Structural and physical investigations of novel germanium compounds: Aryloxides, nanomaterials, and photolysis of oligogermanes

The work described in this dissertation will explore the synthesis and characterization of novel germanium containing compounds in order to gain a better understanding of the organometallic chemistry of germanium. These compounds include germanium bisamides, aryloxogermylenes, polyfunctional aryloxides such as calix[n]arenes and binaphthoxogermanium compounds, and oligogermanes containing up to four germanium atoms.We have found that the germanium bisamides can be trapped using the germylene trapping agent benzil and we have fully characterized those products. The germanium bisamides can also be used as starting materials to synthesize germanium aryloxides via protonolysis of a phenol that contains one or more phenolic groups. Using this method we have prepared the germanium(IV) aryloxides [Ge(OC6H3Ph2-2,6)2(R)(I)] (R = But or Me) where the R = Me derivative was then converted to the triaryloxo species [Ge(OC6H3Ph2-2,6)3(Me)] upon reaction of the iodine containing compound with an extra equivalent of 2,6-diphenylphenol. Using polyfunctional phenols, we prepared and characterized the germanium(II) calix[5]arene complex {calix[5]arene}2Ge2(OSiMe3)4(OH)2, the calix[6]arene complex [(C6H3)6(CH2)6(OSiMe2Ph)6], and the binaphthoxogermanium(II) complex (S,S)-[Ge{OC20H10(OSiMe2Ph)-2'-(SiMe3)2-3,3'}2].Chapter five describes the synthesis of a series of three oligogermanes including a digermane, a trigermane, and a branched neopentyl germane. We have found that these oligogermanes can be used as precursors for the preparation of germanium(0) nanomaterials, and that the size of the resulting nanoparticles correlates with the number of catenated germanium atoms in the precursor compounds. These nanoparticles are fluorescent and the position of the emission maximum is red shifted as the size of the particles increases.Lastly, the sixth chapter will discuss the synthesis, characterization, and photochemistry of a series of six linear oligogermanes. The optical and electronic properties of these compounds were probed using UV/visible spectroscopy and differential pulse voltammetry. The photochemistry of these compounds will be analyzed by photolyzing each compound using UV-C light (280-100 nm) in the presence of acetic acid as a germylene trapping agent. If germylenes :GeR2 are formed, they should be trapped to yield R2Ge(H)OAc. The photolysis products will be characterized by NMR (1H and 13C) spectroscopy, infrared spectroscopy (FTIR), and gas-chromatography mass spectroscopy (GC/MS)

Positioning of the SCRAMBLED Receptor Requires UDP-Glc:sterol Glucosyltransferase 80B1 in \u3cem\u3eArabidopsis\u3c/em\u3e Roots

The biological function of sterol glucosides (SGs), the most abundant sterol derivatives in higher plants, remains uncertain. In an effort to improve our understanding of these membrane lipids we examined phenotypes exhibited by the roots of Arabidopsis (Arabidopsis thaliana) lines carrying insertions in the UDP-Glc:sterol glucosyltransferase genes, UGT80A2 and UGT80B1. We show that although ugt80A2 mutants exhibit significantly lower levels of total SGs they are morphologically indistinguishable from wild-type plants. In contrast, the roots of ugt80B1 mutants are only deficient in stigmasteryl glucosides but exhibit a significant reduction in root hairs. Sub-cellular investigations reveal that the plasma membrane cell fate regulator, SCRAMBLED (SCM), is mislocalized in ugt80B1 mutants, underscoring the aberrant root epidermal cell patterning. Live imaging of roots indicates that SCM:GFP is localized to the cytoplasm in a non cell type dependent manner instead of the hair (H) cell plasma membrane in these mutants. In addition, we provide evidence for the localization of the UGT80B1 enzyme in the plasma membrane. These data lend further support to the notion that deficiencies in specific SGs are sufficient to disrupt normal cell function and point to a possible role for SGs in cargo transport and/or protein targeting to the plasma membrane

FACKEL is a sterol C-14 reductase required for organized cell division and expansion in Arabidopsis embryogenesis

In flowering plants, the developing embryo consists of growing populations of cells whose fates are determined in a position-dependent manner to form the adult organism. Mutations in the FACKEL (FK) gene affect body organization of the Arabidopsis seedling. We report that FK is required for cell division and expansion and is involved in proper organization of the embryo. We isolated FK by positional cloning. Expression analysis in embryos revealed that FK mRNA becomes localized to meristematic zones. FK encodes a predicted integral membrane protein related to the vertebrate lamin B receptor and sterol reductases across species, including yeast sterol C-14 reductase ERG24. We provide functional evidence that FK encodes a sterol C-14 reductase by complementation of erg24. GC/MS analysis confirmed that fk mutations lead to accumulation of intermediates in the biosynthetic pathway preceding the C-14 reductase step. Although fk represents a sterol biosynthetic mutant, the phenotype was not rescued by feeding with brassinosteroids (BRs), the only plant sterol signaling molecules known so far. We propose that synthesis of sterol signals in addition to BRs is important in mediating regulated cell growth and organization during embryonic development. Our results indicate a novel role for sterols in the embryogenesis of plants

Shared functions of plant and mammalian StAR-related lipid transfer (START) domains in modulating transcription factor activity

Abstract Background Steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) domains were first identified from mammalian proteins that bind lipid/sterol ligands via a hydrophobic pocket. In plants, predicted START domains are predominantly found in homeodomain leucine zipper (HD-Zip) transcription factors that are master regulators of cell-type differentiation in development. Here we utilized studies of Arabidopsis in parallel with heterologous expression of START domains in yeast to investigate the hypothesis that START domains are versatile ligand-binding motifs that can modulate transcription factor activity. Results Our results show that deletion of the START domain from Arabidopsis Glabra2 (GL2), a representative HD-Zip transcription factor involved in differentiation of the epidermis, results in a complete loss-of-function phenotype, although the protein is correctly localized to the nucleus. Despite low sequence similarly, the mammalian START domain from StAR can functionally replace the HD-Zip-derived START domain. Embedding the START domain within a synthetic transcription factor in yeast, we found that several mammalian START domains from StAR, MLN64 and PCTP stimulated transcription factor activity, as did START domains from two Arabidopsis HD-Zip transcription factors. Mutation of ligand-binding residues within StAR START reduced this activity, consistent with the yeast assay monitoring ligand-binding. The D182L missense mutation in StAR START was shown to affect GL2 transcription factor activity in maintenance of the leaf trichome cell fate. Analysis of in vivo protein–metabolite interactions by mass spectrometry provided direct evidence for analogous lipid-binding activity in mammalian and plant START domains in the yeast system. Structural modeling predicted similar sized ligand-binding cavities of a subset of plant START domains in comparison to mammalian counterparts. Conclusions The START domain is required for transcription factor activity in HD-Zip proteins from plants, although it is not strictly necessary for the protein’s nuclear localization. START domains from both mammals and plants are modular in that they can bind lipid ligands to regulate transcription factor function in a yeast system. The data provide evidence for an evolutionarily conserved mechanism by which lipid metabolites can orchestrate transcription. We propose a model in which the START domain is used by both plants and mammals to regulate transcription factor activity

Shared functions of plant and mammalian StAR-related lipid transfer (START) domains in modulating transcription factor activity

Background: Steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) domains were first identified from mammalian proteins that bind lipid/sterol ligands via a hydrophobic pocket. In plants, predicted START domains are predominantly found in homeodomain leucine zipper (HD-Zip) transcription factors that are master regulators of cell-type differentiation in development. Here we utilized studies of Arabidopsis in parallel with heterologous expression of START domains in yeast to investigate the hypothesis that START domains are versatile ligand-binding motifs that can modulate transcription factor activity. Results: Our results show that deletion of the START domain from Arabidopsis Glabra2 (GL2), a representative HD-Zip transcription factor involved in differentiation of the epidermis, results in a complete loss-of-function phenotype, although the protein is correctly localized to the nucleus. Despite low sequence similarly, the mammalian START domain from StAR can functionally replace the HD-Zip-derived START domain. Embedding the START domain within a synthetic transcription factor in yeast, we found that several mammalian START domains from StAR, MLN64 and PCTP stimulated transcription factor activity, as did START domains from two Arabidopsis HD-Zip transcription factors. Mutation of ligand-binding residues within StAR START reduced this activity, consistent with the yeast assay monitoring ligand-binding. The D182L missense mutation in StAR START was shown to affect GL2 transcription factor activity in maintenance of the leaf trichome cell fate. Analysis of in vivo protein–metabolite interactions by mass spectrometry provided direct evidence for analogous lipid-binding activity in mammalian and plant START domains in the yeast system. Structural modeling predicted similar sized ligand-binding cavities of a subset of plant START domains in comparison to mammalian counterparts. Conclusions: The START domain is required for transcription factor activity in HD-Zip proteins from plants, although it is not strictly necessary for the protein’s nuclear localization. START domains from both mammals and plants are modular in that they can bind lipid ligands to regulate transcription factor function in a yeast system. The data provide evidence for an evolutionarily conserved mechanism by which lipid metabolites can orchestrate transcription. We propose a model in which the START domain is used by both plants and mammals to regulate transcription factor activity

Nanoscale Metallic Iron for Environmental Remediation: Prospects and Limitations

The amendment of the subsurface with nanoscale metallic iron particles (nano-Fe0) has been discussed in the literature as an efficient in situ technology for groundwater remediation. However, the introduction of this technology was controversial and its efficiency has never been univocally established. This unsatisfying situation has motivated this communication whose objective was a comprehensive discussion of the intrinsic reactivity of nano-Fe0 based on the contemporary knowledge on the mechanism of contaminant removal by Fe0 and a mathematical model. It is showed that due to limitations of the mass transfer of nano-Fe0 to contaminants, available concepts cannot explain the success of nano-Fe0 injection for in situ groundwater remediation. It is recommended to test the possibility of introducing nano-Fe0 to initiate the formation of roll-fronts which propagation would induce the reductive transformation of both dissolved and adsorbed contaminants. Within a roll-front, FeII from nano-Fe0 is the reducing agent for contaminants. FeII is recycled by biotic or abiotic FeIII reduction. While the roll-front concept could explain the success of already implemented reaction zones, more research is needed for a science-based recommendation of nano- Fe0 for subsurface treatment by roll-front

BOLITA, an Arabidopsis AP2/ERF-like transcription factor that affects cell expansion and proliferation/differentiation pathways

The BOLITA (BOL) gene, an AP2/ERF transcription factor, was characterized with the help of an activation tag mutant and overexpression lines in Arabidopsis and tobacco. The leaf size of plants overexpressing BOL was smaller than wild type plants due to a reduction in both cell size and cell number. Moreover, severe overexpressors showed ectopic callus formation in roots. Accordingly, global gene expression analysis using the overexpression mutant reflected the alterations in cell proliferation, differentiation and growth through expression changes in RBR, CYCD, and TCP genes, as well as genes involved in cell expansion (i.e. expansins and the actin remodeling factor ADF5). Furthermore, the expression of hormone signaling (i.e. auxin and cytokinin), biosynthesis (i.e. ethylene and jasmonic acid) and regulatory genes was found to be perturbed in bol-D mutant leave