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Fragestellung: Bei der zementfreien Verankerung von Implantaten kommt es zu einer direkten Interaktion mit dem knöchernen Implantatlager und damit zur Beeinflussung des lokalen Knochenumbaus (Regeneration, Resorption). Für eine Einheilung mit frühzeitiger Wiederbelastung ist die rasche [zum vollständigen Text gelangen Sie über die oben angegebene URL]

In dieser Dissertation haben wir das Verhalten menschlicher Zellen in Raum und Zeit untersucht. Hochwertige Datensätze subzellulärer Regionen in HEK293-Zellen wurden mit Hilfe der BirA* Proximity-Labelling-Aktivität erstellt, wobei die Lokalisierung auf zelluläre Regionen beschränkt wurde, die mit herkömmlichen Methoden nur schwer zu reinigen sind (d. h. die dem Zytosol zugewandten Seiten des ER, Mitochondrien und Plasma-membranen). Wir entwickelten daraufhin einen Ansatz zur Kartierung der Verteilung von Proteinen, die aktiv an RNA binden, und nannten ihn f-XRNAX. Wir stellten hintergrundkorrigierte Proteome für Zellkerne, Zytoplasma und Membranen von HEK293-Zellen her. Überraschenderweise wurden viele nicht-kanonische RBPs in der Membranfraktion identifiziert, und ihre Peptidprofile waren in Regionen mit hoher Dichte an intrinsisch ungeordneten Regionen angereichert, was auf eine möglicherweise schwache, durch diese nicht-strukturellen Motive vermittelte Interaktion mit RNA hinweist. Schließlich konnten wir die unterschiedliche Bindung desselben Proteins an RNA in verschiedenen HEK293-Kompartimenten nachweisen. Im zweiten Teil dieser Arbeit konzentrierten wir uns auf die Bestimmung und Quantifizierung von neu transkribierten RNAs auf Einzelzellebene. Die Kinetik der RNA-Transkription und -Degradation war bis vor kurzem auf Einzelzellebene nicht messbar. Daher haben wir einen neuen Ansatz (SLAM-Drop-seq genannt) entwickelt, indem wir die veröffentlichte SLAM-seq-Methode an Einzelzellen angepasst haben. Wir haben SLAM-Drop-seq verwendet, um die zeitabhängigen RNA-Kinetikraten der Transkription und des Umsatzes für Hunderte von oszillierenden Transkripten während des Zellzyklus von HEK293-Zellen zu schätzen. Wir fanden heraus, dass Gene ihre Expression mit unterschiedlichen Strategien regulieren und spezifische Modi zur Feinabstimmung ihrer kinetischen Raten entlang des Zellzyklus haben., In this PhD dissertation we investigated the behaviour of human cells through space and time. High quality datasets of subcellular regions in HEK293 cells were generated using BirA* proximity labelling activity and restricting its localization at cellular regions difficult to purified with traditional methods (i.e., the cytosol-facing sides of the endoplasmic reticulum, mitochondria, and plasma membranes). We then developed an approach to map the distribution of proteins actively binding to RNA, and named it f-XRNAX. We recovered background-corrected proteomes for nuclei, cytoplasm and membranes of HEK293 cells. Surprisingly, many non-canonical RBPs were identified in the membrane fraction, and their peptide profiles were enriched in regions with high density of intrinsically disordered regions, indicating a possibly weak interaction with RNA mediated by these non-structural motives. Lastly, we provided evidence of the differential binding to RNA of the same protein in different HEK293 compartments. In the second part of this thesis, we focused on the determination and quantification of newly transcribed RNAs at the single-cell level. The kinetics of RNA transcription, processing and degradation were until recently not measurable at the single-cell level. Thus, we have developed a novel approach (called SLAM-Drop-seq ) by adapting the published SLAM-seq method to single cells. We used SLAM Drop-seq to estimate time-dependent RNA kinetics rates of transcription and turnover for hundreds of oscillating transcripts during the cell cycle of HEK293 cells. We found that genes regulate their expression with different strategies and have specific modes to fine-tune their kinetic rates along the cell cycle.

Streptomiceti su višestanične bakterije složenog životnog ciklusa. Najpoznatije su po genetičkom potencijalu za sintezu antibiotika širokog spektra u koje spadaju i tetraciklini od izuzetnog komercijalnog značaja. Već desetljećima se bakterija Streptomyces rimosus koristi za proizvodnju oksitetraciklina (OTC) čija godišnja potrošnja prelazi 108 kg. Biosinteza antibiotika je kontrolirana kompleksnim signalnim putevima koji uključuju i fosforilacije proteina. Kako bi rasvijetlili regulatorne mehanizme uključene u procese odgovorne za rast i sintezu OTC-a u ovoj doktorskoj radnji je primjenom spektrometrije masa po prvi put istražen (fosfo)proteom bakterije S. rimosus G7 i visokog proizvođača OTC-a (soj 23383). Određena je dinamika sinteze (fosfo)proteina tijekom rasta ovih bakterija, a ostvareni rezultati su korelirani s procesima uključenim u biosintezu antibiotika i staničnu diferencijaciju. Identificirano je 4053 proteina (~50% proteoma) te 433 fosfoproteina od kojih je 203 fosfoproteina pronađeno samo kod soja 23383. Funkcionalna karakterizacija i analiza dinamike fosforilacije ovih proteina dali su posve novi uvid u fosfoproteom streptomiceta općenito. Identificirana je do sada neopisana posttranslacijska modifikacija niza proteina koji su važni za staničnu diobu i metabolizam te sintezu antibiotika i odgovor na stres. Streptomycetes are multicellular bacteria with a complex life cycle. They are best known for their great potential for the synthesis of broad-spectrum antibiotics, which include tetracyclines of significant commercial importance. For decades, Streptomyces rimosus has been used to produce oxytetracycline (OTC) whose annual consumption exceeds 108 kg. Antibiotic biosynthesis is controlled by complex signaling pathways that include protein phosphorylation. In order to elucidate the regulatory mechanisms involved in the processes responsible for bacterial growth and OTC synthesis, the (phospho)proteome of S. rimosus G7 and a high OTC producer (strain 23383) was investigated for the first time using mass spectrometry in this doctoral dissertation. The dynamics of (phospho)proteomes during the growth of these bacteria was determined and results were correlated with the processes involved in antibiotic biosynthesis and cell differentiation. In total, 4053 proteins (~ 50% of proteomes) and 433 phosphoproteins were identified, of which 203 phosphoproteins were found only in strain 23383. Functional characterization and analysis of phosphorylation dynamics of these proteins gave a completely new insight into streptomycete phosphoproteins in general. So far undescribed posttranslational modification of a number of proteins important for cell division and metabolism, as well as antibiotic synthesis and stress response, has been identified.

Links between ontogeny and phylogeny in animals have been discussed for more than two centuries. With the uprising of molecular biology and bioinformatics, several studies have revealed the presence of the phylogeny-ontogeny correlation on molecular level in developmental transcriptomes of eukaryotic clades with complex multicellularity. These findings open a possibility to test the phylogeny-ontogeny correlation in more basal organisms, with more obscure development and multicellularity characteristics. Using time-resolved transcriptome and proteome profiles, this study showed that Bacillus subtilis biofilm ontogeny correlates with the evolutionary measures through recapitulation pattern, in a way that evolutionary younger and more diverged genes were increasingly expressed towards the later timepoints of the biofilm growth. Molecular and morphological signatures also revealed that biofilm growth is highly regulated and organized into discrete ontogenetic stages. Together, this suggests that the biofilm formation in Bacillus subtilis is a true developmental process comparable to organismal development in animals, plants and fungi Korelacija između filogenije i ontogenije životinja je predmet rasprave među znanstvenicima od početka 19. stoljeća. Razvojem molekularne biologije i bioinformatike te analizama transkriptoma razvojnih stadija, uočena je prisutnost korelacije filogenije i ontogenije na molekularnoj razini u eukariotskim evolucijskim granama sa složenom višestaničnosti. Ova saznanja potaknula su nas da testiramo korelaciju filogenije i ontogenije u bazalnim organizmima s nejasnim razvojnim i višestaničnim karakteristikama. Analizom transkriptomskih i proteomskih podataka, pokazali smo da ontogenija Bacillus subtilis biofilmova korelira s evolucijskim mjerama u obliku rekapitulacijskog profila, odnosno evolucijski mlađi i divergentniji geni su sve više eksprimirani prema kasnijim stadijima rasta biofilma. Dodatne molekularne analize pokazale su da je rast biofilma reguliran i organiziran u jasno odvojene stadije ontogenije. Zaključno, rezultati pokazuju da je rast Bacillus subtilis biofilmova istinski razvojni proces usporediv s razvojem životinja, biljaka i gljiva.

Es gibt drei Krankheitsbilder, die durch Verzehr von Getreide getriggert werden. Die Zöliakie und die Weizenallergie und die Nicht-Zöliakie Weizensensitivität (NCWS). Während die zugrundeliegenden Auslöser und Mechanismen der ersten beiden Entitäten bereits eingehend untersucht wurden, besteht diesbezüglich bei der NCWS noch Unklarheit. Die Symptome sind sehr unspezifische und es fehlen diagnostische Marker. Als Auslöser stehen neben bakteriell fermentierbaren Kohlenhydraten (FODMAPs) ausgewählte Getreideproteine wie Gluten oder α-Amylase-Trypsin-Inhibitoren (ATIs) im Fokus der Forschung. Ziel der vorliegenden Arbeit war es zum einen, den Einfluss der Wahl des Getreides (Weichweizen, Dinkel, Roggen) und der Herstellung von Brot (Vermahlungsgrad und Wahl zwischen Hefe- und Sauerteig) auf das Vorliegen potenziell immunogener Proteine auf Basis einer Proteomanalyse zu untersuchen. In einem zweiten Schritt sollte die Verträglichkeit ausgewählter Brote in einem transgenen Mausmodell mit intestinalen Entzündungen und in einer Humanstudie an Patienten mit NCWS und subjektiver Dinkeltoleranz auf ihre Verträglichkeit untersucht werden. Dadurch sollten mögliche Auslöser der NCWS eingegrenzt und zugrundeliegende Mechanismen untersucht werden. Im Rahmen des Projektes wurde auf Basis einer quantitativen Proteomics Methode (nano-UHPLC-ESI-MS basiert) die Proteinzusammensetzung von Brot- und Mehlproben analysiert. Zudem wurde auf Basis der Pfam Annotierung eine Liste mit bekannten und potenziell immunogenen Getreideproteinen erstellt, die für die Analyse von Allergenen in Mehl und Brot herangezogen wurde. Dabei zeigte sich, dass weder die absolute Anzahl noch die Abundanz dieser allergenen Proteine abhängig vom Mahlgrad des Mehls oder dem Fermentationsprozess des Teiges waren, was bedeutet, dass sie bei der Brotherstellung nicht selektiv abgebaut werden. Jedoch konnten in Dinkel- und Weizenproben im Vergleich zu Roggenproben solche Proteine in höherer Anzahl und höherer relativer Menge identifiziert werden. Weiter wurden verschiedene Brotsorten aus der Proteomanalyse an einem Mausmodell mit intestinalen Entzündungen untersucht. Dabei konnte keine bessere Verträglichkeit von Roggenbrot im Vergleich zu Dinkel- und Weizenbrot nachgewiesen werden. Stattdessen zeigte sich ein Trend, dass Sauerteigbrot die Darmentzündungen weniger stark negativ beeinflusste, als Hefeteigbrot. Auch stellte sich heraus, dass die Entzündungen unabhängig von Gluten verstärkt wurden. Weder in der Proteomanalyse noch in den Tierversuchen dieses Projektes konnten Unterschiede zwischen Weizen und Dinkel festgestellt werden. In einer Subgruppe von NCWS Patienten wird Dinkelbrot jedoch subjektiv besser vertragen als Weizenbrot, was sowohl auf die Genetik als auch auf die unterschiedliche Herstellung von Weizen- und Dinkelbrot zurückzuführen sein könnte. Um das Phänomen zu verifizieren und ggf. zugrundeliegende Mechanismen zu identifizieren, wurde eine klinische Studie an Patienten dieser Subgruppe durchgehführt. Ziel der verblindeten Studie war es zu untersuchen, ob Dinkelbrot tatsächlich besser vertragen wird als Weizenbrot und ob die Herstellung (16h Teigführung oder 4h Teigführung +Backmittel) einen Einfluss. Nach jedem Brot (jeweils 4 Tage + 3 Tage Washout) wurden die gastrointestinalen Symptome mit dem Irritable Bowel Syndrome Severity Scoring System (IBS-SSS) Fragebogen bewertet. Außerdem wurden extraintestinale Symptome und verschiedene Blut- und Stuhlparameter analysiert. Es zeigte sich, dass Dinkelbrot im Vergleich zu Weizenbrot nach verblindetem Verzehr nicht besser vertagen wurde und dass FODMAP-reiches Brot im Vergleich zu FODMAP-armem Brot nicht schlechter vertragen wird. There are three medical conditions that are triggered by consumption of cereals. Celiac disease, wheat allergy and non-celiac wheat sensitivity (NCWS). While the underlying triggers and mechanisms of the first two entities have been extensively studied, there is still uncertainty in this regard for NCWS. Symptoms are nonspecific and diagnostic markers are lacking. Besides bacterial fermentable carbohydrates (FODMAPs), selected cereal proteins such as gluten or α-amylase trypsin inhibitors (ATIs) are in the focus of research as triggers. The aim of the present work was firstly to investigate the influence of the choice of cereal (common wheat, spelt, rye) and the production of bread (degree of milling and choice between yeast and sourdough) on the presence of potentially immunogenic proteins based on proteomic analysis. In a second step, the tolerability of selected breads should be investigated in a transgenic mouse model with intestinal inflammation and in a human study in patients with NCWS and subjective spelt tolerance. This was to narrow down possible triggers of NCWS and to investigate underlying mechanisms. Within the project, protein composition of bread and flour samples was analyzed based on a quantitative proteomics method (nano-UHPLC-ESI-MS based). In addition, a list of known and potentially immunogenic cereal proteins was generated based on Pfam annotation, which was used for the analysis of allergens in flour and bread. This showed that neither the absolute number nor the abundance of these allergenic proteins were dependent on the degree of milling of the flour or the fermentation process of the dough, which means that they are not selectively degraded during bread production. However, such proteins were identified in higher numbers and higher relative amounts in spelt and wheat samples compared to rye samples. Furthermore, different bread types from the proteome analysis were investigated in a mouse model with intestinal inflammation. This did not demonstrate better tolerability of rye bread compared to spelt and wheat bread. Instead, there was a trend for sourdough bread to have less negative effects on intestinal inflammation compared to yeast dough bread. It also turned out that inflammation was increased independently of gluten. No differences were found between wheat and spelt in either the proteomic analysis or the animal studies in this project. However, in a subgroup of NCWS patients, spelt bread is subjectively better tolerated than wheat bread, which could be due to both genetics and the different production of wheat and spelt bread. In order to verify the phenomenon and identify underlying mechanisms, if any, a clinical study was conducted in patients of this subgroup. The aim of the blinded study was to investigate whether spelt bread is actually better tolerated than wheat bread and whether the production process (16h dough or 1h dough + baking agent) has an influence. After each bread (4 days each + 3 days washout), gastrointestinal symptoms were assessed using the Irritable Bowel Syndrome Severity Scoring System (IBS-SSS) questionnaire. Extraintestinal symptoms and various blood and stool parameters were also analyzed. It was found that spelt bread was not better tolerated compared to wheat bread after blinded consumption and that FODMAP-rich bread was not worse tolerated compared to FODMAP-poor bread.

(English version) Oxygenic photosynthesis is crucial for most forms of the life on the Earth. The splitting of water and evolution of oxygen is conducted by photosystem II (PSII), a multi-subunit pigment- protein complex embedded in the thylakoid membrane. PsbO is an indispensable subunit of PSII, bound to its transmembrane subunits from the luminal side. The main function of PsbO is to stabilise and protect Mn4CaO5 cluster where the water splitting occurs. However, it has probably also some auxiliary functions. These additional functions might be different for isoforms of PsbO proteins, as suggested for Arabidopsis thaliana, which expresses two genes encoding protein isoforms PsbO1 and PsbO2. This thesis studies auxiliary functions of PsbO with a focus on functional differences between PsbO isoforms. We found that besides Arabidopsis thaliana, also many other plant species express two psbO genes. Interestingly, the duplication of psbO gene occurred many times independently, generally at the roots of modern angiosperm families. In spite of this, the PsbO isoforms differ at similar sites in the protein structure, suggesting that similar subfunctionalisation of PsbO isoforms occurred parallelly in various lineages. Biochemical characterisation of PsbO from green alga Chlamydomonas reinhardtii and...

Kvasci su pripadnici carstva gljiva (Fungi) kao i plijesni i mesnate gljive. Eukariotski su organizmi koji se često nazivaju jednostaničnim gljivama, a razmnožavaju se pupanjem. Saccharomyces cerevisiae neinfektivni je mikrob čiji je eukariotski genom bio prvi sekvencioniran. Svojim fenotipskim svojstvima poput učinkovitosti parenja ili sporulacije, doprinio je napretku u istraživanjima. Često korišteni modelni organizam upravo je soj Saccharomyces cerevisiae W303 koji je usko povezan s prvim potpuno sekvencioniranim sojem kvasca S288C te se stoga naziva njegovim potomkom. Mitohondriji, kao organeli većine eukariotskih organizama razlikuju se između različitih vrsta eukariota, po veličini, ali i sadržaju genoma. Cilj ovog rada je analizirati proteom mitohondrija kvasca Saccharomyces cerevisae W303. Stanice kvasca uzgajane su na kvaščevoj podlozi do ulaska u ranu stacionarnu fazu. Iz takvih su stanica izolirani mitohondriji, a iz njih proteini. Ekstrahirani proteini mitohondrija su nakon pripreme analizirani LC/MS metodom. Nakon identifikacije, dobiveni su proteini sistematizirani i utvrđena im je uloga i važnost u funkcioniranju stanice kvasca., Yeasts are members of the fungi kingdom (Fungi), as well as molds and fleshy mushrooms. They are eukaryotic organisms often called unicellular fungi, and they reproduce by budding. Saccharomyces cerevisiae is a non-infectious microbe whose eukaryotic genome was the first to be sequenced. With their phenotypic properties such as mating efficiency or sporulation, they have contributed to scientific research progress. A frequently used model organism is the strain Saccharomyces cerevisiae W303, which is closely related to the first fully sequenced yeast strain S288C and is therefore called its descendant. Mitochondria, as organelles of most eukaryotic organisms, differ between different types of eukaryotes in terms of size and genome content. The aim of this work was to analyze the mitochondrial proteome of the yeast Saccharomyces cerevisae W303. Yeast cells were grown on yeast medium until they reached the early stationary phase. Mitochondria and proteins were isolated from such cells. After preparation, extracted mitochondrial proteins were analyzed by the LC/MS method. After identification, the obtained proteins were systematized and their role and importance in the functioning of the yeast cell was determined.

Cellular proteome profiling revealed that most biomolecules do not exist in isolation, but rather are incorporated into modular complexes. These assembled complexes are usually very large, consisting of 10 subunits on an average and include either proteins alone, or proteins and nucleic acids. Consequently, such macromolecular assemblies rather than individual biopolymers perform the vast majority of cellular activities. The faithful assembly of such molecular assemblies is often aided by trans-acting factors in vivo, to preclude aggregation of complex components and/or non-cognate interactions. A paradigm for an assisted assembly of a macromolecular machine is the formation of the common Sm/LSm core of spliceosomal and histone-mRNA processing U snRNPs. The key assembly factors united in the Protein Arginine Methyltransferase 5 (PRMT5) and the Survival Motor Neuron (SMN) complexes orchestrate the assembly of the Sm/LSm core on the U snRNAs. Assembly is initiated by the PRMT5-complex subunit pICln, which pre-arranges the Sm/LSm proteins into spatial positions occupied in the mature U snRNPs. The SMN complex subsequently binds these Sm/LSm units, displaces pICln and catalyses the Sm ring closure on the Sm-site of the U snRNA. The SMN complex consists of the eponoymous SMN protein linked in a modular network of interactions with eight other proteins, termed Gemins 2-8 and Unrip. Despite functional and structural characterisation of individual protein components and/or sub-complexes of this assembly machinery, coherent understanding of the structural framework of the core SMN complex remained elusive. The current work, employing a combined approach of biochemical and structural studies, aimed to contribute to the understanding of how distinct modules within the SMN complex coalecse to form the macromolecular SMN complex. A novel atomic resolution (1.5 Å) structure of the human Gemin8:7:6 sub-complex, illustrates how the peripheral Gemin7:6 module is tethered to the SMN complex via Gemin8’s C-terminus. In this model, Gemin7 engages with both Gemin6 and Gemin8 via the N- and C-termini of its Sm-fold like domain. This highly conserved interaction mode is reflected in the pronounced sequence conservation and identical biochemical behaviour of similar sub-complexes from divergent species, namely S. pombe and C. elegans. Despite lacking significant sequence similarity to the Sm proteins, the dimeric Gemin7:6 complex share structural resemblance to the Sm heteromers. The hypothesis that the dimeric Gemin7:6 functions as a Sm-surrogate during Sm core assembly could not be confirmed in this work. The functional relevance of the structural mimicry of the dimeric Gemin7:6 sub-complex with the Sm heterodimers therefore still remains unclear. Reduced levels of functional SMN protein is the cause of the devastating neurodegenerative disease, Spinal Muscular Atrophy (SMA). The C-terminal YG-zipper motif of SMN is a major hot-spot for most SMA patient mutations. In this work, adding to the existing inventory of the human and fission yeast YG-box models, a novel 2.2 Å crystal structure of the nematode SMN’s YG-box domain adopting the glycine zipper motif has been reported. Furthermore, it could be assessed that SMA patient mutations mapping to this YG-box domain greatly influences SMN’s self-association competency, a property reflected in both the human and nematode YG-box biochemical handles. The shared molecular architecture and biochemical behaviour of the nematode SMN YG-box domain with its human and fission yeast counterparts, reiterates the pronounced conservation of this oligomerisation motif across divergent organisms. Apart from serving as a multimerization domain, SMN’s YG-box also acts as interaction platform for Gemin8. A systematic investigation of SMA causing missense mutations uncovered that Gemin8’s incorporation into the SMN complex is influenced by the presence of certain SMA patient mutations, albeit independent of SMN’s oligomerisation status. Consequently, loss of Gemin8 association in the presence of SMA patient mutations would also affect the incorporation of Gemin7:6 sub-complex. Gemin8, therefore sculpts the heteromeric SMN complex by bridging the Gemin7:6 and SMN:Gemin2 sub-units, a modular feature shared in both the human and nematode SMN complexes. These findings provide an important foundation and a prospective structural framework for elucidating the core architecture of the SMN complex in the ongoing Cryo-EM studies., Systematische Untersuchungen von zellulären Bestandteilen haben gezeigt, dass viele Proteine nicht isoliert, sondern vielmehr in modularen Komplexen organisiert vorliegen. Mit durchschnittlich zehn Untereinheiten sind diese Komplexe sehr groß, wobei sie entweder ausschließlich aus Proteinen oder aber aus Proteinen und Nukleinsäuren bestehen können. Daher wird der Großteil zellulärer Aktivitäten nicht von einzelnen Biopolymeren, sondern von makromolekularen Komplexen verrichtet. Die Zusammenlagerung dieser Komplexe wird in vivo häufig von Hilfsfaktoren unterstützt, um die Aggregation der Einzelkomponenten und/oder unspezifische Wechselwirkungen zu verhindern. Ein Beispiel für eine derartige Zusammenlagerungshilfe ist die Bildung des Sm/LSm-Cores der mRNA-prozessierenden U snRNPs. Dabei wird die Anlagerung von Sm/LSm Proteinen an die U snRNAs durch eine Anzahl von Hilfsfaktoren orchestriert, die in Protein-Arginin-Methyltransferase 5 (PRMT5)- und dem Survival Motor Neuron (SMN)-Komplexen organisiert sind. Die Zusammenlagerung wird durch die PRMT5-Untereinheit pICln initiiert, die die räumliche Anordnung von Sm/LSm-Proteinen in höher-geordneten Komplexen stabilisiert. Diese werden anschließend auf den SMN-Komplex übertragen, wobei pICln verdrängt und die Verbindung mit der Sm-Seite der U snRNA sichergestellt wird. Der SMN-Komplex besteht aus dem SMN-Protein, das in einem modularen Netzwerk mit acht weiteren Proteinen (Gemins 2-8 und Unrip) interagiert. Auch wenn funktionale und strukturelle Charakterisierungen einzelner Proteinkomponenten und Module dieser Zusammenlagerungs-Maschinerie vorliegen, steht ein tiefergehendes Verständnis des strukturellen Organisation des Gesamt-Komplexes noch aus. In der vorliegenden Arbeit sollte unter Anwendung biochemischer und struktureller Techniken ein Beitrag dazu geleistet werden, die Interaktionen der verschiedenen Komponenten innerhalb des SMN-Komplexes zu verstehen, die so die dreidimensionale Organisation des SMN-Komplexes zu verstehen. Eine neuartige Kristallstruktur des humanen Gemin8:7:6-Subkomplexes bei einer Auflösung von 1.5 Å zeigt, wie der periphere Gemin7:6-Abschnitt durch den C-Terminus von Gemin8 zum SMN-Komplex dirigiert wird. In diesem Modell interagiert Gemin7 sowohl mit Gemin6 als auch Gemin8 über den N- und C-Terminus der Sm-ähnlichen Domäne. Dieser hochkonservierte Interaktionsmodus wird in der erwähnten konservierten Sequenz und dem gleichen biochemischen Verhalten ähnlicher Subkomplexe in divergenten Spezies einschließlich S. pombe und C. elegans widergespiegelt. Obwohl es keine signifikante Übereinstimmung mit der Sequenz von Sm-Proteinen gibt, weist der dimere Gemin7:6-Komplex markante strukturelle Ähnlichkeit mit dem einem Sm-Heterodimer auf. Die Annahme, der dimere Gemin7:6-Subkomplex würde als Hilfsfaktor über die direkte Interaktion mit Sm-Proteinen fungieren konnte in der vorliegenden Arbeit nicht bestätigt werden. Folglich bleibt die Funktion des dimeren Gemin7:6-Subkomplexes im Kontext der SMN-Zusammenlagerungsmaschinerie unklar. Verringerte Mengen des funktionellen SMN-Proteins sind die Ursache für die neurodegenerative Erkrankung Spinale Muskelatrophie (SMA). Das C-terminale YG-Zipper-Motiv von SMN stellt einen Hotspot für die meisten SMA-Mutationen dar. In dieser Arbeit wurde der bereits bekannten YG-Box aus H. sapiens und S. pombe eine neuartige Kristallstruktur der SMN YG-Box aus C. elegans mit einer Auflösung von 2.2 Å hinzugefügt. Zusätzlich wurde gezeigt, dass SMA-verursachende Missense-Mutationen in der YG-Box einen beträchtlichen Einfluss auf die Selbst-Interaktion von SMN haben, was aus biochemischen Versuchen mit der YG-Box aus H. sapiens und C. elegans ersichtlich wurde. Der molekulare Aufbau und das biochemische Verhalten der SMN YG-Box aus C. elegans, S. pombe und H. sapiens betont die Konservierung dieses Oligomerisierungsmotives über mehrere Organismen hinweg. Neben der Funktion als Multimerisationsdomäne dient die YG-Box von SMN auch als Interaktionsplattform für Gemin8. Eine systematische Untersuchung von SMA-verursachenden Missense-Mutationen ergab, dass die Einbindung von Gemin8 in den SMN-Komplex durch definierte Substitutionen massiv beeinflusst wird. Interessanterweise ist dieser Bindungsdefekt unabhängig vom SMN-Oligomerisierungsstatus. Demzufolge würde diese Klasse von SMA-Mutationen spezifisch die Inkorporation des Gemin7:6-Subkomplexes beeinflussen. Die Resultate dieser Arbeit bilden eine wichtige Grundlage für weitere strukturelle Untersuchungen des SMN-Komplexes über Kryo-Elektronenmikroskopie.

Fungi are surrounded by a thick layer of carbohydrates and proteins, which is essential for the cell’s viability – the fungal cell wall. Proteins are incorporated into this organelle in different ways: some are covalently linked to the carbohydrate moiety of the cell wall via Glycosylphosphatidylinositol (GPI)-anchors or alkali-sensitive linkages, others are indirectly attached to the cell wall via disulfide bonds. Cell wall proteins are involved in various cellular functions, such as cell wall biosynthesis, adhesion to external surfaces, or sensing. The GPI-anchored cell wall proteome of the thermophilic model organism Chaetomium thermophilum was identified in the first part of this thesis. First, a prediction of GPI-proteins, anchored to the cell wall and the plasma membrane was done. The prediction was then complemented by mass-spectrometric identification of GPI-anchored cell wall proteins in isolated cell walls. The detected proteins were then analyzed concerning their functions and putative roles and interesting targets for pharmaceutical applications and fundamental research were established, including Gel1/2, Kre9/Knh1, and Ecm33. In addition, the ultrastructure of the C. thermophilum cell wall was analyzed via transmission electron microscopy, revealing short microfibrils in its outer layer and its similarity to the cell wall of S. cerevisiae. The thesis then advances to the analysis of the A-domains of the Candida glabrata adhesins Awp1 and Awp3, which are members of adhesin cluster VI. Although the fungal pathogen lacks certain virulence factors – such as hyphae formation – C. glabrata infections are commonly observed; its large repertoire of adhesins is believed to be the reason therefore. Awp1A and Awp3A both consist of a β helix domain and an α crystallin domain. They are structurally similar to carbohydrate binding proteins, e. g. polysaccharide lyases, but carbohydrate binding could not be observed. A sequence similarity network (SSN) elucidates their high similarity to cluster V adhesins Awp2 and Awp4 and thereby reinforces previous classifications. The structures of Awp1 and Awp3 provide first insights into new types of adhesins in C. glabrata that include the adhesin clusters V and VI. Furthermore, the G-protein coupled receptor Pth11 from C. thermophilum was analyzed. It contains an N-terminal CFEM domain – a domain exclusively found in fungal cell wall and plasma membrane proteins – that is predicted to be the ligand binding site. The CtPth11 CFEM domain consists of five α helices and reveals two potential binding sites, divided by F48. Distinct conformers of F48 allow formation of a tunnel through the domain. A model of the CtPth11 CFEM domain and transmembrane region – based on prediction of neighboring residues via sequence covariation analysis – shows that both potential binding sites are accessible. In a fragment screen, four fragments were bound in the same cavity; three of them could be fitted into their respective electron densities. These hydrophobic fragments are placed in the hydrophobic cavity, with only few additional interactions, which is in accordance with the proposal that Pth11 senses hydrophobic cues on the plant surface., Pilze sind von einer dicken Schicht aus Kohlenhydraten und Proteinen umgeben, die für die Lebensfähigkeit der Zelle essentiell ist – der pilzlichen Zellwand. Proteine sind auf unterschiedliche Arten in dieses Organell integriert: einige sind kovalent an den Kohlenhydratanteil der Zellwand gebunden, entweder über Glycosylphosphatidylinositol (GPI)-Anker oder alkaliempfindliche Bindungen, andere indirekt über Disulfidbindungen. Zellwandproteine sind an unterschiedlichen zellulären Funktionen beteiligt, wie der Zellwandbiosynthese, der Adhäsion an Oberflächen oder der Sensorik. Im ersten Teil dieser Arbeit wurden die GPI-verankerten Proteine des thermophilen Modellorganismus Chaetomium thermophilum identifiziert. Zunächst wurde eine Vorhersage der an Zellwand und Plasmamembran befindlichen GPI-Proteine durchgeführt. Die Vorhersage wurde durch den massenspektrometrischen Nachweis der GPI-verankerten Zellwandproteine in isolierten Zellwänden ergänzt. Die detektierten Proteine wurden hinsichtlich ihrer Funktionen und mutmaßlichen Rollen analysiert. Interessante Targets für pharmazeutische Anwendungen und Grundlagenforschung konnten ermittelt werden, u. a. Gel1/2, Kre9/Knh1 und Ecm33. Zusätzlich wurde die Ultrastruktur der Zellwand von C. thermophilum mittels Transmissionselektronenmikroskopie analysiert, wobei kurze Mikrofibrillen in der äußeren Zellwandschicht und Ähnlichkeit zu der Zellwand von S. cerevisiae festgestellt werden konnten. Die Arbeit behandelt im zweiten Teil die Analyse der A-Domänen der Candida glabrata Adhäsine Awp1 und Awp3, die Mitglieder des Adhäsinclusters VI sind. Obwohl diesem humanpathogenen Pilz bestimmte Virulenzfaktoren - z. B. zur Hyphenbildung - fehlen, werden C. glabrata Infektionen häufig beobachtet, wobei sein großes Repertoire an Adhäsinen einer der wesentlichen Gründe sein sollte. Awp1A und Awp3A bestehen beide aus einer β-Helix-Domäne und einer α-Kristallin-Domäne. Sie ähneln strukturell kohlenhydratbindenden Proteinen, z. B. Polysaccharid-Lyasen. Allerdings konnte keine Bindung von Kohlenhydraten an Awp1-Typ Adhäsinen nachgewiesen werden. Ein Sequenzähnlichkeitsnetzwerk leitet eine hohe Ähnlichkeit zu den Adhäsinen Awp2 und Awp4 des Adhäsinclusters V ab und untermauert damit frühere Klassifizierungen. Die Strukturen von Awp1 und Awp3 geben erste Einblicke in neue Typen von Adhäsinen in C. glabrata, zu denen Adhäsine der Cluster V und VI gehören. Weiterhin wurde der G-Protein-gekoppelte Rezeptor Pth11 aus C. thermophilum analysiert. Er enthält eine N-terminale CFEM-Domäne - diese Domäne kommt ausschließlich in Pilzzellwand- und Plasmamembranproteinen vor -, die als Ligandenbindungsstelle vorhergesagt wurde. Die CFEM-Domäne von CtPth11 besteht aus fünf α-Helices und weist zwei potenzielle Bindungsstellen auf, die durch F48 geteilt werden. Bestimmte Orientierungen des Aminosäurerestes F48 ermöglichen die Bildung eines Tunnels durch die Domäne. Ein Modell der CtPth11-CFEM-Domäne und der Transmembranregion - basierend auf der Vorhersage benachbarter Reste mittels Sequenzkovarianzanalyse - zeigt, dass beide potenziellen Bindungsstellen zugänglich sind. In einem Fragment-Screen wurden vier Fragmente an der gleichen Bindestelle gebunden; drei davon konnten in die jeweiligen Elektronendichten modelliert werden. Diese hydrophoben Fragmente sind in der hydrophoben Bindestelle platziert und weisen nur wenige zusätzliche Interaktionen auf, was zu der Hypothese passt, dass Pth11 hydrophobe Charakteristika auf der Pflanzenoberfläche wahrnimmt.

U ovom radu promatrao se problem traženja proteina iz iste proteinske familije i uspješnost primjene algoritama iz teorije grafova na taj problem. Konkretno, analizirala se isplativost korištenja algoritma za traženje najveće aproksimativne klike koji djeluje kao nadopuna iterativnom pretraživanju u klasifikaciji proteina. Rezultati korištenja navedenog aproksimativnog pristupa usporedeni su sa rezultatima algoritma za traženje egzaktne najveće klike i posebno, sa rezultatima početnog iterativnog pretraživanja proteoma. Jedna od glavnih mjera koja je korištena za usporedbu uspješnosti modela je F1-score, a negdje je u obzir uzeta i vremenska složenost. Postupak je proveden na proteomima talijinog uročnjaka, krumpira, šećerne repe i rajčice. Promatrao se učinak algoritama na manjim i na većim skupovima podataka. Dobiveni rezultati pokazuju da korištenje aproksimativnog algoritma bitno poboljšava uspješnost modela (F1-score), iako nalazi manji broj stvarno pozitivnih (TP) u odnosu na početno iterativno pretraživanje. Takoder, egzaktni algoritam očekivano postiže nešto bolje rezultate od aproksimativnog, ali je na većem broju podataka puno sporiji u izvršavanju. This paper covers the problem of identifying proteins belonging to the same protein family and explores the use of applying known graph theory algorithms to this problem. Specifically, it examines the benefits of maximum clique approximation, which serves as a supplement to iterative searching in protein classification. Results of using the aforementioned approximation approach were compared with the results of using the exact maximum clique algorithm, as well as with the results of the initial proteome iterative search. F1- score was the primary metric used for comparing model performance, while in some cases time complexity was also considered. The procedure was performed on several proteomes, namely thale cress, potato, tomato and sugar beet. Algorithmic effciency was measured on both small and large data sets. Results obtained demonstrate that the use of the approximation algorithm greatly improves model performance (F1-score), although it does retrieve a smaller number of true positives (TP) compared with the initial iterative search. Furthermore, the exact algorithm achieves somewhat better results than the approximation algorithm, but it is considerably slower on large data sets.

Globalno zatopljenje i zasoljavanje tala među najvećim su izazovima današnje poljoprivrede. Poliploidizacija, proces umnožavanja cijelog kromosomskog seta, poznat je evolucijski fenomen koji omogućuje prilagodbu biljaka na nove stresne uvjete. Također, poznato je da su mnoge kultivirane biljke poliploidi. Međutim, točni mehanizmi kako poliploidizacija djeluje u stresnim uvjetima tek se počinju detaljnije ispitivati. U našemu istraživanju nastojali smo odrediti utjecaj toplinskoga i solnog stresa na poliploidne varijante uročnjaka (A. thaliana) u usporedbi s diploidnima. Toplinski stres postignut je izlaganjem biljki temperaturi od 45°C u trajanju od 3h, dok je solni stres postignut izlaganjem biljaka 300 mM otopini natrijeva klorida. Utjecaj stresa na biljke kvantificiran je mjerenjem maksimalne fotosintetske efikasnosti (F v /F m ) i indeksa performansa (PI ABS ) te ukupne koncentracije aminokiseline prolina. Također, utjecaj stresa pratili smo računalnom diferencijalnom analizom proteinskih mrlja na gelovima dobivenima metodom 2D SDS-PAGE, te kasnijom identifikacijom proteina od interesa spektrometrijom masa. Naši rezultati pokazali su značajno veću otpornost fotosintetskog aparata poliploidnih varijanti uročnjaka na toplinski i solni stres od diploidnih. Prolinski test nije se pokazao dovoljno informativnim za kvantifikaciju stresa kod biljaka izloženih toplinskom stresu, dok su kod biljaka izloženih solnom stresu poliploidne varijante pokazale akumulaciju veće koncentracije prolina u odnosu na diploide, sugerirajući bolju otpornost na solni stres. Analizom diferencijalne ekspresije proteina bitnih za stres uočene su znatne razlike u ekspresiji proteina bitnih za proces fotosinteze i mehanizme biljnog odgovora na stres. Budućim istraživanjima s većim brojem tehničkih replika i identificiranih proteina omogućit će se pouzdanija identifikacija i kvantifikacija razlika za stres bitnih proteina s različitom razinom ekspresije u poliploidnim varijantama uročnjaka.

There is a very few papers to provide an overview of the characteristics of the estrous cycle, the relationship of the estrous cycle to physiological manifestations such as the pH of the vaginal environment, as well as the dynamics of the vaginal microbiota in wild mice. The aim of this thesis is to contribute to the understanding of the dynamic relationship between external influences and the physiology of the female reproductive system, to develop a reliable methodology for measuring the pH of the vaginal microenvironment in mice as well as to quantify the overall abundance of some bacterial taxons by comparing sequencing and qPCR methods. The results suggest that the physical presence of the male in the cage has the most significant effect on the prolongation of the estrus phase, in contrast to non-significant olfactory stimulation of the urine. Fluctuation in the pH of the vaginal environment have also been shown to be cyclic, and the qPCR method shows that the composition of the vaginal microbiota, during the estrus phase, differs significantly from other phases of the estrous cycle, as we confirmed by 16S rRNA sequencing. Thus, these results provide a comprehensive view of the variability of the estrous cycle with an emphasis on the variability of the vaginal microbiota and the change in the...

Lipidne kapljice (LK) so pred kratkim priznani citosolni organeli s primarno nalogo shranjevanja maščob. Sestavljene so iz sredice nevtralnih lipidov obdane z enim slojem fosfolipidov in proteinov. Njihovo število, sestava in velikost se nenehno spreminjajo glede na celične potrebe in okolje. Pomembno vlogo imajo pri agresivnih rakavih celicah, saj omogočajo njihovo preživetje v tumorskem okolju, ki je pogosto hipoksično in revno s hranili. Na površini fosfolipidnega monosloja LK je pripetih mnogo proteinov in encimov z različnimi biološkimi funkcijami. V predhodnem delu naše raziskovalne skupine smo ugotovili, da imajo LK pomembno vlogo pri celičnem odzivu na stres, ki ga povzroči pomanjkanje hranil. Predvidevamo, da se dinamične spremembe LK v odzivu na celični stres odražajo v spremenjeni sestavi njihovega proteoma. V tem delu smo želeli ugotoviti, kako se proteom LK spremeni, ko visokoinvazivne celice raka dojke MDA-MB-231 izpostavimo akutnemu pomanjkanju hranil. Intaktne LK smo izolirali iz stradajočih in kontrolnih celic, izvedli izolacijo proteinov, jih razgradili do peptidov in jih analizirali ter kvantificirali z masno spektrometrijo z uporabo pristopa LFQ (ang. "label-free quantification"). Od skupno 381 zaznanih proteinov smo jih 155 (41 %) uvrstili med kontaminante iz drugih celičnih razdelkov, 49 proteinov pa smo glede na dosedanje raziskave lahko povezali z delovanjem LK. Med slednjimi smo zaznali 15 »bona fide« proteinov LK, ki so jim v drugih raziskavah pripisali vezavo na LK. Identificirali smo kar 177 (47 %) proteinov, ki jih dosedanje študije niso povezale z delovanjem LK in predstavljajo nove, potencialno zanimive tarče za nadaljnje raziskave. Akutno stradanje celic raka dojke je močno vplivalo na sestavo proteoma LK. Izpostavili bi 20 proteinov z bistveno spremenjenim izražanjem: G0S2, CDC42, SNX12, CBX3, UBA52, HEBP1, RAC2, AUP1, TBCB, GLRX3, CAST, DBNL, NUCKS1, PTGES3, TXN, PRDX5, TCEAL3, ACSL3, LUC7L2 in HSD17B11. Poleg proteinov vpletenih v presnovo maščobnih kislin, holesterola in metabolizem LK (ACSL3, HSD17B11, HSD17B7, G0S2) smo zaznali proteine, ki sodelujejo pri ubikvitinaciji (UBA52, AUP1), vnetnih odzivih (PTGES3), vzdrževanju redoks ravnotežja v celici (TXN, PRDX5), vezikularnem transportu (TRAPPC3, LZTFL1), organizaciji citoskeleta (TBCB, DBNL) in jedrnih procesih (TCEAL3, NUCKS1). Ti rezultati kažejo na povezavo med dinamičnimi spremembami v proteomu LK in celičnem odzivu na stres. Rezultati tega dela so pomembni za nadaljnje raziskave še neznanih vlog številnih z LK povezanih proteinov pri presnovi in delovanju organela ob stradanju in drugih stresnih pogojih. Lipid droplets (LD) are newly recognized cytosolic organelles primarily involved in lipid storage. LDs are composed of a neutral lipid core, surrounded by a phospholipid monolayer and proteins. Their number, composition and size are constantly changing according to cellular needs and environmental conditions. LDs have important roles in aggressive cancer cells as they enable their survival in the hypoxic and nutrient poor tumour microenvironment. Many proteins and enzymes with different biological functions are bound to the surface of LDs, embedded within the phospholipid monolayer. Our previous studies have identified an important role of LDs in the cellular stress response to starvation. We assume that the dynamic changes in LD metabolism and function caused by cell stress are accompanied by corresponding alterations in their proteome. In this master thesis we set to explore the nutrient deficiency-induced changes in the LD proteome of aggressive MDA-MB-231 breast cancer cells. Intact LD were isolated from starved and control cells, proteins were extracted, digested to peptides and analysed the latter with mass spectrometry using the label-free quantification approach. In total, we identified 381 proteins associated with the LD fraction, 155 (41 %) of which were classified as contaminants from other cell compartments, while 49 proteins were classified as LD-associated according to previous studies. Among the latter, we detected 15 »bona fide« LD proteins, for which a clear interaction with LDs has already been directly demonstrated in previous studies. Importantly, we detected 177 (47 %) proteins without a known connection to LDs, representing a group of novel, potentially interesting targets for further investigation. We also found that the acute starvation conditions imposed on breast cancer cells had a significant effect on the composition of the LD proteome. Notably, significant change in the amounts of the following 20 LD-associated proteins were detected: G0S2, CDC42, SNX12, CBX3, UBA52, HEBP1, RAC2, AUP1, TBCB, GLRX3, CAST, DBNL, NUCKS1, PTGES3, TXN, PRDX5, TCEAL3, ACSL3, LUC7L2 and HSD17B11. Among these, there are proteins involved in fatty acid, cholesterol and LD metabolism (ACSL3, HSD17B11, HSD17B7, G0S2), ubiquitination (UBA52, AUP1), inflammation (PTGES3), cellular redox homeostasis (TXN, PRDX5), vesicular transport (TRAPPC3, LZTFL1), cytoskeletal organization (TBCB, DBNL) and nuclear function (TCEAL3, NUCKS1). These results suggest that the LD proteome is significantly altered in cancer cells exposed to acute nutrient stress and provide an impetus for future studies on the roles of novel LD-associated proteins in cancer cell metabolism and response to stress.

In addition to tissues such as liver, the plasma membrane sodium-dependent citrate transporter, NaCT (SLC13A5), is highly expressed in brain neurons, but its function is not understood. Loss-of-function mutations in the human SLC13A5 gene have been associated with severe neonatal encephalopathy and pharmacoresistant seizures. The molecular mechanisms of these neurological alterations are not clear. We performed a detailed examination of a Slc13a5 deletion mouse model including video-EEG monitoring, behavioral tests, and electrophysiologic, proteomic, and metabolomic analyses of brain and cerebrospinal fluid. The experiments revealed an increased propensity for epileptic seizures, proepileptogenic neuronal excitability changes in the hippocampus, and significant citrate alterations in the CSF and brain tissue of Slc13a5 deficient mice, which may underlie the neurological abnormalities. These data demonstrate that SLC13A5 is involved in brain citrate regulation and suggest that abnormalities in this regulation can induce seizures. The present study is the first to (i) establish the Slc13a5-knockout mouse model as a helpful tool to study the neuronal functions of NaCT and characterize the molecular mechanisms by which functional deficiency of this citrate transporter causes epilepsy and impairs neuronal function; (ii) evaluate all hypotheses that have previously been suggested on theoretical grounds to explain the neurological phenotype of SLC13A5 mutations; and (iii) indicate that alterations in brain citrate levels result in neuronal network excitability and increased seizure propensity.

Introduction: The molecular mechanisms involved in the development and progression of breast cancer have yet to be determined. In the present study, the proteome of cancerous beast and adjacent normal tissues were compared. Materials & Methods: In a cohort study, the cancer tissues and adjacent normal tissues were obtained from 5 female patients with ductal carcinoma in stage 3. The total protein contents of cancer and adjacent normal tissues were extracted. The protein expression levels were examined by Image Master 2D Platinum software following two-dimensional gel electrophoresis. MALDI-TOF MS/MS mass spectrometry was used for proteins identification. Findings: The constant region of Ig gamma-1 chain and beta subunit of hemoglobin were exclusively detected in the cancer and adjacent normal tissues, respectively. The expression of serum albumin and collagen VI alpha chain in the cancer tissue was significantly lower than the normal tissue (P less-than 0.05). In contrast, the expression of a single peptide matching to cytoskeletal type I and II keratin significantly increased in the cancer tissue compared to the normal tissues (P less-than 0.05). Discussion & Conclusion: As the output of our investigation, it seems that proteome of cancerous tissue is extensively different from the adjacent one. Therefore, proteomic approach might be a promising tool for monitoring breast tumorigenesis. However, this needs to be confirmed in future.

Впервые описаны белки, достоверно увеличивающиеся и уменьшающиеся в моче с возрастом в интервале 20-60 лет. Охарактеризованы комбинации белков, связанных с изменением иммунных процессов, нарушением реологии крови, в том числе риском коагулопатии, противоопухолевых защитных механизмов, инсулинового сигнального пути, с изменением характеристик клеточного деления и качества новообразованной ткани. Таким образом, возрастная динамика основных процессов запускает каскад реакций, проявляющихся в замыкании «патологических биохимических кругов», которые формируют предпосылки к развитию заболеваний и, с течением времени, клинические проявления., For the first time proteins are described, reliably increasing and decreasing in urine with age in the range of 20 to 60 years. The combinations of proteins associated with changes in immune processes, violation of blood reology, including the risk of coagulopathy, anticancer defense mechanisms, insulin signaling pathway, changes in cell characteristics are characterized division and quality of the newly formed fabric. Thus, the age dynamics of the main processes triggers a cascade of reactions manifested in the closure of «pathological biochemical circles» that form the prerequisites for the development of diseases and, over time, clinical manifestations., Успехи геронтологии, Выпуск 4 2020

Cereals are among the oldest crops that have been grown and used by humans as important component of their diet. It is an important source of livelihood for the human population and have a wide range of uses, mainly in the food industry. Cereals generally serve as a source of energy in the diet, due to the high starch content. The most commonly grown types of cereals are especially wheat, barley, rye, rice, corn, but there may be other species such as oats and millet. However, even cereals do not avoid the problems associated with the action of abiotic stress factors. Their effect on all plants is manifested by a decrease in vitality, but in crops - such as cereals - mainly by a decrease in yield. Due to the reduction in yield, breeding of resistant cereal genotypes is now in the primary interest of breeders. This work summarizes the basic principles of the action of abiotic stress on plants and explains the reactions of various types of cereals to abiotic stress factors. Key words: proteome, physiology, cereals, abiotic stress, yield

Amastigotes are forms of Leishmania, naturally occurring in vertebrate hosts within phagocytic cells - especially the macrophages. The aim of this project was to compare three types of amastigotes of Leishmania that can be used for experiments under laboratory conditions - namely the axenic amastigotes, cultured extracellularly (without vertebrate phagocytic cells), amastigotes isolated from macrophages infected ex vivo, and "true" amatigotes isolated from lesions of the infected BALB/c mice. Amastigotes were compared with respect to the development in the natural vector and at the proteome level. L. mexicana, the causative agent of cutaneous leishmaniasis in the New World, was chosen for this comparison. In experiments comparing the development of Leishmania in the natural vector Lu. longipalpis we found significantly weaker infections in the sand flies infected with axenic amastigotes compared to other types of amastigotes. In addition to the intensity of infection, we compared the localization of promastigotes in the digestive tract of the phlebotomine sand flies. The following localizations were observed: the abdomen, the thorax, the cardia and the stomodeal valve, which is crucial for infectivity of the sand fly. There was no significant difference in localization in any of the groups of...

Cereals are among the oldest crops that have been grown and used by humans as important component of their diet. It is an important source of livelihood for the human population and have a wide range of uses, mainly in the food industry. Cereals generally serve as a source of energy in the diet, due to the high starch content. The most commonly grown types of cereals are especially wheat, barley, rye, rice, corn, but there may be other species such as oats and millet. However, even cereals do not avoid the problems associated with the action of abiotic stress factors. Their effect on all plants is manifested by a decrease in vitality, but in crops - such as cereals - mainly by a decrease in yield. Due to the reduction in yield, breeding of resistant cereal genotypes is now in the primary interest of breeders. This work summarizes the basic principles of the action of heat/cold stress on plants and explains the reactions of various types of cereals to these abiotic stress factors. Key words: proteome, physiology, cereals, temperature stres, yield, abiotic stres

Ancient protein analysis is providing new insights into the evolutionary relationships between hominin fossils across the Pleistocene. Protein identification commonly relies on the proteolysis of a protein extract using a single protease, trypsin. As with modern proteome studies, alternative or additional proteases have the potential to increase both proteome size and protein sequence recovery. This could enhance the recovery of phylogenetic information from ancient proteomes. Here we identify 18 novel hominin bone specimens from the Kleine Feldhofer Grotte using MALDI-TOF MS peptide mass fingerprinting of collagen type I. Next, we use one of these hominin bone specimens and three Late Pleistocene Equidae specimens identified in a similar manner and present a comparison of the bone proteome size and protein sequence recovery obtained after using nanoLC-MS/MS and parallel proteolysis using six different proteases, including trypsin. We observe that the majority of the preserved bone proteome is inaccessible to trypsin. We also observe that for proteins recovered consistently across several proteases, protein sequence coverage can be increased significantly by combining peptide identifications from two or more proteases. Our results thereby demonstrate that the proteolysis of Pleistocene proteomes by several proteases has clear advantages when addressing evolutionary questions in palaeoproteomics.SignificanceMaximizing proteome and protein sequence recovery of ancient skeletal proteomes is important when analyzing unique hominin fossils. As with modern proteome studies, palaeoproteomic analysis of Pleistocene bone and dentine samples has almost exclusively used trypsin as its only protease, despite the demonstrated advantages of alternative proteases to increase proteome recovery in modern proteome studies. We demonstrate that Pleistocene bone proteomes can be significantly expanded by using additional proteases beside trypsin, and that this also improves sequence coverage of individual proteins. The use of several alternative proteases beside trypsin therefore has major benefits to maximize the phylogenetic information retrieved from ancient skeletal proteomes.

In all organisms, the genetic information of cells is stored in the nucleotide sequence of deoxyribonucleic acid (DNA). The human organism consists of more than 200 different somatic cell types with the same genetic information (genotype). Even though, they drastically differ in their morphology and function (phenotype), which is related to different gene expression levels. Gene expression is controlled by macromolecular interactions and epigenetic modifications on chromatin that are highly locus-specific and drive functional aspects of each locus. Even though, the compositions of macromolecules and modifications on many chromosome loci remain poorly understood, in part due to the lack of locus-specific chromatin purification methods that would allow for targeted, discovery-oriented analyses. In this work, the first enrichment method based on bio-orthogonal conjugation (“click-chemistry) with encoded programmable DNA binding domains (transcription-activator like effectors – TALEs) for purification of user-defined genomic loci was established. This click-mediated enrichment provides complementary potential compared to the existing enzymatic biotinylation strategies used in chromatin enrichment methods in the view of site-specificity and proteome-wide background. This method will enable correlations of local chromatin states with phenotypes as the key to a deeper understanding of the regulation landscape of the eukaryotic genome. As a first outlook experiment, we extended our approach to fusion constructs of specific TALE proteins and ten-eleven translocation (TET) dioxygenases for epigenetic editing in vivo. TETs catalyze the oxidation of 5-methylcytosine (5mC) to the oxidized derivates 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). In combination with the click-mediated enrichment and proteomics analysis, this will enable studying how local epigenetic changes modulate the local chromatin landscape in vivo as basis for alterations in gene expression.

Točnost sinteze proteina određena je procesima koji prethode njihovom nastanku: replikacijom DNA, transkripcijom i translacijom. Greška u bilo kojem od njih dovodi do mistranslacije: uvođenja pogrešne aminokiseline u proteine. Važan faktor u ispravnoj sintezi proteina su aminoacil-tRNA- sintetaze – enzimi koji sparuju aminokiselinu s pripadnom tRNA. Nastala aminoacil-tRNA prenosi se do ribosoma gdje sudjeluje u translaciji. Kako bi osigurale dovoljno visoku točnost translacije, aminoacil-tRNA-sintetaze moraju prepoznati točno određenu aminokiselinu u mnoštvu sličnih supstrata. Neke od njih zato imaju posebnu domenu za popravak pogreške u kojoj hidroliziraju krivo nastale aminoacil-tRNA. Izoleucil-tRNA-sintetaza (IleRS) iz bakterije Escherichia coli ima dva jednako dobra nepripadna supstrata koji strukturno sliče izoleucinu: valin i norvalin. Valin je proteinogena (kanonska) aminokiselina koja je uvijek prisutna u stanici, dok je norvalin neproteinogena (nekanonska) aminokiselina koja se nakuplja u uvjetima nedostatka kisika. Inaktivacija hidrolitičke aktivnosti u domeni za popravak IleRS dovodi do češćeg ugrađivanja norvalina i valina umjesto izoleucina u proteom bakterije (mistranslacija). Supstitucija norvalinom je toksičnija. Kako bi se istražio stanični odgovor uslijed supstitucije izoleucina u proteinima valinom odnosno norvalinom, praćen je utjecaj na vijabilnost i promjene u proteomu bakterije E. coli. Metodama kvantitativne proteomike istraživan je proteomski odgovor, nastanak agregata i vezanje staničnih proteina na DnaK (glavni šaperon u bakteriji E. coli). Razina mistranslacije od 20 % toksičnija je za stanicu od toplinskog stresa. Primijećeno je da tijekom mistranslacije dolazi do povećanja količine šaperona, međutim nije primijećen značajan utjecaj na broj i vrstu DnaK interaktora. Egzogeni dodatak norvalina u medij smanjuje unos izoleucina, valina i leucina u bakteriju. U uvjetima kada je prosječna razina mistranslacije u proteomu niska, u soju koji nema disagregazu ClpB selektivno dolazi do agregacije proteina s većim postotkom mistranslacije. Primarni efekt mistranslacije tako je agregacija nefunkcionalnih proteina, a ne pojačana interakcija s DnaK.

Decreased serum adiponectin levels in type 2 diabetes has been linked to the onset of mitochondrial dysfunction in diabetic complications by impairing AMPK-SIRT1-PGC-1α signaling via impaired adiponectin receptor 1 (AdipoR1) signaling. Here, we aimed to characterize the previously undefined role of disrupted AdipoR1 signaling on the mitochondrial protein composition of cardiac, renal, and hepatic tissues as three organs principally associated with diabetic complications. Comparative proteomics were performed in mitochondria isolated from the heart, kidneys and liver of Adipor1−/− mice. A total of 790, 1,573, and 1,833 proteins were identified in cardiac, renal and hepatic mitochondria, respectively. While 121, 98, and 78 proteins were differentially regulated in cardiac, renal, and hepatic tissue of Adipor1−/− mice, respectively; only 15 proteins were regulated in the same direction across all investigated tissues. Enrichment analysis of differentially expressed proteins revealed disproportionate representation of proteins involved in oxidative phosphorylation conserved across tissue types. Curated pathway analysis identified HNF4, NRF1, LONP, RICTOR, SURF1, insulin receptor, and PGC-1α as candidate upstream regulators. In high fat-fed non-transgenic mice with obesity and insulin resistance, AdipoR1 gene expression was markedly reduced in heart (−70%), kidney (−80%), and liver (−90%) (all P < 0.05) as compared to low fat-fed mice. NRF1 was the only upstream regulator downregulated both in Adipor1−/− mice and in high fat-fed mice, suggesting common mechanisms of regulation. Thus, AdipoR1 signaling regulates mitochondrial protein composition across all investigated tissues in a functionally conserved, yet molecularly distinct, manner. The biological significance and potential implications of impaired AdipoR1 signaling are discussed.

Fragestellung: Die aseptische Lockerung ist eine der häufigsten Indikationen für Revisionsoperationen in der Endoprothetik. Kausal spielen abriebbedingten Osteolysen ('particle disease') sowie die individuelle Knochenqualität hierbei eine wichtige Rolle. Dabei wird eine frühzeitige[zum vollständigen Text gelangen Sie über die oben angegebene URL], Deutscher Kongress für Orthopädie und Unfallchirurgie (DKOU 2019)

Iterativnom pretragom, uz pomoć upita, na proteomu tražimo proteine iz određene klase, nazivamo ih biološki značajnim proteinima. Odgovor na dani upit može se dobiti raznim iterativnim pretraživačima te on može i ne mora sadržavati biološki značajne proteine. U ovom radu analiziramo tehniku poboljšanja točnosti iterativnog pretraživanja. Točnost mjerimo statističkom mjerom F1 score, a tehnika se naziva metoda centralnog elementa. Odgovor na dani upit promatramo kao Markovljev proces na konačnom skupu. Koristeći rezultate iz Markovljevih lanaca pronalazimo težište, centar, odgovora. Postupak iterativne pretrage ponovimo sa centralnim elementom te usporedimo statističke mjere točnosti testa prije i nakon korištenja metode. Tehnika se provodi za različite upite, na proteomima četiri različite biljke - talijin uročnjak, rajčica, krumpir i azijska riža. Nakon analize rezultata za sve kombinacije upita, biljaka i iterativnih pretraživača, dolazimo do zaključka da našom tehnikom poboljšavamo točnost pretrage, a negdje i bitno poboljšavamo. Motif scanning is a very important part of bioinformatics. In this work, we are concerned with improving accuracy of certain iterative scanning tools. The method that we propose - a Central Element Method - is based on interpreting the response to a query as a finite Markov process, and results from Markov chain theory are used to obtain the center. The iterative search process is then repeated with the central element, and the statistical measures of test accuracy before and after are compared. The accuracy is measured by F1 score. The method is applied for various queries, on proteomes of four different plants – thale cress, tomato, potato and Asian rice. It turns out that this technique improves search accuracy and, in some instances, improves it significantly.

Plant leaf tissue proteomics analyses face multiple challenges, including the presence of highly abundant proteins like RuBisCO. This thesis reviews methods in protein analysis and outlines proteome fraction techniques. The experimental part compares the leaf proteome composition of five plants, including three representatives of herbaceous (Arabidopsis thaliana, Solanum lycopersicum, Hordeum vulgare), and two woody plants (Malus domestica and Populus hybrid). Further, peptide fractionations by a high-pH C18 and SCX chromatography, and the role of data processing in proteomics analyses are illustrated.

Die Verletzungen der Kreuzbandstrukturen des Kniegelenkes nehmen eine zentrale Bedeutung in der Orthopädie und Unfallchirurgie ein und weisen aufgrund der hohen Inzidenz eine sozioökonomische Relevanz auf. Eine operative Versorgung der vorderen Kreuzbandruptur wird vor allem bei Instabilitäten des Kniegelenkes durchgeführt, um Folgeschädigungen wie beispielsweise sekundäre Meniskusläsionen oder die Entwicklung einer posttraumatischen Gonarthrose zu vermeiden. Trotz der kontinuierlichen Optimierung der operativen Versorgung nach Ruptur des vorderen Kreuzbandes zeigen sich weiterhin hohe Re-Ruptur-Raten, welche einerseits durch noch unzureichende OP-Techniken und ungenügende Patienten-Compliance und andererseits durch biologisches Transplantatversagen zustande kommen. In diesem Zusammenhang stellen die komplexen biologischen Prozesse (Inflammation, Reparatur, Remodelling) der Knochen-Sehnen-Integration einen wichtigen Forschungsschwerpunkt dar, um die mangelhafte Transplantat-Einheilung zu verstehen und letztendlich die zeitgerechte und stabile ossäre Inkorporation von Sehnen-Transplantaten zu gewährleisten. In diesem Zusammenhang nehmen mit den Osteoblasten und Fibroblasten zwei Zelltypen eine zentrale Rolle im Prozess der ossären Sehnen-Integration und der Bildung extrazellulärer Knochen- und Sehnen-Matrix ein. Dabei legen zahlreiche Studien die Vermutung nahe, dass osteoinduktive Substanzen wie beispielsweise das Bone Morphogenetic Protein-7 (BMP7), welches sich bereits zur Behandlung von aseptischen Pseudarthrosen etabliert hat, die Knochen-Sehnen-Integration positiv beeinflussen. Unklar verbleiben jedoch weiterhin die molekularbiologischen und zellbiologischen Abläufe im Bereich des Knochen-Sehnen-Übergangs und die spezifischen Wirkungsmechanismen besagter osteoinduktiver Proteine. Die vorliegende Arbeit hatte das Ziel, ein standardisiertes Co-Kultur-Modell zur Simulation und Erforschung der Interaktion von Osteoblasten und Fibroblasten aufzubauen und damit eine kontrollierte Analyse der spezifischen Effekte einmaliger und multipler Stimulation mit BMP7 auf die Knochen-Sehnen-Einheilung zu ermöglichen. Im Rahmen dieser Studie konnte ein standardisiertes, an Wang et al. (2007) angelehntes in vitro Co-Kultur-Modell, bestehend aus sekundären osteoblastischen und fibroblastischen Zellen muriner Herkunft in einem temporären Zwei-Kammer-Kultivierungs-System, etabliert werden, welches sich durch eine einfache Handhabung auszeichnet und als Hochdurchsatz-Verfahren eingesetzt werden kann. In diesem Modell diente eine Agarose-Trennschicht zur vorübergehenden Separation der zuvor genannten Zellen, wobei die Entfernung der Trennschicht die Interaktion der beteiligten Zelllinien ermöglichte. Beruhend auf diesem Modell wurden die Auswirkungen einer einfachen und einer mehrfachen Stimulation der osteoblastischen und fibroblastischen Zellen mit rekombinantem humanen BMP7 (rhBMP7) untersucht und die Ergebnisse hinsichtlich statistisch signifikanter Veränderungen ausgewertet. Histologisch zeigte sich in dieser Studie einerseits eine dosisabhängige und andererseits eine von der Anzahl der Stimulationen abhängige förderliche Wirkung der rhBMP7-Applikation. Es konnte mikroskopisch nachgewiesen werden, dass durch die rhBMP7-Hinzugabe eine beschleunigte Migration und Proliferation der osteoblastischen und fibroblastischen Zellen in die Interaktions-Zone resultierte. Diese Beobachtungen deckten sich mit den Ergebnissen der in dieser Studie durchgeführten molekularbiologischen Analysen spezifischer Markergene der Osteogenese. Über qRT-PCR konnte in der vorliegenden Arbeit gezeigt werden, dass eine rhBMP7-Stimulation eine dosisabhängige Steigerung der Alp1-, Col1a1- und Bglap-Genexpression in den osteoblastischen Zellen im Sinne einer gesteigerten Knochenmatrix-Bildung bewirkte. Eine in der Literatur beschriebene Spp1-Genexpressions-Verstärkung in den osteoblastischen Zellen konnte in dieser Studie nicht nachgewiesen werden. Für die fibroblastischen Zellen zeigte sich durch die rhBMP7-Stimulation eine erhöhte Runx2-, Col1a1-, Alp1-, und Bglap-Genexpression. Diese Resultate legten erstaunlicherweise nahe, dass die fibroblastischen Zellen osteogene Eigenschaften ausbilden beziehungsweise annehmen und damit eine Transdifferenzierung zu funktionsfähigen Osteoblasten durchlaufen. Die in dieser Studie präsentierten Ergebnisse lassen vermuten, dass die Knochen-Sehnen-Integration vor allem dadurch zustande kommt, dass in der Kontaktzone zwischen Sehnen- und Knochengewebe einerseits die Osteoblasten vermehrt Knochenmatrix bilden und andererseits die Fibroblasten zu Osteoblasten transdifferenzieren um Knochenmatrix auszubauen und um das Sehnengewebe dadurch ossär zu integrieren. Da viele der molekularbiologischen und zellbiologischen Abläufe der Knochen-Sehnen-Einheilung noch unklar verbleiben, bedarf es weiterer Untersuchungen zur Generierung eines besseren Verständnis für die Prozesse der ossären Sehnen-Integration und zur Entwicklung von therapeutischen Ansätzen., Lesions of the crucial ligaments of the knee joint play an eminent role in the field of orthopaedics and traumatology. Their high incidence makes them highly socio-economically relevant. Surgical treatment of tears of the anterior crucial ligament is required especially when instability occurs. This can prevent possible subsequent damages such as secondary meniscal lesions or posttraumatic gonarthrosis. Despite the continuous optimization of the surgical treatment after tears of the anterior crucial ligament, rerupture rates are still high. These are caused by inadequate surgical technique, insufficient patient compliance and also to failure of the biological integration of the transplant. In this context, complex biological processes (inflammation, reparation, remodelling) of the bone-tendon integration form a central research focus. This can helpt to better understand incorrect or poor transplant incorporation and to ensure a stable and quick bone-tendon-healing. Osteoblasts and fibroblasts play an important role in the process of osseous tendon integration and the formation of extracellular bone or tendon matrix. In this context, numerous studies suggest that osteoinductive substances such as bone morphogenetic protein-7 (BMP7) – which is already approved in the treatment of aseptic pseudoarthrosis – have a positive impact on bone-tendon healing. Yet, the cell biological and molecular biological processes during bone-tendon transition and specific effects of osteoinductive proteins remain unclear. The intention of this thesis was to establish a standardized co-culture model to simulate and investigate the osteoblast-fibroblast interaction. This new method was then applied to analyse the specific effect mechanisms of singular and repetitive stimulation with BMP7 on the bone-tendon healing. Based on the work of Wang et al. (2007), a standardized in vitro co-culture model was successfully established. It consists of secondary murine osteoblast and fibroblast cells incubated in a temporary two-chamber system. More precisely, an agarose divider served as a transient separator of osteoblastic and fibroblastic cells. After the removal of the divider direct interactions of both cell types were possible. Based on this model, the effects of singular and multiple stimulation of the osteoblastic and fibroblastic cells with recombinant human BMP7 (rhBMP7) were examined and analyzed to spot statistically significant changes. The study has shown that the established method provided an easy handling and at the same time showed that the co-culture model could be used as a high-throughput technique. Subsequently the histological analysis of the study showed a dose-dependent and number-dependent effect of the stimulation with rhBMP7. Hence, an accelerated migration and proliferation of the osteoblastic and fibroblastic cells after rhBMP7 application could be observed in the microscopical experiments. These observations were consistent with the results of the molecular biological examinations of specific osteogenic marker genes. Using qRT-PCR, you can see that stimulation with rhBMP7 leads to a dose-dependent increase of Alp1, Col1a1 and Bglap gene expressions in osteoblasts as well as an improved formation of bone matrix. An enhanced Spp1 gene expression as proposed in the common literature was not detectable for the osteoblastic cells in this study. The rhBMP7 application showed an increased gene expression for Runx2, Col1a1, Alp1 and Bglap in the examined fibroblastic cells. Surprisingly suggesting a transdifferentiation of fibroblasts into functional osteoblasts with developing osteoblast specific characteristics. The results of this study indicate that the bone-tendon integration on the one hand is achieved by enhanced bone matrix building of the osteoblasts and on the other hand by transdifferentiation of fibroblasts to functional osteoblastic cells. It seems that both cell types are responsible for the incorporation of tendon tissue into bone tissue including the formation of surrounding bone matrix. Since many cell biological and molecular biological processes of the bone-tendon integration remain unclear, further studies are required to generate a comprehensive understanding for the integration of tendons to bone and to ensure the development of reliable therapeutic approaches.