No description available.

Candida albicans is an opportunistic fungal pathogen that may cause mucutaneous infection and/or disseminated candidasis if the host defense system is impaired (such as those in HIV patients). Cell surface of C. albicans is the frontier where initial interplay between host-pathogen takes place and therefore is of great importance in understanding the mechanism of hostpathogen interaction. MALDI-TOF-MS analysis of intact fungal cells yielded mass signatures for rapid species differentiation, strain grouping and yeast morphogenesis monitoring. Cell surface biotinylations at low temperature (4°C), enzymatic digestion of the intact fungal cell surface proteins ("whole cell shaving"), biotin-avidin affinity enrichment of biotinylated peptides, liquid chromatography mass spectrometry (LC-MS) based proteomic approach were employed for unambiguous identification of cell wall/cell wall associated proteins and the exposed peptide segments of these proteins. SILAC (Stable Isotope Labeling by Amino acids in Cell Culture) based CWP quantification analyses were performed to monitor CWP accumulation level change in response to hyphae induction. Information on surface exposed peptide segments and regulation of cell wall/surface protein during morphogenesis provided new candidates to the pool of potential peptide targets for protective vaccine development. A New type of "fluorous" (fluorinated alkane) affinity gained popularity due to its low level nonspecific protein/peptide binding. Fluorous labeling reagents that target primary amine groups in proteins/peptides were synthesized and characterized. The acid labile linker in the labeling reagents allows cleavage of the bulky fluorous tag moiety and the long oligo ethylene glycol (OEG) spacer after fluorous affinity purification. Upon collision induced decomposition, the labeled peptide ion yielded a characteristic fragment that could be retrieved from the residual portion of fluorous affinity tag, and serve as a marker to indicate that the relevant peptide had been successfully labeled. Results showed that both the protein/peptide labeling and affinity enrichment/separation process were highly efficient.

In eukaryotic cells, the genome is packed into chromatin, a hierarchically organized complex composed of DNA and histone and nonhistone proteins. In this thesis we have addressed the role of chromatin in cellular response to DNA damage (DDR) using various methodologies encompassing functional genomics and proteomics. First, we analyzed histone post-translational modifications (PTM) in the context of specific kind of DNA lesions (ICL-Interstrand Crosslinks) in Fanconi anemia using quantitative proteomics methodology, SILAC (Stable Isotope Labeling of Amino acids during Cell Culture). Using mass spectrometry (MS), we have successfully identified and quantified a number of histone PTM marks in histone H3 and H4, mainly acetylations and methylations,which have shown dependence upon functional FA-pathway. As a next step, we applied a functional genomics approach to study DDR in FA cells. In this analysis we first monitored the expression profile of histone modifying enzymes related to histone acetylations and methylations. Our results suggest some correlations between histone PTMs and gene expression of histone modifying enzymes, although conclusive evidence warrants further investigations. Next, we analyzed the total transcriptome after DNA damage induction in FA mutant and wild type cells. We also included in this analysis IR irradiation, in an attempt to dissociate more generic DDR from more specific changes that are associated with the role of FA pathway to the DNA ICLs. By performing a factorial interaction analysis, we were able to isolate the part of transcriptional response to DNA damage that was requiring functional FA pathway, as well as the genes that were sensitized to DNA damage by the inactivation of FA pathway. In the final part of the thesis, we attempted to solve one of the limitations that we encountered in the histone PTM analysis. The current approaches used to study histone PTMs from particular loci involves classical chromatin immunoprecipitation, which due to involvement of formaldehyde crosslinking render the protein part mostly unavailable for MS-based proteomics. We have proposed a novel methodology, which is based upon the biotin tagging of histones proximal to a protein of interest and subsequent purification of nucleosomes carrying the tagged histone. This methodology does not involve any crosslinking, enabling us to purify histones from specific loci, and subject them to large scale MS-based histone PTM analysis. A time dimension can also be added to our approach, as we can follow the modification status of particular fraction of histones once they get biotinylated. Another advantage is the use of alternate variant histones, which allows us to study the PTM profile of different functional states of chromatin. This methodology certainly has an edge on current techniques to study histone PTMs pattern associated with a particular protein of interest or with particular chromatin state.

Angiotensin II (AngII), the major effector of the renin angiotensin system, mediates kidney disease progression by signalling through AT-1 receptor (AT-1R), but there are no specific measures of renal AngII activity. Accordingly, we sought to define an AngII-regulated proteome in primary human proximal tubular cells (PTEC) in order to identify potential AngII activity markers in the kidney. We utilized stable isotope labelling with amino acids (SILAC) in PTECs to compare proteomes of AngII-treated and control cells. Of 4618 quantified proteins, 83 were differentially regulated. SILAC ratios for 18 candidates were confirmed by Selected Reaction Monitoring (SRM) assays. Both SILAC and SRM revealed the nuclear factor erythroid 2-related 2 (Nrf2) target protein, heme oxygenase-1 (HO-1) as the most significantly upregulated protein in response to AngII stimulation. AngII-dependent regulation of HO-1 gene and protein was further verified by qRT-PCR and ELISA in PTECs. In order to extend these in vitro observations, we utilized a systems biology approach. We thus overlaid a network of significantly enriched gene ontology (GO) terms from our AngII-regulated proteins with a dataset of differentially expressed kidney genes from AngII-treated wild type mice and AT-1R knock-out mice. Five GO terms were enriched both in vitro and in vivo, and all included HO-1. Furthermore, four additional Nrf2 target proteins were functionally important in vitro and in vivo. We then studied HO-1 kidney expression and urinary excretion in AngII-treated wild type mice and mice with PTEC-specific AT-1R gene deletion. Deletion of the AT-1R gene in PTECs lowered both kidney expression and urine excretion of HO-1, confirming AngII/AT-1R mediated regulation of HO-1. In summary, our in vitro experiments identified novel molecular markers of AngII activity in PTECs and the animal studies demonstrated that these markers also reflect AngII activity in PTECs in vivo. These interesting proteins hold promise as specific markers of renal AngII activity in patients and in experimental models.

Zusammenfassung Einleitung: Eimeria tenella ist ein obligat intrazellulärer Parasit und Erreger der Blinddarmkokzidiose beim Huhn. Aufgrund des verbreiteten Einsatzes von Antikokzidia sind seit langem Resistenzentwicklungen bekannt. Die zugrundeliegenden Mechanismen sind komplex und nicht genau bekannt. Für den Nachweis von Resistenz gilt der Tierversuch als „Goldstandard“, er ist aber aufwändig, zeitraubend und ethisch problematisch. Ziele der Arbeit: Es sollte ein für die Erstellung von Sensitivitätsprofilen und Wirksamkeitsprüfungen geeignetes In-vitro-Modell für E. tenella entwickelt und validiert werden. Weiterhin sollte auf mit Antikokzidiaresistenz assoziierte Änderungen im Proteom von Kokzidien untersucht werden. Material, und Methoden: Monolayer von MDBK (Madin-Darby bovine kidney cells) dienten zur Kultivierung von E. tenella für den in vitro ASA (anticoccidial sensitivity assay). Als Parameter wurden die Inhibition (%ISIA, %IRIA) der Invasion durch Sporozoiten (SIA = sporozoite invasion inhibition assay) und der Erregerreproduktion (RIA = reproduction inhibition assay) mittels quantitativer PCR (qPCR) für E. tenella beurteilt. Die minimale Hemmkonzentration (MIC = minimal inhibitory concentration) wurde anhand einer Referenz (sensitiver Laborstamm Houghton, Ref-1) für Monensin (Mon), Salinomycin (Sal), Maduramicin (Mad), Lasalocid (Las) und Toltrazuril (Tol) bestimmt. Auf dieser Basis wurden ein zweiter Laborstamm (Wisconsin, Ref-2) und vier Feldstämme (T-376, FS-1, FS-2 und FS-3) im SIA und RIA getestet. 1.0 × 105 (SIA) und 5.0 × 104 (RIA) Sporozoiten/Well wurden verwendet. Die in vitro Ergebnisse wurden mit den in vivo Daten verglichen. Alle in vitro Versuche wurden in vier Replikaten durchgeführt. Insgesamt wurden 420 Hühner (1. Lebenstag: LT, Cobb-500) in fünf Tierversuche verwendet, um die Empfindlichkeit der E. tenella-Stämme gegen verschiedene Antikokzidia zu untersuchen. In jedem Tierversuch wurden 84 Tiere (12. LT) in 7 Gruppe (n = 12) eingeteilt. Hühner wurden mit 7,5 × 104 Oozysten je Tier am 14. LT infiziert. Die Wirksamkeitsprüfung für den alternativen Naturstoff Allicin erfolgte mittels RIA am Stamm Ref-1. Kontinuierlich in vitro als Tachyzoiten kultivierte T. gondii des RH-Stammes (Sen-RH) wurden unter ansteigenden Mon-Dosen in HFF-Zellen (human foreskin fibroblasts) über 8 Monate auf Resistenz (MonR-RH) selektiert. Es erfolgte eine stabile Isotopenmarkierung (SILAC). Für E. tenella wurden die sensitivenReferenzstämme ebenso wie die Feldstämme variabler Sensitivität isoabarisch-chemisch markiert (Tandem-Massenmarkierung, TMT). Die quantitativen Proteomanalysen erfolgten mit dem nanoUPLC-MS/MS. Ergebnisse: Für Ref-1 betrugen die MIC95 (MIC für eine 95%ige Inhibition) 0,25 (Mon), 0,5 (Sal), 1,0 (Mad), 0,5 (Las) und 5,0 (Tol) μg/ml. Die in vitro Sensitivitätsprofile für Ref-2, FS-1, FS-2, und FS-3 stimmten für die Ionophoren mit den Ergebnissen des Tierversuchs gut überein, für Tol war das aber nicht der Fall. Signifikante Korrelationen wurden für die %IRIA-Werte mit dem Oozystenindex (OI, r = 0,290-0,507; p < 0,05), dem lesion score (LS, r = 0,279-0,345; p < 0.05, für Ref-1, Ref-2 und FS-1) und der Gewichtszunahme im Tierversuch (WGNNC, r = 0.284-0.419; p < 0.05, für Ref-1 und FS-1) festgestellt. Allicin in einer Konzentration von 1,8 mg/ml ergab einen %IRIA von 99,0%, was einer guten Wirkung entspricht. Mittels SILAC wurden 1820 Proteine identifiziert, von denen 1024 Proteine in mehr als vier biologischen Replikaten quantifizierbar waren. Bei 52 Proteinen wurden signifikante Unterschiede zwischen Sen-RH und MonR-RH festgestellt (p < 0,05). Actin, beta-Tubulin cofactor D, Perforin-like Protein 1 (PLP1), und kleine GTPase-vermittelte Signaltransduktionsproteine (GTPases) wurden in T. gondii MonR-RH hochreguliert (p < 0,05). Insgesamt waren 42 Proteine bei der resistenten Mutante MonR-RH hochreguliert. Für E. tenella wurden 97 Proteine identifiziert und 25 Proteine wurden in allen drei biologischen Replikaten quantifiziert. Mon-resistente E. tenella zeigten signifikant hochreguliertes Actin (p < 0,05). Mikronemenproteine wurden in resistenten Toxoplasma (MIC8) und Eimeria (MIC4) herunter reguliert. Schlussfolgerungen: Das entwickelte In-vitro-Verfahren in Kombination mit der qPCR eignet sich zur Bewertung der Sensitivität von E. tenella gegenüber ionophoren Antikokzidia und erbringt Ergebnisse, die mit dem Tierversuch korrelieren. Das Modell kann Tierversuche zum Screening von Wirkstoffen reduzieren, aber nicht vollständig ersetzen, vor allem, wenn die Wirkung (auch) späte Entwicklungsstadien im Zyklus von E. tenella betrifft. Letzteres könnte die mangelnde Aussagekraft für Tol erklären. In T. gondii lässt sich Resistenz in vitro induzieren. Die Proteom-Analyse zeigte eine Verminderung der Invasion (↑ Actin, ↓ MIC8) und des Austritts (↓ PLP1), und eine Aktivierung der Replikation (↑ Actin, ↑ beta-Tubulin cofactor D, und ↑ GTPases proteine). Mon führt zu einer Erhöhung des intrazellulären Na+ gefolgt von einer Erhöhung der Ca++ Konzentrationen. Die Reduzierung der Expression von Proteinen in MonR-RH, die mit Invasion- und Austrittsaktivitäten zusammenhängen, zeigt an, dass dieser Stamm höhere Konzentrationen von Mon verträgt, indem er die ktitische Schwelle der zytosolischen Ca++ Konzentration erhöht ist, die erforderlich ist, um diese Prozesse zu provozieren.

Introduction: Breast cancer is the second highest cause of cancer mortality in women worldwide. Hormonal therapy is considered one of the most effective therapies and is used against luminal-type malignancies. However, 40-50% of tumour cells can develop resistance, thereby limiting the success in breast cancer treatment. In this study, mechanisms of resistance were investigated using a novel multi-stable isotope labelled amino acids (SILAC) proteomics approach in phenotype-specific breast cancer cell lines resistant to endocrine treatment. Method: In vitro chemo-sensitivity (IC50) was determined for MCF7, T47D, MDA-MB-231, MDA-MB-468, MDA-MB-453, BT-20 and MCF-10A breast cell lines using four endocrine-based therapeutic agents (Tamoxifen, 4-Hydroxytamoxifen OHT, Raloxifene, Anastrozole) to select viable strains for resistance studies. MCF7 (luminal-type A) and MDA-MB-231 (triple negative breast cancer, TNBC) were selected and initially subject to OHT or raloxifene exposure with gradual increments for 10 months. WT cells were grown in the absence of drug in parallel as passage controls. Resistant cell lines were assessed by MTT and IF for comparison with parental cell lines. Resistant cell lines, along with the passage control and a SILAC control, were grown in “light” SILAC medium together with WT strains cultured in “heavy” SILAC medium. Proteins were extracted, concentrations determined and analysed by SDS PAGE for quality control. An aliquot of each “light” cell line (resistant, passage control or SILAC control) was combined with an equal amount of “heavy” WT, trypsin digested and analysed by nano-HPLC Orbitrap Fusion mass spectrometry (2D-LC MS/MS). Proteins were identified by database searching using MascotTM. Relative changes (resistant/WT ratio) in protein levels were determined and bioinformatics tools (STRING and UniProt) used to explore significantly changed pathways associated with resistance. Western blotting was used to verify selected target proteins. Results: Four consistently resistant sublines were generated MCF7 OHT Res (2.00-fold more resistant), MCF7 Ralx Res (2.00-fold), MDA-MB-231 OHT Res (1.90-fold change) and MDA-MB-231 Ralx Res (2.00-fold), in addition to two high passage controls. ER expression by IF was decreased in MCF7 OHT Res compared to the WT and MCF7 Ralx Res, whereas CD44 was increased. Proteomic analysis revealed 2247 and 2880 total proteins in MCF7 OHT Res and MCF7 Ralx Res whilst 3471 and 3495 total proteins were identified in MDA-MB-231 OHT Res and MDA-MB-231 Ralx Res, respectively. Bioinformatics tools identified significantly changed pathways included apoptosis, cytoskeleton, cell motility and redox cell homeostasis. Components of the MAPK-signalling cascade were consistently found to be upregulated in resistant cell lines. MAPK1 (ERK2), previously associated with tamoxifen resistance was increased in MDA-MB-231 Ralx Res cell lines by 4.45-fold and confirmed by Western blotting. Sorcin, which contributes to calcium homeostasis and is also linked to multidrug resistance was increased 4.11- and 2.35-fold in MCF7 OHT Res and Ralx Res sub cell lines, respectively. Some results, such as those for c-Jun, were inconsistent between proteomic analysis and Western blotting and require further investigation. Conclusion: The unique resistant cell lines generated here, as well the MCF7 OHT resistant line, provided novel data that give insights into the biological pathways involved in mechanisms of endocrine drug resistance in breast cancer. Proteomics analysis provided extensive data on common functionality and pathways across the resistant cell lines independent of phenotype or SERM. Overall, the results provided interesting targets for re-sensitising resistant breast cancer and the potential to investigate novel combination therapies in the future. / Al-Ahliyya Amman University scholaships

Yes / One of the main obstacles to therapeutic success in colorectal cancer (CRC) is the development of acquired resistance to treatment with drugs such as 5-fluorouracil (5-FU). Whilst some resistance mechanisms are well known, it is clear from the stasis in therapy success rate that much is still unknown. Here, a proteomics approach is taken towards identification of candidate proteins using 5-FU-resistant sublines of human CRC cell lines generated in house. Using a multiplexed stable isotope labelling with amino acids in cell culture (SILAC) strategy, 5-FU-resistant and equivalently passaged sensitive cell lines were compared to parent cell lines by growing in Heavy medium with 2D liquid chromatography and Orbitrap Fusion™ Tribrid™ Mass Spectrometry analysis. Among 3003 commonly quantified proteins, six (CD44, APP, NAGLU, CORO7, AGR2, PLSCR1) were found up-regulated, and six (VPS45, RBMS2, RIOK1, RAP1GDS1, POLR3D, CD55) down-regulated. A total of 11 of the 12 proteins have a known association with drug resistance mechanisms or role in CRC oncogenesis. Validation through immunodetection techniques confirmed high expression of CD44 and CD63, two known drug resistance mediators with elevated proteomics expression results. The information revealed by the sensitivity of this method warrants it as an important tool for elaborating the complexity of acquired drug resistance in CRC. / Sadr ul-Shaheed and the University of Bradford Proteomics Facility were supported by Yorkshire Cancer Research, UK (Cancer Medicine Discovery II, grant B381PA).

No description available.

In the field of proteomics, proteins are analyzed and quantified in high numbers. Protein analysis is of great importance and can for example generate information regarding protein function and involvement in disease. Different strategies for protein analysis and quan- tification have emerged, suitable for different applications. The focus of this thesis lies on protein identification and quantification using different setups and method development has a central role in all included papers. The presented research can be divided into three parts. Part one describes the develop- ment of two different screening methods for His6-tagged recombinant protein fragments. In the first investigation, proteins were purified using immobilized metal ion affinity chro- matography in a 96-well plate format and in the second investigation this was downscaled to nanoliter-scale using the miniaturized sample preparation platform, integrated selective enrichment target (ISET). The aim of these investigations was to develop methods that could work as an initial screening step in high-throughput protein production projects, such as the Human Protein Atlas (HPA) project, for more efficient protein production and purification. In the second part of the thesis, focus lies on quantitative proteomics. Protein fragments were produced with incorporated heavy isotope-labeled amino acids and used as internal standards in absolute protein quantification mass spectrometry experiments. The aim of this investigation was to compare the protein levels obtained using quanti- tative mass spectrometry to mRNA levels obtained by RNA sequencing. Expression of 32 different proteins was studied in six different cell lines and a clear correlation between protein and mRNA levels was observed when analyzing genes on an individual level. The third part of the thesis involves the antibodies generated within the HPA project. In the first investigation a method for validation of antibodies using protein immunoenrichment coupled to mass spectrometry was described. In a second study, a method was developed where antibodies were used to capture tryptic peptides from a digested cell lysate with spiked in heavy isotope-labeled protein fragments, enabling quantification of 20 proteins in a multiplex format. Taken together, the presented research has expanded the pro- teomics toolbox in terms of available methods for protein analysis and quantification in a high-throughput format. / <p>QC 20141022</p>

No description available.