Isolation of intact plastids of the secondary alga \kur{Chromera velia} and treatment of the alga with rifampicin

Photosynthetic organism Chromera velia was discovered recently. Thanks to the fact that it is harmless algae but close relative to apicomplexan parasites it could be used as a helpful-tool for basic research. Apicoplast serves as a target of some antimalarial agents, so my first task was to isolate pure fraction of plastid organelle. And next to apply antibiotic rifampicin at the same doses that are working in Plasmodium falciparum to see whether it would be possible to use C. velia as a model organism in first screening of new antimarial drugs

Evolution of selected enzymes of the shikimate pathway and the haem biosynthetic pathway in Rhodophyta (class Florideophyceae)

Diatoms derived their plastid from red algae through the secondary endosymbiosis. Most of the endosymbiont genes have been transferred from the engulfed alga to the secondary host nucleus, therefore evolution of these genes correspond to the evolution of plastids rather than to the evolution of the host organisms. Similarly, genes coding for ferrochelatase and DAHP synthase from diatoms are closely related to those from plants and green algae. Contrary to this, red algal genes do not cluster within this clade. I tried to amplify and sequence genes coding for ferrochelatase and DAHP synthase from representatives of the class Florideophyceae to investigate their phylogenetic position

Identification of cadavers and skeleton findings

Abstract/Summary The discovery of a cadaver or skeletal remains (hereinafter the "Remains") raises a number of questions, one of which is the matter of the identity of the remains. The answer to this particular question can be found through forensic identification and utilization of one of the methods thereof. The aim of my thesis is to provide a brief overview of the methods which are currently most commonly used for the purposes of identifying Remains and introduce the reader to their basics, options and limitations. In order to ensure a better understanding of the subject, I have included a section concerning the Remains and the post-mortem changes thereof. In this thesis, I will focus only on those parts of scientific fields, which are relevant for identification of the Remains, and, similarly with respect to information systems, I target only the information being collected and analyzed by such systems in cases of Remains whose identity is not know. In this thesis, I have chosen to proceed from general terms, division of forensic identification, activities being carried out upon the discovery of the Remains, to the individual methods of identification, information system and practical case studies. The thesis is divided into ten chapters. In the first chapter, I address the subject of general terms..

Identification of cadavers and skeleton findings

Abstrakt/Shrnutí Nález mrtvoly či kostrového nálezu (dále jen "ostatky") vyvolá řadu otázek, jednou z nich je i otázka totožnosti ostatků. Právě na tuto otázku nám může dát odpověď kriminalistická identifikace za pomoci některé ze svých metod. Cílem mé diplomové práce je vytvoření stručného přehledu v současné době nejpoužívanějších metod sloužících k identifikaci ostatků a představit čtenáři jejich základy, možnosti a omezení. Pro lepší pochopení tématu jsem zařadila i část týkající se ostatků a jejich změn po smrti. V této práci se zaměřím pouze na ty části vědních oborů, které jsou relevantní pro identifikaci ostatků, stejně tak jako u informačních systémů se zaměřuji pouze na informace, které systémy shromažďují a vyhodnocují v případech týkajících se ostatků neznámé totožnosti. Ve své práci jsem zvolila postup od obecných pojmů, dělení kriminalistické identifikace, činností při nálezu až po jednotlivé metody identifikace, informační systémy a příklady z praxe. Práce je členěna do deseti kapitol. V první kapitole se zabývám základními pojmy souvisejícími se smrtí. V druhé kapitole přiblížím pojem kriminalistická identifikace, její druhy a metody. Do této kapitoly jsem zařadila i statistické údaje od roku 2015. Ve třetí kapitole se zabývám postupem při nálezu ostatků, ohledáním a pitvou. Do kapitol čtyři...Abstract/Summary The discovery of a cadaver or skeletal remains (hereinafter the "Remains") raises a number of questions, one of which is the matter of the identity of the remains. The answer to this particular question can be found through forensic identification and utilization of one of the methods thereof. The aim of my thesis is to provide a brief overview of the methods which are currently most commonly used for the purposes of identifying Remains and introduce the reader to their basics, options and limitations. In order to ensure a better understanding of the subject, I have included a section concerning the Remains and the post-mortem changes thereof. In this thesis, I will focus only on those parts of scientific fields, which are relevant for identification of the Remains, and, similarly with respect to information systems, I target only the information being collected and analyzed by such systems in cases of Remains whose identity is not know. In this thesis, I have chosen to proceed from general terms, division of forensic identification, activities being carried out upon the discovery of the Remains, to the individual methods of identification, information system and practical case studies. The thesis is divided into ten chapters. In the first chapter, I address the subject of general terms...Department of Criminal LawKatedra trestního právaPrávnická fakultaFaculty of La

Screening Cyanobacteria for Apoptosis Induction in Human Cancer Cell Lines: Discovery of a Novel Compound Nocuolin A

Cancer-related diseases are mostly associated with reduced or inappropriate cell death. This thesis focuses on secondary cyanobacterial metabolites which induce apoptosis in human cancer cells in vitro and thus may serve as potential drug hits. Screening and selection of active natural extracts clearly precede activity-guided isolation of a bioactive compound itself. Summarizing the results of phenotypic screening of cyanobacterial extracts for inducers of apoptosis, I show that adjustment of measurement the activity of key apoptotic enzymes, caspases, per cell significantly enlarges the pool of detected hits. This could be of particular importance, since this correction is relevant for complex natural extracts as well as chemical libraries of pure compounds, and moreover applicable all the way from small-sized screens to high-throughput ones. Further, I investigated the apoptosis inducing activity of nocuolin A (NoA) a new cyanobacterial compound isolated and described by our group. NoA shows remarkable characteristics regarding its structure (1,2,3-oxadiazine heterocycle), biosynthetic origin and also its biological activity. It induces caspase-dependent apoptosis and shows potency against a panel of nine human cancer cell lines, which makes NoA a pharmaceutically interesting compound. I also bring the first insights into elucidation of its mode of action in cancer cells in vitro

The cyanobacterial metabolite nocuolin a is a natural oxadiazine that triggers apoptosis in human cancer cells - Fig 3

<p><b>Predicted and experimental FTIR spectra of cyclic nocuolin A form (A) and hypothetical linearized structure (B).</b> In the case of cyclic NoA form the theoretically predicted <i>ab initio</i> spectrum (red line) show exceptionally good overlap with the experimental (black line). Substantially different was the predicted spectrum obtained for hypothetical linear NoA form. The main vibration bands and corresponding structures are highlighted. For details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172850#pone.0172850.s011" target="_blank">S11</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172850#pone.0172850.s012" target="_blank">S12</a> Figs.</p

Chemical structure of nocuolin A.

<p>Panel (A) demonstrates nocuolin A molecule with highlighted spin systems A, B, and C as obtained by NMR—COSY and ¹H-¹³C HSQC-TOSCY. Panel (B) displays the crucial NMR correlations. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172850#pone.0172850.s001" target="_blank">S1</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172850#pone.0172850.s012" target="_blank">S12</a> Figs.High resolution mass spectrometry (HRMS) measurement of NoA provided ions corresponding to a protonated molecule [M+H]⁺, sodium adduct [M+Na]⁺, potassium adduct [M+K]⁺, and sodium adduct of a dimer [2M+Na]⁺ (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172850#pone.0172850.s002" target="_blank">S2 Fig</a>), which allowed us to determine the exact mass of the protonated molecule (299.2233) and to calculate the neutral formula to C₁₆H₃₀N₂O₃ with high accuracy (∆ 1.2 ppm in FTMS). In parallel we have found production of this compound in extracts of two other cyanobacterial strains, <i>Anabaena</i> sp. PCC 7108 (axenic strain) and <i>Nodularia</i> sp. HBU26, via HPLC-HRMS/MS measurements (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172850#pone.0172850.s001" target="_blank">S1B and S1C Fig</a>). The analysis revealed the occurrence of a molecular ion perfectly matching that of NoA in molecular mass, fragmentation pattern and retention behaviour in extracts of both strains (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172850#pone.0172850.s001" target="_blank">S1 Fig</a>). However, due to the extremely low level of NoA production in PCC 7108 and HBU26 we have performed the structure elucidation on the compound purified from strain <i>Nostoc</i> sp. CCAP 1453/38.</p

MS fragments of nocuolin A demonstrating the connection of the A/B and C spin systems obtained by NMR.

<p>We suppose the formation of secondary linear resonance structure with interrupted N-O bond of the oxadiazine ring and formation of additional N-N double bond during the ionization process. For the full MS spectra see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172850#pone.0172850.s002" target="_blank">S2 Fig</a> and for detail fragmentation see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172850#pone.0172850.s010" target="_blank">S10 Fig</a>.</p

Nocuolin A triggers caspase-dependent apoptosis.

<p>(A) NoA-induced cell death morphologically resembles apoptosis (blebbing) as illustrated by the time-lapse microscopy images obtained with HeLa cells exposed to 6.7 μM NoA over 24 hours. (B, C, D) The cell death is caspase-dependent. (B) The protease activity of caspase-3/7 for the DEVD sequence was measured as a luminescence signal at various time points. The relative luminescence units were normalised per cell (nRLU). NoA (a/b/c) at 6.7 μM stands for three independent experiments. (C) The enhanced activity of caspase-3/7 (nRLU) was reduced when the cells treated with NoA and positive controls, were pre-treated with caspase inhibitor Z-VAD-FMK (10 μM). (D) NoA treatment (6.7 μM) resulted in weak enhancement of activated caspase-3 fragments (19/17 kDa) and a consistent increase in PARP-1 cleavage. These apoptosis markers were completely absent when the caspase inhibitor Q-VD-OPh (10 μM) was added prior to NoA (lines 5,7,9,11). As a positive control, taxol (B) and staurosporine (STS) (C, D) were used both at concentration 1 μM and for indicated time.</p

NMR data for nocuolin A.

<p>The ¹H, ¹³C, and ¹⁵N NMR data are presented (600.23 MHz for ¹H, 150.93 MHz for ¹³C, 60.82 ppm for ¹⁵N, CD₃CN, 303.2 K). ¹⁵N NMR of ¹⁵N labelled sample (70.93 MHz, CD₃CN, 293.2 K) <i>δ</i> 222, 302. Asterisk (*) denotes HSQC readouts.</p