CARACTERISATION IN VITRO ET IN VIVO DE NOUVEAUX AGENTS PYRROLO-PYRIMIDINE CIBLANT LES MICROTUBULES POUR LE TRAITEMENT DES CANCERSEn dépit de l’émergence des thérapies ciblées et de l’immunothérapie, la chimiothérapie reste un gold-standard pour le traitement de nombreux cancers. Parmi les agents chimiothérapeutiques conventionnels, les poisons du fuseau interférant avec la dynamique des microtubules (taxanes, vinca-alcaloïdes) sont très largement utilisés. Cependant, leurs nombreux effets indésirables et l’émergence de chimiorésistance limitent leur efficacité et soulignent la nécessité d’identifier de nouveaux inhibiteurs de la tubuline.Notre équipe a réalisé un criblage cellulaire à haut-débit sur plus de 7500 molécules chimiques et identifié une famille de composés pyrrolo-pyrimidine, active sur les formes de cancer pulmonaire résistantes à l’apoptose et aux thérapies ciblées. Notre objectif était de caractériser in vitro et in vivo 15 molécules de cette famille afin d’identifier à terme un potentiel candidat-médicament pour le traitement des cancers résistants.Des essais préliminaires de cytotoxicité et d’apoptose sur différentes lignées cancéreuses pulmonaires ont permis de sélectionner, parmi les 15 composés pyrrolo-pyrimidine, 2 molécules prometteuses : PP-2 et PP-13. PP-2 et PP-13 ont des effets cytotoxiques sur de nombreuses lignées cancéreuses humaines, incluant les lignées résistantes aux thérapies ciblées. En perturbant l’organisation et la dynamique des microtubules, PP-2 et PP-13 induisent le blocage transitoire des cellules en prométaphase puis aboutissent aux phénomènes de catastrophe mitotique, de glissement mitotique ou de division asymétrique. Des études mécanistiques avancées montrent que PP-2 et PP-13 sont des agents poisons du fuseau et entrent en compétition avec la colchicine pour la liaison sur la tubuline. Contrairement aux antimitotiques conventionnels, PP-2 et PP-13 ne sont pas sensibles aux mécanismes de chimiorésistance par surexpression de pompes d’efflux. De plus, à l’IC50, ces 2 composés n’affectent pas le réseau microtubulaire des cellules à l’interphase suggérant un effet toxique (et principalement neurotoxique) moindre. La molécule PP-13 semble être la molécule anticancéreuse la plus prometteuse en raison de son IC50 10 fois inférieure à celle de PP-2 dans les différentes lignées cancéreuses étudiées et d’une affinité pour la tubuline 2 fois plus élevée. In vivo, PP-13 réduit significativement la croissance tumorale et étastatique. L’ensemble de ces résultats suggère que PP-13 pourrait être une alternative intéressante pour le traitement de nombreux cancers y compris des cancers chimiorésistants. / CHARACTERIZATION OF NEW MICROTUBULE-TARGETING AGENTS WITH A PYRROLO-PYRIMIDINE STRUCTURE FOR THE TREATMENT OF CANCERSDespite the emergence of targeted therapies and immunotherapy, chemotherapy remains a gold-standard for the treatment of numerous malignancies. Spindle poisons that interfere with microtubule dynamics (taxanes, vinca alkaloids) are commonly used in chemotherapy drug combinations. However, their troublesome side effects and the emergence of resistance highlight the need for identifying alternative agents. Thanks to a high throughput cell-based assay, we screened agents able to restore apoptosis in apoptosis-resistant lung cancer cells. We selected 15 molecules belonging to the pyrrolopyrimidine family and investigated their anti-cancer effects in vitro and in vivo. Our aim was to identify a potential drug-candidate for the treatment of resistant cancers.From cytotoxicity and apoptosis preliminary assays, we selected the 2 most promising molecules (PP-2, PP-13) among the 15 pyrrolopyrimidine compounds. PP-2 and PP-13 exert cytotoxic effects on a large panel of human cancer cell lines, including targeted therapy-resistant cell lines. By interfering with mitotic spindle organization and microtubule dynamics, PP-2 and PP-13 impair the congression of the chromosomes, promote spindle assembly checkpoint-dependent mitotic blockade and finally lead to asymmetric division, mitotic slippage and direct apoptotic death. PP-2 and PP-13 directly target tubulin and compete with colchicine for the binding to tubulin. Unlike conventional antimitotic agents, PP-2 and PP-13 are not sensitive to chemoresistance mechanisms involving the overexpression of efflux pumps. Moreover, at IC50, these two compounds do not affect the microtubule network during interphase suggesting a less toxic (mainly neurotoxic) effect. Among these two compounds, the PP-13 molecule appears to be the most interesting anti-cancer molecule because of its IC50 10-fold lower than PP-2 and its 2-fold higher affinity for tubulin. In vivo, PP-13 significantly reduces tumor and metastasis growth. All these results suggest that PP-13 might be a potential alternative for the treatment of many cancers including chemoresistant cancers.

Human mesenchymal stem cells (hMSCs) have been identified as a promising cell-based therapy candidate to treat a number of unmet clinical indications, however, in vitro expansion will be required to increase the available number of cells and meet this demand. Scalable manufacturing processes, amenable to closed, single-use and automated technology, must therefore be developed in order to produce safe, effective and affordable hMSC therapies. To address this challenge, a controlled serum-free end-to-end microcarrier process has been developed for hMSCs, which is amenable to large-scale manufacture and therefore increasing economies of scale. Preliminary studies in monolayer culture assessed the level of variability in growth between five hMSC donors, which was found to have a variance of 25.3 % after 30 days in culture. This variance was subsequently reduced to 4.5% by the development of a serum-free monolayer culture process with the maintenance of critical hMSC characteristics and an increased number of population doublings. In order to transfer this into a scalable system, the serum and serum-free expansion processes were transferred into suspension by the addition of plastic microcarriers in 100 mL spinner flasks without control of pH or dissolved oxygen (DO). This achieved a maximum cell density of 0.08 ± 0.01 · 106 cells.mL-1 in FBS-based medium, 0.12 ± 0.01 · 106 cells.mL-1 in HPL-based medium and 0.27 ± 0.03 · 106 cells.mL-1 in serum free medium after six days. In order to drive consistency and yield into the manufacturing process, a process control system was developed for the FBS-based microcarrier expansion process in a 100 mL DASbox bioreactor platform to control DO, pH, impeller rate and temperature. Reduced impeller rates and DO concentrations were found to be beneficial, with a final cell density of 0.11 ± 0.02 · 106 cells.mL-1 and improved post-harvest outgrowth and colony-forming unit (CFU) potential compared to uncontrolled microcarrier and monolayer culture. This controlled bioreactor expansion process was then applied to the previously developed serum-free microcarrier process, eventually achieving a final cell density of 1.04 ± 0.07 · 106 cells.mL-1, whilst retaining key post-harvest hMSC characteristics. Following the controlled serum-free expansion and harvest of hMSCs, a downstream and cryopreservation process was developed to assess the impact of prolonged holding times and subsequent unit-operations on hMSC quality characteristics. This showed that hMSCs are able to maintain key characteristics throughout the entire end-to-end process, demonstrating their potential for commercial scale manufacture.

The intervertebral disc (IVD), situated between adjoining vertebrae, consists of the gelatinous nucleus pulposus (NP) in the centre surrounded by the tougher annulus fibrosus (AF). Its main roles are to distribute loads and to act as joints. With aging, degenerative disc disease (DDD) occurs due to an imbalance in anabolic and catabolic events in the IVD, which results in a loss of function. Lower back pain (LBP) affects 84% of people at some point in their lifetime and is strongly associated with DDD. Current LBP treatments have limited long term efficacy and are symptomatic rather than curative. Cell-based therapies are regarded to hold great potential for the treatment of DDD as it has been hypothesised that they could regenerate the damaged tissue and alleviate LBP. A number of natural and synthetic biomaterials have been investigated as NP tissue engineering scaffolds with varying results. In this study, a self assembling peptide hydrogel (SAPH) was investigated for its potential as a cell carrier and/or scaffold for NP tissue engineering. SAPHs display the advantages of natural polymer hydrogels such as biocompatibility and biodegradability whilst combining the advantages of synthetic materials such as controlled structural and mechanical propertiesCharacterisation determined that the SAPH nanofibrous architecture had features that were of similar scale to extracellular matrix (ECM) components of the human NP. The mechanical properties of the SAPH could be optimised to closely match the native tissue. The system could shear thin and self-heal making the system ideally suited to delivery via minimally invasive procedure. The three dimensional (3D) culture of bovine NP cells (bNPCs) in the SAPH demonstrated that the NP phenotype could be restored after de-differentiation during monolayer culture. Gene expression results demonstrated that ‘traditional’ and ‘novel’ NP markers were highly expressed throughout in vitro culture. Cell viability was high, cell population remained stable and bNPCs adopted the characteristic rounded morphology of native NPCs. Finally, type II collagen and aggrecan, the main ECM components of the NP, were deposited with increasing production over culture period. Growth differentiation factor 6 (GDF-6) has been identified as the most promising current growth factor for inducing discogenic differentiation from human bone marrow mesenchymal stem cell (h-BMMSCs). After samples were stimulated with GDF-6, gene expression results confirmed that a NP-like phenotype could be induced with high expression of ‘traditional’ and ‘novel’ NP markers. Cell viability was high, cell population remained stable and NP associated ECM components were deposited with cells displaying a rounded morphology. Interestingly, when h-BMMSCs were cultured without GDF-6, it was strongly suggested that spontaneous discogenic differentiation occurred after culture in the SAPHs as ‘traditional’ and ‘novel’ NP markers were highly expressed, morphology was comparable to native NPCs and type II collagen and aggrecan were deposited extracellularly. If these findings were accurate then this is the first study to demonstrate that a NP-like phenotype could be induced from MSCs without use of an exogenous growth factor or a discogenic bioactive motif. Despite exciting and novel results, further work is required to confirm the potential of SAPHs for NP tissue engineering scaffolds.

Rift Valley fever virus (RVFV) is a mosquito-borne virus which has the ability to infect a large variety of animals including humans in Africa and Arabian Peninsula. The abortion rate among these animals are close to 100%, and young animals develop severe disease which often are lethal. In humans, Rift Valley fever (RVF) presents in most cases as a mild illness with influenza-like symptoms. However, in about 8% of the cases it progresses into a more severe disease with a high case fatality rate. Since there is such a high abortion rate among infected animals, a link between human miscarriage and RVFV has been suggested, but never proven. We could in paper I for the first time show an association between acute RVFV infection and miscarriage in humans. We observed an increase in pregnant women arriving at the Port Sudan Hospital with fever of unknown origin, and several of the patients experienced miscarriage. When we analysed their blood samples for several viral diseases we found that many had an acute RVFV infection and of these, 54% experienced a miscarriage. The odds of having a miscarriage was 7 times higher for RVFV patients compared to the RVFV negative women of which only 12% miscarried. These results indicated that RVFV infection could be a contributing factor to miscarriage. RVFV is an enveloped virus containing the viral glycoproteins n and c (Gn and Gc respectively), where Gn most likely is responsible for the initial cellular contact. The protein DC-SIGN on dendritic cells and the glycosaminoglycan heparan sulfate has been suggested as cellular receptors for RVFV, however other mechanisms are probably also involved in binding and entry. Charge is a driving force for molecular interaction and has been shown to be important for cellular attachment of several viruses, and in paper II we could show that when the charge around the cells was altered, the infection was affected. We also showed that Gn most likely has a positive charge at a physiological pH. When we added negatively charged molecules to the viral particles before infection, we observed a decreased infection efficiency, which we also observed after removal of carbohydrate structures from the cell surface. Our results suggested that the cellular interaction partner for initial attachment is a negatively charged carbohydrate. Further investigations into the mechanisms of RVFV cellular interactions has to be undertaken in order to understand, and ultimately prevent, infection and disease. There is currently no vaccine approved for human use and no specific treatments for RVF, so there is a great need for developing safe effective drugs targeting this virus. We designed a whole-cell based high-throughput screen (HTS) assay which we used to screen libraries of small molecular compounds for anti-RVFV properties. After dose-response and toxicity analysis of the initial hits, we identified six safe and effective inhibitors of RVFV infection that with further testing could become drug candidates for treatment of RVF. This study demonstrated the application of HTS using a whole-cell virus replication reporter gene assay as an effective method to identify novel compounds with potential antiviral activity against RVFV.

Controlar a proliferação celular de tumores é um objetivo que vem sendo perseguido há décadas, com moderado sucesso na maioria dos casos. Dentre os diversos tipos de tumores que atingem a humanidade, alguns gliomas são considerados os mais fatais, por haver pouca ou nenhuma alternativa de tratamento efetivo. Agentes que apresentam propriedades anti-tumorais, como glicocorticóides (GC) e a forma all-trans do ácido retinóico (ATRA) são utilizados como adjuvantes no tratamento de alguns tipos de glioma. Entretanto, apesar de serem moléculas bastante conhecidas, pouco se sabe sobre seu mecanismo de ação como anti-tumoral. Para endereçar este problema, nosso laboratório se propôs a isolar e caracterizar genes regulados por estes agentes, utilizando modelos celulares, como as linhagens C6 e ST1 de glioma de rato, e as linhagens T98G e A172 de glioma humano. A linhagem C6 apresenta características de células transformadas e tumorais em cultura, e responde a GC, ou ATRA, com inibição de crescimento e achatamento celular. A linhagem ST1, variante derivado da C6, é hiper-responsiva ao tratamento com GC e, aparentemente, mais responsiva ao tratamento com ATRA, passando por um processo de completa reversão fenotípica tumoral-normal, devido ao expressivo aumento do tempo de dobramento, diminuição da densidade de saturação, recuperação da dependência de fatores de crescimento presentes no soro fetal bovino e da dependência de ancoragem para proliferação e perda do potencial tumorigênico, além de sofrer alterações morfológicas, como um maior achatamento celular e reorganização em feixes paralelos, que a aproximam do fenótipo normal. No presente trabalho buscou-se alterações moleculares induzidas por ATRA em células ST1, para melhor compreender a cascata de eventos desencadeada por ação deste fármaco. Em paralelo foram realizados estudos da ação de ATRA sobre as células T98G, buscando-se correlacionar os dados obtidos em modelo celular murino com modelos humanos. Para tanto, duas metodologias de estudo foram aplicadas: a) análise proteômica através de eletroforese bidimensional de proteínas (2D-PAGE), acoplada à espectrometria de massa (MALDI-TOF), para gerar perfis de expressão protéica na ausência e na presença de ATRA, permitindo comparação e identificação de proteínas moduladas no processo; b) construção de vetores plasmideais e retrovirais para super-expressar ou bloquear a expressão de um inibidor de serina protease de rato (serpinb6), descrito previamente no laboratório como estando potencialmente envolvido no processo de reversão fenotípica de ST1 induzido por ATRA. A abordagem proteômica permitiu a identificação de sete proteínas potencialmente reguladas por ATRA no modelo celular ST1, como as proteínas envolvidas em proliferação celular (c-Fos e SCGF), as proteínas de citoesqueleto (actina e tubulina), as proteínas envolvidas em estresse celular (GRP78 e Hsc70) e a proteína TCTP, classicamente reprimida em processos de reversão do fenótipo tumoral. O uso de construções plasmideais e retrovirais permitiu a obtenção de populações celulares que super-expressam serpinb6 e a análise de fenótipo destas células indicou que serpinb6 também pode ter função citoprotetora em células ST1, o que a coloca junto com as proteínas GRP78 e Hsc70 identificadas, evidenciando a importância desta classe de proteínas no processo estudado. / Control of tumor cell proliferation is an objective that has been pursued for decades, with modest or no success in the majority of the cases. Among the several kinds of tumors that develop in humans, some gliomas are considered the most fatal, due to the lack of alternatives for effective treatment. Anti-tumor agents, such as glucocorticoids (GC) or all-trans retinoic acid (ATRA) are used in combination with other drugs in some glioma cases. However, besides being very known molecules, their anti-tumor mechanism is not completely understood. In order to address this problem, our laboratory decided to isolate and characterize genes regulated by these agents, using cellular models, such as the C6 and ST1 rat glioma cell lines and the T98G and A172 human glioma models. The C6 cell line is fully transformed and tumoral in culture and responds to GC or ATRA treatment, showing growth inhibition and cell flattening. The ST1 variant is hyper-responsive to the treatment with GC and, apparently, more responsive to the treatment with ATRA, when compared to C6. Upon treatment with these agents, it undergoes a complete tumoral to normal phenotypic reversion, characterized by an increase in doubling time, decrease of saturation density in culture, recovery of dependence of serum factors for proliferation and anchorage for colony formation, besides inhability to form tumors in nude mice and morphological changes. Here we present the efforts undertaken towards better understanding of the molecular changes induced by ATRA in ST1 cells. Aiming at the correlation of the data obtained from a rat model with human models, all the studies were performed in parallel with the T98G human glioma cell model. To this end, two study methodologies were applied: a) proteomic analysis through bidimensional electrophoresis coupled to MALDI-TOF identification, to generate protein expression profiles in the presence and absence of ATRA, allowing comparison and identification of proteins modulated in the process; b) construction of plasmid and retroviral vectors to overexpress or block the expression of a serine protease inhibitor (serpinb6), previously described in the laboratory as being potentially involved in the process of tumoral to normal phenotypic reversion promoted by ATRA in ST1. The proteomics approach allowed the identification of seven proteins potentially regulated by ATRA in ST1, such as the proteins involved in cell proliferation (c-Fos and SCGF), cytoskeleton organization (actin and tubulin), cellular stress (GRP78 and Hsc70) and the tumor related protein TCTP, classically repressed in tumoral to normal reversions, and related to the three groups of proteins mentioned above. By using plasmid and retroviral vectors it was possible to obtain recombinant cell populations over-expressing serpinb6. The phenotype analysis of these populations indicated that serpinb6 can also have cell protection effects in ST1, which would classify it together with GRP78 and Hsc70 as an anti-stress protein highlighting the importance of this protein class in the process studied.

Despite spectacular progress in biophysics, molecular biology and biochemistry our ability to predict the dynamic behavior of multicellular systems under different conditions is very limited. An important reason for this is that still not enough is known about how cells change their physical and biological properties by genetic or metabolic regulation, and which of these changes affect the cell behavior. For this reason, it is difficult to predict the system behavior of multicellular systems in case the cell behavior changes, for example, as a consequence of regulation or differentiation. The rules that underlie the regulation processes have been determined on the time scale of evolution, by selection on the phenotypic level of cells or cell populations. We illustrate by detailed computer simulations in a multi-scale approach how cell behavior controlled by regulatory networks may emerge as a consequence of an evolutionary process, if either the cells, or populations of cells are subject to selection on particular features. We consider two examples, migration strategies of single cells searching a signal source, or aggregation of two or more cells within minimal multiscale models of biological evolution. Both can be found for example in the life cycle of the slime mold Dictyostelium discoideum. However, phenotypic changes that can lead to completely different modes of migration have also been observed in cells of multi-cellular organisms, for example, as a consequence of a specialization in stem cells or the de-differentiation in tumor cells. The regulatory networks are represented by Boolean networks and encoded by binary strings. The latter may be considered as encoding the genetic information (the genotype) and are subject to mutations and crossovers. The cell behavior reflects the phenotype. We find that cells adopt naturally observed migration strategies, controlled by networks that show robustness and redundancy. The model simplicity allow us to unambiguously analyze the regulatory networks and the resulting phenotypes by different measures and by knockouts of regulatory elements. We illustrate that in order to maintain a cells' phenotype in case of a knockout, the cell may have to be able to deal with contradictory information. In summary, both the cell phenotype as well as the emerged regulatory network behave as their biological counterparts observed in nature.

The present study deals with the comparison and establishment of methods for the detection of antibodies against recombinant human growth hormone (rhGH). Therefore, different methods for the detection of hGH-Abs were evaluated and compared in order to establish a test system that can be used for the detection of neutralizing antibodies against hGH, which could be developed under rhGH treatment. This manuscript describes in detail the validation of a newly developed biological assay, the neutralizing hGH-antibody assay (NAb assay). Therefore, a cell line transfected with the growth hormone receptor, that proliferates in the presence of hGH, was used. This proliferation was quantified by an increase of the optical density (OD/ absorbance) after addition of a colorimetric reagent, whereas the presence of hGH-antibodies leads to an inhibition of cell proliferation. To validate the test system for the detection of hGH-antibodies, we tested serum samples of 4 patients suffering from neurosecretory dysfunction (NSD) and samples taken from 6 patients with growth hormone deficiency (GHD) which were treated with rhGH and were highly suspected for a-hGH antibodies. These samples were tested in two different immunological assays, capable to screen sera for anti-hGH immunreactivity in the case of hGH-insensitivity during GH treatment. Using the NAb assay the neutralizing activity of specific hGH-antibodies was proved in serum samples of NSD and GHD type 1A patients. In case of neutralizing hGH-antibody activity, a clinically based decision can be made whether rhGH therapy should be stopped or the rhGH dosis should be increased. By the use of our test system, we offer the measurement of anti-hGH-antibody activity to other laboratories in cases when secondary hGH-insensitivity is assumed or observed.

Van Kempen et al. described high natalizumab concentrations in their natalizumab-treated multiple sclerosis (MS) patients at time of re-dosing. Based on the literature research the authors consider a natalizumab concentration above 2 μg/mL to be sufficient for an adequate alpha-4 integrin receptor saturation of above 70%. Recently, we have demonstrated similar results using our new cell-based immunoassay to evaluate free natalizumab concentration, cell-bound natalizumab, and alpha-4 integrin receptor saturation as the key pharmacokinetic/pharmacodynamic parameters of natalizumab treatment in different in vivo settings. We investigated the effects of treatment interval extension or treatment cessation.

Cancer as a disease causes about 8.8 million deaths worldwide per year, a number that will largely increase in the next decades. Although the cellular processes involved in tumor emergence are more and more understood, the implications of specific changes at the cellular scale on tumor emergence at the tissue scale remain elusive. Main reasons for this lack of understanding are that the cellular processes are often hardly observable especially in the early phase of tumor development and that the interplay between cellular and tissue scale is difficult to deduce. Cell-based mathematical models provide a valuable tool to investigate in which way observable phenomena on the tissue scale develop by cellular processes. The implications of these models can elucidate underlying mechanisms and generate quantitative predictions that can be experimentally validated. In this thesis, we infer the role of genetic and phenotypic cell changes on tumor development with the help of cell-based Markov chain models which are calibrated by tissue-scale data. In the first part, we utilize data on the diagnosed fractions of benign and malignant tumor subtypes to unravel the consequences of genetic cell changes on tumor development. We introduce extensions of Moran models to investigate two specific biological questions. First, we evaluate the tumor regression behavior of pilocytic astrocytoma which represents the most common brain tumor in children and young adults. We formulate a Moran model with two absorbing states representing different subtypes of this tumor, derive the absorption probabilities in these states and calculate the tumor regression probability within the model. This analysis allows to predict the chance for tumor regression in dependency of the remaining tumor size and implies a different clinical resection strategy for pilocytic astrocytoma compared to other brain tumors. Second, we shed light on the hardly observable early cellular dynamics of tumor development and its consequences on the emergence of different tumor subtypes on the tissue scale. For this purpose, we utilize spatial and non-spatial Moran models with two absorbing states which describe both benign and malignant tumor subtypes and estimate lower and upper bounds for the range of cellular competition in different tissues. Our results suggest the existence of small and tissue-specific tumor-originating niches in which the fate of tumor development is decided long before a tumor manifests. These findings might help to identify the tumor-originating cell types for different cancer types. From a theoretical point of view, the novel analytical results regarding the absorption behavior of our extended Moran models contribute to a better understanding of this model class and have several applications also beyond the scope of this thesis. The second part is devoted to the investigation of the role of phenotypic plasticity of cancer cells in tumor development. In order to understand how phenotypic heterogeneity in tumors arises we describe cell state changes by a Markov chain model. This model allows to quantify the cell state transitions leading to the observed heterogeneity from experimental tissue-scale data on the evolution of cell state proportions. In order to bridge the gap between mathematical modeling and the analysis of such data, we developed an R package called CellTrans which is freely available. This package automatizes the whole process of mathematical modeling and can be utilized to (i) infer the transition probabilities between different cell states, (ii) predict cell line compositions at a certain time, (iii) predict equilibrium cell state compositions and (iv) estimate the time needed to reach this equilibrium. We utilize publicly available data on the evolution of cell compositions to demonstrate the applicability of CellTrans. Moreover, we apply CellTrans to investigate the observed cellular phenotypic heterogeneity in glioblastoma. For this purpose, we use data on the evolution of glioblastoma cell line compositions to infer to which extent the heterogeneity in these tumors can be explained by hierarchical phenotypic transitions. We also demonstrate in which way our newly developed R package can be utilized to analyze the influence of different micro-environmental conditions on cell state proportions. Summarized, this thesis contributes to gain a better understanding of the consequences of both genetic and phenotypic cell changes on tumor development with the help of Markov chain models which are motivated by the specific underlying biological questions. Moreover, the analysis of the novel Moran models provides new theoretical results, in particular regarding the absorption behavior of the underlying stochastic processes.

Food contact materials are materials that are intended to come into contact with food, and we are exposed to different types of chemicals that exist in the packages on a daily basis. In this study, a battery of effect‑based in vitro cellular bioassays was used to evaluate the presence of bioactive compounds in commonly used food contact materials made of paper and cardboard, retrieved from the Swedish market. Sample extracts were tested at concentrations 0.3, 1, 3 and 10 mg food contact material/mL cell culture medium. The use of effect-based bioassays allowed for screening of multiple health-relevant endpoints in a non-targeted approach. Hence, taking unknown substances and mixtures into consideration when addressing potential toxicity of the materials. In essence, detection of bioactivity could be considered as moderate to high in assays of positive effects. Antiandrogenic and antiestrogenic effects were found in 72% of the samples, followed by 47% bioactivity in the Nrf2 assay. No androgenic effect was detected. Usage of effect-based bioassays allows for high sensitivity and low detection limits, and these can be used as a first approach to evaluate package materials to ensure the safety of consumers. / Livsmedelsförpackningar är material som är avsedda att komma i kontakt med livsmedel. Vi exponeras för olika typer av kemikalier som existerar i dessa förpackningar varje dag. I denna studie användes ett batteri av effektbaserade in vitro bioanalytiska metoder för att undersöka förekomst av bioaktiva ämnen i vanligen använda livsmedelsförpackningar tillverkade av papper och kartong, insamlade från den svenska marknaden. Provextrakten testades i koncentrationerna 0.3, 1, 3 och 10 mg livsmedelsförpackning/mL cellmedium. Att använda effektbaserade bioanalyser möjliggör undersökning av flertalet hälsorelevanta effekter genom en icke-riktad strategi. På så sätt tas okända substanser och komplexa blandningar i beaktande. Andelen bioaktiva prover kan anses måttlig till hög för positiva analyser. Antiandrogena och antiöstrogena effekter detekterades i 72% av proverna, följt av 47% bioaktivitet i Nrf2 analysen. Ingen agonistisk androgen effekt observerades. Att använda effektbaserade bioanalyser möjliggör hög sensitivitet och detektion vid låga koncentrationer, därför kan dessa användas som ett första steg för att evaluera förpackningsmaterial för att säkra konsumenternas hälsa.