The molecular biology of macrophage activating and deactivating cytokines and their receptors was discussed. Comparison of IFN-γ amino acid sequences of several mammalian species reveals a low conservation of amino acids. The interaction of IFN-γ with its receptor system is complicated and coherent with the species specificity of IFN-γ. Identification by PCR of an IFN-γ-like gene in the trout genome was not possible. In contrast with IFN-γ, TGF-β is very conserved in its amino acid sequence. The PCR-amplification of a TGF-β fragment from amphibian, <I>Xenopus</I>, and rainbow trout cDNA libraries was possible. Two oligonucleotide primers were used in PCRs to amplify a 360 bp fragment of trout Mhc class II β chain. Using these two oligos, this 360 bp fragment could be amplified from trout spleen cDNA library and HK leucocytes. cDNA synthesized from RNA extracted from ConA/PMA stimulated HK leucocytes was used as template DNA in PCR, and a class II specific fragment was amplified. This class II fragment could not be amplified from HK macrophages treated with a MAF containing supernatant, although HK macrophages treated with a control supernatant did express class II molecules. This could suggest that priming or activation of trout macrophages results in a decreased expression of Mhc class II antigens. A novel method for analysing 5'nucleotidase activity of head kidney macrophages was optimised for use with cell monolayers, with respect to the effect of cell numbers, temperature and substrate concentration. Both lysed and whole cells could be used for determination of 5'nucleotidase activity. Maximal 5'nucleotidase activity was found in the range of 27°C to 33°C and using a substrate concentration of ≥ 1 μmol AMP ml^-1 for whole cells and ≥ 1.5 μmol AMP ml^-1 for lysed cells. 5'nucleotidase activity was also correlated with respiratory burst activity in cells treated with a variety of supernatants containing MAF activity. A significant inverse relationship between these two activities was found. MAF-treated cells were also found to lose 5'nucleotidase activity faster than control cells in the presence of cycloheximide, suggesting such cells may have a higher membrane turnover of this enzyme.

FGF2 is expressed in renal podocytes as they differentiate <I>in-vivo</I>, as demonstrated by specific antibody staining within developing glomeruli of chicken metanephros. Mice lacking FGF2 have been generated in the laboratory by targeted deletion and kidneys of <I>Fgf2</I> deficient mice appear to develop normally. Detailed histological analysis of adult kidneys from these mice however has revealed low frequency glomerular abnormalities. Additionally, a few obvious cases of glomerulosclerosis with severe podocyte damage were observed specifically in mutant mice. Taken together these observations indicate FGF2 plays a role in podocyte development and/or function. A novel culture system that allows the induction of podocyte cell differentiation <I>in-vivo</I> has recently been developed. The system is based on isolation of conditionally immortalised podocyte cells derived from H-2K<SUP>b</SUP>tsA58 transgenic (immorto) mice. Isolation of podocyte cells from renal glomeruli of wild-type and FGF2 deficient immorto mice was performed to address the functional relevance of FGF2 in podocyte development. Conditionally immortalised wild-type podocyte cells (wild-type MPCs) display characteristic features of podocytes <I>in-vivo</I>, however Fgf2 null MPCs show striking morphological and molecular abnormalities. Mutant podocyte cells do not undergo the epithelial-to-mesenchymal transformation (EMT) associated with normal podocyte maturation and, correspondingly, fail to differentiate. The EMT mediator <I>Slug</I> is up-regulated as wild-type cells differentiate but is missing in mutant cells, suggesting this transcription factor acts downstream of FGF signalling to mediate EMT associate maturation of podocytes. <I>Fgf7</I> and <I>Fgf10</I> expressions are lost in <I>Fgf2</I> deficient MPC cells, but not in <I>Fgf2</I> deficient kidney cortex, affording an explanation to the emergence of a stronger defect <I>in-vitro</I>.

No description available.

<p>Abstract</p><p>Transcription factors control numerous vital processes in the cell through their ability to control gene expression. Dysfunctional regulation by transcription factors lead to disorders and disease. Transcription factors regulate gene expression by binding to DNA sequences (motifs) on the genome and altering chromatin. DNase-seq footprinting is a well-established assay for identification of DNA sequences that bind to transcription factors. We developed computational techniques to analyze footprints and predict transcription factor binding. These transcription factor specific predictive models are able to correct for DNase sequence bias and characterize variation in DNA binding sequence. We found that DNase-seq footprints are able to identify cell-type or condition specific transcription factor activity and may offer information about the type of the interaction between DNA and transcription factor. Our DNase-seq footprint model is able to accurately discover high confidence transcription factor binding sites and discover alternative interactions between transcription factors and DNA. DNase-seq footprints can be used with ChIP-seq data to discover true binding sites and better understand transcription regulation.</p> / Dissertation

Objective: Patient-reported outcomes (PROs) are measures collected from a patient to determine how he/she feels or functions in regards to a health condition. Longitudinal PROs, which are collected at multiple occasions from the same individual, may be affected by response shift (RS). RS is a change in a person’s self-evaluation of a target construct. Latent variable models (LVMs) are statistical models that relate observed variables to latent variables (LV). LVMs are used to analyze PROs and detect RS. LVs are random variables whose realizations are not observable. Factor scores are estimates of LVs for each individual and can be estimated from parameter estimates of LVMs. Factor scoring methods to estimate factor scores include: Thurstone, Bartlett, and sum scores. This simulation study examines the effects of RS on factor scores used to test for change in the LV means and recommend a factor scoring method least affected by RS. Methods: Data from two time points were fit to three confirmatory factor analysis (CFA) models. CFA models are a type of LVM. Each CFA model had different sets of parameters that were invariant over time. The unconstrained (Uncon) CFA model had no invariant parameters, the constrained (Con) model had all the parameters invariant, and the partially constrained (Pcon) model had some of the parameters invariant over time. Factor scores were estimated and tested for change over time via paired t-test. The Type I error, power, and factor loading (the regression coefficient between an observed and LV) and factor score bias were estimated to determine if RS influenced the test of change over time and factor score estimation. Results: The results depended on the true LV mean. The Type I error and power were similar for all factor scoring methods and CFA models when the LV mean was 0 at time 1. For LV mean of 0.5 at time 1 the Type I error and power increased as RS increased for all factor scores except for scores estimated from the Uncon model and Bartlett method. The biases of the factor loadings were unaffected by RS when estimated from an Uncon model. The factor scores estimated from the Uncon model and the Bartlett and sum scores method had the smallest factor score biases. Conclusion: The factor scores estimated from the Uncon model and the Bartlett method was least affected by RS and performed best in all measures of Type I error, statistical power, factor loading and factor score bias. Estimating factor scores from PROs data that ignores RS may result in erroneous (or biased) estimates.

The key to the co-rotational (CR) finite element is the separation between the rigid body motion and the deformational motion. It is this separation which makes it superior to other methods in the analysis of large displacement problems. Since the dynamic analysis of a guyed transmission line system contains large displacements from the vibration of the cable, it is considered appropriate to utilize the technique in the analysis. This thesis re-formulates and simplifies the CR method for such a purpose. Numerical tests show that the time step required for convergence in the present technique is ten times less than that is required for convergence in ANSYS. In the construction of the equation for the prediction of the longitudinal load factor (LLF) for the A402-M guyed transmission line due to cable break events, the tower is modelled using a simplified model of a detailed lattice tower. The simplified model considers latticed tower segment as an equivalent beam segment. The use of the simplified model enables to perform the broken wire dynamic analysis of the ten-span transmission line system within a day or two on a personal computer. Two initiating events are considered: all conductors on one arm break and all cables in one span break. Based on the analysis results, it is found that the LLFs for the all cables break event for the A402-M tower are 5% less than that calculated using the EPRI equation. It is therefore recommended that either the LLFs derived from the EPRI equation or from the proposed equation be used in the design of a guyed transmission tower for the broken wire event. The developed procedure can also be used to predict the LLF for the other type transmission line systems.

Objective: To determine whether serum phthalate concentrations differ in men and women with infertility compared to those without infertility in Saskatchewan Hypothesis: Serum phthalate concentrations will be greater in men and women with infertility compared to fertile men and women Setting: Patients undergoing assisted reproduction for the treatment of infertility; healthy volunteers recruited from the community Recruitment and sample collection: Infertile couples were recruited prior to in vitro fertilization (IVF) therapy for treatment of unexplained infertility (n=15), polycystic ovarian syndrome (PCOS, n=13), and male factor infertility (n=12); fertile men (n=15) and women (n=15) were recruited using poster advertisements. Blood samples were collected by venipuncture for phthalate analysis. Main outcome measures: Serum phthalates concentrations (ng/mL) Design: Prospective cohort pilot study Methods: In infertile couples, blood samples were collected on the following 3 days of the IVF cycle: early during ovarian stimulation, day of oocyte retrieval and day of embryo transfer. In healthy volunteers, 3 blood samples were collected over a 2 week period. Liquid chromatography and tandem mass spectrometry (LC-MS/MS) was conducted to quantify concentrations of four phthalates: di-n-butyl phthalate (DBP), diethyl phthalate (DEP), di-(2-ethylhexyl) phthalate (DEHP) and diisodecyl phthalate (DIDP). Phthalate concentrations were compared among the four study groups using non-parametric Kruskal-Wallis and Mann-Whitney U post hoc tests. Results: Serum DEHP and DEP concentrations did not differ among control, unexplained, PCOS, and male factor infertility groups in both men and women (p>0.05). DBP in women did not differ among study groups (p=0.205). In contrast, DBP was lesser in men with unexplained, PCOS, and male factor infertility compared to controls (p < 0.05). Similarly, DIDP was lesser in women of couples with unexplained, PCOS and male factor infertility groups compared to fertile women (p < 0.05). Less DIDP was detected in men with unexplained and male factor infertility compared to the control group (p < 0.05) Conclusion: Serum phthalate concentrations in serum were lesser or not different in infertility patients undergoing IVF compared to fertile volunteers. These findings do not support the notion that serum phthalate concentrations are associated with human infertility. Further research is needed to determine whether phthalate concentration in blood cells and adipose tissue differ in infertile versus fertile men and women.

Purpose: When the prevalence of physical teen dating violence (TDV) victimization is examined at the state level, significant variation exists; the prevalence ranges from 7.4% in Oklahoma and Vermont to 17.8% in Louisiana. Using U.S. states as the unit of analysis, this study sought to determine whether gender inequality is a societal level risk factor for TDV victimization. Method: Data measuring physical TDV victimization were obtained from the 2009 YRBS. To measure the level of gender inequality in each state, the Gender Inequality Index (GII) was calculated using the procedure described in the United Nations’ Human Development Report. Pearson’s correlation coefficients were calculated to determine the association between TDV victimization, the GII, and the indicators of the GII. Results: Of the 40 states included in analyses, the GII was significantly associated with the state prevalence of both total TDV victimization (r=.323, p=.042) and female TDV victimization (r=.353, p=.026). Subsequent to removal of the outlying case of Oklahoma, the GII was also significantly associated with male TDV victimization (r=.366, p=.022). Several individual GII indicators were significantly associated with TDV victimization after removing the outlying case. Ordinary least squares regression was used to create a model for TDV victimization and gender inequality. Conclusion: This is the first study to examine societal level gender inequality as a risk factor for state level TDV victimization using nationally representative data on school youth. As policy-makers implement TDV prevention policy at the state level, further research understanding potential macro-level risk factors is particularly important.

It is becoming increasingly apparent that many of the genes in the class III region of the human Major Histocompatibility Complex encode proteins involved in the immune and inflammatory responses. Furthermore, genetic studies have indicated that genes within the class III region, particularly the telomeric segment containing the TNF gene, could contribute to susceptibility to diseases of immune-related aetiology. To further characterise this region and to identify candidate disease susceptibility genes, two overlapping cosmids, TN62 and TN82, covering an ~82kb segment of DNA around the TNF gene were selected for sequence analysis. The eight known genes in this region have been precisely positioned with the order: Gl/AIF-1, 1C7, LST1 (B144), LTB, TNF, LTA, IKBL, BAT1 (centromere to telomere) and their genomic structures have been defined. Comparison of the Gl genomic region with previously described cDNA and genomic sequences, together with the results of RT-PCR, indicates that three alternative transcripts, Gl, Allograft Inflammatory Factor-1 and Interferon-γ Responsive Transcript-1, are all derived from this gene. The completion of the sequence of 1C7 (D6S2570) has revealed that this gene encodes a putative novel member of the immunoglobulin superfamily. A number of alternatively spliced transcripts of 1C7 were identified by RT-PCR, all of which are expressed in immune-related cell lines. Alternative splicing within the immunoglobulin domain- encoding region was seen to result in possible set switching between an IgV domain and an IgC2 domain. In addition to this, a previously unidentified gene, homologous to a number of V- ATPase G-subunits, has been located 1kb telomeric of IKBL. Lastly, the pseudogene UCRH-L and an AIF-1 gene fragment have been identified in the intergenic region between AIF-1 and 1C7. In order to assess the contribution of loci in this region to disease susceptibility, the genes AIF-1, 1C7, ATP6G and the BAT1 promoter region were subjected to mutation analysis. A total of 28 polymorphisms have been identified, 8 in AIF-1, 10 in 1C7, 7 in ATP6G and 3 in the BAT1 promoter region. Work is at present underway to genotype a number of the identified polymorphisms in control DNAs and in DNA samples from patients with combined variable immunodeficiency (CVID). The information generated from this analysis will bring us closer to explaining the reported linkage of CVID with the telomeric end of the human MHC class III region.

No description available.