Breast cancer is a leading cause of cancer death and in 2009, the American Cancer Society estimates that over 192,000 new cases of breast cancer will be diagnosed, and over 40,000 women will die from breast cancer. Estrogen (E2) is required for normal female development and reproduction, but long-term exposure is carcinogenic and considered a risk factor for breast cancer. Membrane ion channels are essential for cell proliferation and are suggested to have a role in cancer, especially potassium channels. In the present study, we investigate the effects of estrogen and the estrogen antagonist ICI182780 on G-protein inwardly rectifying potassium channels (GIRK) in estrogen responsive MCF-7 breast cancer cells. GIRK1 and GIRK2 specific channels are thought to play a major role in rapid channel activation. We found increases in GIRK1 and GIRK2 membrane protein levels in response to estrogen treatment, as well as increases in intracellular potassium concentrations and cellular proliferation. ICI182780 treatment increased GIRK1, GIRK2 and GIRK4 membrane protein levels but resulted in an initial decrease in intracellular potassium concentration and decreased cell proliferation. GIRK1 RNAi knockdown decreased estrogen receptor alpha protein levels and activation. In addition, estrogen treatment resulted in increased phosphorylation of specific members of GPCR and MAPK signaling pathways that have been shown to be responsive to GIRK1 knockdown. Using microarray analysis of nontreated and E2 treated MCF7 cells, we observed 489 differentially expressed genes (283 upregulated and 206 downregulated) that were comprised largely of transcription and cell cycle associated genes. This study identified several human cell cycle associated genes that are both responsive to E2 treatment and are functionally correlated with GIRKs. Five genes were selected for further analysis by real time PCR. Our data suggests that specific GIRK channel composition at the cell membrane may be stimulated by estrogen exposure or downstream targets of estrogen signaling and may contribute to increased cell proliferation in MCF-7 breast cancer cells. Taken together, these data add further support of GIRK involvement in cancer progression and identify some potential biological roles of GIRKs in cancer biology beyond the initilal findings of GIRK1 assciation in metastatic breast cancer.

This paper reports the solution of an optimization problem for spines of cylindrical profile that transfer heat by convection when the spine material has constant thermal conductivity. The volume enclosed by the spine is used as a method of rejecting power from the base surface maintained at a specified temperature. The ultimate goal of this thesis is to find a cylindrical spine of minimum profile volumne which transfers a maximum amount of heat by convection when the thermal conductivity is constant. We compute the geometrical and thermal properties of the optimal cylindrical spine.

Mammalian C3 is a complement protein which consists of an α chain (125kDa) and β chain (75kDa) held together by a disulfide bond. The a chain contains a conserved thiolester site which provides the molecule with opsonic properties. The protein is synthesized as a single pro-C3 molecule which is post-translationally modified. C3 genes have been identified in organisms from different phyla, however, the shark C3 gene remains to be cloned. Sequence data from the shark will contribute to understanding further the evolution of this key protein. To obtain additional sequence data for shark C3 genes a cDNA library was constructed and screened with a DIG-labeled C3 probe. Fifty clones were isolated and sequenced. Analysis identified four sequences that yielded positive alignments with C3 of a variety of organisms including human C3. Deduced amino acid sequence analysis confirmed a β/α cut site (RRRR), the CR3 and properdin binding sites, the catalytic histidine, and the reactive thiolester sequence. In the shark there are at least two C3-like genes as the gene sequence obtained is distinct from that previously described.

A central question of evolutionary biology concerns the study of evolvability. A growing body of theory implicates the pattern of genetic effect interactions underlying a trait, referred to as the genetic architecture of a trait, as an important component of evolutionary dynamics. The model organism, Drosophila melanogaster, belongs to a group of fly lineages, the Acalyptrate Diptera, that exhibit evolutionary stasis in their wing shape. The small variation in wing shape between the member species of such a disparate group, indicates either a uniform stabilizing selection of wing shape for all species or a lack of evolvability in the trait. This thesis investigates the genetic architecture of wing shape in D. melanogaster as a possible constraint on its evolvability. I conduct a line-cross analysis for estimating the genetic effects of various percentages of alleles from a parental population in the genetic background of another parental population; and, a chromosome substitution analysis for estimating the genetic effects of various X-chromosomes substituted into different genetic backgrounds. For both experiments, I use fly populations established by Houle et al. that had undergone thirty generations of artificial selection on an index of wing shape. The line-cross analysis suggests that the two lines selected to increase or decrease the wing shape index score had decanalized genetic architectures with respect to their second generation hybrids. This is consistent with the idea that wing shape evolutionary stasis is due, in part, to low evolvability of the trait. The chromosome substitution experiment was hampered by the extinction of over fifty percent of the substitution lines in the experimental design, and so the results had little power to estimate the genetic architecture of the trait. However, the best estimate of the analysis suggests a negative directional epistasis, which is also consistent with the hypothesis that the wings are canalized. / A Thesis submitted to the Department of Biological Sciences in partial fulfillment of the requirements for the degree of Master of Science. / Spring Semester, 2007. / January 25, 2007. / genetic architecture, epistasis, directional epistasis, evolvability, Drosophila / Includes bibliographical references. / Thomas F. Hansen, Professor Directing Thesis; Richard Bertram, Committee Member; Lloyd M. Epstein, Committee Member; David Houle, Committee Member.

The process of normal wound repair after tissue injury follows a closely regulated sequence involving inflammation, the recruitment, activation and proliferation of fibroblasts and the secretion of extracellular matrix, which culminates in healing and termination of the proliferative and secretory processes. In pathological fibrosis, the normal healing and termination stages are disregulated and fibroblast activity continues unabated. This persistent "activated" state of the fibroblasts is the cause of the excessive accumulation of ECM, predominantly fibrillar collagens type I and III, which results in the disruption of the normal tissue function . In cirrhosis, the end stage of liver fibrosis, type I collagen represents up to 50% of liver proteins. Hepatic stellate cells (HSCs) are the major cell type responsible for collagen synthesis in the liver. In normal liver, quiescent HSCs store vitamin A, but only express trace amounts of type I collagen. Upon a fibrogenic stimulus, HSCs become activated, a process in which they lose vitamin A, proliferate, change morphologically into myofibroblasts, and increase their synthesis of extracellular matrix proteins. In order to understand the pathophysiology of cirrhosis, as well as other fibroproliferative disorders which can effect the heart, lungs, and skin, it is critical to elucidate the molecular mechanisms which regulate the expression and synthesis of type I collagen. In the first aim of this dissertation, we examined the role of the RNA-binding protein RBMS3 in regulating type I collagen expression. RBMS3 expression increases upon activation of HSCs, and is also increased in liver fibrosis. Through our research, we showed that RBMS3 specifically interacts with a conserved 60 nucleotide sequence in the 3' UTR of the homeobox transcription factor Prx1. Prx1, which is also upregulated in activated HSCs and liver fibrosis, transactivates the collagen á1(I) promoter and stimulates transcription of the gene. The binding of RBMS3 to the 3' UTR of the Prx1 mRNA results in the stabilization of the mRNA and increased protein synthesis. Since Prx1 is a transcription factor which increases collagen gene transcription, this mechanism may promote the profibrotic phenotype of HSCs. In the second aim of the dissertation, we focused on the posttranscriptional regulation of type I collagen expression. In the 5' UTR of á1(I), á2(I), and á1(III) collagen mRNA there is a stem-loop structure that encompasses the translation initiation codon. This 5' stem-loop is strongly conserved in evolution differing by only two nucleotides between fish and human collagen mRNAs. In order to study the role of the 5' stem-loop, our collaborators designed a transgenic mouse in which the 5' stem-loop structure of the collagen á1(I) gene was abolished. The collagen á1(I) mRNA stability and protein synthesis in fibroblasts from these transgenic mice was significantly decreased. Since it was clear that the 5' stem-loop was essential for proper type I collagen synthesis, we examined the role that 5' stem-loop binding proteins have in regulating this mechanism. Through the use of 2-D gel SDS-PAGE and MALDI-TOF MS, we identified several 5' stem-loop associated proteins which included LARP6, non-muscle myosin IIb, nucleolin, and vimentin. We discovered that LARP6, through direct binding of the collagen 5' stem-loop, enables the aggregation of type I collagen mRNAs into large complexes. Additional proteins identified in these RNA-protein complexes are components of stress granules, which regulate RNA metabolism. This type I collagen mRNA stress granule formation, along with non-muscle myosin IIb, may facilitate the mechanism by which the coordinated translation of á1(I) and á2(I) collagen mRNAs can occur. Overall, my dissertation research has accomplished two major findings. The first finding is how the transcription of the collagen á1(I) gene is regulated through the binding of RBMS3 to the mRNA encoding transcription factor Prx1. The second finding is how the translation of type I collagen is regulated by the LARP6 mediated aggregation of collagen mRNAs and their association with non-muscle myosin. Elucidation of these mechanisms may help in the development of antifibrotic drugs. / A Dissertation submitted to the Department of Biomedical Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Fall Semester, 2008. / August 7, 2008. / RNA, Liver Fibrosis, Biomedical Science / Includes bibliographical references. / Branko Stefanovic, Professor Directing Dissertation; Ken Roux, Outside Committee Member; Yanchang Wang, Committee Member; James Olcese, Committee Member.

N-methyl-D-aspartate receptors (NMDARs) are glutamate-gated ion channels that play key roles in excitatory synaptic transmission and are involved in numerous neurological disorders as well. The number of neuronal surface NMDARs are not static and can be altered in response to neuronal activity and sensory input. Endocytosis is well known to decrease the number of surface receptors and down-regulate receptor-mediated functions. However, whether a subset of receptor endocytosis regulates the activity of remaining surface (non-internalized) receptors remains unknown. It is shown from this study that the dynamic endocytosis of NMDARs induced by either the activation of group 1 metabotropic glutamate receptors (mGluRs) or the stimulation of the glycine binding site of NMDARs inhibits the activity of remaining surface NMDARs. This effect can be prevented by the modulation of the functional properties of surface NMDARs, for example, inactivating the surface NMDARs by application of the NMDAR antagonist AP5 or blocking Na+ influx. Furthermore, it is shown from this study that the endocytosis of NMDARs enhances the serine phosphorylation of surface NMDAR NR2A subunits. Coincidentally, the inactivation of surface NMDARs or the blockade of Na+ influx produces similar enhancement of serine phosphorylation in NMDARs as well. It is identi ed, by site directed mutagenesis, phosphorylation of NR2A serine 1416 as an essential mechanism in mediating NMDAR endocytosis-induced functional changes in surface receptors. In this study, protein kinase D (PKD/PKCu) is identified to be activated following NMDAR endocytosis, and intracellular application of PKD/PKCu successfully inhibits NMDAR-mediated whole-cell currents. Moreover, the blocking Na+ influx essentially eliminates the NMDAR endocytosis-induced down-regulation of NMDA-evoked whole-cell and synaptic responses. Taken together, the data from this study provide clues on understanding that the reduction of whole-cell responses induced by NMDAR endocytosis is attributed to the inhibition of the activity of remaining surface NMDARs. In addition, the modulation of the functional properties of surface receptors is shown to be an effective way to prevent functional change in surface receptors induced by recptor endocytosis. / A Dissertation submitted to the Department of Biomedical Science in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Spring Semester, 2011. / December 10, 2010. / Includes bibliographical references. / Xian-Min Yu, Professor Directing Dissertation; Paul Trombley, University Representative; Charles Ouimet, Committee Member; Choogon Lee, Committee Member.

Protein phosphorylation ' a reversible covalent process ' has evolved as one of the major posttranslational modifications that targets numerous transcription factors. Apoptosis (programmed cell death) is an irreversible intrinsic mechanism essential for removing unwanted cells, and maintaining tissue homeostasis in multicellular organisms. Activation of caspases is a major event during programmed cell death by which more than 280 proteins are cleaved. YY1 (Yin-Yang 1) is a multifunctional zinc finger transcription factor that has been implicated in different cellular processes such as proliferation, embryogenesis, differentiation, development, tumorigenesis, and apoptosis. YY1 has been shown to be a phosphoprotein and several studies have reported that phosphorylation regulates its activities. Also, YY1 was found to be one of only a few transcription regulators that is a target for cleavage by caspases in vitro and in vivo as well, but very little is known about the mechanisms that regulate its cleavage during cell death. Here, we identify serine 118 in the N-terminal domain of YY1, as the site of CK2' phosphorylation, proximal to a caspase cleavage site. CK2 inhibitors, as well as knockdown of CK2' by siRNA, reduce S118 phosphorylation in vivo and enhance YY1 cleavage under apoptotic conditions, whereas increasing CK2' activity by overexpression in vivo elevates S118 phosphorylation. A serine to alanine substitution at serine 118 also increases the cleavage of YY1 during apoptosis when compared to wild-type YY1. Taken together, we have discovered a regulatory link between YY1 phosphorylation at serine 118 and regulation of its cleavage during programmed cell death. In response to genotoxic stress, eukaryotic cells activate a set of DNA-damage kinase cascades that are initiated by sensor proteins such as ATM, ATR or DNA-PK. These sensor proteins phosphorylate various targets including Chk1, Chk2, and p53. To study the signaling pathway(s) regulating the cleavage of YY1 under apoptotic conditions, we have used ATR-deficient fibroblasts, as wells as knockout cell lines where Chk2, DNA-PKcs, p53 and ATM are ablated. Cisplatin, x a DNA damaging agent, was added to these cells to induce cell death. Cisplatin-induced YY1 cleavage and apoptosis are found to be independent of DNA-PK and Chk2, delayed in ATM and p53 knockout cell lines, and suppressed in ATR-deficient fibroblasts and DLD-Chk1 heterozygous cells. Consistently, inhibition of ATR with a selective ATR inhibitor prevents cisplatin-induced YY1 cleavage. Together, these results suggest a critical role of ATR-Chk1 in regulating YY1 cleavage by caspases during apoptosis. / A Dissertation submitted to the Department of Biomedical Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Summer Semester, 2011. / June 24, 2011. / Includes bibliographical references. / Myra M. Hurt, Professor Directing Dissertation; Hank Bass, Outside Committee Member; Akash Gunjan, Committee Member; Yanchang Wang, Committee Member; Cathy Levenson, Committee Member.

In the female rat, secretion of prolactin is tightly controlled by a complex interplay of both stimulatory and inhibitory hypothalamic factors. In addition, the profile of prolactin release in the ovariectomized rat treated with estradiol exhibits circadian rhythmicity, which requires an intact suprachiasmatic nucleus (SCN). This suggests that photic cues influence the hypothalamic factors coordinating the release of prolactin, namely oxytocin (arising from the paraventricular and periventricular nucleus of the hypothalamus) and dopamine (arising from neuroendocrine dopaminergic neurons in the periventricular nucleus and the arcuate nucleus). How photic cues regulate the secretion of prolactin, however, has yet to be explicated in animals treated with estradiol. Recent research has determined that the SCN sends efferents of vasoactive intestinal polypeptide to neuroendocrine dopaminergic neurons as well as to the paraventricular and periventricular nuclei of the hypothalamus. The first purpose of this study was to elucidate the mechanism regulating the rhythmicity of prolactin release. To determine this, vasoactive intestinal polypeptide synthesis was disrupted using vasoactive intestinal polypeptide antisense deoxyoligonucleotides infused into the dorsal border of the SCN. This treatment caused a phase-advance in the pattern of prolactin secretion in estradiol-treated ovariectomized rats. Using immunohistochemistry, oxytocin and dopamine activity was established through double labeling with FOS-related antigens (a marker of neuronal activity). In both populations of neurons (oxytocin neurons and neuroendocrine dopaminergic neurons), a phase advance was also observed. In oxytocin neurons, disruption of vasoactive intestinal polypeptide phase-advanced the increase in activity that normally accompanies the prolactin surge. Whereas, in dopamine neurons, disruption of vasoactive intestinal polypeptide phase-advanced the decrease in activity that is required to allow for the prolactin surge. Furthermore, clock gene expression has been localized to neuroendocrine dopaminergic neurons. In all three populations of neuroendocrine dopaminergic neurons, the expression of the clock gene period 2 oscillates. Disruption of vasoactive intestinal polypeptide disrupted this pattern of activity. Thus, the SCN influences the precise timing of the estradiol-induced prolactin surge via vasoactive intestinal polypeptide projections to oxytocinergic and dopaminergic neurons, and possibly does so by entraining clock gene expression in these neurons. Once entrained, oxytocin exerts its stimulatory effects at a previously unknown site. Using a selective oxytocin antagonist that does not cross the blood brain barrier, the pituitary was determined to be the site of oxytocin stimulation. Interestingly, the oxytocin receptor density is upregulated in response to estradiol; therefore, suckling- and estradiol-induced prolactin surges require higher doses of oxytocin antagonist than cervically-stimulated ovariectomized animals. Furthermore, in the presence of progesterone, the oxytocin antagonist was incapable of blocking the estradiol-induced surge; raising the possibility that progesterone may act through mechanisms independent of oxytocin to stimulate prolactin secretion. Therefore, the work in this dissertation provides additional mechanisms involved in the temporal and physiological control of estradiol-induced prolactin secretion. / A Dissertation submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Summer Semester, 2009. / July 3, 2009. / Endocrinology, Hypothalamus, Hormone / Includes bibliographical references. / Marc E. Freeman, Professor Directing Dissertation; Penny J. Gilmer, Outside Committee Member; Debra A. Fadool, Committee Member; Elaine Hull, Committee Member; Michael Meredith, Committee Member.

Although zinc has been implicated in the functionality of the hippocampus since the 1970's, the recent discovery of adult neuronal stem cells in this region of the brain provides the potential for novel zinc-regulated hippocampal processes. The current work addressed the role of zinc in the proliferation, maintenance, and survival of neuronal precursor cells. First, this work employed a genome-wide analysis of the effect of dietary zinc deficiency in the hippocampus. The data revealed that 3 weeks of dietary zinc deficiency resulted in the down-regulation of nearly 400 genes, many of which were associated with synaptic plasticity (stau2, syn1), neurotransmitter receptors (grin1, gabrb3), neurogenesis (iguana, id2, nek9), and cell viability (sod2, stat3). Furthermore, using a candidate gene approach, the current work shows a vital role of zinc in p53-mediated mechanisms governing proliferation and apoptosis of neuronal precursor cells. For example, zinc deficient cells show increased nuclear and mitochondrial translocation of the tumor suppressor p53. Using a dominant negative construct to ensure p53 regulation, this work shows that nuclear p53 is responsible for the downstream target genes responsible for cell cycle arrest (reprimo, lats2). These data coincide with a decrease in BrdU-labeling. The current work also highlights initial protective responses governed by the transcription factor p53. However, zinc deficient neuronal precursors also show a p53-dependent increase in mitochondrial reactive oxygen species which could be mediated by a decreased expression of glutathione peroxidase mRNA and mitochondrial localized p53. If the deficiency is severe or prolonged nuclear p53 regulates expression of apoptotic genes (rb1, tgf-β) whereas, mitochondrial p53 mediates interactions with Bcl-family proteins to initiate a loss in mitochondrial membrane potential. Ultimately, the current work adds to the essential role of zinc in the hippocampus and identifies a novel mechanism for zinc in the regulation of neuronal precursor proliferation, maintenance, and survival. / A Dissertation submitted to the Department of Biomedical Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Summer Semester, 2010. / June 11, 2010. / Zinc, Neurogenesis, Apoptosis, p53, Proliferation, Stem Cells, Hippocampus / Includes bibliographical references. / Cathy W. Levenson, Professor Directing Dissertation; J. Michael Overton, Committee Member; Charles Ouimet, Committee Member; Shridhar Sathe, Committee Member; Laura Keller, University Representative.

Fibrosis is characterized by excessive synthesis of type I collagen which impedes the normal function of an affected organ. Type I collagen is the most abundant protein in the human body, produced by the folding of two a1(I) polypeptides and one a2(I) polypeptides into the triple helix. Expression of collagen 1a(I) gene is predominantly regulated at the level of mRNA stability and translation. A conserved stem'Cloop structure is found in the 5' untranslated region of collagen mRNAs. The 5'stem-loop structure which is critical for the coordinated translation encompasses the start codon in collagen mRNAs and regulates collagen synthesis by the binding of RNA binding proteins. Assembly of collagen heterotrimer occurs on the membrane of endoplasmic reticulum (ER) while a1(I) and a2(I) mRNAs are associated with polysomes. I have found that the binding of 5'stem-loop binding proteins LARP6 and nonmuscle myosin II to the 5'stem-loop regulates coordinated translation of collagen mRNAs and is required for collagen triple helix formation. My work describes that the coordinated translation of collagen mRNAs increases local concentration of the chains necessary for productive folding. Chapter 1 presents the identification of 5'stem- loop binding protein and its function in regulating collagen synthesis. To identify 5'stem-loop binding proteins we performed expressional cloning and cloned La ribonucleoprotein domain family member 6 (LARP6) as the protein which binds to both 5'stem- loop of collagen a1(1) and a2(1) mRNAs a sequence specific manner. LARP6 has a distinctive bipartite RNA binding domain (amino acids 32-45 and 218-300) which is not found in other members of the La super family. The RNA binding domain of.LARP6 interacts with the two single-stranded regions of the 5'stem-loop. The Kd for binding of LARP6 to the 5'stem-loop is 1.4 nM. The binding is to the single stranded regions of the bulge of 5'stem-loop RNA, in both the nucleus and the cytoplasm. Recombinant LARP6 has a similar binding affinity and specificity to the 5'stem-loop as the endogenous LARP6. The C-terminal region of LARP6 has a nuclear localization signal, which allows LARP6 to accumulate in the nucleus. Combination of gain of function by adenoviral delivery and loss of function by using either dominant negative forms of LARP6 or siRNA directly against the RNA sequence of LARP6 were employed to determine the function of LARP6. Over expression of LARP6 decreased synthesis of collagen protein, however it did not change collagen mRNA steady state level. Also, collagen mRNAs were redistributed from polysomal fractions to the fractions representing the free polysomes in LARP6 overexpressed cells. This suggests that overexpression of LARP6 blocked ribosomal loading on collagen mRNAs and inhibited collagen mRNA translation. Endogenous LARP6 and overexpressed LARP6 were not associated with polysomes. These results suggested that LARP6 prevents premature translation of collagen mRNAs. Knocking down LARP6 by small interfering RNA also decreased steady state level of collagen polypeptide in the cell as well as the section rate of the protein. However, collagen mRNA stability was not affected, nor was the degradation of collagen protein by proteasome. Therefore, it is likely that collagen mRNA translation had been inhibited by the depletion of LARP6. We describe that collagen protein is synthesized at discrete regions of the endoplasmic reticulum. Using a collagen-GFP (green fluorescent protein) reporter protein, we could reproduce this focal pattern of synthesis, but it was observed only when the reporter was encoded by mRNA with the 5'stem-loop and in the presence of LARP6. When the reporter was encoded by mRNA without the 5' stem-loop, or in the absence of LARP6, it accumulated diffusely throughout the endoplasmic reticulum. This indicates that LARP6 activity is needed for focal synthesis of collagen polypeptides. We postulated that the LARP6-dependent mechanism increases local concentration of collagen polypeptides for more efficient folding of the collagen heterotrimer. In Chapter 2, we describe that nonmuscle myosin IIB plays an important role in regulating collagen synthesis. First, we identified nonmuscle myosin II B as LARP6 binding protein by tobramycin affinity purification and confirmed the binding specificity of myosin II B to LARP6 and to collagen mRNAs. We showed that the C terminus of LARP6 was required for the binding to nonmuscle myosin IIB, and that this binding was not RNA dependent. Secondly, we identify the role of nonmuscle myosin IIB in regulating type I collagen synthesis. Nonmuscle myosin II filaments are required for the secretion of collagen a2(I) peptide and the colocalization of a1(I) and a2(I) peptides in the cell. The motor activity of myosin II as well as the integrity of the filaments is involved in this process. We also discovered that the effect of myosin II on type I collagen synthesis was mediated by 5'stem-loop of LARP6 and through the protein-protein interaction with LARP6. At the end, we determined that nonmuscle myosin IIB filaments are involved in directing collagen mRNAs to polysomes for translation. Therefore, we concluded that nonmuscle myosin II interacts with LARP6 and collagen 5'stem-loop to regulates coordinate translation of collagen mRNAs. In Chapter 3, we explain the role of LARP6 and nonmuscle myosin II in collagen synthesis stimulated by cytokines and growth factors TGF-beta and ouabain. Cytokines and growth factors including TGF-beta and ouabain are stimulators of fibrosis and they increase collagen synthesis in scleroderma skin fibroblasts, hepatic stellate cells, kidney fibroblasts and cardiac fibroblasts. When we down regulated LARP6 or disrupted nonmuscle myosin II filaments, the stimulation of type I collagen secretion by TGF-beta 1 and ouabain was diminished. Therefore, we concluded that LARP6 and nonmuscle myosin II regulate inducible collagen synthesis in fibrosis. This dissertation describes the specific regulation of type I collagen synthesis in fibrosis. The first finding was that collagen protein synthesis took place of discrete regions on the ER membrane through by the binding of LARP6 to 5'stem-loop. The second finding was that coordinated synthesis of type I collagen polypeptide requires nonmuscle myosin II. The third finding discovered that profibrotic cytokines like TGF-beta and ouabain induced collagen synthesis through LARP6 and nonmuscle myosin II mechanism in human scleroderma skin fibroblasts and rat cardiac fibroblasts. In conclusion, we have discovered that LARP6 and nonmuscle myosin II regulated collagen synthesis. This pathway may contribute to excess collagen deposition in fibrosis. This information will help to find future anti-fibrotic therapy. / A Dissertation submitted to the Department of Biomedical Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Spring Semester, 2010. / March 15, 2010. / Includes bibliographical references. / Branko Stefanovic, Professor Directing Dissertation; Hengli Tang, University Representative; Myra M. Hurt, Committee Member; Yanchang Wang, Committee Member.