<p> Early light induced proteins (ELIPs) are widely distributed in the plant kingdom. Members of the extended light harvesting complex (LHC) superfamily, <i> ELIP</i>s are expressed in the nucleus and the ELIP protein is transiently localized to the thylakoid membranes. Significant increase in expression of <i> ELIP</i>s has been reported in response to stresses such as high light, high and low temperature, exposure to UV and salinity. ELIP expression also increases at transitional stages of chloroplast development such as deetiolation, conversion to chromoplast, and senescence.</p><p> In search of <i>cis-</i>regulatory regions, the <i>A. thaliana ELIP1</i> gene promoter has been investigated in our lab. A double-SORLIP1 element was identified as a critical <i>cis-</i>regulatory region common in the promoters of <i>A. thaliana ELIP1</i> (At3g22840) and <i>ELIP2</i> (At4g14690). Point mutations in the double-SORLIP1 element led to significant decline in expression.</p><p> Due to the importance of the double-SORLIP1 element, a yeast-one-hybrid assay was set up to find the specific DNA-binding proteins that bind to this region. Light harvesting complex II (LHCA2) and the ortholog of sugar beet HS1 PRO-1 2, heat-shock-like protein 2 (HSPRO2), showed a high specificity in binding to the double-SORLIP1 element. Investigation of <i>lhca2</i> and <i>hspro2 Arabidopsis</i> mutants did not show any significant difference in high light induced expression of <i>ELIP1</i> or <i> ELIP2</i> as compared to wild type. However, the high frequency of LHCA2 clones selected by yeast-one-hybrid assay and the high specificity in binding to the double-SORLIP1 element cannot be ignored.</p><p> After reviewing the literature, I hypothesized that LHCA2 may be a new retrograde signal that regulates expression of <i>ELIP</i> genes. However, more experimental evidence is needed to support this proposed function. The potential regulatory role of HSPRO2 is also discussed.</p>

Chloroplast protein degradation during senescence is delayed in autophagy mutants

<p> We have used the <i>C. elegans</i> primordial gonad to understand how stem cells assemble into a niche during development. The <i>C. elegans </i> primordial gonad contains two somatic gonad precursor cells (SGPs) and two primordial germ cells (PGCs). The primordial gonad assembles during embryogenesis when PGCs and SGPs come together adjacent the intestine. </p><p> As a first step in understanding niche assembly, we investigated how PGCs move to the site where the primordial gonad forms. PGCs and somatic cells move into the interior during gastrulation. Because somatic cells require transcription to ingress whereas PGCs are transcriptionally quiescent, we hypothesized that somatic cells might push or pull the PGCs into the embryo. We used videomicroscopy to identify cells that contact the PGCs, and used laser killing to determine if the contacting cells are required for PGC ingression. The PGCs are surrounding by adjacent mesodermal cells and internal endodermal cells. We found that the only contacting cells necessary for PGC ingression were the endodermal cells, which ingress into the embryo an hour before the PGCs. Killing or altering the fate of the endodermal cells prevented PGC ingression but not ingression of other somatic cells. Using fluorescent membrane markers and live imaging, we showed that PGCs and endodermal cells maintain contact throughout gastrulation, and that endodermal cells move dorsally as PGCs ingress form the ventral surface. PGCs express high levels of E-cadherin/HMR-1, and knocking down E-cadherin/HMR-1 caused PGCs to detach from endodermal cells and remain on the surface of the embryo. Finally, we show that the enrichment of HMR-1 protein in the PGCs is not due to transcriptional upregulation, but is instead due to an increase in protein expression mediated by the hmr-1 3' UTR. We propose that PGCs upregulate E-cadherin/HMR-1 to maintain tight adhesion with endodermal cells, which pull the PGCs into the embryo and position them at the site of primordial gonad assembly. Our results highlight the importance of germ cell - gut interactions during development and of E-cadherin-mediated adhesion in niche formation.</p>

<p> The cellular stress response (CSR) is one of the most highly conserved mechanisms among all organisms. Cellular stress can be defined as damage or the threat of damage to proteins, macromolecules and/or DNA. The response to damage can involve cell cycle regulation, protein chaperoning, DNA repair or, if macromolecular damage is too severe, apoptotic mechanisms can be initiated. This thesis details experiments that were designed to examine the cellular response to non-lethal environmental stressors at the protein level, using two fish species as study models. Two proteins that can cause cell cycle arrest and apoptosis mechanisms were examined. Expression of the CCAAT enhancer binding protein-delta (C/EBP-&delta;) was examined in the zebrafish, <i>Danio rerio,</i> exposed to acute, non-lethal hypoxic conditions. While C/EBP-&delta; was expressed constitutively in control individuals during all time points, exposure to hypoxic conditions did not have a consistent significant effect on C/EBP-&delta; expression (two-way ANOVA, P>0.05) in zebrafish white muscle tissue. In a second study, the expression of the growth arrest and DNA damage 45-alpha protein (gadd45-&alpha;), a mediator of cell cycle arrest and perhaps apoptosis was examined in heat-stressed liver tissue of an extremely cold-adapted Antarctic fish, <i>Trematomus bernacchii.</i> Gadd45-&alpha; levels were higher in fish exposure to 2&deg;C across all time points (one-way ANOVA; P&lt;0.05). The findings in these two studies expand our understanding of the CSR and how two genes that are involved in cell cycle regulation respond to acute, non-lethal environmental stress.</p>

<p> Coordinated movement of cardiac valves controls unidirectional flow of the blood with every heart beat. Cardiac valves are composed of thin, pliable leaflets that withstand compressive tension, fluid shear stress, and bending stress as blood flows through them. The structure and the mechanical properties of the valves render them durable during the lifetime of human beings. However, changes in hemodynamic environment, inflammatory responses, and congenital valvular defects can all cause valves to undergo irreversible structural changes, one of which is calcific aortic stenosis (CAS). CAS affects 2-3% of the population over 65 years old in the western world, and the only effective treatment is valve replacement surgery. CAS is characterized by tissue stiffening and the formation of calcified nodules, the development of which is associated with abnormal differentiation of resident fibroblasts known as valvular interstitial cells (VICs). Upon tissue injury, VICs are activated to myofibroblasts which deposit excessive collagen and stiffen the matrix. Understanding how the pathogenic phenotype of VICs is regulated by cues from the matrix may lead to new therapeutic treatments for CAS. In this thesis, I examined how matrix elasticity and TGF-&beta;1 regulate VIC phenotypes. First, I characterized the VIC population from porcine aortic valves and showed that this population is relatively homogeneous. When I cultured these primary cells on different substrates, I found that poly(ethylene glycol) hydrogels mimicked the native valve matrix better than tissue culture polystyrene plates with respect to preserving the quiescent fibroblast phenotype. At the level of signaling, I demonstrated that this is mediated through an elasticity-regulated PI3K/AKT pathway. Additionally, I showed that reduced matrix rigidity redirected activated valvular myofibroblasts into dormant fibroblasts without inducing significant apoptosis. Finally, I examined the effect of TGF-&beta;1 on VIC gene expression over time with microarray-based gene expression profiling and found that TGF-&beta;1 up-regulated cell-cell contact proteins (e.g., OB-cadherin, N-cadherin) in order to regulate valvular myofibroblast activation. Collectively, my thesis work revealed novel mechanosensing mechanisms employed by VICs to respond to matrix elasticity and explored the complex interactions among multiple extracellular cues, including matrix elasticity, TGF-&beta;1 and cell-cell adhesion, to direct the cellular fate of VICs.</p>

<p> Acidic aqueous-phase pretreatment is a promising approach that has been directed at maximizing intermediates yields (e.g. sugars, sugar degradation products, and lignin) from biomass for fuel and chemical production. This dissertation explores the kinetic fundamentals of biomass hydrolysis in acidic aqueous-phase with different catalysts (e.g. sulfuric acid, metal chlorides), operating conditions (e.g. temperature, time pressure), and equipment configurations (e.g. batch, flowthough). </p><p> The kinetic analysis revealed that crystalline cellulose is insusceptible to hydrolysis compared with agarose at low temperature (e.g.140 &deg;C), while it decomposed rapidly at elevated temperature (e.g. 220 &deg;C). Higher temperature with reduced time was desirable for glucose production whereas lower temperature with prolonged time was preferred for xylose generation. In acidic conditions, furfural and levulinic acid were stable whereas 5-hydroxymethylfurfural was susceptible to decomposition with high rate constant. MgCl₂ can promote the cleavage of C-O-C bond in polysaccharides (e.g. agarose) and enhance the subsequent dehydration reaction to 5-hydroxymethylfurfural. Unlike transition metal chlorides and H2SO₄, MgCl₂ has little ability to induce retro aldol and rehydration reactions to generate byproducts like lactic acid and levulinic acid. Mg²⁺ possessing hgiher activity than other alkali and alkaline earth metal chlorides (Na⁺ and Ca2⁺) resulted in 40.7% yield and 49.1% selectivity of 5-hydroxymethylfurfural. </p><p> Dissolution of biomass was significantly enhance using acidic hot water flowthrough pretreatment at 200&mdash;280&deg;C. Significant cellulose removal accompanied with the transformation of cellulose I to cellulose II and amorphous cellulose were observed when temperature was above 240 &deg;C for water-only and 220 &deg;C for dilute acid. Approximately100% of the xylan and &sim;90% of the cellulose were solubilized and recovered. Up to 15% of the lignin was solubilized, while the remaining lignin was insoluble. Over 90% sugar yields were obtained from pretreated whole slurries using less than 10 FPU/g cellulase plus hemicellulase enzyme. </p><p> A kinetic model was developed to depict the biomass degradation in flowthrough system. This model predicted the sugar generation more precisely than the conventional homogeneous first-order reaction models. Mass transfer limitations were minimized using 4mm biomass particle sizes with 4g biomass loading at 25mL/min flow rate, produced hydrolyzate slurries with 13g/L potential sugar concentrations.</p>

<p> The abstract is not available for copy and paste.</p>

<p> Fungi are thought to be one of the most diverse groups of organisms in the Arctic. They drive mineral and energy cycles and influence the occurrence of other organisms as mutualists (mycorrhizae, endophytes, lichens), decomposers and pathogens. Nevertheless, information on fungal biodiversity and distribution patterns in relation to environments across the Arctic is still sparse. Molecular methods were used to examine the diversity and community structures of ectomycorrhizal fungi (EMF) associated with two principal arctic host plants, <i> Salix arctica</i> and <i>Dryas integrifolia,</i> as well as total soil fungal communities of adjacent disturbed and undisturbed areas of patterned-ground features across the five bioclimatic subzones (A-E) of the North American Arctic. Key findings include the following: (1) More diverse fungal communities had been observed than previously known. These communities encompass nearly all fungal phyla and included all fungal guilds. However, a few species-rich fungal families dominated these fungal communities. (2) Surprisingly, species richness did not decline with latitude. (3) The most abundant fungal taxa were widely distributed in and beyond the Arctic. Yet root (EMF) and soil fungal communities showed niche preferences in regard to bioclimatic subzones. Furthermore, disturbed and undisturbed patterned ground features harbored different soil fungal communities with the exception of the coldest subzone A. In contrast, EMF community composition was not affected by host plant identity. (4) Fungal communities in the warmest subzone E were distinct from the other arctic subzones and the majority of taxa matched fungi from the boreal forest. (5) Key drivers of fungal community and guild composition along the bioclimatic gradient included regional climate, pH as well as vegetation composition and productivity across the subzones. At the local scale of patterned-ground features, fungal communities were correlated with vegetation composition and microclimate. With a warming climate, I would expect an enhanced colonization of patterned-ground features by vascular plants that would then affect fungal community structure not only at the species level, but also at the level of fungal guilds. In particular I would expect increases in fungi that are symbiotic with plants and a northward shift of both plant and fungal taxa.</p>

<p> The RNA-binding proteins U2AF and PTB play important roles in gene expression in many eukaryotic species. Although U2AF and PTB have been well-studied, their functional requirements have not been investigated on a genome-wide scale. In this thesis, I analyze RNA expression data to determine the requirement of the general splicing factor U2AF in <i>S. pombe</i> and to identify genes misregulated in Drosophila PTB mutants. I find that many introns are insensitive to U2AF inactivation in a <i>Schizosaccharomyces pombe</i> U2AF59 mutant, <i>prp2.1.</i> Bioinformatics analysis indicates that U2AF-insensitive introns have stronger 5' splice sites and higher A/U composition. The importance of intronic nucleotide composition was further investigated using wild type RNA expression data sets. I show that nucleotide composition is a relatively important factor for regulated intron retention in a variety of species. I also analyzed the RNA-binding protein PTB using RNA Seq data to reveal genes misregulated in PTB mutants in <i>D. melanogaster.</i> I identify misregulation of alternative splicing in PTB mutants and putative PTB binding sites. In the PTB embryonic lethal mutant, which shows dorsoventral patterning defects, I show that dorsal fate genes are significantly up-regulated. I present a model to link PTB to dorsal closure defects. This thesis provides the first genome-wide analysis of U2AF in <i>S. pombe</i> and PTB in <i>Drosophila melanogaster. </i></p>

Thesis (Ph. D.)--University of California, San Francisco, 2008. / Source: Dissertation Abstracts International, Volume: 68-11, Section: B, page: 7314. Adviser: Kevan M. Shokat.