Browsing by Subject "proteomics"

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  • Partanen, Asta (Helsingfors universitet, 2013)
    Cyanobacteria are aquatic and terrestrial organisms that have photosynthetic cababilities. Cyanobacteria are classified to five orders according to their morphology. These are Chroococcales, Pleurocapsales, Oscillatoriales, Nostocales and Stigonematales. Anabaena sp. 90 is a filamentous cyanobacterium in the order of Nostocales. A sample of this cyanobacterium was isolated from the lake Vesijärvi in 1986. Anabaena sp. 90 produces a variety of bioactive peptides such as microcystins, anabaenopeptins and anabaenopeptilides which are the most commonly studied of the peptides produced by the strain. Majority of the bioactive peptides are produced by non-ribosomal peptide synthetases (NRPS). The way these compounds affect cyanobacteria is currently unclear. Anabaenopeptilide deficient mutant (apd-) variety has been developed from the Anabaena sp 90 wild type (WT). The mutant is unable to produce anabaenopeptilides. The aim of my Master´s thesis was to study the changes in proteomes of WT and apd- strains by conducting a six day cultivation experiment. The purpose was to evaluate the possible differences in the amount of proteins that the different cultivation stocks were able to produce. The secondary purpose was to examine the transcription of anabaenopeptilide synthetase genes. Proteomic study was performed using a procedure called two dimensional differential gel electrophoresis (2D DIGE). The differences in the proteomes´ statistics were examined by using the Student´s t test. Transcription of anabaenopeptilide synthetase genes were studied using a reverse transcriptase polymerase chain reaction (RT-PCR). The data collected from these studies concluded that there were differences in the proteomes of WT and apd- strains. In eighteen cases there were significant differences in the protein amounts between the strains. The single protein counts were often higher in the apd- than in the WT strain. Anabaenopeptilide synthetase genes were transcribed in both strains which suggests that the mutation in the apd- strain did not hinder the ability of the strain to produce anabaenopeptilide transcripts. The identification of significant proteins would yield more detailed knowledge of the metabolical functions of WT and apd- strains.
  • Teikari, Jonna (Helsingfors universitet, 2013)
    Cyanobacteria are phototrophic organisms. They usually occur in water but many species also live in terrestrial habitats, e.g. in symbiotic relationships with fungus. Inorganic phosphorus (Pi) is usually considered to be a limiting factor for the growth of cyanobacteria living in water, since cyanobacteria can use only Pi as a direct source of phosphorus. It has been shown that cyanobacteria have pho-regulon similar to that of Escherichia coli. Pho-regulon can transport and assimilate inorganic phosphate. Cyanobacteria usually produce a wide range of bioactive compounds, which can e.g. be toxic or prevent growth of other bacteria, fungi or yeast. Many of these compounds are produced by non-ribosomal peptide synthetases (NRPS). The aim of this study was to investigate changes in Anabaena sp. 90 proteome, and differences in amounts of bioactive compounds produced by the strain, while growing it in media with high (5,5 mg/l) or low (0,05 mg/ml) Pi concentration. Before moving the culture into two different Pi media, phosphorus storages of the culture were emptied by growing the strain in the media without Pi. In this study, 2D differential gel electrophoresis (2D-DIGE) coupled with LC/MS was used to study proteomes of the organism. Bioactive compounds were also analyzed by LC/MS. Anabaena sp. 90 was chosen because of its fully sequenced and annotated genome. The strain has been found to produce microcystins, anabaenopeptins and anabaenopeptilides. Eleven protein spots with significantly increased or decreased protein quantities were identified in the low Pi media. Ten of them were identified as proteins, which participate in bacterial stringent response. Stringent response is activated when culture is achieving the stationary phase. These stringent response proteins participate in the amino-acid metabolism and translation. One of the proteins was shown to be a ribosomal protein. In addition, the identified proteins included ribulose-bisphosphate carboxylase oxygenase (RuBisCO), which had a significantly lower concentration in the cells in low-phosphorous media. There were no significant differences in amounts of bioactive compounds when growing the culture in low and high Pi media. More replicates could be used, when the study of bioactive compounds is repeated.
  • Ottman, Noora; Huuskonen, Laura; Reunanen, Justus; Boeren, Sjef; Klievink, Judith; Smidt, Hauke; Belzer, Clara; de Vos, Willem M. (2016)
    Akkermansia muciniphila is a common member of the human gut microbiota and belongs to the Planctomycetes-Verrucomicrobia-Chlamydiae superphylum. Decreased levels of A. muciniphila have been associated with many diseases, and thus it is considered to be a beneficial resident of the intestinal mucus layer. Surface-exposed molecules produced by this organism likely play important roles in colonization and communication with other microbes and the host, but the protein composition of the outer membrane (OM) has not been characterized thus far. Herein we set out to identify and characterize A. muciniphila proteins using an integrated approach of proteomics and computational analysis. Sarkosyl extraction and sucrose density-gradient centrifugation methods were used to enrich and fractionate the OM proteome of A. muciniphila. Proteins from these fractions were identified by LC-MS/MS and candidates for OM proteins derived from the experimental approach were subjected to computational screening to verify their location in the cell. In total we identified 79 putative OM and membrane-associated extracellular proteins, and 23 of those were found to differ in abundance between cells of A. muciniphila grown on the natural substrate, mucin, and those grown on the non-mucus sugar, glucose. The identified OM proteins included highly abundant proteins involved in secretion and transport, as well as proteins predicted to take part in formation of the pili-like structures observed in A. muciniphila. The most abundant OM protein was a 95-kD protein, termed PilQ, annotated as a type IV pili secretin and predicted to be involved in the production of pili in A. muciniphila. To verify its location we purified the His-Tag labeled N-terminal domain of PilQ and generated rabbit polyclonal antibodies. Immunoelectron microscopy of thin sections immunolabeled with these antibodies demonstrated the OM localization of PilQ, testifying for its predicted function as a type IV pili secretin in A. muciniphila. As pili structures are known to be involved in the modulation of host immune responses, this provides support for the involvement of OM proteins in the host interaction of A. muciniphila. In conclusion, the characterization of A. muciniphila OM proteome provides valuable information that can be used for further functional and immunological studies.
  • Tavakoli, Shirin; Kari, Otto; Turunen, Tiina; Lajunen, Tatu; Schmitt, Mechthild; Lehtinen, Julia; Tasaka, Fumitaka; Parkkila, Petteri; Ndika, Joseph; Viitala, Tapani; Alenius, Harri; Urtti, Arto; Subrizi, Astrid (2021)
  • Ndika, Joseph; Airaksinen, Liisa; Suojalehto, Hille; Karisola, Piia; Fyhrquist, Nanna; Puustinen, Anne; Alenius, Harri (2017)
    Background: Seasonal allergic rhinitis (SAR) caused by intermittent exposure to seasonal pollen causes itching, nasal congestion, and repeated sneezing, with profound effects on quality of life, work productivity, and school performance. Although both the genotype and environmental factors can contribute to the immunologic basis of allergic reactions, the molecular underpinnings associated with the pathogenesis of allergic rhinitis are not entirely clear. Methods: To address these questions, nasal epithelial brushings were collected from 29 patients with SAR and 31 control subjects during and after the pollen season. We then implemented an orbitrap-based, bottom-up, label-free quantitative proteomics approach, followed by multivariate analyses to identify differentially abundant (DA) proteins among the 4 sample groups. Results: We identified a total of 133 DA proteins for which the most significantly overrepresented functional category was found to be interferon 1 signaling. Two proteins, cystatin 1 and myeloblastin, the former of which protects against protease activity of allergens and the latter with a role in epithelial barrier function, were DA in patients with SAR and control subjects, irrespective of season. Moreover, interferon-inducible protein with tetratricopeptide repeats 1, cystatin 1, and interferon-inducible protein with tetratricopeptide repeats 3 were found to be differentially regulated between patients with SAR and control subjects, with inverse abundance dynamics during the transition from fall to spring. Conclusion: We identified type 1 interferon-regulated proteins as biomarkers in patients with SAR, potentially playing an important role in its pathogenesis. Moreover, when compared with patients with SAR, healthy subjects exhibit an antagonistic proteomic response across seasons, which might prove to be a therapeutic target for disease prevention.
  • Cypryk, Wojciech (Helsingin yliopisto, 2016)
    Extracellular vesicles (EVs) are small, membranous entities secreted from most eukaryotic cells at both homeostatic and stress conditions. Carrying active biological molecules nucleic acids, lipids and proteins EVs serve as important means of intercellular communication. In the immune system, EVs circulting in body fluids play important modulatory roles in coordination of responses. EVs are integral part of secretome all proteins secreted by cells. EVs provide means for secretion of proteins that are not trafficked through conventional, ER/Golgi-mediated mechanisms. Analysis of EV proteome provides basis for fundamental discoveries in understanding the biogenesis, secretion and delivery of these nanosized messengers to target cells. Macrophages are principal tissue-resident effector cells of innate immune system, performing surveillance of their neighborhood in search for danger. Macrophages express pattern recognition receptors (PRRs) which recognize conserved pathogen-associated molecular patterns (PAMPs) conserved range of molecules expressed by pathogens. PRRs also recognize endogeneous ligands that appear in the human body in other dangerous conditions and diseases. These are collectively called danger-associated molecular patterns (DAMPs). Recognition of PAMPs and DAMPs by PRRs triggers intracellular signaling, leading to activation of macrophage defense mechanisms. These begin with inflammation and secretion of pro-inflammatory cytokines, but many other proteins are also actively secreted by activated macrophages. Although inflammatory cytokine secretion is a well-studied process, very few studies investigating overall and EV-mediated protein secretion from human macrophages were performed. This thesis aims at broadening our understanding of EV-mediated protein secretion from human macrophages activated in response to selected innate immune activators, namely extracellular adenosine triphosphate (ATP), a potent DAMP released during cell damage; (1,3)-β-glucan, a polysaccharide component of fungal cell wall, activating dectin-1-mediated signaling and influenza A virus (IAV) - common seasonal pathogenic virus targeting lung epithelia and macrophages. Total secretome and purified EVs were analyzed using different high-throughput mass spectrometry-based methods and further explored using bioinformatic and biochemical studies. The presented results provide evidence that EV-mediated protein secretion is an integral part of responses of human macrophages to studied stimuli. Its activation is demonstrated with quantitative and comparative proteomic approaches. The secreted vesicles are characterized by a broad size range consistent with both exosomes and microvesicles, demonstrating that both types of vesicular structures are involved in protein secretion. The EVs carry distinct set of immunologically important proteins, and bioinformatic analysis suggests that the secreted EVs may exert immunomodulatory effects on recipient cells. It is shown that during IAV infection, EVs are mediators of pro-inflammatory and antiviral cytokine release, thus they may serve as protective capsules of targeted cytokine delivery. Proteomic analysis identified also a broad set of DAMPs unconventionally secreted in association with EVs, further extrapolating their function towards danger signaling in cellular immune responses. The study on ATP-mediated responses further investigates the intracellular signaling involving calpains, abundant cytosolic proteases, identifying their crucial roles downstream P2X7 receptor: in EV release, as well as inflammasome activation and IL-1β secretion. The data presented here indicate that EVs serve as unconventional means for secretion of a broad range of proteins secreted in PRR-mediated responses in human macrophages. Bioinformatic and functional analysis identifies potential processes involved in their generation as well as their roles in intercellular communication. Together, the presented thesis contributes to our understanding of unconventional, EV-mediated protein secretion in macrophage responses towards common, physiologically releveant threats. The studies presented here will serve as basis for further detailed functional analysis of the roles of EVs in communication between macrophages and other immune system cells. It will also lay the grounds for future studies involving EV-mediated macrophage responses in patients with fungal and viral infections.
  • Holm, Matilda; Joenväärä, Sakari; Saraswat, Mayank; Mustonen, Harri; Tohmola, Tiialotta; Ristimäki, Ari; Renkonen, Risto; Haglund, Caj (2019)
    Abstract Colorectal cancer (CRC) stands for 10% of the worldwide cancer burden and has recently become the second most common cause of cancer death. The 5-year survival rate depends mainly on stage at diagnosis. Mass spectrometric proteomic analysis is widely used to study the plasma proteome, which is complex and contains multitudes of proteins. In this study, we have used Ultra Performance Liquid Chromatography-Ultra Definition Mass Spectrometry (UPLC-UDMSE)-based proteomics to analyze plasma samples from 76 CRC patients. We identified several plasma proteins, such as CP, TVP23C, FETUB, and IGFBP3, of which altered levels led to significant differences in survival, as seen by Cox regression and Kaplan-Meier analysis. Additionally, during Cox regression analysis, samples were adjusted for age and/or tumor stage, enabling stringent analysis. These proteins, although in need of further validation, could be of use during patient follow-up, as their levels can non-invasively be measured from blood samples, and could be of use in predicting patient outcome. Several of these proteins additionally have roles in metabolism and inflammation, two processes central to the development and progression of cancer, further indicating their importance in cancer.
  • Mlocicki, Daniel; Sulima, Anna; Bień, Justyna; Näreaho, Anu; Zawistowska-Deniziak, Anna; Basałaj, Katarzyna; Sałamatin, Rusłan; Conn, David Bruce; Savijoki, Kirsi (2018)
    In cestodiasis, mechanical and molecular contact between the parasite and the host activates the immune response of the host and may result in inflammatory processes, leading to ulceration and intestinal dysfunctions. The aim of the present study was to identify antigenic proteins of the adult cestode Hymenolepis diminuta by subjecting the total protein extracts from adult tapeworms to 2DE immunoblotting (two-dimensional electrophoresis combined with immunoblotting) using sera collected from experimentally infected rats. A total of 36 protein spots cross-reacting with the rat sera were identified using LC-MS/MS. As a result, 68 proteins, including certain structural muscle proteins (actin, myosin, and paramyosin) and moonlighters (heat shock proteins, kinases, phosphatases, and glycolytic enzymes) were identified; most of these were predicted to possess binding and/or catalytic activity required in various metabolic and cellular processes, and reported here as potential antigens of the adult cestode for the first time. As several of these antigens can also be found at the cell surface, the surface-associated proteins were extracted and subjected to in-solution digestion for LC-MS/MS identification (surfaceomics). As a result, a total of 76 proteins were identified, from which 31 proteins, based on 2DE immunoblotting, were predicted to be immunogenic. These included structural proteins actin, myosin and tubulin as well as certain moonlighting proteins (heat-shock chaperones) while enzymes with diverse catalytic activities were found as the most dominating group of proteins. In conclusion, the present study shed new light into the complexity of the enteric cestodiasis by showing that the H. diminuta somatic proteins exposed to the host possess immunomodulatory functions, and that the immune response of the host could be stimulated by diverse mechanisms, involving also those triggering protein export via yet unknown pathways.
  • Sillanpää, Annika (Helsingin yliopisto, 2020)
    The scientific data has demonstrated that surface-associated proteins have a significant role affecting the adaptation to GIT environment, adherence to the intestinal mucus and other potential health benefits occurring through cross-talk of propionibacteria and the host. The reported achievements on the complementary proteomic approaches optimize the accuracy of surface protein identification and surface proteins related to anti-inflammatory activities and adhesion has been identified from Propionibacterium freudenreichii strains. Thus, the aim of this thesis was to compare the effect of atmospheric conditions on surfaceome expression of the P. freudenreichii type strain DSM 20271. Bacterial cultures cultivated in aerobic and anaerobic atmosphere were harvested at the mid-exponential phase of growth. Samples were subjected to gel-free proteomic analysis, based on direct analysis of peptides acquired by trypsin cell-surface shaving followed by identification of released surface-attached proteins and peptides using LC-MS/MS and label-free quantification. It was demonstrated in this work that different atmospheric conditions highly influenced the protein expression patterns. Overall, the expression of more than a hundred proteins were affected by the change of environmental condition, of which the majority were predicted not to include either classical nor non-classical secretion motifs. It is still unresolved question if these cytoplasmic proteins in the shaved-fraction could be transported to the bacterial surface area by uncharacterized mechanisms to serve a specific moonlighting function. Few of the identified proteins, most of which were up-regulated in aerobic growth condition, were already described in other bacterial species to be involved with general stress response (ClpP, ClpB, and Ctc) and reduction of reactive oxygen species (Tpx, AhpC, and AhpF).
  • Sutinen, Elina M.; Korolainen, Minna A.; Hayrinen, Jukka; Alafuzoff, Irina; Petratos, Steven; Salminen, Antero; Soininen, Hilkka; Pirttila, Tuula; Ojala, Johanna O. (2014)
  • Talman, Virpi; Teppo, Jaakko Sakari; Pöhö, Päivi Anneli; Movahedi, Parisa; Vaikkinen, Anu; Karhu, Suvi Tuuli; Trošt, Kajetan; Suvitaival, Tommi; Heikkonen, Jukka; Pahikkala, Tapio; Kotiaho, Ahti Antti Tapio; Kostiainen, Risto Kalervo; Varjosalo, Markku Tapio; Ruskoaho, Heikki Juhani (2018)
    Background The molecular mechanisms mediating postnatal loss of cardiac regeneration in mammals are not fully understood. We aimed to provide an integrated resource of mRNA, protein, and metabolite changes in the neonatal heart for identification of metabolism‐related mechanisms associated with cardiac regeneration. Methods and Results Methods and results Mouse ventricular tissue samples taken on postnatal day 1 (P01), P04, P09, and P23 were analyzed with RNA sequencing and global proteomics and metabolomics. Gene ontology analysis, KEGG pathway analysis, and fuzzy c‐means clustering were used to identify up‐ or downregulated biological processes and metabolic pathways on all 3 levels, and Ingenuity pathway analysis (Qiagen) was used to identify upstream regulators. Differential expression was observed for 8547 mRNAs and for 1199 of 2285 quantified proteins. Furthermore, 151 metabolites with significant changes were identified. Differentially regulated metabolic pathways include branched chain amino acid degradation (upregulated at P23), fatty acid metabolism (upregulated at P04 and P09; downregulated at P23) as well as the HMGCS (HMG‐CoA [hydroxymethylglutaryl‐coenzyme A] synthase)–mediated mevalonate pathway and ketogenesis (transiently activated). Pharmacological inhibition of HMGCS in primary neonatal cardiomyocytes reduced the percentage of BrdU‐positive cardiomyocytes, providing evidence that the mevalonate and ketogenesis routes may participate in regulating the cardiomyocyte cell cycle. Conclusions This study is the first systems‐level resource combining data from genomewide transcriptomics with global quantitative proteomics and untargeted metabolomics analyses in the mouse heart throughout the early postnatal period. These integrated data of molecular changes associated with the loss of cardiac regeneration may open up new possibilities for the development of regenerative therapies
  • Soderholm, Sandra; Fu, Yu; Gaelings, Lana; Belanov, Sergey; Yetukuri, Laxma; Berlinkov, Mikhail; Cheltsov, Anton V.; Anders, Simon; Aittokallio, Tero; Nyman, Tuula A.; Matikainen, Sampsa; Kainov, Denis E. (2016)
    Human influenza A viruses (IAVs) cause global pandemics and epidemics. These viruses evolve rapidly, making current treatment options ineffective. To identify novel modulators of IAV-host interactions, we re-analyzed our recent transcriptomics, metabolomics, proteomics, phosphoproteomics, and genomics/virtual ligand screening data. We identified 713 potential modulators targeting 199 cellular and two viral proteins. Anti-influenza activity for 48 of them has been reported previously, whereas the antiviral efficacy of the 665 remains unknown. Studying anti-influenza efficacy and immuno/neuro-modulating properties of these compounds and their combinations as well as potential viral and host resistance to them may lead to the discovery of novel modulators of IAV-host interactions, which might be more effective than the currently available anti-influenza therapeutics.
  • Gallud, Audrey; Delaval, Mathilde; Kinaret, Pia; Marwah, Veer Singh; Fortino, Vittorio; Ytterberg, Jimmy; Zubarev, Roman; Skoog, Tiina; Kere, Juha; Correia, Manuel; Loeschner, Katrin; Al-Ahmady, Zahraa; Kostarelos, Kostas; Ruiz, Jaime; Astruc, Didier; Monopoli, Marco; Handy, Richard; Moya, Sergio; Savolainen, Kai; Alenius, Harri; Greco, Dario; Fadeel, Bengt (2020)
    Despite considerable efforts, the properties that drive the cytotoxicity of engineered nanomaterials (ENMs) remain poorly understood. Here, the authors inverstigate a panel of 31 ENMs with different core chemistries and a variety of surface modifications using conventional in vitro assays coupled with omics-based approaches. Cytotoxicity screening and multiplex-based cytokine profiling reveals a good concordance between primary human monocyte-derived macrophages and the human monocyte-like cell line THP-1. Proteomics analysis following a low-dose exposure of cells suggests a nonspecific stress response to ENMs, while microarray-based profiling reveals significant changes in gene expression as a function of both surface modification and core chemistry. Pathway analysis highlights that the ENMs with cationic surfaces that are shown to elicit cytotoxicity downregulated DNA replication and cell cycle responses, while inflammatory responses are upregulated. These findings are validated using cell-based assays. Notably, certain small, PEGylated ENMs are found to be noncytotoxic yet they induce transcriptional responses reminiscent of viruses. In sum, using a multiparametric approach, it is shown that surface chemistry is a key determinant of cellular responses to ENMs. The data also reveal that cytotoxicity, determined by conventional in vitro assays, does not necessarily correlate with transcriptional effects of ENMs.
  • Suojalehto, Hille; Wolff, Henrik; Lindström, Irmeli; Puustinen, Anne (2018)
    Background The mechanisms of work-related asthma (WRA) are incompletely delineated. Nasal cell samples may be informative about processes in the lower airways. Our aim was to determine the nasal protein expression profiles of WRA caused by different kind of exposures. Methods We collected nasal brush samples from 82 nonsmoking participants, including healthy controls and WRA patients exposed to (i) protein allergens, (ii) isocyanates and (iii) welding fumes the day after relevant exposure. The proteome changes in samples were analysed by two-dimensional difference gel electrophoresis, and the differentially regulated proteins found were identified by mass spectrometry. Immunological comparison was carried out using Western blot. Results We detected an average of 2500 spots per protein gel. Altogether, 228 protein spots were chosen for identification, yielding 77 different proteins. Compared to the controls, exposure to protein allergens had the largest effects on the proteome. Hierarchical clustering revealed that protein allergen- and isocyanate-related asthma had similar profiles, whereas asthma related to welding fumes differed. The highly overrepresented functional categories in the asthma groups were defence response, protease inhibitor activity, inflammatory and calcium signalling, complement activation and cellular response to oxidative stress. Immunological analysis confirmed the found abundance differences in galectin 10 and protein S100-A9 between the groups. Conclusions Work-related asthma patients exposed to protein allergens and isocyanates elicit similar nasal proteome responses and the profiles of welders and healthy controls were alike. Revealed biological activities of the protein expression changes are associated with allergic inflammation and asthma.
  • Hellinen, Laura; Sato, Kazuki; Reinisalo, Mika; Kidron, Heidi; Rilla, Kirsi; Tachikawa, Masanori; Uchida, Yasuo; Terasaki, Tetsuya; Urtti, Arto (2019)
    PURPOSE. Retinal pigment epithelium (RPE) limits the xenobiotic entry from the systemic blood stream to the eye. RPE surface transporters can be important in ocular drug distribution, but it has been unclear whether they are expressed on the apical, basal, or both cellular surfaces. In this paper, we provide quantitative comparison of apical and basolateral RPE surface proteomes. METHODS. We separated the apical and basolateral membranes of differentiated human fetal RPE (hfRPE) cells by combining apical membrane peeling and sucrose density gradient centrifugation. The membrane fractions were analyzed with quantitative targeted absolute proteomics (QTAP) and sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH-MS) to reveal the membrane protein localization on the RPE cell surfaces. We quantitated 15 transporters in unfractionated RPE cells and scaled their expression to tissue level. RESULTS. Several proteins involved in visual cycle, cell adhesion, and ion and nutrient transport were expressed on the hfRPE plasma membranes. Most drug transporters showed similar abundance on both RPE surfaces, whereas large neutral amino acids transporter 1 (LAT1), p-glycoprotein (P-gp), and monocarboxylate transporter 1 (MCT1) showed modest apical enrichment. Many solute carriers (SLC) that are potential prodrug targets were present on both cellular surfaces, whereas putative sodium-coupled neutral amino acid transporter 7 (SNAT7) and riboflavin transporter (RFT3) were enriched on the basolateral and sodium- and chloride-dependent neutral and basic amino acid transporter (ATB(0+)) on the apical membrane. CONCLUSIONS. Comprehensive quantitative information of the RPE surface proteomes was reported for the first time. The scientific community can use the data to further increase understanding of the RPE functions. In addition, we provide insights for transporter protein localization in the human RPE and the significance for ocular pharmacokinetics.
  • Järvinen, Hanna (Helsingfors universitet, 2014)
    Reverse-phase protein microarray, RPMA, is a novel and promising technology for proteomic profiling. The low sample consumption, high-throughput format, high sensitivity and good precision make RPMA attractive tool for clinical use. In RPMA, cellular lysates obtained from various sources (e.g. clinical samples, cell lines) are arrayed onto a substratum as a small spots such that an array is comprised of hundreds to thousands of different samples. The array is incubated with a capture molecule (e.g. antibody) that is validated to recognize the analyte of interest. Signal is created by labelled secondary antibody and the signal is detected by colorimetry, chemiluminescence or fluorescence methods. The literature part introduces the RPMA technology and its applications. RPMA have been utilized in versatile applications for example in cell signal pathway profiling, drug discovery and discovery and validation of biomarkers. In the future, it is hoped to allow individual therapy regimes and the evaluation of treatment efficacy. The aim of the experimental part was to culture various cell lines and prepare lysates for RPMA. The lysates were prepared of ARPE-19, HepG2, Hepa-RG, SKOV-3-ip1, SKOV-3, Caco-2, hCMEC/D3, HCE and D-407 cell cultures. The lysates were stored in -80 °C for subsequent use in RPMAs. The purpose was to optimize the method and based on the optimization studies, to print one RPMA. Cell lysates were arrayed onto nitrocellulose coated glass slide using Nano-Plotter (Gesim)-device which allows automated sample printing. β-actin and α-tubulin proteins were assessed from the samples. To create the signal, fluorescence dye was used, and detected at the visible wavelengths. Based on this study, more optimization is required. The detection method used in the RPMA was not optimal, but the experiment showed promising potential. By taking into account the development issues, the method performance can be significantly improved. Of these issues, perhaps the most important is to use infrared region for the signal detection instead of visible wavelengths.
  • Reigada, Inés; San-Martin-Galindo, Paola; Gilbert-Girard, Shella; Chiaro, Jacopo; Cerullo, Vincenzo; Savijoki, Kirsi; Nyman, Tuula A.; Fallarero, Adyary; Miettinen, Ilkka (2021)
    Bacterial biofilms are an important underlying cause for chronic infections. By switching into the biofilm state, bacteria can evade host defenses and withstand antibiotic chemotherapy. Despite the fact that biofilms at clinical and environmental settings are mostly composed of multiple microbial species, biofilm research has largely been focused on single-species biofilms. In this study, we investigated the interaction between two clinically relevant bacterial pathogens (Staphylococcus aureus and Pseudomonas aeruginosa) by label-free quantitative proteomics focusing on proteins associated with the bacterial cell surfaces (surfaceome) and proteins exported/released to the extracellular space (exoproteome). The changes observed in the surfaceome and exoproteome of P. aeruginosa pointed toward higher motility and lower pigment production when co-cultured with S. aureus. In S. aureus, lower abundances of proteins related to cell wall biosynthesis and cell division, suggesting increased persistence, were observed in the dual-species biofilm. Complementary phenotypic analyses confirmed the higher motility and the lower pigment production in P. aeruginosa when co-cultured with S. aureus. Higher antimicrobial tolerance associated with the co-culture setting was additionally observed in both species. To the best of our knowledge, this study is among the first systematic explorations providing insights into the dynamics of both the surfaceome and exoproteome of S. aureus and P. aeruginosa dual-species biofilms.
  • Ján, Labuda; Richard P., Bowater; Miroslav, Fojta; Günter, Gauglitz; Zdeněk, Glatz; Ivan, Hapala; Jan, Havliš; Ferenc, Kilar; Aniko, Kilar; Lenka, Malinovská; Siren, Heli Marja Marita; Petr, Skládal; Federico, Torta; Martin, Valachovič; Michaela, Wimmerová; Zbyněk, Zdráhal; David Brynn, Hibbert (2018)
    Recommendations are given concerning the terminology of methods of bioanalytical chemistry. With respect to dynamic development particularly in the analysis and investigation of biomacromolecules, terms related to bioanalytical samples, enzymatic methods, immunoanalytical methods, methods used in genomics and nucleic acid analysis, proteomics, metabolomics, glycomics, lipidomics, and biomolecules interaction studies are introduced.
  • Ishchenko, Tatiana (Helsingfors universitet, 2014)
    Propionibacterium freudenreichii is commonly known as a Swiss-type cheese ripening culture, and is also frequently applied in the industrial production of vitamin B12 (cobalamin). One way to increase vitamin B12 productivity is the supplementation of medium with 5,6-dimethylbenzimidazole (DMBI), the lower ligand of cobalamin. The aim of this thesis was to investigate the effect of exogenous DMBI on vitamin B12 synthesis and protein production in type strain of P. freudenreichii ssp. freudenreichii. Results showed that DMBI supplementation improved cobalamin production 2.7- and 2.0-fold in cultures grown in anaerobic and two-step processes, respectively. Two-step incubation enabled the achievement of significantly higher levels of cobalamin both in the absence and presence of DMBI. The highest cobalamin level of 0.6 ?g/ml was achieved by the combination of DMBI supplementation with the two-step process. Proteome maps displayed 474 protein spots which is less than 20% of P. freudenreichii predicted proteome. No significant differences in protein production were found between cultures grown in the absence and presence of DMBI. Only two protein spots were more abundant in presence of DMBI and four spots showed higher abundance in absence of DMBI. None of detected proteins from these spots were found to be directly involved in cobalamin biosynthesis.