Nonlinear tests for genetic studies of complex disease

Linkage and association studies are major analytical tools to search for susceptibility genes for complex diseases. With the availability of large collection of single nucleotide polymorphisms (SNPs) and the rapid progresses for high throughput genotyping technologies, together with the ambitious goals of the International HapMap Project, genetic markers covering the whole genome will be available for genome-wide linkage and association studies. In order not to inflate the type I error rate in performing genome-wide linkage and association studies, multiple adjustment for the significant level for each independent linkage and/or association test is required, and this has led to the suggestion of genome-wide significant cut-off as low as 5 × 10 −7. Almost no linkage and/or association study can meet such a stringent threshold by the standard statistical methods. Developing new statistics with high power is urgently needed to tackle this problem. This dissertation proposes and explores a class of novel test statistics that can be used in both population-based and family-based genetic data by employing a completely new strategy, which uses nonlinear transformation of the sample means to construct test statistics for linkage and association studies. Extensive simulation studies are used to illustrate the properties of the nonlinear test statistics. Power calculations are performed using both analytical and empirical methods. Finally, real data sets are analyzed with the nonlinear test statistics. Results show that the nonlinear test statistics have correct type I error rates, and most of the studied nonlinear test statistics have higher power than the standard chi-square test. This dissertation introduces a new idea to design novel test statistics with high power and might open new ways to mapping susceptibility genes for complex diseases. ^

The Role of IL-6 in Adenosine-Mediated Pulmonary Fibrosis

Adenosine is a purinergic signaling molecule that regulates various aspects of inflammation and has been implicated in the pathogenesis of chronic lung diseases. Previous studies have demonstrated that adenosine up-regulates IL-6 production through the engagement of the A2B adenosine receptor in various cell types, including alveolar macrophages. IL-6 is elevated in mouse models and humans with chronic lung disease, suggesting a potential role in disease progression. Furthermore, chronic elevation of adenosine in the lungs of adenosine deaminase deficient (Ada-/-) mice leads to the development of pulmonary inflammation, alveolar destruction, and fibrosis, in conjunction with IL-6 elevation. Thus, it was hypothesized that IL-6 contributes to pulmonary inflammation and fibrosis in this model. To test this hypothesis, Ada/IL-6 double knockout mice (Ada/IL-6-/-) were generated to assess the consequences of genetically removing IL-6 on adenosine-dependent pulmonary injury. Ada/IL-6-/- mice exhibited a significant reduction in inflammation, alveolar destruction, and pulmonary fibrosis. Next, Ada-/- mice were treated systematically with IL-6 neutralizing antibodies to test the efficacy of blocking IL-6 on chronic lung disease. These treatments were associated with decreased pulmonary inflammation, alveolar destruction, and fibrosis. To determine the role of IL-6 in a second model of pulmonary fibrosis, wild type mice and IL-6-/- mice were subjected to intraperitoneal injections of bleomycin twice a week for four weeks. Results demonstrated that IL-6-/- mice developed reduced pulmonary fibrosis. To examine a therapeutic approach in this model, wild type mice exposed to bleomycin were treated with IL-6 neutralizing antibodies. Similar results were observed as with Ada-/- mice, namely diminished pulmonary inflammation and fibrosis. In both models, elevations in IL-6 were associated with increased phosphorylated STAT-3 in the nuclei of numerous cell types in the airways, including type II alveolar epithelial cells (AEC). Genetic removal and neutralization of IL-6 in both models was associated with decreased STAT-3 activation in type II AEC. The mechanism of activation in these cells that lack the membrane bound IL-6Ra suggests IL-6 trans-signaling may play a role in regulating fibrosis. Characterization of this mechanism demonstrated that the soluble IL-6Ra (sIL-6Ra) is upregulated in both models during chronic conditions. In vitro studies in MLE-12 alveolar epithelial cells confirmed that IL-6, in combination with the sIL-6Ra, activates STAT-3 and TWIST in association with enhancement of epithelial-to-mesenchymal transition, which can contribute to fibrosis. Similarly, patients with idiopathic pulmonary fibrosis demonstrated a similar pattern of increased IL-6 expression, STAT-3 activation, and sIL-6Ra increases. These findings demonstrate that adenosine-dependent elevations in IL-6 contribute to the development and progression of pulmonary inflammation and fibrosis. The implications from these studies are that adenosine and/or IL-6 neutralizing agents represent novel therapeutic targets for the treatment of pulmonary disorders where fibrosis is a detrimental component.

The development and characterization of two types of chronic responses in irradiated mouse colon

The hypothesis to be tested is that there are two distinct types of chronic responses in irradiated normal tissues, each resulting from damage to different cell populations in the tissue. The first is a sequala of chronic epithelial depletion in which the tissue's integrity cannot be maintained, i.e. a "consequential" chronic response. The other response is due to cell loss in the connective tissue and/or vascular stroma, i.e. a "primary" chronic response. The purpose of this study was to test the hypothesis in the murine colon by first, establishing a model of each chronic response and then, by determining whether the responses differed in timing of expression, histology, and expression of specific collagen types. The model of late damage used was colonic obstructions/strictures induced by a single dose of 27 Gy ("consequential" response) and two equal doses of 14.75 Gy (t = 10 days) ("primary" response). "Consequential" lesions appeared as early as 5 weeks after 27 Gy and were characterized by a deep mucosal ulceration and a thickened fibrotic serosa containing excessive accumulations of collagen types I and III. Both types were commingled in the scar at the base of the ulcer. Fibroblasts were synthesizing pro-collagen types I and III mRNA 10 weeks prior to measurable increases in collagen. A significant decrease in the ratio of collagen types I:III was associated with the "consequential" response at 4-5 months post-irradiation. The "primary" response, on the other hand, did not appear until 40 weeks after the split dose even though the total dose delivered was approximately the same as that for the "consequential" response. The "primary" response was characterized with an intact mucosa and a thickened fibrotic submucosa which contained excessive amounts of only collagen type I. An increased number of fibroblasts were synthesizing pro-collagen type I mRNA nearly 25 weeks before collagen type I levels were increased. The "primary" response lesion had a significantly elevated collagen type I:III ratio at 10-13 months post-irradiation. These data show a clear difference between the two chronic response and suggest that not all chronic responses share a common pathogenesis, but depend on the cell population in the tissue that is damaged. ^

Mechanistic studies of mouse skin tumor promotion by chrysarobin

Since the anthrone chrysarobin oxidizes and generates free radicals, investigations were conducted to assess a possible role for free radicals or reactive oxygen species (ROS) in skin tumor promotion by chrysarobin. Epidermal glutathione levels were not noticeably altered by chrysarobin, nor did a glutathione-depleting agent enhance promotion by chrysarobin. Multiple applications of chrysarobin increased lipid peroxide levels in mouse epidermis two-fold as compared with controls. The antioxidant $\alpha$-tocopherol and the lipoxygenase inhibitor nordihydroguaiaretic acid both inhibited production of lipid peroxides by chrysarobin. The antioxidants $\alpha$-tocopherol acetate and ascorbyl palmitate effectively inhibited promotion and promoter-related effects induced by chrysarobin. Since prooxidant states can lead to increases in intracellular Ca$\sp{2+}$, the effect of two Ca$\sp{2+}$ antagonists, verapamil and TMB-8, on chrysarobin-induced promotion and promoter-related effects were investigated. Both Ca$\sp{2+}$ antagonists inhibited promotion and promoter-related effects induced by chrysarobin, suggesting a possible role for intracellular Ca$\sp{2+}$ alterations in chrysarobin-tumor promotion. Since radical generating compounds are reported to possess the ability to enhance progression of papillomas to squamous cell carcinomas (SCCs), the effects of chrysarobin on papilloma development were tested. Growth kinetics and regression of papillomas generated with limited promotion with chrysarobin were similar to what was reported for the nonradical generating promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) (Aldaz et al., 1991). To test the chrysarobin's ability to enhance progression of pre-existing papillomas to SCCs, tumors were generated by initiation with dimethylbenz (a) anthracene and promotion with TPA. Then mice were treated with chrysarobin, TPA or acetone for 45 weeks. When mice treated with chrysarobin were compared to mice treated continually with TPA with similar numbers of papillomas, the number of papillomas that progressed to SCCs was similar, suggesting that papilloma burden influences the progression of papillomas to SCCs, rather than radical production. In summary, the present study suggests that chrysarobin produces oxidative stress in mouse epidermis as indicated by the generation of lipid peroxides. Antioxidants inhibited production of lipid peroxides and tumor promotion by chrysarobin. Collectively, these data suggest a role for free radicals or ROS in tumor promotion by chrysarobin. ^

Phenotypic and genotypic analysis of mouse hepatitis virus (JHM) complementation group A temperature-sensitive mutants

Temperature sensitive (ts) mutant viruses have helped elucidate replication processes in many viral systems. Several panels of replication-defective ts mutants in which viral RNA synthesis is abolished at the nonpermissive temperature (RNA$\sp{-})$ have been isolated for Mouse Hepatitis Virus, MHV (Robb et al., 1979; Koolen et al., 1983; Martin et al., 1988; Schaad et al., 1990). However, no one had investigated genetic or phenotypic relationships between these different mutant panels. In order to determine how the panel of MHV-JHM RNA$\sp{-}$ ts mutants (Robb et al., 1979) were genetically related to other described MHV RNA$\sp{-}$ ts mutants, the MHV-JHM mutants were tested for complementation with representatives from two different sets of MHV-A59 ts mutants (Koolen et al., 1983; Schaad et al., 1990). The three ts mutant panels together were found to comprise eight genetically distinct complementation groups. Of these eight complementation groups, three complementation classes are unique to their particular mutant panel; genetically equivalent mutants were not observed within the other two mutant panels. Two complementation groups were common to all three mutant panels. The three remaining complementation groups overlapped two of the three mutant sets. Mutants MHV-JHM tsA204 and MHV-A59 ts261 were shown to be within one of these overlapping complementation groups. The phenotype of the MHV-JHM mutants within this complementation class has been previously characterized (Leibowitz et al., 1982; Leibowitz et al, 1990). When these mutants were grown at the permissive temperature, then shifted up to the nonpermissive temperature at the start of RNA synthesis, genome-length RNA and leader RNA fragments accumulated, but no subgenomic mRNA was synthesized. MHV-A59 ts261 produced leader RNA fragments identical to those observed with MHV-JHM tsA204. Thus, these two MHV RNA$\sp{-}$ ts mutants that were genetically equivalent by complementation testing were phenotypically similar as well. Recombination frequencies obtained from crosses of MHV-A59 ts261 with several of the gene 1 MHV-A59 mutants indicated that the causal mutation(s) of MHV-A59 ts261 was located near the overlapping junction of ORF1a and ORF1b, in the 3$\sp\prime$ end of ORF1a, or the 5$\sp\prime$ end of ORF1b. Sequence analysis of this junction and 1400 nucleotides into the 5$\sp\prime$ end of ORF1b of MHV-A59 ts261 revealed one nucleotide change from the wildtype MHV-A59. This substitution at nucleotide 13,598 (A to G) was a silent mutation in the ORF1a reading frame, but resulted in an amino acid change in ORF1b gene product (I to V). This amino acid change would be expressed only in the readthrough translation product produced upon successful ribosome frameshifting. A revertant of MHV-A59 ts261 (R2) also retained this guanidine residue, but had a second substitution at nucleotide 14,475 in ORF1b. This mutation results in the substitution of valine for an isoleucine.^ The data presented here suggest that the mutation in MHV-A59 ts261 (nucleotide 13,598) would be responsible for the MHV-JHM complementation group A phenotype. A second-site reversion at nucleotide 14,475 may correct this defect in the revertant. Sequencing of gene 1 immediately upstream of nucleotide 13,296 and downstream of nucleotide 15,010 must be conducted to test this hypothesis. ^

Functional analysis of MRF4 during mouse embryogenesis

MRF4 is one of four skeletal muscle specific regulatory genes, (the other three genes being MyoD, myf5, and myogenin), each of which has the unique ability to orchestrate an entire program of muscle-specific transcription when introduced into diverse cell types. These findings have led to the notion that these factors function as master regulators of muscle cell fate. Analysis of mice lacking MyoD, myf5, and myogenin have further defined their roles in the commitment and differentiation of myotomal progenitor cells. Current data strongly supports the model that MyoD and myf5 share functional redundancy in determining the muscle cell lineage, while myogenin acts downstream of MyoD and myf5, to initiate myoblast differentiation. Unlike other myogenic bHLH genes, MRF4 is expressed predominantly in the adult, suggesting that it may function to regulate adult muscle maturation and maintenance. To test this hypothesis and to eventually incorporate MRF4 into a general model for muscle specification, differentiation, maturation and maintenance, I deleted the MRF4 gene. MRF4-null mice are viable and fertile, however, they show mild rib anomalies. In addition, the expression of myogenin is dramatically upregulated only in the adult, suggesting that myogenin may compensate for the loss of MRF4 in the adult, and MRF4 may normally suppress the expression of myogenin after birth. MRF4 is also required during muscle regeneration after injury.^ To determine the degree of genetic redundancy between MRF4-myogenin; and MRF4-MyoD, I crossed the MRF4-null mice with MyoD- and myogenin-null mice respectively. There are no additional muscle phenotypes in double-null progeny from a MRF4 and myogenin cross, suggesting that the existence of residual fibers in myogenin-null mice is not due to the presence of MRF4. MRF4 expression also cannot account for the ability of myogenin-null myoblasts to differentiate in vitro. However, the combination of the MRF4-null mutation with the myogenin-null mutation results in a novel rib phenotype. This result suggests that MRF4 modifies the myogenin-null rib phenotype, and MRF4 and myogenin play redundant roles in rib development.^ MRF4 also shares dosage effects with MyoD during mouse development. (MyoD+/$-$;MRF4$-$/$-$)mice are fertile and viable, while (MyoD$-$/$-$;MRF4+/$-$) mice die between birth and two weeks after birth, and have a small skeletal structure. The double homozygous mice for MRF4 and MyoD mutations are embryonic lethal and die at around E10.5. These results suggest that MRF4 and MyoD share overlapping functions during mouse embryogenesis. ^

The volume effect in irradiated mouse colorectum

Damage of the colorectum is the dose-limiting normal tissue complication following radiotherapy of prostate and cervical cancers. One approach for decreasing complications is to physically reduce the treatment volume. Mathematical models have been previously developed to describe the change in associated toxicity with a change in irradiated volume, i.e. the "volume effect", for serial-type normal tissues including the colorectum. The first goal of this thesis was to test the hypothesis that there would not be a threshold length in the development of obstruction after irradiation of mouse colorectum, as predicted by the Probability model of the volume effect. The second goal was to examine if there were differences in the threshold and in the incidence of colorectal obstruction after irradiation of two mouse strains, C57B1/6 (C57) and C3Hf/Kam (C3H), previously found to be fibrosis-prone and-resistant, respectively, after lung irradiation due, in part, to genetic differences. The hypothesis examined was that differences in incidence between strains were due to the differential expression of the fibrogenic cytokines $\rm TGF\beta$ and $\rm TNF\alpha.$ Various lengths of C57 and C3H mouse colorectum were irradiated and the incidence of colorectal obstruction was followed up to 15 months. A threshold length was observed for both mouse strains, in contradiction of model predictions. The mechanism of the threshold was epithelial regeneration after irradiation. C57 mice had significantly higher incidence of colorectal obstruction compared to C3H mice, especially at smaller irradiated lengths. Colorectal tissue was obtained at various times after irradiation and prepared for histology, immunohistochemistry and RNase protection assay for measurement of $\rm TGF\beta 1,$ 2, 3 and $\rm TNF\alpha$ mRNA. Distinct strain differences in the histological time of appearance and spatial locations of fibrosis were observed. However, there were no consistent strain difference in mRNA levels or immunolocalization for any of the cytokines examined. The data indicate the need for volume effect models that account for biologically important processes, such as the effect of epithelial regeneration after irradiation. As well, changes in fibrogenic cytokines at the mRNA level do not contribute to the strain difference in radiation-induced colorectal obstruction. ^

Cost-benefit analysis: The ``gold standard'' re-examined

The dissertation reviews the recommendations of the Panel on Cost Effectiveness in Health and Medicine (Panel) convened by the US Public Health Service in 1993 in four areas: aggregation of costs and benefits, methods of estimating resources used, definition of population impacted and perspective used in cost benefit analysis. Financial data from a clinical trial was used to test whether different approaches in each of the above four areas would change the net benefit resulting from a cost benefit analysis. Differences in aggregation of cost and benefit resulted in the same net benefit, but not the same cost/benefit ratios. Differences in resource use estimation methods, population subgroups definitions and perspectives all produced different net benefits. Difference in perspective resulted in different and often opposing decisions as to whether the proposed intervention from the clinical trial should be implemented. ^

In vivo definition of genetic and cellular aspects of mammalian sexual differentiation

The differentiation of the reproductive organs is an essential developmental process required for the proper transmission of the genetic material. Müllerian inhibiting substance (MIS) is produced by testes and is necessary for the regression of the Müllerian ducts: the anlagen of the uterus, fallopian tubes and cervix. In vitro and standard transgenic mouse studies indicate that the nuclear hormone receptor Steroidogenic factor 1 (SF-1) and the transcription factor SOX9 play an essential role in the regulation of Mis. To test this hypothesis, mutations in the endogenous SF-1 and SOX9 binding sites in the mouse Mis promoter were introduced by gene targeting in embryonic stem (ES) cells. In disagreement with cell culture and transgenic mouse studies, male mice homozygous for the mutant SF-1 binding site correctly initiated Mis transcription in the fetal testes, although at significantly reduced levels. Surprisingly, sufficient Mis was produced for complete elimination of the Müllerian duct system. However, when the SF-1 binding site mutation was combined with an Mis -null allele, the further decrease in Mis levels led to a partial retention of uterine tissue, but only at a distance from the testes. In contrast, males homozygous for the mutant SOX9 binding site did not initiate Mis transcription, resulting in pseudohermaphrodites with a uterus and oviducts. These studies suggest an essential role for SOX9 in the initiation of Mis transcription, whereas SF-1 appears to act as a quantitative regulator of Mis transcript levels perhaps for influencing non-Müllerian duct tissues. ^ The Mis type II receptor, a member of the TGF- b superfamily, is also required for the proper regression of the Müllerian ducts. Mis type II receptor-deficient human males and their murine counterparts develop as pseudohermaphrodites. A lacZ reporter cassette was introduced into the mouse Mis type II receptor gene, by homologous recombination in ES cells. Expression studies, based on b -galactosidase activity, show marked expression of the MIS type II receptor in the postnatal Sertoli cells of the testis as well as in the prenatal and postnatal granulosa cells of the ovary. Expression is also seen in the mesenchymal cells surrounding the Müllerian duct and in the longitudinal muscle layer of the uterus. ^

The role of Lmx1b in the control of dorsoventral compartmentalization in the distal mesenchyme of the developing mouse limb

Lmx1b encodes a LIM-homeodomain transcription factor required for dorso-ventral (D-V) patterning in the mesenchyme of the vertebrate limb. In the absence of Lmx1b function, limbs exhibit a bi-ventral pattern indicating that Lmx1b is required for cells to adopt a dorsal cell fate. However, how Lmx1b specifies dorsal cell fates in the mesenchyme of the distal limb is unknown. Lmx1b is initially expressed throughout the dorsal and ventral limb bud mesenchyme, then becomes dorsally restricted around E10.5. At this stage, there is a sharp boundary between Lmx1b expressing and Lmx1b non-expressing cells. How the dorso-ventral Lmx1b expression boundary is formed and maintained is currently unknown. One mechanism that may contribute to establishing and/or maintaining the Lmx1b expression boundary is if the dorsal mesenchyme is a lineage-based compartment, where different groups of non-mingling cells are separated. Compartment formation has been proposed to rely on compartment-specific selector gene activity which functions to restrict cells to a compartment and specifies the identity of cells within that compartment. Based on the evidence that the dorsal limb identity relies on the expression of Lmx1b in the dorsal half of the limb mesenchyme, we hypothesized that Lmx1b might function as a dorsal limb bud mesenchyme selector gene to set up a dorsal compartment. To test this hypothesis, we developed an inducible CreERT2/ loxP based fate mapping approach that permanently marks Lmx1b wild-type and mutant cells and examined the distribution of their descendents in the developing limb. Our data is the first to show that dorso-ventral lineage compartments exist in the limb bud mesenchyme. Furthermore, Lmx1b is required for maintenance of the dorso-ventral compartment lineage boundary. The behavior of Lmx1b mutant cells that cross into the ventral mesenchyme, as well as previous chimera analysis in which mutant cells spread evenly in the ventral limb and form patches in the dorsal side, suggest that cell affinity differences prevent intermingling of dorsal and ventral cells. ^

A physiological role of cytochromes P450 4Fs: Mouse model

Cytochromes P450 4Fs (CYP4F) are a subfamily of enzymes involved in arachidonic acid metabolism with highest catalytic activity towards leukotriene B 4 (LTB4), a potent chemoattractant involved in prompting inflammation. CYP4F-mediated metabolism of LTB4 leads to inactive ω-hydroxy products incapable of initiating chemotaxis and the inflammatory stimuli that result in the influx of inflammatory cells. Our hypothesis is based on the catalytic ability of CYP4Fs to inactivate pro-inflammatory LTB4 which assures these enzymes a pivotal role in the process of inflammation resolution. ^ To test this hypothesis and evaluate the changes in CYP4F expression under complex inflammatory conditions, we designed two mouse models, one challenged with lipopolysaccharide (LPS) as a sterile model of sepsis and the other challenged with a systemic live bacterial infection of Citrobacter rodentium, an equivalent of the human enterobacterium E. coli pathogen invasion. Based on the evidence that Peroxisome Proliferator Activated Receptors (PPARs) play an active role in inflammation regulation, we also examined PPARs as a regulation mechanism in CYP4F expression during inflammation using PPARα knockout mice under LPS challenge. Using the Citrobacter rodentium model of inflammation, we studied CYP4F levels to compare them to those in LPS challenged animals. LPS-triggered inflammation signal is mediated by Toll-like 4 (TLR4) receptors which specifically respond to LPS in association with several other proteins. Using TLR4 knockout mice challenged with Citrobacter rodentium we addressed possible mediation of CYP4F expression regulation via these receptors. ^ Our results show isoform- and tissue-specific CYP4F expression in all the tissues examined. The Citrobacter rodentium inflammation model revealed significant reduction in liver expression of CYP4F14 and CYP4F15 and an up-regulation of gene expression of CYP4F16 and CYP4F18. TLR4 knockout studies showed that the decrease in hepatic CYP4F15 expression is TLR4-dependent. CYP4F expression in kidney shows down-regulation of CYP4F14 and CYP4F15 and up-regulation of CYP4F18 expression. In the LPS inflammation model, we showed similar patterns of CYP4F changes as in Citrobacter rodentium -infected mice. The renal profile of CYP4Fs in PPARα knockout mice with LPS challenge showed CYP4F15 down-regulation to be PPARα dependent. Our study confirmed tissue- and isoform-specific regulation of CYP4F isoforms in the course of inflammation. ^

Ser88 phosphorylation regulates dynein light chain 1 (DLC1) function in the mouse mammary gland

Dynein light chain 1 (DLC1) is a highly conserved and ubiquitously expressed protein which might have critical cellular function as total loss of DLC1 caused Drosophila embryonic death. Despite many proteins and RNAs interaction with it identified, DLC1's function(s) and regulation are largely unknown. Recently, DLC1 was identified as a physiological substrate of P21-activate kinase 1(Pak1) kinase from a human mammary cDNA library in a yeast-2-hybridization screening assay. Studies in primary human tumors and cell culture implicated that DLC1 could promote mammary cancerous phenotypes, and more importantly, Ser88 phosphorylation of DLC1by Pak1 kinase was found to be essential for DLC1's tumorigenic activities. Based on the above tissue culture studies, we hypothesized that Ser88 phosphorylation regulates DLC1. ^ To test this hypothesis, we generated two transgenic mouse models: MMTV-DLC1 and MMTV-DLC1-S88A mice with mammary specific expression of the DLC1 and DLC1-S88A cDNAs. Both of the transgenic mice mammary glands showed rare tumor incidence which indicated DLC1 alone may not be sufficient for tumorigenesis in vivo. However, these mice showed a significant alteration of mammary development. Mammary glands from the MMTV-DLC1 mice had hyperbranching and alveolar hyperplasia, with elevated cell proliferation. Intriguingly, these phenotypes were not seen in the mammary glands from the MMTV-S88A mice. Furthermore, while MMTV-DLC1 glands were normal during involution, MMTV-S88A mice showed accelerated mammary involution with increase apoptosis and altered expression of involution-associated genes. Further analysis of the MMTV-S88A glands showed they had increased steady state level of Bim protein which might be responsible for the early involution. Finally, our in vitro data showed that Ser88 phosphorylation abolished DLC1 dimer and consequently might disturb its interaction with Bim and destabilize Bim. ^ Collectively, our findings provided in vivo evidence that Ser88 phosphorylation of DLC1 can regulate DLC1's function. In addition, Ser88 phosphorylation might be critical for DLC1 dimer-monomer transition. ^

Expression study of the atherosclerosis mouse aorta reveals significant disturbance of calcium signaling pathway

Atherosclerosis is a complex disease resulting from interactions of genetic and environmental risk factors leading to heart failure and stroke. Using an atherosclerotic mouse model (ldlr-/-, apobec1-/- designated as LDb), we performed microarray analysis to identify candidate genes and pathways, which are most perturbed in changes in the following risk factors: genetics (control C57BL/6 vs. LDb mice), shearstress (lesion-prone vs. lesion-resistant regions in LDb mice), diet (chow vs. high fat fed LDb mice) and age (2-month-old vs. 8-month old LDb mice). ^ Atherosclerotic lesion quantification and lipid profile studies were performed to assess the disease phenotype. A microarray study was performed on lesion-prone and lesion-resistant regions of each aorta. Briefly, 32 male C57BL/6 and LDb mice (n =16/each) were fed on either chow or high fat diet, sacrificed at 2- and 8-months old, and RNA isolated from the aortic lesion-prone and aortic lesion-resistant segments. Using 64 Affymetrix Murine 430 2.0 chips, we profiled differentially expressed genes with the cut off value of FDR ≤ 0.15 for t-test, and q <0.0001 for the ANOVA. The data were normalized using two normalization methods---invariant probe sets (Loess) and Quantile normalization, the statistical analysis was performed using t-tests and ANOVA, and pathway characterization was done using Pathway Express (Wayne State). The result identified the calcium signaling pathway as the most significant overrepresented pathway, followed by focal adhesion. In the calcium signaling pathway, 56 genes were found to be significantly differentially expressed out of 180 genes listed in the KEGG calcium signaling pathway. Nineteen of these genes were consistently identified by both statistical tests, 11 of which were unique to the test, and 26 were unique to the ANOVA test, using the cutoffs noted above. ^ In conclusion, this finding suggested that hypercholesterolemia drives the disease progression by altering the expression of calcium channels and regulators which subsequently results in cell differentiation, growth, adhesion, cytoskeletal change and death. Clinically, this pathway may serve as an important target for future therapeutic intervention, and thus the calcium signaling pathway may serve as an important target for future diagnostic and therapeutic intervention. ^

Mouse retinal development: Role of cell adhesion and mechanism of gene regulation

Cell differentiation and pattern formation are fundamental processes in animal development that are under intense investigation. The mouse retina is a good model to study these processes because it has seven distinct cell types, and three well-laminated nuclear layers that form during embryonic and postnatal life. β-catenin functions as both the nuclear effector for the canonical Wnt pathway and a cell adhesion molecule, and is required for the development of various organs. To study the function of β-catenin in retinal development, I used a Cre-loxP system to conditionally ablate β-catenin in the developing retina. Deletion of β-catenin led to disrupted laminar structure but did not affect the differentiation of any of the seven cell types. Eliminating β-catenin did not reduce progenitor cell proliferation, although enhanced apoptosis was observed. Further analysis showed that disruption of cell adhesion was the major cause of the observed patterning defects. Overexpression of β-catenin during retinal development also disrupted the normal retinal lamination and caused a transdifferentiation of neurons into pigmented cells. The results indicate that β-catenin functions as a cell adhesion molecule but not as a Wnt pathway component during retinal neurogenesis, and is essential for lamination but not cell differentiation. The results further imply that retinal lamination and cell differentiation are genetically separable processes. ^ Sonic hedgehog (shh) is expressed in retinal ganglion cells under the control of transcription factor Pou4f2 during retinal development. Previous studies identified a phylogenetically conserved region in the first intron of shh containing a Pou4f2 binding site. Transgenic reporter mice in which reporter gene expression was driven by this region showed that this element can direct gene expression specifically in the retina, but expression was not limited to the ganglion cells. From these data I hypothesized that this element is required for shh expression in the retina but is not sufficient for specific ganglion cell expression. To further test this hypothesis, I created a conditional allele by flanking this region with two loxP sites. Lines carrying this allele will be crossed with retinal-specific Cre lines to remove this element in the retina. My hypothesis predicts that alteration in shh expression and subsequent retinal defects will occur in the retinas of these mice. ^

Use of short term test systems for the prediction of the hazard represented by potential chemical carcinogens

It has been hypothesized that results from the short term bioassays will ultimately provide information that will be useful for human health hazard assessment. Although toxicologic test systems have become increasingly refined, to date, no investigator has been able to provide qualitative or quantitative methods which would support the use of short term tests in this capacity.^ Historically, the validity of the short term tests have been assessed using the framework of the epidemiologic/medical screens. In this context, the results of the carcinogen (long term) bioassay is generally used as the standard. However, this approach is widely recognized as being biased and, because it employs qualitative data, cannot be used in the setting of priorities. In contrast, the goal of this research was to address the problem of evaluating the utility of the short term tests for hazard assessment using an alternative method of investigation.^ Chemical carcinogens were selected from the list of carcinogens published by the International Agency for Research on Carcinogens (IARC). Tumorigenicity and mutagenicity data on fifty-two chemicals were obtained from the Registry of Toxic Effects of Chemical Substances (RTECS) and were analyzed using a relative potency approach. The relative potency framework allows for the standardization of data "relative" to a reference compound. To avoid any bias associated with the choice of the reference compound, fourteen different compounds were used.^ The data were evaluated in a format which allowed for a comparison of the ranking of the mutagenic relative potencies of the compounds (as estimated using short term data) vs. the ranking of the tumorigenic relative potencies (as estimated from the chronic bioassays). The results were statistically significant (p $<$.05) for data standardized to thirteen of the fourteen reference compounds. Although this was a preliminary investigation, it offers evidence that the short term test systems may be of utility in ranking the hazards represented by chemicals which may be human carcinogens. ^