Endocannabinoids are derivatives of arachidonate-based lipids that activatea network of signaling pathways in eukaryotes...

Breast cancer is a world health problem and is a leading cause of cancer-related death among women in the United States. However, breast cancer risks were reported to be reduced through exposure to Vitamin D through its Receptors identified as the p53 target gene. The purpose of this study was to assess the associations between VDR gene polymorphisms knowledge/awareness and decisions to reduce breast cancer risks and likelihood of mammogram screening among women in Texas. Data from survey were used. Roy adaptation model was the theoretical framework that guided this quasi- experimental, quantitative research. The dependent variables were decisions to reduce breast cancer risks and likelihood of mammogram screening. The independent variables were knowledge about VDR gene polymorphisms and exposure to vitamin D. The covariates were level of education, awareness, lifestyle, breast self-exams, mammograms, age, early menarche, late menopause, and family history of breast cancer. The chi-square test and regression analysis were used to test the stated research hypotheses and to answer the research questions. Knowledge of VDR gene polymorphisms and exposure to vitamin D were not significantly associated with breast cancer risk, ï?£2 (3, N= 250) =3.84, p > 0.05. Also, awareness of the risk factors for breast cancer was not significantly associated with decisions to go for mammogram screenings or to enroll in breast cancer risk-reduction programs, ï?£2 (3, N= 250) =1.58, p > 0.05. To advocate for the promotion of awareness of the importance of pharmacogenetic testing for VDR gene polymorphisms for early detection of breast cancer, which would help to undertake appropriate therapeutic measures in a timely manner to prevent cancer metastasis, further research is warranted.

Aflatoxin B1 (AFB1) is a potent dietary hepatocarcinogen and may be a lung carcinogen when inhaled. To study the relative ability of lung and liver to metabolize AFB1, a susceptible (Swiss-Webster rat) and resistant species (Syrian golden hamster) were pretreated with inducing agents in order to identify specific AFB1 metabolizing enzymes in each tissue. Analysis of AFB1-exo-epoxide (AFBO) formation, O-dealkynation assays, and protein immunoblots demonstrated that cytochrome P450 (CYP) 1A proteins were overexpressed in both the lung and liver of hamsters pretreated with 3-methylchyolanthrene (3-MC). Only CYP1A1 was expressed in the lung and there was no indication that this protein was involved in AFB1 activation. CYP1A2, on the other hand, was induced in the liver and this correlated well with both increasing protein activity and AFBO formation. It would appear that CYP1A2 is important in activating AFB1 in hamster liver. Although the hamster is resistant as compared to the rat, AFBO formation was higher in both the lung and liver of the hamster compared to the rat. Glutathione S-transferase (GST) Yc subunits were detected in the lung and liver of both species but were not induced by the inducing agents used in these experiments. Following intratracheal injections of [3H]AFB1, in the rat, specific activity was localized in the liver. Only a fraction of the activity was detected in the lung. Of four inducing agents used, only pretratment with phenobarbital (PB) showed increased AFB1-DNA binding in either lung or liver. This correlated with increased CYP2B1 protein levels in both lung and liver, as well as increased CYP2B1 activity and AFBO formation in the liver. Cooxidation of AFB1 by purified prostaglandin H-synthase was shown to produce AFBO but microsomal fractions from rabbit lung and liver failed to show detectable levels of AFBO formation by this cooxidative pathway. Neither purified 5-lipoxygenase or cytosolic fractions from rabbit lung or liver showed detectable levels of LOX mediated cooxidation of AFB1 to AFBO. These studies demonstrate that hamster resembles the human in regard to AFB1 activation in the liver, but that a different as yet unknown enzyme is responsible for hamster lung AFB1 activation. Further evidence that the rat is a poor model for human AFB1 metabolism was demonstrated with the fact that rat activates AFB1 with CYP2B1, a protein unknown in humans.

The interferon (lFN) signalling and RNA interference (RNAi) pathways are the major antiviral pathways in animals and plants, respectively. Although the mechanism of RNAi remains to be completely characterised, the genes that encode the proteins involved in this process have been identified in the plant, fungi and animal kingdoms (Fagard et al., 2000, Grishok et aI., 2000, Hall et al., 2003, Kanellopoulou et al., 2005, Kolb et al., 2005); with comparative analyses indicating that RNAi is an evolutionarily conserved mechanism. Several studies have identified RNAi suppressors encoded by animal viruses, suggesting an antiviral role for the RNAi pathway in animals as well as plants (Andersson et al., 2005, Bennasser et al., 2006, Garcia et al., 2006, Li et al., 2004, Lichner et al., 2003, Lingel et al., 2005, Lu & Cullen, 2004, Wang et al., 2006). However, most of these studies were performed in non-mammalian systems and as yet, there is no direct evidence indicating that the RNAi pathway plays a significant antiviral role during the infection of mammalian cells. Interestingly, several viruses have now been shown to express their own microRNA (miRNA) in infected cells (Grey et al., 2005, Pfeffer et al., 2005, Pfeffer et al., 2004, Samols et al., 2005, Sullivan et al., 2005). Further, in the case of hepatitis C virus (HCV), there is evidence that the virus usurps the host cell miRNAs to enhance viral replication (Jopling et al., 2005). The principal aim of this project was to investigate the role of RNAi in mammalian cells during viral infection, particularly infection with the influenza A virus. This thesis is divided into six major chapters followed by a brief general discussion. Chapter 1 contains a general introduction to the RNAi pathway. It describes the history of the discovery of RNAi and summarizes the known and proposed antiviral roles of the RNAi pathway in plants and mammalian cells. Chapter 2 describes the general materials and methods used for this project. There are four main result chapters, each dealing with a specific experimental system. Each chapter is divided into a brief introduction, specific materials and methods used, followed by presentation of the experimental results and a brief discussion. Chapter 3 describes the development of an in vitro Dicer activity assay to study the effect of viral proteins on the activity of the mammalian Dicer protein. It was demonstrated that crude cell lysates derived from influenza A virus infected cells impaired the activity of Dicer and this observation was not due to degradation of the Dicer protein by virally-induced proteases. Chapter 4 describes the use of a GFP reporter assay for screening potential RNAi suppressors. This assay is suitable for studying viral proteins in isolation. The effect of the influenza NS1 protein on the RNAi pathway in HEK293 cells was investigated and it was shown that NS1 could exert modest, but nevertheless significant, suppression of the RNAi pathway. Northern studies, performed to examine the processing of shRNA in the presence of NS1, demonstrated that NSI suppressed the RNAi mechanism through interfering with the maturation ofshRNA into siRNA. Chapter 5 describes the effect of over-expressing components of the RNAi pathway on influenza A virus infection. In these experiments, Exportin 5, which encodes a protein involved in the transport of pre-miRNA/shRNA into the cytoplasm, was over-expressed during influenza A virus infection. Reduced viral infection was observed in cells over-expressing Exportin 5, suggesting that this treatment protects cells from virus infection. Chapter 6 describes the expressed small RNA profile during influenza A virus infection in MDCK cells. Novel canine miRNA homologues were identified through cloning and sequencing. No definitive evidence for virally-derived siRNA/miRNA was found but a general reduction of endogenous miRNA expression was detected.

We have developed a system to hunt and reuse special gene integration sites that allow for high and stable gene expression. A vector, named pRGFP8, was constructed. The plasmid pRGFP8 contains a reporter gene, gfp2 and two extraneous DNA fragments. The gene gfp2 makes it possible to screen the high expression regions on the chromosome. The extraneous DNA fragments can help to create the unique loci on the chromosome and increase the gene targeting frequency by increasing the homology. After transfection into Chinese hamster ovary cells (CHO) cells, the linearized pRGFP8 can integrate into the chromosome of the host cells and form the unique sites. With FACS, 90 millions transfected cells were sorted and the cells with strongest GFP expression were isolated, and then 8 stable high expression GFP CHO cell lines were selected as candidates for the new host cell. Taking the unique site created by pRGFP8 on the chromosome in the new host cells as a targeting locus, the gfp2 gene was replaced with the gene of interest, human ifngamma, by transfecting the targeting plasmid pRIH-IFN. Then using FACS, the cells with the dimmest GFP fluorescence were selected. These cells showed they had strong abilities to produce the protein of interest, IFN-gamma. During the gene targeting experiment, we found there is positive correlation between the fluorescence density of the GFP CHO host cells and the specific production rate of IFN-gamma. This result shows that the strategy in our expression system is correct: the production of the interesting protein increases with the increase fluorescence of the GFP host cells. This system, the new host cell lines and the targeting vector, can be utilized for highly expressing the gene of interest. More importantly, by using FACS, we can fully screen all the transfected cells, which can reduce the chances of losing the best cells. / Singapore-MIT Alliance (SMA)

High-resolution physically ordered gene maps for the horse (Equus caballus, ECA) are essential to the identification of genes associated with hereditary diseases and traits of interest like fertility, coat color, and disease resistance or susceptibility. Such maps also serve as foundations for genome comparisons across species and form the basis to study chromosome evolution. In this study seven equine chromosomes (ECA6, 7, 10, 15, 18, 21 and X) corresponding to human chromosomes (HSA) 2, 19 and X were selected for high-resolution mapping on the basis of their potential involvement in diseases and conditions of importance to horses. To accomplish this, gene- and sequence-specific markers were generated and genotyped on the TAMU 5000rad horse x hamster RH panel. Additionally, screening of a BAC library by overgoes and subsequent STS content mapping and fingerprinting approaches were used to assemble and verify a BAC contig along a ~5 Mb span on ECA21. Dense gene maps were generated for each of the seven equine chromosomes by adding 408 new markers (285 type I and 123 type II) to the current maps of these chromosomes, thereby greatly improving overall map resolution to one mapped marker every 960kb on average (range: 700 kb – 1.3 Mb). Moreover, the contig on ECA21 contained 47 markers (42 genes and 5 microsatellites) as well as 106 STS markers distributed along 207 BAC clones. Comparisons of these maps with other species revealed a remarkably high level of horse-human X chromosome conservation, as well as two evolutionary breakpoints unique to Perissodactyls or Equids for the equine homologues of HSA19 and HSA2, one of which has been more precisely localized by the ECA21 contig. Thus, high resolution maps developed for these chromosomes i) provide a basis to map traits of interest rapidly to specific chromosomal regions, ii) facilitate searches for candidate genes for these traits by fine comparisons of the equine regions with corresponding segments in other species, and iii) enable understanding the evolution of the chromosomes. Expansion of this work to the entire equine genome will be important for developing novel strategies for diagnosis, prevention, and treatment of equine diseases.

No description available.

The interferon (lFN) signalling and RNA interference (RNAi) pathways are the major antiviral pathways in animals and plants, respectively. Although the mechanism of RNAi remains to be completely characterised, the genes that encode the proteins involved in this process have been identified in the plant, fungi and animal kingdoms (Fagard et al., 2000, Grishok et aI., 2000, Hall et al., 2003, Kanellopoulou et al., 2005, Kolb et al., 2005); with comparative analyses indicating that RNAi is an evolutionarily conserved mechanism. Several studies have identified RNAi suppressors encoded by animal viruses, suggesting an antiviral role for the RNAi pathway in animals as well as plants (Andersson et al., 2005, Bennasser et al., 2006, Garcia et al., 2006, Li et al., 2004, Lichner et al., 2003, Lingel et al., 2005, Lu & Cullen, 2004, Wang et al., 2006). However, most of these studies were performed in non-mammalian systems and as yet, there is no direct evidence indicating that the RNAi pathway plays a significant antiviral role during the infection of mammalian cells. Interestingly, several viruses have now been shown to express their own microRNA (miRNA) in infected cells (Grey et al., 2005, Pfeffer et al., 2005, Pfeffer et al., 2004, Samols et al., 2005, Sullivan et al., 2005). Further, in the case of hepatitis C virus (HCV), there is evidence that the virus usurps the host cell miRNAs to enhance viral replication (Jopling et al., 2005). The principal aim of this project was to investigate the role of RNAi in mammalian cells during viral infection, particularly infection with the influenza A virus. This thesis is divided into six major chapters followed by a brief general discussion. Chapter 1 contains a general introduction to the RNAi pathway. It describes the history of the discovery of RNAi and summarizes the known and proposed antiviral roles of the RNAi pathway in plants and mammalian cells. Chapter 2 describes the general materials and methods used for this project. There are four main result chapters, each dealing with a specific experimental system. Each chapter is divided into a brief introduction, specific materials and methods used, followed by presentation of the experimental results and a brief discussion. Chapter 3 describes the development of an in vitro Dicer activity assay to study the effect of viral proteins on the activity of the mammalian Dicer protein. It was demonstrated that crude cell lysates derived from influenza A virus infected cells impaired the activity of Dicer and this observation was not due to degradation of the Dicer protein by virally-induced proteases. Chapter 4 describes the use of a GFP reporter assay for screening potential RNAi suppressors. This assay is suitable for studying viral proteins in isolation. The effect of the influenza NS1 protein on the RNAi pathway in HEK293 cells was investigated and it was shown that NS1 could exert modest, but nevertheless significant, suppression of the RNAi pathway. Northern studies, performed to examine the processing of shRNA in the presence of NS1, demonstrated that NSI suppressed the RNAi mechanism through interfering with the maturation ofshRNA into siRNA. Chapter 5 describes the effect of over-expressing components of the RNAi pathway on influenza A virus infection. In these experiments, Exportin 5, which encodes a protein involved in the transport of pre-miRNA/shRNA into the cytoplasm, was over-expressed during influenza A virus infection. Reduced viral infection was observed in cells over-expressing Exportin 5, suggesting that this treatment protects cells from virus infection. Chapter 6 describes the expressed small RNA profile during influenza A virus infection in MDCK cells. Novel canine miRNA homologues were identified through cloning and sequencing. No definitive evidence for virally-derived siRNA/miRNA was found but a general reduction of endogenous miRNA expression was detected.

In combating variable pathogens, mammalian immune systems have evolved diverse families of ligands and receptors. Epitomizing this strategy are the polymorphic major histocompatibility complex class I genes (termed HLA class I in humans) that encode ligands for highly variable natural killer (NK) cell receptors (in humans, the killer cell immunoglobulin-like receptors or KIR). Technological advances are poised to allow sequencing of these polymorphic genes, the most variable in the human genome, at the highest possible accuracy and resolution. However, studies that correlate immunogenetic polymorphisms with functional changes are in their infancy and often limited to those variants that combine high ligand avidity and high frequency in Caucasians. As a result, there is a paucity of information regarding the true scope of functional human immunogenetic diversity. This not only restricts our understanding of the evolution and function of the human immune system, but also underserves non-Caucasian populations with respect to disease association studies and therapeutic advances. The work presented in this thesis details original research and methodological advances that begin to address these functional shortfalls, the goal being to improve our understanding of the relationship between immunogenetic diversity, protein structure and immune function.

Understanding the molecular mechanisms that govern productivity and growth of recombinant host cells is essential to devise informed approaches to increase commercial viability and availability of biopharmaceuticals. This work has focused on the roles of the mammalian target of rapamycin complex 1 (mTORC1) signalling pathway in CHO cells, the most widely used expression system in the biopharmaceutical industry. mTORC1 is a master regulator of cell growth, protein synthesis and metabolism in response to availability of nutrients, oxygen and growth factors. Therefore, it was hypothesised that increased activity of mTORC1 enhances growth and productivity of recombinant CHO cells. The study of a recombinant GS-CHO cell line in the serum-free suspension batch culture indicated a gradual decrease in the activity of mTORC1, as defined by the decreased extent of site-specific phosphorylation of two widely ascribed downstream target proteins (ribosomal protein S6 kinase 1 (S6K1) and 4E-BP1, an inhibitor of translation initiation). The decline in the activity of mTORC1 paralleled decreased growth rate, recombinant protein specific productivity and global protein translation. To further clarify the role of the mTOR pathway in cell growth and protein production, cells in batch culture were treated with rapamycin, a specific inhibitor of mTORC1. Treatment with rapamycin stalled the growth of the CHO cell line transiently, but recombinant protein specific productivity, longevity of batch culture, and final antibody titre were greater than control. Rapamycin addition produced discriminating effects on downstream signalling targets, implicating distinct roles for these targets in control of growth and protein synthesis. Engineering the mTORC1 pathway by overexpression of specific components of this pathway (S6K1 and Rheb) generated increased growth and extended viability. Greater proliferation was not associated with improved productivity suggesting highly proliferative phenotypes that prioritise cell growth over synthesis and secretion of recombinant antibody in the recombinant GS-CHO cells examined. Therefore, the engineering of mTORC1 pathway may be beneficial to increase robustness or adaptation to stressed conditions (such as serum- free suspension growth, low nutrition availability and hypoxia).