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<p>The incidence and prevalence of people suffering from end stage renal disease is increasing. Proteinuria, particularly the presence of albumin in urine is concerning because proteinuria is associated with the progression to end stage renal disease (ESRD). Understanding the mechanisms involved in damaging the glomerular filtration barrier is essential. Transforming growth factor beta (TGFB) is a key cytokine in mediating glomerulosclerosis and proteinuria. Not much is known about the downstream pathways that mediates the renal damage and proteinuria.</p> <p>I hypothesize that TGFB induces proteinuria through podocyte de-differentiation and this occurs through SMAD dependent and independent pathways.</p> <p>Methods: I used adenovirus mediated gene transfer of TGFB1 to rat renal artery to study the effects of TGFB1 on renal structure and functions. To study the importance of SMAD3 in mediating downstream effects of TGFB1 in proteinuria and podocyte effacement, I used an anti-glomerular basement membrane model in SMAD3+/+ and SMAD3-/- mice to induce glomerulonephritis and proteinuria.</p> <p>Results: Transient TGFB1 overexpression via AdTGFB1 induced significant proteinuria, podocyte foot process effacement, nephrin down-regulation, and nephrinuria. The expression of synaptopodin was also significantly down-regulated by TGFB1. TGFB1 increased the expression of the angiopoietin receptor, Tie2, in podocyte cell culture. In cultured podocytes, TGFB1 downregulated the gene and protein expression of both nephrin and synaptopodin. These findings suggest that locally produced TGFB1 can cause podocyte de-differentiation marked by a loss of synaptopodin, nephrin, and foot process effacement; this process is partly regulated by angiopoietins. This process represents a novel pathway that may explain proteinuria in a variety of common renal diseases.</p> <p>Both SMAD3+/+ and SMAD3-/- mice had proteinuria after induction of anti-GBM glomerulonephritis, though to a lesser extent in SMAD3-/- mice. SMAD3-/- and SMAD3+/+ mice developed significant glomerulonephritis with progressive interstitial fibrosis and chronic renal impairment. The SMAD3+/+ mice were found to be more prone to fibrotic changes, interstitial damage and tubular and glomerulosclerosis than the SMAD3-/- mice. This suggests that TGFB1 signals through pathways other than SMAD3 such as those triggered by hypoxia.</p> <p>Conclusion: I have shown that TGFB1 upregulation via AdTGFB1 induces proteinuria through podocyte dedifferentiation and FP effacement. Angiopoietins are essential for TGFB1 mediated podocyte injury. The effects of TGFB are partially mediated through SMAD3 as there is residual podocyte effacement and proteinuria in the SMAD3-/- mice. Hence there are SMAD3 dependent and independent pathways involved in proteinuria.</p> / Doctor of Science (PhD)

Axon degeneration can result from primary damage due to a variety of causes, and in some instances, its effects can further propagate damage to vicinal neurons. When an axon has been damaged or transected, Wallerian degeneration is the apoptotic-like mechanism that is initiated, ultimately leading to the death of the neuron. The post-injury cellular inflammatory response is recruited to clear the degraded debris and is also responsible for activating the cascade events leading to additional cell death in surrounding neurons. A unique strain of mutant mice were discovered to express what is called the Wallerian Degeneration Slow (Wld') gene, which produces a chimeric nuclear protein that has been observed to dramatically delay both the onset of axon degeneration and the initiation of the post-injury cellular inflammatory response . Recent studies seem _to indicate the neuroprotective phenotype induced by the Wld' protein is the result of it modulating levels of genetic and protein expression in the damaged neuron. This thesis will review what is known about the Wld' protein and discuss how it offers an in-depth look at the molecular mechanisms behind neurodegenerative injury and disease states. In addition, we will discuss our efforts of isolating and purifying plasmids with the Wld' gene that have been cloned in order to create a stable cell line to aid in the future study for the characterization of neuroprotective mechanisms and the molecular pathways of neurodegeneration .

<p> Pregnane X receptor (PXR) is a major transcriptional regulator of xenobiotic metabolism and transport pathways in the liver and intestines, which are critical for protecting organisms against potentially harmful xenobiotic and endobiotic compounds. Inadvertent activation of drug metabolism pathways through PXR is known to contribute to drug resistance, adverse drug-drug interactions, and drug toxicity in humans. In both humans and rodents, PXR has been implicated in non-alcoholic fatty liver disease, diabetes, obesity, inflammatory bowel disease, and cancer. Because of PXR's important functions, it has been a therapeutic target of interest for a long time.</p><p> Recent mechanistic studies have shown that PXR is modulated by multiple PTMs. In this thesis work, we conducted the first detailed examination of PXR regulation by acetylation. We found that PXR is efficiently acetylated <i> in vitro</i> and <i>in vivo</i> in multiple cell lines (293T, HepG2, LS174T). Acetylation and deacetylation are mediated by p300 and SIRT1, respectively. We found that PXR is directly acetylated by p300 at K109 by LC-MS/MS analysis. The K109Q acetylation mimicking mutant displayed reduced transcriptional activity and reduced ability to induce <i>cyp3A4</i> target gene mRNA and protein compared to the WT and the K109R acetylation-defective mutant. The diminished activity of the K109Q mutant appears to be due to impaired heterodimerization with RXRa and impaired binding of the PXR-RXRa heterodimer to DNA response elements. Furthermore, PXR acetylation appears to have an effect at the phenotypic level, in that pharmacological modulation of PXR acetylation levels can modulate its lipogenic function in mouse primary hepatocytes independent of a ligand. Moreover, the K109Q mutant displays impaired chemoprotective function based on morphological assessment of cells overexpressing K109Q and challenged with indomethacin, suggesting that K109 acetylation downregulates PXR's chemoprotective and perhaps anti-apoptotic functions, although this must be explored further. Notably, the K109R mutant displayed the WT phenotype, further supporting that acetylation itself, not just any arbitrary mutation confers the effect. Altogether, the data suggests that acetylation at K109 represents an overall "loss of function" effect on PXR activity. Implications of our findings are discussed in the context of known roles for PXR in transcription, health, and disease.</p>

<p> This work focuses on members of the Boletales. This order is comprised of a morphological and ecologically diverse set of species. While the vast majority of species are pileate-stipitate with pores and have a mutualistic nutritional strategy ectomycorrhal (ECM), there are resupinate and gilled species, and saprotrophs and mycoparasites as well. In the first chapter, we review the ecological niche occupied by <i>Boletinellus merulioides. </i> This species was originally considered to be ECM, the symbiont to <i> Fraxinus americana.</i> This hypothesis was rejected, and replaced by the possibility of a mutualism with an <i>F. americana</i> aphid pest, <i>Prociphilus fraxinifolii.</i> We present the first study that observed all three species, since the original publication, the first molecular data for each species, and isotopic fractionation results for <i> B. merulioides</i> and <i>P. fraxinifolii.</i> Additionally, we describe a new morphology for sclerotia of <i>B. merulioides.</i> In total, we are unable to reject the possibility of a facultative mutualism between <i>B. merulioides</i> and <i>P. fraxinifolii.</i></p><p> Chapters two through five review systematics in the <i>Boletineae. </i> Chapter two presents the most comprehensive phylogenetic review of the <i>Boletineae,</i> at the time publication, and remains one of the most inclusive <i>Boletineae phylogenies.</i> Three genes, nuclear large subunit, translation elongation factor 1-alpha, and DNA directed RNA polymerase II largest subunit, were used. This chapter is a summary of <i> Boletineae</i> taxonomy and morphological characteristics, with a clade by clade analysis. We present compelling evidence for the mycoparasitic nutritional mode of <i>Buchwaldoboletus lignicola.</i> Additionally, we found that <i>Chalciporus piperatus,</i> a close relative of <i> B. lignicola</i>, is likely to be a mycoparasite. We present strong evidence that the genus <i>Boletus</i> is limited to single clade that contains approximately 10% of the validly published <i>Boletus</i> species. </p><p> A subset of the taxa sampled in chapter two was used in the phylogenies presented in chapters three, four, and five. Each of these chapters reviews the phylogenetic placement of traditionally problematic species/genera; <i> Surotius eximius, Harrya chromapes</i> and allies, and the Boletaceae species with longitudinally striated spores. These groups have been in multiple. Our results show that <i>Sutorius</i> and <i>Harrya</i> species are distinct from other Boletacaea species and that the longitudinally striated species have been lumped together. By correcting taxonomic confusion and using a multigene data set we are able to resolve these problematic species, and provide a path for future systematics and evolutionary analysis.</p>

<p> The focus of the present research is the synthesis of polypeptides for the study of protein folding and misfolding and for the development of novel polypeptide-based optical antennas in nanotechnology. It is hypothesized that simple polypeptides can be used as models to mimic <i>in vivo</i> folding of globular proteins. Desired repetitive polypeptides were genetically encoded and expressed in <i>E. coli</i> using conventional methods and characterized using a variety of spectroscopic (including circular dichroism (CD), deep UV resonance Raman (DUVRR), UV-vis and fluorescence) and microscopic (atomic force microscopy (AFM) and transmission electron microscopy (TEM)) techniques. The polypeptides predominantly formed bilayer, fibrillar structures with a cross &beta;-core. <b>YEHK21-YE8</b>, a chimeric polypeptide, folded within three days. The folding/fibrillation of the chimeric construct illuminates the controlling factors and hence suggests the importance of those factors in amyloidogenic diseases. <b>YE8</b> and <b>YE8</b> derivatives illustrated the role of proline in &beta;-sheet formation. The EW polypeptide models elucidated the influence of tryptophan residues and the degree of polymerization on folding. The study of <b>EW14C1</b> and <b>EW21C1</b> demonstrated light-harvesting properties when labeled with a suitable dye.</p>

<p> Multidrug resistant <i>Acinetobacter baumannii,</i> a common etiologic agent of severe nosocomial infections in compromised hosts, usually harbors <i>aac(6&rsquo;)-Ib.</i> This gene codes for an aminoglycoside acetyltransferase that modifies amikacin and other aminoglycosides of clinical relevance. The goal of this work was to interfere with expression of this resistance gene and induce susceptibility to amikacin in resistant pathogens. In vitro translation assays led to the identification of an antisense oligodeoxynucleotide (ODN4) that targets the initiation of translation region of <i>aac(6&rsquo;)-Ib </i> mRNA. An isosequential nuclease-resistant chimeric oligomer composed of 2&rsquo;,4&rsquo;-bridged nucleic acid-NC (BNA^NC) residues and deoxynucleotides (BNA^NC-DNA) covalently bound to the cell-penetrating peptide (RXR)₄XB (where &ldquo;X&rdquo; and &ldquo;B&rdquo; stand for 6-aminohexanoic acid and &beta;-alanine, respectively). This compound, called CPPBD4, inhibited translation at similar levels observed with ODN4. Addition of a combination of Amikacin and CPPBD4 to a culture of an <i>Acinetobacter baumannii</i> clinical strain harboring <i> aac(6&rsquo;)-Ib</i> resulted in growth inhibition indicating that CPPBD4 reached the cytosol and interfere with the expression of the resistance enzyme. </p>

<p> Bronchopulmonary dysplasia (BPD) is a serious respiratory complication for the preterm newborn characterized clinically by prolonged respiratory distress and histologically by alveolar simplification and decreased pulmonary vasculature. The development of BPD is well linked to oxidative stress suffered by the newborn as a result of a preterm fetal-neonatal transition, supplemental oxygen, infection, increased inflammation, and mechanical ventilation. Damage suffered by oxidative stress may be through direct mechanisms or through alteration of redox&not;sensitive pathways involved in cell death, cell survival, differentiation, and proliferation. Redox&not;sensitive modifications regulating protein function and redox-sensitive pathways have mainly been ascribed to oxidative modification of cysteine thiols. As their modification is critical for protein function, maintenance of the thiol redox status is crucial. Thioredoxin-1 (Trx1) functions in maintenance of thiol redox homeostasis, and its redox activity is intimately linked to antioxidant, cytoprotection, proliferation responses, and cytoprotection. While Trx1 targets of redox regulation have been identified, we hypothesize that additional protein may be redox regulated and that Trx1 target profiles may change during oxidative stress. Therefore a novel immunoprecipitation approach, identified as the substrate trap approach, was developed to identify Trx1 targets. The following demonstrates the use of the substrate trap approach for identification of Trx1 redox targets and further application of the approach to identify alterations in target profiles in response to oxidative stress. Use of nuclear targeted substrate trap was successfully employed to enrich from nuclear Trx1 targets. As a final component the characterization of the Trx1 system in mouse from late embryonic development through the first week of life animals were exposed to room air or hyperoxia (model of BPD). Characterization indicates impairment of the Trx1 system in response to hyperoxic injury. As Trx1 is known to regulate proliferation, cell death, survival, differentiation pathways, impairment of the Trx1 system during early neonatal development may potentiate hyperoxic injury and alterations in lung development. Better understanding of Trx1 interactions occur through the substrate trap in a physiological model of BPD will help elucidate redox-signaling pathways involved in BPD pathogenesis.</p>

<p> Beta-arrestins are adaptor proteins that can scaffold a number of signaling proteins to promote localized activity within the cell. Downstream of some GPCRs, &beta;-arrestins can promote activation of the actin filament severing protein, cofilin, through two mechanisms: one involving inhibition of LIM Kinase (LIMK) which negatively regulates cofilin activity through phosphorylation on serine 3. The mechanism by which &beta;-arrestin-1 regulates LIMK activity has not been elucidated; however, it has been shown to be important for cell migration downstream of protease-activate-receptor-2 (PAR-2), dendritic spine formation and opioid receptor function. Here my work demonstrate that &beta;-arrestin-1 directly binds both cofilin and LIMK, and inhibits LIMK activity directly and investigate the mechanism by which inhibition of kinase activity occurs. Using serial truncations and site-directed mutagenesis, I identify crucial residues for cofilin and LIMK interaction within amino acids 1-99 of &beta;-arrestin-1 and show that charged residues at 50 and 51 are crucial for binding to LIMK and R51 is required for LIMK inhibition, PAR2 stimulated cofilin dephosphorylation and cell migration. Additionally, our work reveals that amino acids 1-99 aminos of &beta;-arrestin-1 bind both cofilin and LIMK with a higher apparent affinity than the full length and blocks PAR2-stimulated cofilin dephosphorylaton in HEK293 cells, suggesting it functions as a selective dominant negative &beta;-arrestin-1, inhibiting specifically the cofilin pathway. Thus, residues in the N-terminus of &beta;-arrestin-1 are involved in LIMK inhibition and cofilin activation and this, in turn, is important for cell migration downstream of PAR-2.</p>

<p> Most plants harbor microbial symbionts, which often affect host performance and fitness. Endophytic <i>Epichlo&euml;</i> species are systemic fungal microbial symbionts of many cool-season pooid grasses. Benefits to the host from <i>Epichlo&euml;</i> infection include increased resistance to stressful environmental factors, such as drought and limited soil nutrients, due to morphological and physiological changes. The major benefit of <i> Epichloe</i> infection is enhanced protection against herbivory due to production of fungal alkaloids. The fungal alkaloids have varying activity against invertebrate or mammalian grazers. Although <i>Epichlo&euml; </i> endophytes are well-studied in agronomic grasses such as tall fescue and perennial ryegrass, little is known about the how the presence of different endophyte species and their frequencies and distribution are related to environmental factors in native grasses. Using two native grasses to eastern [<i>Poa alsodes</i> (Grove Bluegrass)] and western [<i>Achnatherum robustum </i> (Sleepygrass)] North America, I addressed the following questions: 1) how are endophyte species distributed among populations along a latitudinal gradient, 2) what fungal alkaloids are produced by different endophyte species, 3) how do fungal alkaloids affect insect herbivores, and 4) what are the effects of different endophytes on host plant growth? (Abstract shortened by ProQuest.) </p>