Background: Cancer is a common disease, and the choice of treatment becomes more difficult over time due to chemotherapy resistant in cancer cells. To improve the in vitroassay and the individual cancer treatment, a luminescence-based endpoint assay, CellTiter Glo 2.0 was compared with the currently in use fluorescence endpoint assay, fluorometric microculture cytotoxic assay.  Aim: The aim of this study was to validate and compare the CellTiter Glo 2.0 assay with awell-established method (FMCA) and MTT [3-(4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2Htetrazolium bromide] assay. Moreover, investigate whether the generated data can be used as a reference database for validation of patient samples in the future.   Materials and methods: The validation was performed on peripheral blood mononuclear cells from different healthy donors and two cell lines (HCT116-wt and HT-29) of colorectal cancer carcinoma were ordered frozen from American Type Culture Collection. Analysis was also done in solid samples (ovarian and kidney cancer cells). To get as correct evaluation as possible all materials were analyzed in parallel between the two methods.  Results and conclusion: A clear trend was observed when using CellTiter Glo 2.0 assay,post FMCA directly on tumor cells. This setup, makes it possible to collect reference data in the future. In addition, a high spread of the survival index data was noted between the two methods. The reason is still unknown but could be due to the low number of tested tumor cells, therefore more tumor cells need to be tested in future studies

Panther Fusion® system är ett fullständigt automatiserat in vitro diagnostiksystem som har högt flöde av prov-till-resultat och tillåter utförandet av in vitro diagnostiska tester genom användning av realtids Polymerase Chain Reaction (realtids-PCR). Syftet med detta projekt är att validera om det finns möjlighet att använda ett kit från Amplidiag på Panther fusion open access för att diagnostisera vankomycin resistenta enterokocker (VRE) samt att utvärdera analys direkt på E-swab prover istället för anrikningsbuljong. Det är viktigt att kunna detektera vanA-och vanB-generna i prover för att VRE hyser dessa gener. Sensitivitet och effektivitet av Panther fusion open access undersöktes genom att analysera proverna parallellt med Bio-Rad CFX384/96-PCR som är rutinmetoden för VRE-diagnostik på Klinisk mikrobiologi i Lund. Specificitet av en tidigare publicerad PCR-mix (PCR-mix) som kan detektera vanA och vanB i prover jämfördes med Amplidiag assay mix 2 som används i rutinmetoden. För att kunna utvärdera skillnaden av specificiteten och känsligheten mellan E-swab och anrikningsbuljong utfördes analysen på både E-swab och anrikningsbulong med både Panther fusion open access och Bio-Rad CFX384/96-PCR. MgCl2- koncentration optimerades till 3,0 mM. Sensitivitet och effektivitet av Panther fusion open access var högre med PCR-mix än Amplidiag assay mix 2 och uppvisade optimalt linjäritet. Specificitetstest av PCR-mixen visade att den bara kunde detektera vanA och vanB. Resultaten av spikade VRE-negativa patientprover (E-swab) visade sig vara orimliga då flera av proverna och kontrollerna gav resultat för båda generna. För att ersätta VRE-diagnostik på Bio-Rad CFX384/96 med Panther fusion open access behöver ytterligare experiment genomföras direkt på E-swab och vankomycine variabla enterokocker (VVE)-prover på Panther fusion open access. / Panther Fusion® system is a fully automated in vitro diagnostic system that has a high flow of sample-to-results and allows the performance of in vitro diagnostics tests using real-time Polymerase Chain Reaction (real-time PCR). The purpose of this project is to validate whether it is possible to use a kit from Amplidiag on Panther fusion open access to diagnose vancomycin resistant enterococcus (VRE). In addition, this project aims to evaluate and analyze E-swab samples directly instead of enrichment broth. It is important to be able to detect VanA and VanB genes in samples because VRE harbors these genes. Sensitivity and efficiency of Panther fusion open access were tested by analyzing the samples simultaneously with Bio-Rad CFX384/96 which is the routine method for VRE diagnostic at Clinical Microbiology in Lund. Specificity of a previously published PCR-mix (PCR-mix) that can detect VanA and VanB genes in samples was compared with Amplidiag assay mix 2 used in the routine method. In order to evaluate the difference in specificity and sensitivity between E-swab and enrichment broth, the analysis was performed on both E-swab and enrichment broth with both Panther fusion open access and Bio-Rad CFX384/96-PCR. MgCl2 concentration was optimized to 3,0 mM. Both sensitivity and efficiency of Panther fusion open access were higher with PCR-mix than those with Amplidiag assay mix 2 and it showed optimal linearity. Specificity test of PCR-mix detected only VanA and VanB genes. The results of inoculated the negative VRE-patient samples (E-swab) indicated that they were unreasonable because some of the samples and controls gave results of both genes. Replacement of the VRE-diagnostic method on Bio-Rad CFX384/96 with Panther fusion open access requires performing additional experiments directly on E-swab and vancomycin variable enterococcus (VVE) samples using Panther fusion open access.

As the aging population is increasing worldwide, so is the prevalence of neurodegenerativediseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD), frontotemporal dementia(FTD) and amyotrophic lateral sclerosis (ALS). Reliable biomarkers able to aid the diagnosis anddifferentiation of these diseases are needed in order to start the right treatment as early as possible.Due to its representative state of the central nervous system, cerebrospinal fluid (CSF) is afavorable sample material for biomarker discovery within neurodegenerative diseases. Alteredprotein levels of this body fluid might serve as a biomarker, but further validation of earlierfindings is needed. The aim of this project was to validate earlier studies suggesting potentialprotein biomarkers in CSF. From a list of 80 potential biomarkers in the CSF of patient samples,eight were chosen to be included in this validation effort. By utilizing a suspension bead array ina sandwich assay setup, 21 antibodies were tested in an initial screening. Antibody pairs that couldmeasure the protein levels in a dilution dependent manner was further optimized before individualpatient samples were analyzed. Sandwich assays targeting the three proteins Amphiphysin(AMPH), Chitotriosidase-1 (CHIT1) and Beta-synuclein (SNCB) were successfully developed andcorrelated to earlier generated data using a suspension bead array with a single binder setup.Therefore, the earlier findings of elevated levels of AMPH and SNCB in AD patients and CHIT1in ALS patients were successfully validated. / Prevalensen av neurodegenerativa sjukdomar såsom Alzheimers sjukdom (AD), Parkinsonssjukdom (PD), frontallobsdemens (FTD) och amyotrofisk lateralskleros (ALS) ökar i takt med denåldrande populationen. Pålitliga biomarkörer som kan hjälpa till vid diagnostiseringen av dessasjukdomar behövs för att starta rätt behandling så tidigt som möjligt. Ryggmärgsvätska, enkroppsvätska tillhörande det centrala nervsystemet, kan ge en inblick i det centrala nervsystemetstillstånd. Förändrade proteinnivåer i denna kroppsvätska skulle därför kunna fungera sombiomarkörer. Målet i detta projekt var att validera tidigare föreslagna proteinbiomarkörer iryggmärgsvätska. Utifrån en lista av 80 tidigare analyserade proteiner i ryggmärgsvätska hospatienter, inkluderades åtta proteiner i detta valideringsförsök. En antikroppsbaserad så kalladsandwich assay användes i en suspension bead array för att testa 21 stycken antikroppar i ett initialtscreeningsförsök. Antikroppspar som kunde mäta proteinnivåer på ett spädningsberoende vis i detinitiala screeningsförsöket optimerades vidare innan den utvecklade sandwich assayn användes föratt analysera proteinnivåer i individuella prover. Sandwich assays gentemot Amphiphysin(AMPH), Chitotriosidase-1 (CHIT1) och Beta-synuclein (SNCB) kunde bli framtagna ochkorrelerade gentemot tidigare genererat data från en single binder assay på ett framgångsrikt sätt.Projektet kunde därmed validera tidigare fynd som indikerat förhöjda nivåer av AMPH och SNCBi AD patienter, samt förhöjda nivåer av CHIT1 i ALS patienter.

M. Tech. (Department of Biotechnology, Faculty of Applied and Computer Sciences), Vaal University of Technology. / Obesity has steadily increased and represents a major public health problem worldwide, reducing quality of life and causing a range of health problems. Obesity has emerged as the fifth leading risk of global deaths. Annually, 2.8 million adults die as a result of being overweight or obese. The increase of obesity remains inexplicable in terms of genetic susceptibility to obesity. The genetic loci identified by genome-wide association studies (GWASs) explains about 2% of the heritability for obesity. Perhaps other factors such as epigenetics may be involved in the increase of obesity and may offer solutions for the management of obesity. Epigenetics is defined as a heritable change in gene expression without altering the genome sequences. It may help in providing a logical explanation between the genome and environment which shapes obesity risk and may help to explain the "missing heritability". Epigenetics may affect two mechanisms, namely: i) DNA methylation,and ii) histone modifications. DNA methylation might give scientists a link to the rise in obesity.The study aimed to investigate the effect of sugars used as sweeteners in sugar-sweetened beverages (SSB) on DNA methylation in a Caco-2 cell line in vitro. Four major objectives were pursued in the study which were to:(1) stimulate the Caco-2 cells with varying concentrations of sugar sweeteners and assess the morphological changes of the cells; (2) evaluate the cytotoxicity of different concentrations of the sugar sweetener on the Caco-2 cell line using the Alamar blue and LDH assay; (3) obtain genomic DNA from the treated Caco-2 cell line and perform bisulfite conversion and rest; and (4) amplify the WT1, MEG3, TNFRSF9, ATP10A, and CD44 obesity-associated genes and ascertain their degree of methylation. Caco-2 cells were stimulated with sugar sweeteners at varying concentrations (low, medium and high) for an incubation period of 62 days,and images of the cells were captured for morphological characterisation. The incubation condition entailed cells plated in a 12 or 96 well plate, incubated in a humidified 5% CO2 incubator at 37 °C and there is nutrient renewal every three days.Alamar blue, a cell proliferation colourimetric assay and lactate dehydrogenase assays (LDH), a homogenous membrane fluorimetric assay were used for the cytotoxicity studies. The results of the characterisation showed that different concentrations of sugar sweeteners affected the morphology of the cells as the incubation period progressed. The cytotoxicity results of both LDH and Alamar blue depicted low concentration of sweeteners that had low-to-moderate toxicity and the medium and high concentration of the sweeteners had a moderate to high toxicity on the Caco-2 cells. DNA from the Caco-2 cells was extracted. Techniques used to study DNA methylation such as bisulfite conversion, PCR amplification and restriction enzymes that have differential sensitivity to 5-methyl-cytosine were performed. The quality of DNA extracted was good. The bisulfite conversion was conducted andno amplification was observed, as a contingency plan Normal PCR was performed to amplify the CpG islands, and there was amplification. In conclusion, the study showed that a low concentration of a sugar sweetener (fructose: glucose) used in beverages had low toxicity to the Caco-2 cell line and prolonged exposure of the low concentration might have an adverse effect on the cells' morphology. At medium concentrations, the sugar sweetener used in beverages had medium toxicity to Caco-2 cells; prolonged exposure may lead to morphological changes. These findings indicated that control of dietary glucose intake is an important strategy in combating the development of obesity and type-2 diabetes. DNA methylation could not be established.

Ever increasing applications of nanomaterials (materials with one or more dimension less than 100 nm) has raised awareness of their potential genotoxicity. They have unique physico¿chemical properties and so could have unpredictable effects. Zinc oxide (ZnO) and titanium dioxide (TiO2) are widely used in a number of commercial products. There are published studies indicating that some forms of these compounds may be photo-clastogenic in mammalian cells. What has not been investigated before is the effect of nanoparticles from these compounds in human germ cells. Thus the present study has examined their effects in the presence and absence of UV light in human sperm and compared responses to those obtained with human lymphocytes using the Comet assay to measure DNA damage. The effect of nanoparticles (40-70nm range) was studied in human sperm and lymphocytes in the dark, after pre-irradiation with UV and simultaneous irradiation with UV. The studies do provide some evidence that there are photo-genotoxic events in sperm and lymphocytes in the absence of overt toxicity. The cytotoxic and genotoxic potentials of ZnO and TiO2 as well as their effect on phosphotyrosine expression, were examined in the human epithelial cervical carcinoma cells (Hela cells). This was done to try and determine the underlying molecular events resulting from their exposure to ZnO and TiO2 nanoparticles occurring at the same time as DNA is damaged. Concentration- and time-dependent cytotoxicity, and an increase in DNA and cytogenetic damage with increasing nanoparticle concentrations were reported in this study. Mainly for zinc oxide, genotoxicity was clearly associated with an increase in tyrosine phosphorylation. Nanotechnology has raced ahead of nanotoxicology and little is known of the effects of nanoparticles in human systems, let alone in diseased individuals. Therefore, the effects of TiO2 nanoparticles in peripheral blood lymphocytes from patients with respiratory diseases (lung cancer, chronic obstructive pulmonary disease (COPD) and asthma) were compared with those in healthy individuals using genotoxic endpoints to determine whether there are any differences in sensitivity to nano-chemical insult between the patient and control groups. The results have shown concentration dependent genotoxic effects of TiO2 in both respiratory patient and control groups in the Comet assay and an increasing pattern of cytogenetic damage measured in the micronucleus assay without being statistically significant except when compared with the untreated controls of healthy individuals. Furthermore, modulation of ras p21 expression was investigated. Regardless of TiO2 treatment, only lung cancer and COPD patients expressed measurable ras p21 levels that showed modulation as the result of nanoparticle treatment. Results have suggested that both ZnO and TiO2 nanoparticles can be genotoxic over a range of concentrations without either photoa-ctivation or being cytotoxic.

For the past few decades, the popularity of graphene oxide (GO) nanomaterials (NMs) has increased exceedingly due to their biomedical applications in drug delivery of anti-cancer drugs. Their unique physicochemical properties such as high surface area and good surface chemistry with unbound surface functional groups (e.g. hydroxyl - OH, carboxyl /ketone C=O, epoxy/alkoxy C-O, aromatic group C=C, etc) which enable covalent bonding with organic molecules (e.g. RNA, DNA) make GO NMs as excellent candidates in drug delivery nanocarriers. Despite the overwhelming biomedical applications, there are concerns about their genotoxicity on human DNA. Published genotoxicity studies on GO NMs were performed using non-commercial GO with 2-3 layers of GO sheets, synthesized in various laboratories with the potential for inter-laboratory variabilities. However, what has not been studied before is the effects of the commercial GO (15-20 sheets; 4-10% edge-oxidized; 1 mg/mL) in vitro on DNA damage in human whole blood and peripheral blood lymphocytes (PBL) from real-life patients diagnosed with chronic pulmonary diseases [asthma, chronic obstructive pulmonary disease (COPD), and lung cancer], and genotoxic endpoints compared with those from healthy control individuals to determine whether there are any differences in GO sensitivity. Thus, in the present study, we had characterized GO NMs using Zetasizer Nano for Dynamic Light Scattering (DLS) and zeta potential (ZP) in the aqueous solution, and electron microscopy using the Scanning Electron Microscope (SEM) and Transmission Electron Microscope (TEM) in the dry state, respectively. Cytotoxicity studies were conducted on human PBL from healthy individuals and patients (asthma, COPD, and lung cancer) using the Methylthiazolyldiphenyl-tetrazolium bromide (MTT) and Neutral Red Uptake (NRU) assays, respectively. The genotoxicity (DNA damage) and cytogenetic effects (chromosome aberration parameters) induced by GO NMs on human whole blood from healthy individuals and patients were studied using the Alkaline Comet Assay and Cytokinesis-blocked Micronucleus (CBMN) assay, respectively. Our results showed concentration-dependent increases in cytotoxicity, genotoxicity, and chromosome aberrations, with blood samples from COPD and lung cancer patients being more sensitive to DNA damage insults compared with asthma patients and healthy control individuals. Furthermore, the relative gene and protein expressions of TP53, CDKN1A/p21, and BCL-2 relative to GAPDH on human PBL were studied using the Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) and Western Blot techniques, respectively. Our results have shown altered gene and protein expression levels. Specifically, GO-induced cytotoxicity, genotoxicity, and micronuclei aberrations were associated with TP53 upregulation - a biomarker of DNA damage - in both patients and healthy individuals. These effects show that GO NMs have promising roles in drug delivery applications when formulated to deliver drug payload to COPD and cancer cells. However, the fact that cytotoxicity, genotoxicity, chromosome instability, and gene/protein expressions - biomarkers of cancer risk - were observed in healthy individuals are of concern to public health, especially in occupational exposures at micro levels at the workplace.

Introduction: Malignant Melanoma (MM), caused by UV radiation-induced DNA damage, is the most invasive form of skin cancer and has an increasing incidence worldwide. The hallmarks of MM include the presence of reactive oxygen species (ROS) and excessive proliferation of tumour cells. Many treatments are available or under investigation as anticancer therapeutics such as cell therapy, immunotherapy, gene therapy and nanotechnology-based strategies but they all have severe complications and side effects that limit their wider use. Methods: The present in vitro study has evaluated the genotoxic and cytotoxic effects of Se and CBSC-derived exosomes, individually and in combination, on lymphocytes from MM patients and healthy controls, and on the CHL-1 melanoma cell line. The comet assay and cell counting kit-8 (CCK-8) assay were used to measure genotoxicity and cytotoxicity, respectively, in all cell types. Molecular mechanisms underlying the observed effects were explored using transcriptional and protein expression profiling of key cell cycle and apoptosis genes, by employing the RT qPCR and Western blotting techniques. Conclusion: Selenium displays antioxidant and genoprotective effects in human lymphocytes, especially in MM patients. Both Se (10 μM) and CBSC-derived exosomes (120 μL) are well tolerated in lymphocytes, but show significant genotoxicity and cytotoxicity towards the CHL-1 cell line, with combined administration exhibiting a synergistic effect.

No / Drinking water disinfection by-products (DBPs) are generated by the chemical disinfection of water and may pose hazards to public health. Two major classes of DBPs are found in finished drinking water: haloacetic acids (HAAs) and trihalomethanes (THMs). HAAs are formed following disinfection with chlorine, which reacts with iodide and bromide in the water. Previously the HAAs were shown to be cytotoxic, genotoxic, mutagenic, teratogenic and carcinogenic. OBJECTIVES: To determine the effect of HAAs in human somatic and germ cells and whether oxidative stress is involved in genotoxic action. In the present study both somatic and germ cells have been examined as peripheral blood lymphocytes and sperm. The effects of three HAA compounds: iodoacetic acid (IAA), bromoacetic acid (BAA) and chloroacetic acid (CAA) were investigated. After determining appropriate concentration responses, oxygen radical involvement with the antioxidants, butylated hydroxanisole (BHA) and the enzyme catalase, were investigated in the single cell gel electrophoresis (Comet) assay under alkaline conditions, >pH 13 and the micronucleus assay. In the Comet assay, BHA and catalase were able to reduce DNA damage in each cell type compared to HAA alone. In the micronucleus assay, micronuclei (MNi) were found in peripheral lymphocytes exposed to all three HAAs and catalase and BHA were in general, able to reduce MNi induction, suggesting oxygen radicals play a role in both assays. These observations are of concern to public health since both human somatic and germ cells show similar genotoxic responses.

Hintergrund: Um akute Rejektionen nach Nierentransplantation früh erkennen und behandeln zu können, gleichzeitig die Nebenwirkungen einer immunsuppressiven Therapie zu minimieren, wäre die Etablierung eines “Immunmonitorings”, welches zu jedem Zeitpunkt Hinweise auf die Aktivierung des Immunsystems des Empfängers gegen das Transplantat gibt, wünschenswert. Methodik: In dieser Studie wurden die Anzahl der spenderreaktiven, IFNgamma-produzierenden T-Zellen von 52 nierentransplantierten Patienten zu verschiedenen Zeitpunkten vor (prä-TX) und nach Transplantation (post-TX) im ELISpot-Assay gemessen und in Zusammenhang mit der klinischen Entwicklung gebracht. Außerdem wurde das Assay auf Reproduzierbarkeit untersucht und versucht zu optimieren. Ergebnisse: Eine stark erhöhte Anzahl spenderreaktiver Zellen prä-TX (>200 IFNgamma-spots/3*100000 PBMZ, n = 5) war immer mit einer akuten Rejektion des Transplantats assoziiert. Post-TX korrelierte die Anzahl der spenderreaktiven, IFNgamma-produzierenden Zellen mit der Nierenfunktion ein Jahr nach Transplantation. Diese Korrelation wurde in den Wochen 2 und 3 post-TX und bei Patienten ohne akute Rejektion, besonders deutlich. Hinsichtlich der methodischen Optimierung hat sich die magnetische Depletion CD2pos-Zellen als effektiv gezeigt, die IFNgamma-Sekretion von Stimulatorzellen zu unterbinden. Um die Reproduzierbarkeit des Assays zu verbessern sollten Stimulatorzellen im Überschuss und Empfänger-T-Zellen in einer konstanten Anzahl eingesetzt werden. Dabei sollte die Gesamtzellzahl über 1000000 Zellen/ml betragen. Conclusion: Das ELISpot-Assay ist zur Erkennung klinisch relevanter T-Zellsensibilisierungen vor und nach Transplantation geeignet. Vor einem Einsatz in der klinischen Routine sollten jedoch einige methodische Verbesserungen vorgenommen werden. / Background: In order to perform early diagnosis and treatment of acute rejections after renal transplantation while minimizing side effects of immunosuppression, an immune monitoring tool is needed, which gives information on the activation state of the immune system of the transplant recipient against the allograft at any given time. Methods: In this study, frequencies of donor-reactive, IFNgamma-producing T cells where measured in 52 renal transplant recipients at different time points before (pre-TX) and after transplantation (post-TX) using the ELISPOT-assay. The frequencies were correlated with clinical outcome. Also, the reproducibility of the assay and possibilities of optimization were tested. Results: Highly elevated frequencies of donor-reactive cells pre-TX (>200 IFNgamma-spots/3*100000 PBMC´s, n = 5) were always associated with acute rejection episodes after transplantation. Post-TX frequencies of donor-reactive, IFNgamma-producing cells correlated significantly with graft function one year post-TX. This correlation was strongest for frequencies in week 2 and 3 post-TX and in patients without acute rejection. Regarding the methodical optimization, magnetic CD2pos-cell depletion of donor leucocytes proved useful to inhibit IFNgamma secretion of stimulating cells. To improve reproducibility of the assay stimulating cells should be used as a surplus, a constant number of responding T cells should be chosen, and overall cell concentration should exceed 1000000 cells/ml. Conclusion: The ELISPOT-assay is a useful tool to detect clinically relevant T cell sensibilisation pre- and post-TX. Before it is routinely used some methodical alterations must be performed.

Salmonella enterica gehört zu den gramnegativen Bakterien und ist der Erreger von verschiedenen Darmerkrankungen, von Gastroenteritis bis systemische Infektionen, und jährlich die Ursache für hunderttausende Todesfälle. In den letzten 30 Jahren wurden grundlegende Mechanismen der Invasion und des intrazellulären Wachstums von Salmonella gelöst: Salmonella verfügt über eine Reihe von Virulenzfaktoren (Effektorproteine), die mittels Typ-III-Sekretionssystemen (T3SS) zur molekularen Manipulation der Wirtszelle ausgeschüttet werden. Allerdings sind die Funktionen einiger dieser Effektorproteine nur unzureichend charakterisiert. Darüber hinaus hat die Rolle von RNA-Protein-Wechselwirkungen in bakteriellen Prozessen, einschließlich Infektionen, an Bedeutung gewonnen. Eine der am häufigsten verwendeten Techniken zur Untersuchung von Effektorproteinen ist der Gentamicin Protection Assay (GPA), eine einfache Methode, die den Salmonella-Infektionsprozess in vitro nachbildet. Da der GPA jedoch starken Schwankungen unterliegt und eine Endpunktmessungen darstellt, ist dieser unzureichend, wenn gleichzeitig mehrere Bedingungen oder zeitabhängige Dynamiken untersucht werden müssen. Um diese Einschränkungen zu umgeben, wurde InFluence entwickelt, eine Hochdurchsatz-Fluoreszenz-Mikroskopiemethode. Diese Methode ermöglicht Einblicke in die von Salmonellen besiedelten intrazellulären Nischen, und erwies sich als nützliches Instrument zur Charakterisierung von nicht nur von Effektorproteinen, sondern auch von Wirtsproteinen im Infektionsprozess. Darüber hinaus haben wir zur Entwicklung von TriPepSVM, einer Support Vector Machine (SVM), beigetragen. Dieser Algorithmus, der zusammen mit der AG Marsico (ICB - Helmholtz Zentrum München) entwickelt wurde, sagt RNA-Bindeproteine voraus, mithilfe von Tripeptiden in intrinsisch ungefalteten Regionen (IDR). 66 RBPs hat TriPepSVM in Salmonella vorausgesagt, wovon drei im Rahmen dieser Arbeit experimentell validiert wurden. / Salmonella enterica is a species of Gram-negative bacteria and the causative agent of enteric diseases, ranging from gastroenteritis to systemic infections, causing hundreds of thousands of deaths every year. In the last 30 years the basic mechanisms underpinning invasion and intracellular growth have been unravelled: Salmonella produces tens of virulence factors - termed “effectors” - which are secreted by two distinct Type III Secretion Systems (T3SS) for the hijacking of the host cell molecular machinery via protein-protein interactions. Although the biochemical activities of many effectors have been characterised, the functions of some have remained elusive. In addition, post-transcriptional regulation has emerged to prominence in bacteria, with RNA-protein interactions playing a pivotal role in many bacterial functions, including infection. One of the most frequently used techniques to study Salmonella effectors is the Gentamicin Protection Assay (GPA), a simple method which replicates the infection process in vitro; however, as GPA is subject to high levels of variation and only generates end-point measurements, the method can be inadequate when studying multiple conditions at once or following time-dependent dynamics. InFluence, a high-throughput, fluorescence microscopy-based analysis pipeline, was designed to address these issues. InFluence offered insights into the intracellular niches occupied by Salmonella, and proved to be a useful tool in assessing not only the contribution to the infection process of effectors, but that of host proteins too. In addition, we contributed to the development of TriPepSVM, a support vector machine-based (SVM) method developed with the Marsico group (Institute of Computational Biology (ICB) - Helmholtz Zentrum Munchen) for predicting RNA-binding proteins (RBPs) using tripeptides that are frequent in Intrinsic Disordered Regions (IDRs). TriPepSVM predicted 66 new RBPs in Salmonella, and three were experimentally validated.