The F1 -ATPase from Trypanosoma brucei is elaborated by three copies of an additional p18-subunit.

The F-ATPases (also called the F1 Fo -ATPases or ATP synthases) are multi-subunit membrane-bound molecular machines that produce ATP in bacteria and in eukaryotic mitochondria and chloroplasts. The structures and enzymic mechanisms of their F1 -catalytic domains are highly conserved in all species investigated hitherto. However, there is evidence that the F-ATPases from the group of protozoa known as Euglenozoa have novel features. Therefore, we have isolated pure and active F1 -ATPase from the euglenozoan parasite, Trypanosoma brucei, and characterized it. All of the usual eukaryotic subunits (α, β, γ, δ, and ε) were present in the enzyme, and, in addition, two unique features were detected. First, each of the three α-subunits in the F1 -domain has been cleaved by proteolysis in vivo at two sites eight residues apart, producing two assembled fragments. Second, the T. brucei F1 -ATPase has an additional subunit, called p18, present in three copies per complex. Suppression of expression of p18 affected in vitro growth of both the insect and infectious mammalian forms of T. brucei. It also reduced the levels of monomeric and multimeric F-ATPase complexes and diminished the in vivo hydrolytic activity of the enzyme significantly. These observations imply that p18 plays a role in the assembly of the F1 domain. These unique features of the F1 -ATPase extend the list of special characteristics of the F-ATPase from T. brucei, and also, demonstrate that the architecture of the F1 -ATPase complex is not strictly conserved in eukaryotes

Application of Neural Networks for Capital Market Trading

The developments in the area of machine learning, data processing and the application of self-learning algorithms couldn´t been ignored by people interested in trading on stock market. Artificial neural network, as one of the areas of artificial intelligence, is a very powerful computing tool, which can, based on the right settings and a sufficient amount of input data, reach above-average results. The purpose of this work is to describe on what theoretical basis are neural networks constructed and practically verify their contribution in the field of stock market price prediction.Vývoj v oblasti strojového zpracování dat a aplikaci samoučících se algoritmů nemohl zůstat bez povšimnutí ani v oblasti obchodování na akciových trzích. Umělé neuronové sítě, které jsou jednou z oblastí umělé inteligence, jsou velmi mocným výpočetním nástrojem, který při vhodném nastavení a s dostatečným množstvím vstupních dat, může podávat nadprůměrné výsledky. Smyslem této práce je popsat na jakém teoretickém základu jsou neuronové sítě konstruovány a dále prakticky ověřit jejich přínos v oblasti predikce cen na akciových trzích

FoF1-ATP synthase/ATPase in the parasitic protist, \kur{Trypanosoma brucei}

This thesis primarily focuses on the FoF1-ATP synthase/ATPase complex in the parasitic protist, Trypanosoma brucei. Instead of its normal aerobic function to synthesize ATP, it is required to hydrolyze ATP to maintain the m in the infective bloodstream stage of T. brucei and the related parasite, T. b. evansi. To better understand the composition, structure and function of this druggable target, my work focused on deciphering the function of three of the unique Euglenozoa specific subunits that comprise this complex molecular machine. Furthermore, the ADP/ATP carrier, which provides substrates for the FoF1-ATP synthase/ATPase, was functionally characterized and evaluated if it is physically associated with the complexes of the oxidative phosphorylation pathway

How the Payment Model Influences the Waste Number and Structure

The purpose of this work is to evaluate a potential relation between the number of communal and separated waste produced by municipalities in the Czech Republic and related pricing method. I am trying to demonstrate that municipalities using fixed charges, which is still the dominant pricing method in the Czech Republic, produce more communal and less separated waste than municipalities using the variable charges. The survey consists data from 22 municipalities in 2015. There are three hypothesis for verification. The first one is based on fact that if municipality use fix charging than the number of communal waste would be higher than if they use variable charging. The second one suggest if municipality use variable charging, the habitants will be more motivated to separate waste and the total number of separated waste would be bigger. The last one is comparing the total number of separated waste with total waste produced

ATPaseTb2, a unique membrane-bound FoF1-ATPase component, is essential in bloodstream and dyskinetoplastic trypanosomes.

In the infectious stage of Trypanosoma brucei, an important parasite of humans and livestock, the mitochondrial (mt) membrane potential (Δψm) is uniquely maintained by the ATP hydrolytic activity and subsequent proton pumping of the essential FoF1-ATPase. Intriguingly, this multiprotein complex contains several trypanosome-specific subunits of unknown function. Here, we demonstrate that one of the largest novel subunits, ATPaseTb2, is membrane-bound and localizes with monomeric and multimeric assemblies of the FoF1-ATPase. Moreover, RNAi silencing of ATPaseTb2 quickly leads to a significant decrease of the Δψm that manifests as a decreased growth phenotype, indicating that the FoF1-ATPase is impaired. To further explore the function of this protein, we employed a trypanosoma strain that lacks mtDNA (dyskinetoplastic, Dk) and thus subunit a, an essential component of the proton pore in the membrane Fo-moiety. These Dk cells generate the Δψm by combining the hydrolytic activity of the matrix-facing F1-ATPase and the electrogenic exchange of ATP4- for ADP3- by the ATP/ADP carrier (AAC). Surprisingly, in addition to the expected presence of F1-ATPase, the monomeric and multimeric FoF1-ATPase complexes were identified. In fact, the immunoprecipitation of a F1-ATPase subunit demonstrated that ATPaseTb2 was a component of these complexes. Furthermore, RNAi studies established that the membrane-bound ATPaseTb2 subunit is essential for maintaining normal growth and the Δψm of Dk cells. Thus, even in the absence of subunit a, a portion of the FoF1-ATPase is assembled in Dk cells

ATPaseTb2 is a bona fide subunit of the monomeric and multimeric F_oF₁-ATPases.

<p>A) ATPaseTb2_TAP and p18_TAP tagged complexes were purified from BF cells using one-step IgG affinity chromatography from non-induced (NON) and 2 days induced (IND) cells containing the regulatable ectopic TAP tagged protein. The tagged protein complexes were eluated by TEV protease, fractionated on SDS-PAGE and examined by western blot analyzes. The presence of the tagged ATPaseTb2 and p18 subunits was verified using a <i>c</i>-myc antibody (top panels: ATPaseTb2_TAP and p18_TAP). The known F_oF₁-ATPase subunits (sub β, p18 and ATPase_Tb1) were detected using specific antibodies. The lack of signal for AAC serves as a control for specificity of the used method. The applicable sizes of the protein marker are indicated on the left. B) p18_TAP tagged complexes were purified from dyskinetoplastic <i>T</i>. <i>b</i>. <i>evansi</i> as described above for BF cells and subjected to the same set of antibodies. C) The sedimentation profile of F₁- and F_oF₁-ATPase complexes was determined using glycerol gradient sedimentation. Hypotonically purified mitochondria from PF427, BF427, Dk164 and <i>T</i>. <i>b</i>. <i>evansi</i> cells were lysed with 1% Triton X-100 and fractionated on a 10–30% glycerol gradient. The glycerol gradient fractions were collected, fractionated by SDS-PAGE and analyzed by western blots. Western analyzes with an anti-β antibody depicted the sedimentation profile of the F₁-ATPase and monomeric/multimeric F_oF₁-ATP synthase complexes, whereas the anti- ATPaseTb2 antibody only immunodecorates this protein within the monomeric/multimeric F_oF₁-ATPase complexes. The subdivision of the various structural forms of the F_oF₁-ATPase complexes are underlined as determined in [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004660#ppat.1004660.ref026" target="_blank">26</a>]. The glycerol gradient fractions and the sizes of the protein marker are indicated. Nonspecific bands visible in Dk164 and <i>T</i>. <i>b</i>. <i>evansi</i> gradients are indicated by asterisks. D) The native F₁- and F_oF₁-ATPase complexes were visualized using hrCNE. Purified mitochondria from PF427, BF427, Dk164 and <i>T</i>.<i>b</i>.<i>evansi</i> cells were lysed with digitonin (4 mg/mg), fractionated on a 3%-12% hrCNE and blotted onto a nitrocellulose membrane. The F₁-ATPase (F₁), the F₁-ATPase bound with the c-ring (F_1+C) and the monomeric F_oF₁/multimeric (F_oF₁)_n complexes were all visualized using specific polyclonal antibodies against either subunit β or ATPaseTb2. The size of ferritin from the equine spleen (440 kDa) is indicated on the left.</p

ATPaseTb2 depletion does not appreciably affect F₁-ATPase activity in BF <i>T</i>.

<p><b><i>brucei</i> cells, but it does significantly diminish the stability of F</b>_<b>o</b><b>F</b>_<b>1</b><b>-ATPase complexes</b>. A) Employing the Sumner ATPase assay, the F₁-ATPase hydrolytic activity was measured in ATPaseTb2 RNAi cells either not induced (NON) or induced for 2 days (IND2). Crude mt vesicles were obtained by digitonin extraction and the ATPase activity was assayed by measuring the release of free phosphates. The specific F₁-ATPase inhibitor, azide (AZ, 2 mM), was added as indicated. The total amount of free-phosphate created from all ATPase enzymes present in the sample was set at 100% (hatched column). The azide-sensitive activity representing the F₁-ATPase is depicted in dark grey. The results are means ± s.d. (n = 4). B) In-gel ATP hydrolysis activity of F_oF₁-ATPase was visualized after ATPaseTb2 reduction. Mitochondria from RNAi non-induced cells (NON) and cells induced for 2 (IND2) and 3 (IND3) days were lysed with 2% dodecyl maltoside. Equal amounts of lysed mitochondrial proteins (100 μg) were fractionated on a 2%-12% BNE and the F_oF₁-ATPase activity was visualized by in-gel histochemical staining resulting in a white lead phosphate precipitate. Positions of F₁-ATPase and monomeric F_oF₁-ATPase are depicted. The size of equine spleen ferritin (440 kDa) is indicated. C) The stability of F_oF₁-ATPase complexes upon ATPaseTb2 silencing was examined using hrCNE. Mitochondria from RNAi non-induced cells (NON) and cells induced for 2 (IND2) and 3 (IND3) days were lysed by digitonin (4 mg/mg). Equal amounts of lysed mitochondrial proteins (20 μg) were fractionated on a 3%-12% hrCNE, blotted onto a nitrocellulose membrane and probed with the anti-p18 antibody. Positions of F₁-ATPase and monomeric and dimeric F_oF₁-ATPases are depicted by arrows. The size of ferritin from equine spleen (440 kDa) is indicated. D) The sedimentation profile of F_oF₁-ATPase complexes was examined using western blot analysis of glycerol gradient fractions. Mitochondria from RNAi non-induced cells (NON) and cells induced for 2 days (IND2) were lysed with 1% Triton X-100. An equal amount of the cleared lysates (3,3 mg) were loaded on a manually poured 10–30% glycerol gradient. Western analyzes with anti-β and anti-p18 antibodies depicted the sedimentation profile of the F_oF₁-ATPase complexes. The manually fractionated glycerol gradient fractions are labelled and sizes of the protein marker are indicated.</p

Cell growth, Δψ_m maintenance and the stability of FoF1-ATPase complexes are all affected by the loss of ATPaseTb2 in dyskinetoplastic <b><i>T.b. evansi</i></b>.

<p>A) Growth curves of the non-induced (NON) and induced (IND) ATPaseTb2 RNAi Dk <i>T</i>. <i>b</i>. <i>evansi</i> cell lines were measured for 10 days. Cells were maintained in the exponential growth phase (between 10⁵ and 10⁶ cells/ml) and the cumulative cell number represents the cell density adjusted by the daily dilution factor. The figure is representative of three independent RNAi-inductions. B) The steady-state abundance of ATPaseTb2 in non-induced (NON) cells and cells harvested 1, 2, 3 and 4 days post RNAi induction (IND1, IND2, IND3, IND4) was determined by western blot analysis using a specific ATPaseTb2 antibody. Mt Hsp70 served as a loading control. The numbers underneath the ATPaseTb2 panel represent the abundance of immunodetected protein expressed as a percentage of the non-induced samples after normalizing to the loading control. The pertinent sizes of the protein marker are indicated on the left. The figure is a representative western blot from three independent RNAi-inductions. C) The Δψ_m was measured in non-induced (NON) ATPaseTb2 RNAi cells and cells induced for 2 and 3 days (IND2 and IND3) by flow cytometry using Mitotracker Red CMX-Ros. The results are means ± s.d. (n = 3). *P< 0.05, Student’s <i>t</i>-test. D) The F₁-ATPase hydrolytic activity was measured for induced (IND3) or non-induced Dk ATPaseTb2 RNAi cells as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004660#ppat.1004660.g005" target="_blank">Fig. 5A</a>. The results are means ± s.d. (n = 4). E) The stability of F_oF₁-ATPase complexes upon ATPaseTb2 depletion was examined using hrCNE. Mitochondria from wild type BF427, ATPaseTb2 RNAi <i>T</i>. <i>b</i>. <i>evansi</i> non-induced cells (NON) and cells induced for 3 days (IND3) were lysed by digitonin (4 mg/mg). Equal amounts of lysed mitochondrial proteins (20 μg) were fractionated on a 3%-12% hrCNE, blotted onto a nitrocellulose membrane and probed with an anti-β antibody. Positions of F₁-ATPase and monomeric and multimeric F_oF₁-ATPases are depicted by arrows. The size of the equine spleen ferritin (440 kDa) is indicated on the left. F) The sedimentation profile of F_oF₁-ATPase complexes was examined using western blot analysis of glycerol gradient fractions. Mitochondria from RNAi non-induced cells (NON) and cells induced for 3 and 4 days (IND3 and IND4) were lysed by 1% Triton X-100 and an equal amount of the cleared samples (1.3 mg) were loaded on a 10–30% glycerol gradient. Western analyzes with anti-β and anti-p18 antibodies depicted the sedimentation profile of the F_oF₁-ATPase complexes. The glycerol gradient fractions are labelled and the relevant sizes of the protein marker are indicated. A nonspecific band only visible in Dk164 gradients is indicated by an asterisk.</p