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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.

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.

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.

Miyelin kılıfı, bilginin nöronlar üzerinde verimli transferini sağlar ve nöronların işlevi ve sağ kalımı için elzemdir. Miyelin gelişiminde gözlenen bozuklukların veya miyelin hasarının (demiyelinizasyon), elektriksel sinyal iletimini sekteye uğratarak, nöronları atrofiye iterek ve kalıcı fonksiyonel bozukluğa neden olarak multipl skleroz (MS) gibi birçok nörolojik hastalığın kökeninde yattığı bilinmektedir. Bu çalışmada merkezi sinir sisteminde demiyelinizasyonu anlamak için oligodendrosit ve makrofaj hücreleri arasındaki protein-protein etkileşimlerinin belirleyerek MS'de etkili olan ve ilaçlar tarafından hedeflenebilecek yeni sinyal yolaklarının tanımlanmasını amaçladık. Bu amaçlara ulaşmak için, büyük veri yöntemlerini kullanarak oligodendrositler ile makrofaj hücreleri arasındaki protein-protein etkileşimlerinin bir atlası oluşturuldu. Benzersiz bir şekilde, metodolojimiz, tek bir hücre tipi içindeki proteinlerden ziyade hücre-hücre interakt eden proteinleri analiz etti. Ayrıca, MS hastalarının ve kontrollerinin gen ifade profilleri, protein etkileşim verileri ile birlikte değerlendirildi. Sonuç olarak, MS ile ilişkili membran ve hücre dışı proteinler tanımlanmıştır. Bu proteinlerden nörogenezde etkili ve reseptör-ligand gibi özel ilişkisi olan tirozin kinaz ailesinden efrin A7 reseptörü (EphA7) seçilerek hücre kültürü ve fare beyin kesitlerinde boyanıp mikroskopta incelendi. İncelemeler sonucunda EphA7'nin makrofaj hücrelerinde in vivo ve in vitro ifade olduğu gözlemlendi. EphA7'nin bilinen ligandlarından olan EfrinA1, A3, A4 ve A5'in oligodendrosit hücrelerinde farelerde in vivo ve in vitro ifade olduğu gözlemlendi. EphA7'nin oligodendrositlerde eskpresyonu gözlemlenmezken, makrofajlarda efrinA1, efrinA3, A4 ve A5'in ifade edildiği gözlemlendi. Ayrıca efrin ligandlarının astrositlerde de ifade edildiği gözlemlendi. Sonuç olarak EphA7'nin makrofajlarda/mikroglialarda ifade edildiği gözlemlendi. EfrinA1, A3, A4 ve A5 ligandlarının oligodendrosit ve astrosit hücrelerinde ifade edildiği gözlemlendi.Anahtar Kelimeler: Bağışıklık Hücreleri, Multipl Skleroz, Oligodentrosit, Proteom Myelin sheath facilitates efficient transfer of information over neurons and is essential for the function and survival of neurons. Thus, incorrect development of myelin or myelin damage is at the root of many neurological diseases such as multiple sclerosis (MS). Dysmyelination or demyelination disrupts electrical signal transmission, causing neuronal atrophy and permanent functional damage. In order to investigate demyelination in the central nervous system, we aimed to identify the novel MS-associated signaling pathways by identifying protein-protein interactions between oligodendrocyte and immune cells. To achieve this goal, a map of protein-protein interactions between oligodendrocytes and immune system cells was generated using large data methodologies. Uniquely, our methodology analyzed cell-to-cell interacting proteins rather than proteins within a single cell type. In addition, gene expression profiles of MS patients and controls were evaluated along with the protein interaction data. Consequently, MS-associated membrane and extracellular proteins were identified. Ephrin A7 receptor (EphA7) is a member of the tyrosine kinase family and functions in neurogenesis emerged as the top candidate protein. I examined EphA7's expression in cell culture and mouse brain sections using microscopy. EphA7 was expressed macrophages in vitro and in vivo in mouse. Moreover, Ephrins A1, A3, A4, and A5, which are known ligands of EphA7, were expressed in oligodendrocytes in vitro and in vivo in mouse. EphA7 was not present in oligodendrocytes. Ephrins A1, A3, A4, and A5 were expressed in macrophages and Ephrins A1, A3, A4, and A5 was also expressed in astrocytes. As a result, it was observed that EphA7 was expressed in macrophages/microglia. EfrinA1, A3, A4 and A5 ligands were observed to be expressed in oligodendrocyte and astrocyte cells.Key words: Immune Cells, Multiple Sclerosis, Oligodendrocyte, Proteom 82

Proteomics is analyzing the total amount of proteins expressed in a certain time point, in an organism, a tissue or a cell, by using protein separation and identification techniques. From 1994, proteomics was first introduced by Marc. R. Wilkins, to now proteomics advanced and begins to use in several areas instead of clinical medicine. Food industry is one of these areas. Proteomics is a powerful tool used in food quality, food safety, genetically modified organisms, effects of storage and packaging on foods and food allergens researches. The aim of this review is to define proteomics and proteomics techniques and usage in food industry area., Proteomiks, bir organizma, doku veya hücrede herhangi bir anda bulunan proteinlerin tümünün, geniş çaplı protein ayırma ve tanımlama teknikleri kullanılarak analiz edilmesidir. 1994’te Avustralyalı araştırmacı Marc. R. Wilkins tarafından ortaya konmasının ardından günümüze kadar büyük gelişme göstererek sadece tıpta değil birçok alanda kullanılmaya başlanmıştır. Gıda endüstrisi de bu alanlardan biridir. Proteomiks, gıda kalitesi, gıda güvenliği, genetiği değiştirilmiş gıdalar, depolama ve paketleme gibi işlemlerin gıdalar üzerine etkileri, gıda alerjenleri gibi konularda yapılan araştırmalarda tercih edilen bir yöntem haline gelmiştir. Bu derlemede proteomiksin ne olduğu, nasıl uygulandığı ve gıda endüstrisinde hangi alanlarda kullanıldığı hakkında genel bilgiler vermek amaçlanmıştır.

Die Leber ist das zentrale Stoffwechselorgan aller Wirbeltiere. Die kleinste funktionelle Einheit der Leber stellt das von periportaler (PP) nach perizentraler (PZ) Seite blutdurchflossene Leberläppchen (Lobulus) mit seinen metabolisch aktiven Hepatozyten dar. Obwohl die Hepatozyten morphologisch nicht unterscheidbar sind, sind ihre Enzymausstattungen und damit ihre Aufgaben entlang der Porto-Zentral-Achse divergent, weshalb der Begriff der metabolischen Zonierung geprägt wurde. Systematische Studien zur Untersuchung der Zonierung beruhten bis dato lediglich auf RNA-Expressionsanalysen der verschiedenen Bereiche des Leberläppchens. Um detaillierte Unterschiede in den funktionellen Eigenschaften der Hepatozyten zu erfassen, wurde in dieser Arbeit eine weitreichende Analyse des Proteoms von PZ und PP Hepatozyten durchgeführt. Diese beinhaltete die Analyse von posttanslationalen Modifikationen wodurch zentrale regulatorische Mechanismen in der Zellen abgebildet werden konnten. Um diese breite Abdeckung zu erreichen wurde neben der (1) Massenspektrometrie (MS) in einem Full-MS Ansatz sowie einer (2) MS-basierten Immunoaffintätsanalyse (3) das antikörperbasierte hochdurchsatz Western Blot System DigiWest eingesetzt. Eine Weiterentwicklung stellt die in dieser Arbeit etablierte und als (4) LiquiWest bezeichnete Technologie dar. Sie beruht auf dem DigiWest, besitzt jedoch durch zahlreiche Optimierungen sowie Automatisierungslösungen bei gleichbleibender Datenqualität und signifikant reduzierter Eingriffszeit eine erhöhte Reproduzierbarkeit sowie einen 5-fach höheren Durchsatz. Mit Hilfe der genannten Technologien konnten zonierte Hepatozyten auf ihre Proteinexpression hin detailliert analysiert werden. Von den insgesamt über 2000 detektierten Proteinen sowie deren Modifikationen wurden 120 als differenziell exprimiert detektiert. Dabei zeigten Stoffwechselvorgänge wie oxidoreduktive Prozesse, die Gluconeogenese, der Aminosäureabbau aber auch regulatorische Prozesse wie der MAP-Kinase- und der TGF-beta Signalweg eine signifikant periportale Zonierung. Dahingegen waren die Glycolyse, der Citratzyklus, der Purin- und Fremdstoffmetabolismus und vor allem der Wnt-Signalweg perizentral ausgeprägt. Um den Einfluss der Kultivierung auf den Differenzierungszustand von Hepatozyten zu beleuchten, wurde eine neue, schonende und gleichzeitig spezifische Trennmethode eingesetzt. Die Trennung nutzte die charakteristische Expression der Glutaminsynthetase (GS) in PZ Hepatozyten und erlaubte in einer gentechnisch veränderten Maus durch Reportergenexpression die Anreicherung von GS positiven (GS+) oder GS negativen (GS-) Zellen. Durch kontrollierte Zugabe von Wnt-Agonisten wurde ein in vivo naher Zustand in der Kultur eingestellt. Bei der Analyse von über 100 Proteinen aus verschiedenen Signalwegen zeigte sich trotz dieser Kulturbedingungen eine Dedifferenzierung über mehrere Tage hinweg.

In dieser Arbeit wurde die Analyse von Protein Cross-Links systematisch untersucht. Dabei wurde neben der Messung mittels LC-MS/MS auch die Auswertung mit speziellen Suchalgorithmen näher betrachtet. Die drei häufigsten Suchprogramme pLink, xQuest und StavroX wurden dabei miteinander verglichen. Mit diesen Ergebnissen wurden auch Kriterien für die Annahme oder Ablehnung von identifizierten Cross-Links festgelegt. Neben klassischem, chemischem Cross-Linking wurde auch versucht, photochemisches Cross-Linking zu optimieren. Zunächst wurde die Aktivierung der photoreaktiven Komponente mit einem gepulsten Laser getestet. Aufgrund des mangelnden Erfolgs wurde daraufhin die notwendige Aktivierungszeit des Reagenzes mit einer UV-Lampe bestimmt, um den Misserfolg des ersten Versuchs zu erklären. Darüber hinaus wurde die Anreicherung von Cross-Link Peptiden optimiert, um deren Identifizierung zu erleichtern. Anhand des bestehenden ChaFRADIC-Protokolls mit Kationenaustauschchromatographie wurden drei neue Anreicherungs-Protokolle für Cross- Link Peptide entwickelt. Da der ursprüngliche ChaFRADIC-Ansatz nicht auf Cross-Link Peptide übertragen werden konnte, wurden neue Modifikationen, Proteasen und Chromatographie-Techniken getestet und erfolgreich eingesetzt. Alle drei entwickelten Anreicherungsmethoden ermöglichten die Identifikation zusätzlicher Cross-Links in BSA mit unterschiedlicher Effizienz. Daneben wurden in Kooperation mit weiteren Forschungsgruppen weitere Proteine mittels Massenspektrometrie analysiert, um neue strukturelle Informationen zu erhalten. Erstmalig wurde der BBSome-Komplex aus sechs Proteinen mit Cross-Linking untersucht, um die Interaktion zwischen den einzelnen Protein-Untereinheiten zu bestimmen. Zusätzlich wurde die Interaktion zweier bakterieller Peptide (Cdc42 mit IbpA) über einen neuartigen ATPCross- Linker massenspektrometrisch bestimmt. Abschließend wurde in dieser Arbeit ein Peptid-Standard für Cross-Link Peptide entwickelt. Über ein neuartiges Reaktionsschema konnte eine große Anzahl von Cross-Link Peptide mit beliebiger Sequenz hergestellt werden. Eine Mischung aus 20 Standard-Peptiden wurde anschließend verwendet, um verschiedenen Fragmentierungsmethoden für Cross-Link Peptide miteinander zu vergleichen und die optimale Messmethode zu bestimmen.

In this study primary hepatocytes were characterized on a metabolic and proteomic level in different cultivation setups, namely collagen sandwich (SW) and monolayer (ML) culture. The impact of insulin and glucose on the central carbon metabolism and hepatic function was addressed as well, also integrating species differences between murine and human hepatocytes. Overall, the central carbon metabolism was very robust in regard of the used cultivation condition. Hepatic function and the handling of stress as induced by ammonia, however, were clearly affected, with a rapid loss of hepatic function in the ML culture. Proteome analysis revealed a development away from freshly isolated hepatocytes in both setups, which was stronger in the ML culture. Differential handling of oxidative stress marked the strongest difference between the used cultivation setups. If exposed to insulin, human and murine hepatocytes reacted by an increased uptake of amino acids and glucose. 13C tracer experiments revealed a differential metabolic usage of carbon sources by both species, depending on the available amount of glucose. Hepatic function was differentially regulated by insulin, but also by glucose and the cultivation condition. Thereby, certain species-specific differences were detected, especially cytochrome P450 activity and insulin clearance. The susceptibility of primary hepatocytes to various factors has to be kept in mind when planning experiments and when comparing different studies. In dieser Arbeit wurden primäre Hepatozyten in Collagen Monolayer- (ML) und Sandwich-Kulturen (SW) proteomisch und metabolisch charakterisiert. Der Einfluss von Insulin und Glukose auf den Zentralstoffwechsel und die Funktionalität der Hepatozyten wurde ebenfalls untersucht, wobei auch Speziesunterschiede zwischen humanen und murinen Hepatozyten addressiert werden konnten. Der Zentralstoffwechsel erwies sich als sehr robust gegenüber den unterschiedlichen Kultivierungsformen, die Funktionalität der Zellen und ihr Umgang mit Stress, z.B. durch Ammonium, wurden jedoch stark beeinträchtigt. In der ML-Kultur kam es zu einem beträchtlichen Funktionsverlust der Hepatozyten. Die Proteomanalyse zeigte, dass sich die Zellen in beiden Kulturen von frisch isolierten Hepatozyten weg entwickelten, was stärker in der ML-Kultur war. Der größte Unterschied zwischen den Kultivierungsformen zeigte sich im Umgang mit oxidativem Stress. Insulin führte zu einer vermehrten Aufnahme von Aminosäuren und Glukose. Eine unterschiedliche Verwendung von Kohlenstoffquellen in Abhängigkeit von der Glukosekonzentration konnte durch 13C-Markierungsexperimente gezeigt werden. Die hepatische Funktionalität wurde durch alle Faktoren beeinflusst, wobei diese sich in den beiden Spezies unterschiedlich auswirkten. Diese Empfindlichkeit von Hepatozyten gegenüber den verschiedenen Aspekten der Kultivierungsbedingungen muss beim Planen von Experimenten und beim Vergleichen von Studienergebnissen stets bedacht werden.

Chlamydia trachomatis ist ein obligat intrazelluläres Humanpathogen, welches sich, nach der Einnistung in seiner Wirtszelle, innerhalb einer membranumhüllten Vakuole, der sogenannten Inklusion, befindet. Die Inklusionsmembran stellt dabei die primäre Kontaktfläche für Pathogen–Wirtszell-Interaktionen dar und definiert dadurch die Nische, in welcher sich die Bakterien vermehren können. Zum ersten Mal beschreiben wir in dieser Arbeit die Isolation und biochemische Charakterisierung der Inklusion von C. trachomatis, mittels herkömmlicher Organellaufreiniungsverfahren und massenspektrometrischer Proteomanalyse an einem zentralen Zeitpunkt der Infektion. Die relative Quantifizierung von Proteinen mittels stabiler isotopenmarkierter Aminosäuren in Zellkultur (SILAC) und die zusätzliche markierungsfreie Quantifizierung erlaubten uns die Darstellung dieses subzellulären Proteoms mit hohem Konfidenzniveau. Mithilfe dieser Methoden haben wir über dreihundert Wirtszellproteine identifiziert und quantifiziert, welche noch nicht als inklusionslokalisiert bekannt waren. Die globale Analyse dieser Daten bestätigte die Rekrutierung vieler Proteine, welche in verschiedenen Membrantransportwegen involviert sind. Zudem fanden sich viele Proteine des retrograden Transportweges, welcher bislang nicht im Zusammenhang mit Chlamydien-Infektionen untersucht wurde. Die detaillierte Analyse dieser Proteine zeigte, dass Sorting Nexine sehr spezifisch zur Inklusion rekrutiert werden. Zudem haben wir mithilfe des unlängst beschriebenen Inhibitors Retro-2 gezeigt, dass retrograder Transport essenziell für die effiziente intrazelluläre Replikation von C. trachomatis ist. Zusammengefasst haben wir eine große Anzahl zuvor unbekannter inklusionsassoziierter Proteine identifiziert und quantifiziert, und auf Basis unserer Daten schlagen wir den retrograden Transportweg als neues Potenzielles Angriffsziel von Therapeutika zur Behandlung von C. trachomatis Infektionen vor., Chlamydia trachomatis is an obligate intracellular human pathogen which, after invasion of its host cell, resides within a membrane bounded vacuole called the inclusion. The inclusion membrane represents the main host-pathogen interface of Chlamydiae and thereby defines the environment in which the bacteria thrive. Here we describe for the first time the isolation and biochemical characterization of the mid-infection inclusion of C. trachomatis using mass spectrometry based proteomics in combination with traditional organelle purification techniques. Relative quantification by Stable isotope labeling by amino acids in cell culture (SILAC) and additional label free quantification allowed the generation of a high confidence subcellular proteome. Using this approach, we were able to identify and quantify over three hundred host cell proteins that were not reported previously to be inclusion localized. By analyzing the obtained data on a global scale, we were able to confirm previous reports on the recruitment of proteins implied in several membrane trafficking pathways. Detailed analysis of proteins identified to be involved in retrograde trafficking, a pathway that has not been described previously in the context of chlamydial infections, showed that sorting nexins are specifically recruited to the inclusion. We furthermore identified retrograde trafficking to be essential for the efficient intracellular replication of C. trachomatis using the recently described inhibitor Retro-2, which drastically reduced bacterial progeny formation upon treatment. Taken together, we identified and quantified a large number of previously unknown inclusion associated proteins, and we propose retrograde trafficking as a novel potential drug target for the treatment of infections with C. trachomatis.

Genomik, genom ve genlerle ilgili bilginin açıklanmasına yönelik yapılan çalışmalardır. Genomik çalışmalarda, bir hücre veya dokunun tüm genleri, topluca değerlendirilmekte ve yorumlanmaktadır. Bunun için güçlü bir biyoinformatif desteğe ihtiyaç duyulmaktadır. Genomik çalışmalar, yapısal ve fonksiyonel olmak üzere iki grupta incelenmektedir. Yapısal genomik, organizmanın genetik bilgilerinin ortaya çıkar tılmasını sağlamaktadır. Ama genlerin fonksiyonları ile ilgili bilgi vermez. Fonksiyonel genomik, genlerin fonksiyonlarının öğrenilmesinin yanı sıra, organizma açısından önemlerinin anlaşılmasına aracılık eder. Ancak, genomik bilgi proteinlerle ilgili bilgi ile desteklenmelidir. Proteinler ile ilgiliyi bilgiyi topluca sağlamayı amaçlayan disipline ise proteomik denmektedir. Proteomik, proteomun yapı, yerleşim, miktar, diğer moleküllerle olan etkileşim vb özelliklerinin incelenmesini sağlar ve biyoinformatif desteğe ihtiyaç duyar. Proteinler çeşitli özelliklerinden dolayı genler kadar kolay çalışılmasa da, genomik ve proteomik, tıp ve tıp dışı birçok alanda, çeşitli amaçlarla kullanılmaktadır. Bu derleme ile genomik ve proteomik çalışmalar hakkında farkındalık oluşturmak amaçlanmıştır. Aynı zamanda geleneksel biyokimyasal-genetik yöntemler ile farklılıkları da vurgulanmak istenmiştir. Genomics is a total practice for the description of informations about genome and genes. Genomics studies provide assessment and interpre tation of total genome of a cell or a tissue. A powerful bioinformatics is one of the major components of genomics. Genomics is divided into two groups as structural and functional genomics. Structural genomics provides genetic informations of organism, but not functions of genes. Functions and importance of genes can be discovered through functional genomics. Genomics have to be supported with informations of proteins. Proteomics is discipline which obtains total information of proteins. It investigates features of proteome. Genomics and proteomics can be implemented for different scientific areas and purposes. In this rewiev, we aim to raise awareness about genomics and proteomics. Also, we want to underline the differences between these methods and traditional genetical-biochemical methods.

Escherichia coli, originally known as Bacterium coli commune, was identified in 1885 by the German pediatrician, Theodor Escherich. They are Gram-negative, straight and rod-shaped bacteria from the Enterobacteriaceae family. Due to its rapid growth rate, simple nutritional requirements and established genetic manipulation techniques, E. coli has become a model organism in the field of basic biomolecular sciences for understanding various biological phenomena. As a result, well established information about E. coli's genetics and several completed genome sequences are available. Among them, the genome sequences of two closely related K-12 non-pathogenic strains, MG1655 and W3110 have been accurately determined. Resequencing of PCR products of selected regions indicates that there is only eight true insertion/deletion or base differences between the two strains in addition to the 13 sites where differences are due to insertion sequences, defective prophages and two sites due to the W3110 inversion between the ribosomal RNA genes rrnD and rrnE. The rate of nucleotide changes between both the strains is estimated to be relatively low with almost identical genome structures. Hence the following important questions arise: - Is this high degree of similarity at the nucleotide level reflected in the metabolic phenotype? - Do sub-strains with almost identical genome structures exhibit similar behaviour in cellular metabolism? - How do global aspects of cell metabolism, protein synthesis and gene expression differ among closely related sub-strains of the same species, revealing possible complexities of cellular metabolism? To address these queries, we analyzed the growth behaviour in strictly controlled conditions and analysed the global proteome and transcriptome pools of these closely related E. coli sub-strains W3110 and MG1655. We applied the conventional 2-dimensional polyacrylamide gel electrophoresis for global proteomic profiling which is still the major method for global proteome analysis. Global changes in the gene expression levels were analysed using microarrays, thus providing quantitative information about the gene expression levels. Being the most extensively studied model organism, E. coli is frequently used in molecular evolutionary studies. A few potential reasons for this are: its capacity to propagate and reproduce quickly, facilitating the evolution experiments for many generations in a short time span, and capability to store the evolved and ancestor strains, allowing for direct comparison between them. As a result, several studies have used gene expression and proteome profiling methods to study molecular evolution, but these studies were confined to a single type of evolution process and were focused on a single molecular aspect that characterizes a cell (transcript or protein abundance). Metabolome profiling has been frequently applied for obtaining quantitative information on metabolites for studies on mutational or environmental effects, but not in an evolutionary context. In our study, we depicted a complete picture of molecular evolution processes in the laboratory among the two strains MG1655 and DH10B under three different evolutionary conditions in all three functional levels of the cell (transcriptome, proteome and metabolome). These data sets obtained from the three functional levels would be of vital importance for viewing a global picture of the experimental sample in question. To eliminate the possibility of the strain-dependent phenomenon of evolution and to examine the parallelism of the laboratory evolution processes, we examined all the evolutionary processes in two strains. The major questions that arose during our study were: - What are the transcriptome, proteome and metabolome changes occurring during the excess-nutrient adaptive evolution process? - Which genes, proteins and metabolites are vitally involved in the prolonged stationary phase evolution process? - What are the transcript, protein and metabolite changes occurring due to the pleiotropic effects due to environmental shift? - To what extent are the changes occurring during these evolutionary processes seen in both strains? - Among both the strains, is the path of evolution similar in these evolutionary processes (parallelism)? By global protein profiling technologies and integrating the multidimensional datasets generated, we aimed to find vital genes, proteins and metabolites involved in the evolutionary processes in three conditions in two E. coli K-12 strains. These generated datasets from all the three functional levels would be an initial resource for the systems biology of microbial evolution.

A lot of epidemiological and placebo-controlled randomised studies and a set of very different animal- and cell-models have already supported the tumourgenesis inhibitory and apoptosis inducing effect of nonsteroidal anti-inflammatory drugs (NSAIDs). In this context various mechanisms of inhibition of proliferation and induction of apoptosis are discussed. Within the scope of the present work the described effects of the NSAIDs at the example of the non-selective Cox-1/Cox-2-inhibitor flufenamic acid was examined in a neuroblastoma cell line and was confirmed. Thus relatively unspecific experiments like MTT-assays and analyses of the cellmorphology have verified the inhibitory effect of flufenamic acid regarding the viability, activity and proliferation of the cells of the neuroblastoma cell line KELLY. More specific investigations like apoptosis-assays with acridineorange and ethidiumbromide (AO/EB-staining) and FACS-analyses with propidiumiodide have shown that under the influence of flufenamic acid in adequate concentrations the neuroblastoma cells induce apoptosis. For the proteome analyses a concentration of 500 µM flufenamic acid was chosen. In this concentration clear effects were to be observed at the described experiments. Besides, this concentration clearly lay about the concentration necessary for the inhibition of the cyclooxygenases basing on the presumption that the effect is also to be led back on cyclooxygenase independent mechanisms which respond only to higher concentrations. Thus with proteome analyses of the neuroblastoma cells which were treated more than 3 h, 6 h, 9 h and 12 h with 500 µM flufenamic acid twelve proteins were differently regulated compared to the control group. A connection of these proteins with the inhibition of the cyclooxygenases has been unknown up to now. Only three, namely Hsp75, Hsc70/54 and Lbp, are mentioned directly or indirectly related to NSAIDs. The group of these identified proteins must be called rather heterogeneous. Thus the three proteins Hsp75, Hsc70/54 and TCP-1ε are known to be molecular chaperones. Four other proteins, Eno-1, PK-M1/M2, PDC-E2 and MDH1, are directly or indirectly involved in the production and supply of energy by means of glycolysis. RbP0, eEF2 and sEF2b are regulating enzymes of the translation. And p47 is involved as a co-factor of p97 in fusion of membranes, whereas Lbp act as a laminin receptor. In spite of the partly very different functions most of these with flufenamic acid regulated proteins were already mentioned regarding apoptosis, cell proliferation and cancer. A connection between the described observations at the cellular level, the known effects of NSAIDs on apoptosis and proliferation and the identified regulated proteins at molecular level is obvious. On the one hand the results of this work offer new starting points for further investigations of the mechanisms by which NSAIDs induce apoptosis and inhibit proliferation. On the other hand some of the regulated proteins could be pharmacological targets for the therapy of cancer., In vielen epidemiologischen und Placebo-kontrollierten randomisierten Studien, in unterschiedlichsten Tier- und Zell-Modellen wurde bereits die Tumorgenese-hemmende und Apoptose-induzierende Wirkung der Wirkstoffklasse der NSAR belegt. Dabei wurden und werden verschiedene Mechanismen, die im Zusammenhang mit der Hemmung der Proliferation und Einleitung des apoptotischen Zelltods stehen, diskutiert. Im Rahmen der vorliegenden Arbeit wurde die beschriebene Wirkung der NSAR am Beispiel des nicht-selektiven Cox-1/Cox-2-Hemmers Flufenaminsäure an einer Neuroblastom-Zelllinie untersucht und bestätigt. So haben relativ unspezifische Versuche, wie MTT-Tests und Untersuchungen der Zellmorphologie, die hemmende Wirkung von Flufenaminsäure auf die Vitalität, Aktivität und Proliferation der Neuroblastomzellen der Zelllinie KELLY belegt. Spezifischere Untersuchungen, wie Apoptose-Tests mit Acridinorange und Ethidiumbromid (AO/EB-Färbung) und FACS-Analysen mit Propidiumjodid, haben gezeigt, dass die Neuroblastomzellen unter dem Einfluss von Flufenaminsäure bei entsprechender Konzentration die Apoptose einleiten. Die für die Proteomanalysen gewählte Konzentration von 500 µM Flufenaminsäure, für die bei den beschriebenen Versuchen deutliche Effekte zu beobachten waren, lag dabei deutlich über der für die Cyclooxygenase-Hemmung notwendigen Konzentration, was auf der Annahme beruht, dass die pro-apoptotische Wirkung auch auf Cyclooxygenase-unabhängige Mechanismen, die erst auf höhere Konzentrationen ansprechen, zurückzuführen ist. So wurden bei den Proteomanalysen der Neuroblastomzellen, die über 3 h, 6 h, 9 h und 12 h mit 500 µM Flufenaminsäure behandelt wurden, zwölf Proteine differentiell zur Kontroll-Gruppe reguliert, für die ein Zusammenhang mit der Hemmung der Cyclooxygenasen bisher nicht bekannt ist, und von denen nur drei, nämlich Hsp75, Hsc70/54 und Lbp, direkt oder indirekt in Verbindung mit einem NSAR erwähnt werden. Die Gruppe dieser identifizierten Proteine kann man als recht heterogen bezeichnen. So sind die drei Proteine Hsp75, Hsc70/54 und TCP-1ε als molekulare Chaperone bekannt, vier andere Proteine, Eno-1, PK-M1/M2, PDC-E2 und MDH1, sind direkt oder indirekt über die Glykolyse an der Energiegewinnung und -bereitstellung beteiligt. RbP0, eEF2 und sEF2b sind regulierende Enzyme der Translation, während p47 als Co-Faktor von p97 an Membranfusionen beteiligt ist und Lbp als Laminin-Rezeptor fungiert. Trotz der zum Teil sehr unterschiedlichen Funktionen wurden die meisten dieser durch Flufenaminsäure regulierten Proteine bereits in Verbindung mit Apoptose, Zell-Proliferation oder Krebs erwähnt. Ein Zusammenhang zwischen den beschriebenen Beobachtungen auf zellulärer Ebene, der bekannten pro-apoptotischen und anti-proliferativen Wirkung von NSAR und den identifizierten regulierten Proteinen auf molekularer Ebene ist naheliegend. Die Ergebnisse dieser Arbeit bieten also einerseits neue Ansatzpunkte für die weitere Erforschung der Mechanismen, durch die NSAR die Apoptose einleiten und die Proliferation hemmen können. Andererseits könnten einige der regulierten Proteine pharmakologische Targets für die Behandlung von Krebserkrankungen darstellen.