SKIP and BIR/Survivin are evolutionarily conserved proteins. SKIP is a known transcription and splicing cofactor while BIR-1/Survivin regulates cell division, gene expression and development. Loss of function of C. elegans SKIP (SKP-1) and BIR-1 induces overlapping developmental phenotypes. In order to uncover the possible interactions of SKP-1 and BIR-1 on the protein level, we screened the complete C. elegans mRNA library using the yeast two-hybrid system. These experiments identified partially overlapping categories of proteins as SKP-1 and BIR-1 interactors. The interacting proteins included ribosomal proteins, transcription factors, translation factors and cytoskeletal and motor proteins suggesting involvement of the two studied proteins in multiple protein complexes. To visualize the effect of BIR-1 on the proteome of C. elegans we induced a short time pulse BIR-1 overexpression in synchronized L1 larvae. This led to a dramatic alteration of the whole proteome pattern indicating that BIR-1 alone has the capacity to alter the chromatographic profile of many target proteins including proteins found to be interactors in yeast two hybrid screens. The results were validated for ribosomal proteins RPS-3, RPL-5, non-muscle myosin and TAC-1, a transcription cofactor and a centrosome associated...

Proteomics is the large-scale study of proteins, particularly their structures and functions. Proteomics has been utilized in medicine for investigation of disease mechanisms and biomarker discovery. Instrumental methods cover sample preparation, protein and peptide separation and mass spectrometry. At present, there is no proteomic method that can be used as universal for every sample. Analytical methods need to be adapted and optimized for certain samples. The aim of this work was to create methodic procedures and to interpret results of experimental and clinical research. The first part of the thesis includes experiments utilizing proteomics to study changes in the plasma proteome clinically relevant porcine model of sepsis-induced peritonitis. Proteomic analyzes were also starting methodological strategies in experiments aimed at kidney physiology and pathophysiology of acute kidney injury during sepsis. Renal biopsies were analyzed in order to study the time course of proteome changes caused by sepsis and surgery. The second part of the thesis contains experiments studying biocompatibility. A method for elution of proteins interacting with adsorbents used in extracorporeal liver support system and with hemodialyzer capillaries was prepared. Analysis of proteins adsorbed to polysulfone...

PB2 ist ein essentieller Bestandteil der trimeren RNA abhängigen RNA Polymerase von Influenzaviren und ist bekannt für seine Schlüsselrolle in der Bestimmung des Viruswirtsspektrums. Diese Arbeit diente der Identifizierung neuer Interaktionspartner von PB2 eines saisonalen und eines hochpathogenen Influenzavirus Stammes im Kontext infizierter humaner alveolar Epithelzellen (A549) unter Einsatz massenspektrometrischer Analysen. Die anschließende Untersuchung ausgewählter zellulärer Interaktoren hatte zum Ziel, deren Einfluss auf den Replikationszyklus der Influenzaviren zu bestimmen, sowie Unterschiede in ihrer Relevanz für das saisonale und das hochpathogene Virus aufzuzeigen. Die Erzeugung und Nutzung von Influenzaviren die einen Strep-tag an ihrem PB2 Protein tragen ermöglichte eine Anreicherung von PB2 und seiner Interaktionspartner. Die anschließende massenspektrometrische Analyse identifizierte 22 potentielle PB2 Interaktionspartner. Eine Auswahl an 13 Proteinen wurde tiefer gehend analysiert und eine Komplexbildung mit PB2 konnte für 9 Proteine bestätigt werden. Darüber hinaus zeigten 11 Proteine einen Polymerase stimulierenden bzw. hemmenden Effekt. Das Polymerase stimulierende Protein HSPA8 wurde zur weiteren Untersuchung ausgewählt. Während ein Einfluss von HSPA8 auf den hochpathogenen Influenzastamm nicht abschließend geklärt werden konnte, wurde seine Bedeutung für den Vermehrungszyklus des saisonalen Stammes aufgezeigt. Die Überexpression von HSPA8 führte zu einer Steigerung der Polymerase-Aktivität, wohingegen die Erniedrigung des HSPA8 Spiegels in einer Verringerung der viralen Replikation und der Polymerase-Aktivität resultierte. Interessanterweise führte die Erniedrigung des HSPA8 Spiegels auch zu stark verminderter PB2-Expression, jedoch nur im Falle des saisonalen Influenzastammes. Dieser Befund deutet auf eine Rolle von HSPA8 als PB2-Chaperon, notwendig für Proteinstabilität von saisonalen aber nicht hochpathogenen Influenzaviren, hin. / PB2 is an essential component of the influenza virus trimeric RNA dependent RNA polymerase and is known to play a key role in virus host range determination. Here, a combined affinity-purification/mass spectrometric approach was performed to identify novel interaction partners of PB2 of seasonal and highly pathogenic viral strains in infected human alveolar epithelial cells (A549). The subsequent analysis of selected cellular interaction partners aimed to determine the influence of these proteins on the replication cycle, as well as to determine differences in their relevance for the seasonal and the highly pathogenic influenza virus strain. Generation and use of recombinant influenza viruses carrying a Strep-tag at their PB2 protein allowed for enrichment of PB2 and its interaction partners. The subsequent mass spectrometric analysis identified 22 potential PB2 interaction partners. A selection of 13 proteins was further analyzed, and co-precipitation with PB2 was confirmed for 9 proteins. Moreover, an inhibitory or stimulatory effect on polymerase activity was observed for 11 proteins. The polymerase stimulating protein HSPA8 was selected for further investigation. While the influence of HSPA8 on the highly pathogenic strain remained unclear, its importance for seasonal influenza virus life cycle was demonstrated. Overexpression of HSPA8 resulted in increased polymerase activity while HSP8 knock down resulted in reduction of viral replication and viral polymerase activity. Intriguingly, the knock down of HSPA8 led to a strong decrease of PB2 protein expression. However, this was only observed for seasonal PB2. These results indicate a role of HSPA8 as a PB2 chaperone, necessary for protein stability of seasonal but not highly pathogenic influenza virus.

No description available.

Ziel der vorliegenden Arbeit war die Identifizierung genetischer Faktoren, die das Plasmaproteom regulieren. Die Untersuchungen wurden im Modellsystem einer F2-Kreuzung zweier Inzucht-Mausstämme (FVB.LDLR-/-, C57BL/6.LDLR-/-) durchgeführt, die sich in ihrer Atheroskleroseausprägung unterscheiden. Von jedem der 453 Tiere der F2-Generation wurden Plasmaproteomprofile mittels Massenspektrometrie (MALDI-TOF) generiert. Diese Spektren wurden in zwei unabhängigen Datenanalysen ausgewertet und eine Kopplungsanalyse (QTL-Analyse, quantitative trait loci) der Phänotypen mit jeweils 192 genetischen Markern in jedem der F2-Tiere durchgeführt. So wurden die Datensätze von Proteom und Genom miteinander kombiniert, um Genorte, die mit unterschiedlich regulierten Proteinen in Verbindung stehen, zu identifizieren. Dieser Ansatz ist bisher in der Literatur nicht beschrieben worden. In der vorliegenden Arbeit wird sowohl die Methodik der statistischen Auswertung als auch die weitere Analyse der generierten Daten beschrieben. Es wurden zahlreiche hochsignifikante Kopplungssignale gefunden, von denen zwei durch die Identifizierung von Proteinen verifiziert werden konnten. Es handelt sich hierbei um das Apo-A2 des HDL auf Chromosom 1 und Hämoglobin subunit alpha auf Chromosom 11. Eine Kolokalisation der gefundenen Proteine mit Loci der Atherosklerosedisposition konnte nicht identifiziert werden. Dieser Ansatz zeigt erstmals, dass eine hypothesenfreie Verbindung proteomischer und genomischer Daten möglich ist und zur Identifizierung genetisch regulierter Plasmaproteine beitragen kann.

No description available.

Fundação de Amparo a Pesquisa do Estado de Minas Gerais / The royal jelly (RJ), a secretion produced by the hipopharingeal and mandibular glands of the nurse honeybees, have a variety of pharmacological activities, aside of being necessary for the castes differentiation and the queen Apis mellifera longevity. To identify the N-glycoproteins of two samples of RJ provinient from distinct origin and conditions, we used the concanavalin A affinity chromatography (ConA) associated to the mass spectrometry. The investigated samples of RJ were the brazilian RJ (GRB), produced experimentally in local apiary, and the Chinese RJ (GRC), mattered from China and commercially available. The protein profile in SDS-PAGE 1D was characteristic for GRB and GRC. The N-glycoproteins fractions eluted from the ConA column had presented nine polypeptides for GRB and 12 for GRC, with relative molecular mass (Mr) varying between 130 and 15 kDa. It was observed that the immediately aftercollects freezing of the GRB did not inhibited the appearance of bands with Mr lesser than 49 kDa. A total of 21 bands was excised from gel and digested with tripisin for analysis in MALDI-TOF MS. The bioinformatic analysis revealed that all the 16 proteins identified in the GRB and GRC samples belong to the A. mellifera apalbumins family (Apa-1, Apa-2 and Apa-3). The Apa-2 was the protein that had greater prevalence between the N-glycoproteins of the RJ, presenting Mr varying between 110 and 25 kDa. The fact that the Apa-2 had been identified with distinct Mrs can be related to glycosilation, oligomerization or degradation of this apalbumin. Therefore, the ConA affinity chromatography associated with the proteomic analysis made possible the identification of three members of the family of RJ main proteins (Apa-1, Apa-2 and Apa-3). Moreover, the comparative study of the GRB and GRC glycoprotein composition suggests similar standard between these samples and can be useful in future studies that evaluate the biological functions of the apalbumins pos-translational modifications. / A geléia real (GR), uma secreção produzida pelas glândulas hipofaringeal e mandibular das abelhas operárias, possui uma variedade de atividades farmacológicas, além de ser fundamental para a diferenciação das castas e longevidade da rainha de Apis mellifera. Com o objetivo de identificar alguns dos componentes bioativos deste produto da colméia, utilizou-se a cromatografia de afinidade de concanavalina A (ConA) associada à espectrometria de massa para identificar as N-glicoproteínas de duas amostras de GR obtidas de origem e condições distintas. As amostras de GR investigadas foram a GR brasileira (GRB), produzida experimentalmente em apiário local, e a GR chinesa (GRC), importada da China e disponível comercialmente. O perfil de proteínas em SDSPAGE 1D foi característico para GRB e GRC, com um maior número de polipeptídeos para GRC. As frações das N-glicoproteínas eluídas da coluna de ConA apresentaram nove polipeptídeos para GRB e 12 para GRC, com massa molecular variando entre 130 e 15 kDa. Observou-se que o congelamento imediatamente pós-coleta da GRB não impediu o aparecimento de polipeptídeos com Mr menor que 49 kDa. Um total de 21 bandas foi excisado do gel e digerido por tripsina para análise em MALDI-TOF MS. Para cada perfil de massa dos peptídeos (PMF) obtido, realizou-se a busca em banco de dados utilizando-se o software Mascot. Esta análise revelou que as 16 proteínas identificadas nas amostras de GRB e GRC pertencem à família das apalbuminas (Apa-1, Apa-2 e Apa-3) da abelha A. mellifera. A Apa-2 foi a proteína que teve maior prevalência entre as N-glicoproteínas da GR, apresentando Mr variando entre 110 e 25 kDa. O fato da Apa-2 ter sido identificada com Mrs distintas pode estar relacionado a fenômenos de glicosilação, oligomerização ou degradação desta apalbumina. Portanto, a cromatografia de afinidade de ConA associada a análise proteômica possibilitou a identificação de três membros da família das principais proteínas da GR (Apa-1, -2 e -3). Além disso, o estudo comparativo da composição de glicoproteínas da GRB e GRC sugere padrão similar entre estas amostras e pode ser útil em estudos futuros que avaliem as funções biológicas das modificações pós-traducionais das apalbuminas. / Mestre em Genética e Bioquímica

Fish sperm cryopreservation becomes highly attractive due to increasing number of potential applications and it has been considerably developed over the past decades. Cryopreservation of fish sperm is expected to improve the broodstock management in hatcheries, simplify hybridization and selective breeding, and serve as a useful tool for biodiversity and conservation of endangered species programs. Nevertheless, its status nowadays is highly contentious due to absence of specific techniques and standardization in the developed protocols. Moreover, the heterogeneity of cryoresistance in sperm of some fish individuals and, strong impairment of the sperm quality and survival after cryopreservation, to date, present some challenges inhibiting wide applicability of this approach. Therefore, a better understanding of the mechanisms of spermatozoa cryo-injuries and the development of new and more effective methods for cryopreservation of fish sperm are of primary importance. In the current work, the causes of individual variability in sperm cryoresistance, influence of cryopreservation process on sperm quality parameters regarding application of uncontrolled cooling devices were investigated. One of the study outputs is the founding the possible causes of sperm cryoresistance heterogeneity in common carp (Cyprinus carpio) males. As a result, a negative correlation between changes in the contents of major lipid classes and sperm motility after cryopreservation was found. In particular, the increased level of phospholipid, cholesterol and free fatty acid in samples with low post-thaw motility percentage were observed and related to decrease in sperm cryoresistance in common carp sperm. In the investigation of the consequence of the application of uncontrolled cooling devices on post-thaw sperm parameters of sterlet sperm, it has been shown that sterlet sperm could be cryopreserved by both Styrofoam box and dry shipper devices studied. However, due to inability to precisely control cooling process in these devices, the cooling conditions can be changed by varying straw number during freezing in Styrofoam box or by straw position during freezing in dry shipper. As a consequence, these changes shift the optimal freezing rates needed for successful sturgeon sperm cryopreservation resulting in decrease in post-thaw sperm motility and fertilization ability. These results suggest the way of standardization of fish sperm cryopreservation protocols by selecting appropriate straw number or straws position when uncontrolled cooling devices are used. Finally, to explore the cryopreservation effects on fish spermatozoa, the Percoll density gradient centrifugation technique was elaborated and applied for the first time to sterlet (Acipenser ruthenus) sperm. That technique allows to select and analyse only the sperm population that survived cryopreservation procedure and saved high motility parameters. It was found that this spermatozoa subpopulation possesses minimal changes in protein profile in comparison to native sperm. Moreover, the differences in many proteins which were found in sperm suspension after cryopreservation without separation is related to the presence of non-viable and sperm wreckage subpopulations in the frozen-thawed sperm suspension. Collectively, all of these detected changes in sperm motility parameters, viability, and protein profiles of viable spermatozoa after freeze-thaw process suggest some explanations for the mechanism responsible for cryodamage of sperm and provides the background for further development of an efficient cryopreservation protocols. However, it should be also further studied if the Percoll separation technique could serve as a useful tool for both better understanding of non-lethal sperm cryodamages and improvement of assisted reproduction practice in sturgeon species.

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.

No description available.