different frequency regimes (1, 4 and 6 months apart). The early childhood oral health impact scale (ECOHIS) questionnaire was completed at each study visit. Statistical analyses included descriptive and parametric methods. A p-value ≤ 0.05 was statistically significant. Results: Eighty-two children (58.5% male) were recruited with a mean age of 44.3±14.2 months. Statistically significant improvement in total ECOHIS scores for all children was observed following treatment (baseline mean scores 4.64±4.74 vs. second visit score 3.26±4.36 vs. third visit score 3.25±3.77, p=0.006 and p=0.01, respectively). The following domains showed the most improvement with SDF treatment: parental distress, and family function. No statistically significant difference in ECOHIS scores were observed between the three frequency groups at baseline, second visit, and final visit (p=0.24, p=0.71 and 0.26) Conclusions: Children exhibited improved overall OHRQoL following two applications of 38% SDF and 5% FV, although there were no significant differences observed between the treatment groups. The results from our study demonstrate that SDF is a suitable non-restorative caries management option for children with ECC as it is associated with improved oral health related quality of life.

Objective: To investigate the effectiveness of silver diamine fluoride (SDF) to arrest early childhood caries (ECC) in very young children randomized to three different application frequency groups (regimens) Methods: Children with active dentinal carious lesions (ICDAS 5 or 6) in primary teeth without any signs of pulpal involvement were randomized into three different application frequency groups; 1, 4, or 6 months apart. Children underwent treatment with 38% SDF at baseline visit and again at a second visit. Treated lesions were assessed at the second and third visits to determine arrest success. Participants were considered completely successful (CS) if all treated lesions were arrested and incompletely successful (IS) if at least one lesion was not arrested. Statistical analyses included descriptive and bivariate analyses. A p value ≤ 0.05 was significant. Results: A total of 84 children with 486 carious lesions were recruited into the study with 28 children in each group. The overall proportion of completely successful treated children at visit 2 and final visit were 40% and 68% respectively. Results of McNemar’s test showed a significant increase in complete success from 40% at second visit to 68% at visit 3 (p-value <0.001) across all groups for 84 study participants. Within each group, significant improvement in complete success at child-level was also noted with p-values of 0.041,0.023 and 0.004 for Regimen 1, 2 and 3 respectively. Participants in one month group showed a change of complete success from 36% at second visit to 71% at third visit (p-value 0.004). No significant differences were noted between the groups at second visit and final visit (p >0.05) Conclusions: Findings in this study suggested that two applications of SDF at different frequencies showed similar frequency of successfully treated cases at the child level. No difference was noted in success rates comparing three groups either at second or third visit. Results from our study

Introduction: Silver diamine fluoride (SDF) is a simple and non-invasive agent to arrest caries in children. The purpose of this study is to investigate changes to the oral bacteriome and mycobiome in young children treated with SDF Methods: Plaque samples were collected from 45 children with early childhood caries (ECC). Frequency regimens of SDF were two applications 4 months apart (4M), two applications 6 months apart (6M), and two applications 1 month apart (1M). DNA was extracted and sent to Genome Quebec Innovation Center for library preparation and paired-end Illumina MiSeq PE300 sequencing of the V4 region of bacterial 16S rRNA and fungal ITS1 rRNA genes. Sequencing data was analyzed using QIIME2 2018.11. Results: A total of 195 carious lesions in 44 children were treated at baseline and followed over two subsequent study visits. The overall arrest rates were 77.69% at Visit 2 and 93.61% at Visit 3 respectively. Arrest rates were higher for all lesions after two applications of SDF. The alpha diversity and beta diversity analysis showed no significant differences in the supragingival bacteriome and mycobiome for all three regimens. Taxonomic assignment showed that Streptococcus, Corynebacterium and Actinomyces were the most abundant genera for Bacteria and Candida, Blumeria, and Malassezia were the most abundant genera for Fungi. Streptococcus mutans and Veillonella dispar both showed a decrease in relative abundance, but these changes were a trend and not found to be statistically significant. Candida albicans was highly abundant in all groups regardless of number of visits or regimens. The differential abundance analysis showed significant changes in both bacterial and fungal species, particularly Lactobacillus spp., Bifidobacterium spp. and Candida spp. Our study found a vast diversity of fungal species and differential abundance analysis suggested that SDF treatment may have an effect on the abundance of specific fungi. Conclusion: SDF was an effective m

Early childhood caries (ECC) and its more severe form S-ECC are prevalent diseases linked to various etiologic factors, including sex, microbiota, diet, and genetics. Recent studies have highlighted the role of inter-kingdom interactions involving fungi and cariogenic bacteria in the onset of dental caries. However, sex-specific and oral site-specific differences in the oral microbiome are poorly characterized. Evidence suggests that there is a plausible relationship between genetic variants in taste genes and enhanced ECC risk or protection. This has been attributed to the modulatory effect of bitter taste receptors on taste preferences and oral innate immune responses. Nevertheless, previous caries studies focused on select sweet and bitter taste receptors (T1Rs and T2Rs), with minimal or no attention to other taste signal transducing proteins such as free fatty acid receptors (FFARs), salt and sour ion channels (ENaCs and OTOP1), and carbonic anhydrases (CAs). Therefore, this study investigated whether variants in multiple taste genes play a role in S-ECC and the oral microbiome. The first part of the thesis focuses on characterizing the oral microbiome, identifying sex-specific and site-specific bacterial and fungal species that are associated with S-ECC. The second part evaluates the association of taste genetic variants with S-ECC and the dental plaque microbial composition. The third part investigates the role of seven genes, involved in downstream taste signaling, in S-ECC and dental biofilm composition. These studies demonstrate that sex- and site-specific differences in the oral microbiome exist. A significant difference in the abundance of Neisseria was observed between males and females with S-ECC. Streptococcus mutans, Candida albicans, and Candida dubliniensis, among others, were enriched in the dental plaque, compared to oral swabs, of children with S-ECC. Also, genetic variants in genes encoding proteins involved in taste sensations (SCNN1D, CA6, TAS

The fundamental framework of steroidogenesis across steroidogenic cells is similar, especially the initial mitochondrial steps involving cholesterol translocation across outer mitochondrial membrane (OMM) by the START domain containing proteins (e.g., StAR) and subsequent conversion to pregnenolone by the enzyme P450scc in the inner mitochondrial membrane (IMM). Thus, cholesterol and mitochondria are essential and highly interconnected in steroidogenesis. However, our understanding of this conserved process in steroidogenesis remains limited. Particularly, the transport of cholesterol from the OMM to the IMM. Moreover, growing evidence suggest an important role of autophagy/lipophagy and mitochondrial dynamics in regulating steroidogenic cholesterol homeostasis. However, a potential role the intracellular cholesterol pool in itself in the regulation steroidogenic events remains unexplored. I investigated an unexpected testicular phenotype of two transgenic mouse models and found that the male transgenic mice that expressed a mutant form of a mitochondrial protein prohibitin-1 (PHB1Tyr114Phe) from the Fabp-4 gene promoter displayed smaller testes, higher testosterone levels and lower gonadotropin levels as compared with age matched PHB-1 overexpressing and wild-type mice. Additionally, I found StAR and P450scc as interacting partners of PHB1 in Leydig cell mitochondria. Further analysis of testis and Leydig cells from the transgenic mice revealed that PHB1 plays a regulatory role in coordinating cell signaling, cholesterol homeostasis, and mitochondrial biology pertaining to steroidogenesis. Moreover, I tested the hypothesis that intracellular cholesterol pool in steroidogenic cells plays a role in regulating cell-intrinsic factors and events pertaining steroidogenesis. I found that the depletion of intracellular cholesterol pool in steroidogenic cells (i.e., MA-10, Y-1 and BeWo cells) induces autophagy, affects mitochondrial dynamics, and upregulates steroidogenic f

Introduction: Early childhood caries (ECC) is a widespread, multifactorial disease. Dental decay is a result of low-pH demineralization caused by acidogenic bacteria living in oral biofilms. Nutrition is a key determinant of ECC, as these bacteria require fermentable carbohydrates to grow. Veillonella are important bacteria that facilitate the activity of acidogenic bacteria by removing excess lactate products. A majority of oral microbiome studies focus on the importance of well-known cariogenic bacteria, such as Streptococcus mutans, while few studies have characterized Veillonella in ECC. The purpose of this study was to gain a greater understanding of the relationship between nutrition, oral Veillonella, and ECC. Methods: A case-control study design was used. The nutritional profiles of 158 children (75 caries-free, 83 with ECC) from Manitoba were determined via NutriSTEP (Nutrition Screening Tool for Every Preschooler and Toddler). Veillonella species were assessed using relative abundance data from 16S rRNA sequencing of dental plaque samples. A multivariable logistic regression model was used to estimate the association of ECC with NutriSTEP scores and relative abundance of Veillonella. Results: Caries-affected children tended to be older, established residents, Indigenous, live in rural or remote areas, and have less-educated parents (p ≤ 0.05). Children with ECC had higher total NutriSTEP scores (23.7 ± 6.8) than children without ECC (21.1 ± 6.6; p ≤ 0.05). Relative abundance data revealed significantly higher levels of Veillonella dispar, Streptococcus mutans, and Streptococcus salivarius in caries-affected children (p ≤ 0.05). Children with a high abundance of Veillonella parvula (OR = 2.33; 95% CI: 1.10, 4.91) and greater nutritional risk/NutriSTEP scores (OR = 1.08; 95% CI: 1.02, 1.14) were more likely to experience ECC than children with insignificant levels of the bacteria and lower nutritional risk/NutriSTEP scores. Conclusions: Our findings reflect

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.