@phdthesis{Ghandour2020,
  author    = {Ghandour, Rabea},
  title     = {Identification of chloroplast translational feedback regulation and establishment of aptamer based mRNA purification to unravel involved regulatory factors},
  doi       = {10.25932/publishup-48289},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-482896},
  school      = {Universit{\"a}t Potsdam},
  pages     = {XIII, 173},
  year      = {2020},
  abstract  = {After endosymbiosis, chloroplasts lost most of their genome. Many former endosymbiotic genes are now nucleus-encoded and the products are re-imported post-translationally. Consequently, photosynthetic complexes are built of nucleus- and plastid-encoded subunits in a well-defined stoichiometry. In Chlamydomonas, the translation of chloroplast-encoded photosynthetic core subunits is feedback-regulated by the assembly state of the complexes they reside in. This process is called Control by Epistasy of Synthesis (CES) and enables the efficient production of photosynthetic core subunits in stoichiometric amounts. In chloroplasts of embryophytes, only Rubisco subunits have been shown to be feedback-regulated. That opens the question if there is additional CES regulation in embryophytes. I analyzed chloroplast gene expression in tobacco and Arabidopsis mutants with assembly defects for each photosynthetic complex to broadly answer this question. My results (i) confirmed CES within Rubisco and hint to potential translational feedback regulation in the synthesis of (ii) cytochrome b6f (Cyt b6f) and (iii) photosystem II (PSII) subunits. This work suggests a CES network in PSII that links psbD, psbA, psbB, psbE, and potentially psbH expression by a feedback mechanism that at least partially differs from that described in Chlamydomonas. Intriguingly, in the Cyt b6f complex, a positive feedback regulation that coordinates the synthesis of PetA and PetB was observed, which was not previously reported in Chlamydomonas. No evidence for CES interactions was found in the expression of NDH and ATP synthase subunits of embryophytes. Altogether, this work provides solid evidence for novel assembly-dependent feedback regulation mechanisms controlling the expression of photosynthetic genes in chloroplasts of embryophytes. In order to obtain a comprehensive inventory of the rbcL and psbA RNA-binding proteomes (including factors that regulate their expression, especially factors involved in CES), an aptamer based affinity purification method was adapted and refined for the specific purification these transcripts from tobacco chloroplasts. To this end, three different aptamers (MS2, Sephadex ,and streptavidin binding) were stably introduced into the 3' UTRs of psbA and rbcL by chloroplast transformation. RNA aptamer based purification and subsequent chip analysis (RAP Chip) demonstrated a strong enrichment of psbA and rbcL transcripts and currently, ongoing mass spectrometry analyses shall reveal potential regulatory factors. Furthermore, the suborganellar localization of MS2 tagged psbA and rbcL transcripts was analyzed by a combined affinity, immunology, and electron microscopy approach and demonstrated the potential of aptamer tags for the examination of the spatial distribution of chloroplast transcripts.},
  language  = {en}
}
@article{CywinskiOlejkoLoehmannsroeben2015,
  author    = {Cywinski, Piotr J. and Olejko, Lydia and L{\"o}hmannsr{\"o}ben, Hans-Gerd},
  title     = {A time-resolved luminescent competitive assay to detect L-selectin using aptamers as recognition elements},
  series = {Analytica chimica acta : an international journal devoted to all branches of analytical chemistry},
  volume    = {887},
  journal   = {Analytica chimica acta : an international journal devoted to all branches of analytical chemistry},
  publisher = {Elsevier},
  address   = {Amsterdam},
  issn      = {0003-2670},
  doi       = {10.1016/j.aca.2015.06.045},
  pages     = {209 -- 215},
  year      = {2015},
  abstract  = {L-selectin is a protein with potential importance for numerous diseases and clinical disorders. In this paper, we present a new aptamer-based luminescent assay developed to detect L-selectin. The sensing system working principle is based on Forster Resonance Energy Transfer (FRET) from a donor terbium complex (TbC) to an acceptor cyanine dye (Cy5). In the present approach, the biotinylated aptamer is combined with Cy5-labelled streptavidin (Cy5-Strep) to yield an aptamer-based acceptor construct (Apta-Cy5-Strep), while L-selectin is conjugated using luminescent TbC. Upon aptamer binding to the TbC-labelled L-selectin (L-selectin-TbC), permanent donor-acceptor proximity is established which allows for radiationless energy transfer to occur. However, when unlabelled L-selectin is added, it competes with the L-selectin-TbC and the FRET signal decreases as the L-selectin concentration increases. FRET from the TbC to Cy5 was observed with time-gated time-resolved luminescence spectroscopy. A significant change in the corrected luminescence signal was observed in the dynamic range of 10 -500 ng/mL L-selectin, the concentration range relevant for accelerated cognitive decline of Alzheimer's disease, with a limit of detection (LOD) equal to 10 ng/mL. The aptasensor-based assay is homogeneous and can be realized within one hour. Therefore, this method has the potential to become an alternative to tedious heterogeneous analytical methods, e.g. based on enzyme-linked immunosorbent assay (ELISA). (C) 2015 Elsevier B.V. All rights reserved.},
  language  = {en}
}