Signal Transducers and Activators of Transcription (STATs) are latent transcription factors that mediate several cellular responses. This protein family consists of seven members, STAT1 – 6 including two closely related molecules, STAT5a and STAT5b, that show 96% amino acid sequence homology and are critical for lymphoid, myeloid and erythroid cell development and function. Activated STAT proteins dimerize and translocate to the nucleus, where they bind to high-affinity DNA motifs to modulate gene expression. We recently identified STAT5b as the critical regulator of IgE-mediated cytokine production in mast cells. STAT5b knockout (KO) cells show decreased sensitivity to IgE-mediated passive systemic anaphylaxis accompanied with decreased production of IL-6 and IL-13 compared to wild type counterparts. Interestingly, STAT5b KO mice demonstrated elevated levels of serum IgE but a normal response to histamine-mediated passive systemic anaphylaxis. The current work demonstrates that STAT5b regulates mast cell function both in vivo and in vitro. Additionally, activated STAT proteins can also form tetramers through an N-terminal domain-mediated oligomerization process when bound to low-affinity tandem motifs. Dr. Warren Leonard’s laboratory generated STAT5a-STAT5b double knock-in (DKI) mice in which STAT5 proteins are phosphorylated and can form dimers but not tetramers. We have now found that bone marrow-derived mast cells from STAT5 DKI mice are defective in IgE-induced cytokine and chemokine production and exhibit defective stem cell factor (SCF)-induced migration and survival responses in vitro. Similarly, IgE-mediated passive systemic anaphylaxis is decreased in STAT5 DKI mice. These data indicate that Stat5 tetramers are critical for some aspects of mast cell function in allergic and inflammatory disease.

Eine Vielzahl präklinischer Schlaganfallstudien zeigte die neuroprotektive und neuroregenerative Wirkung des hämatopoetischen Wachstumsfaktors G-CSF (Granulozyten-Kolonie stimulierender Faktor). Ein Wirkungsmechanismus des G-CSF ist die Mobilisation von protektiven Knochen-markzellen in die ischämische Läsion, wobei diese zeitverzögert nach G-CSF-Gabe stattfindet. Eine zusätzliche frühzeitige Transplantation mononukleärer Knochenmarkzellen (BM MNC) könnte diese therapeutische Lücke füllen. Ziel der vorliegenden Studie war es, die Wirksamkeit dieser Kombinations-therapie in einem Schlaganfallmodell der spontan hypertensiven Ratte (SHR) zu testen. Syngene BM MNC wurden aus dem Knochenmark von SHRs durch immunmagnetische Depletion der Granulozyten isoliert. Nach Verschluss der Arteria cerebri media wurde den Tieren über insgesamt 5 Tage G-CSF verabreicht und zusätzlich zu einem frühen (6h nach Schlaganfall) oder späteren (48h nach Schlaganfall) Zeitpunkt BM MNC intravenös appliziert. Unbehandelte Schlaganfalltiere sowie Tiere mit alleiniger G-CSF-Therapie dienten als Kontrolle. Das Infarktvolumen wurde weder durch die alleinige G-CSF-Gabe noch durch die zusätzliche Zelltherapie verändert. Dennoch wiesen Tiere mit G-CSF-Einzeltherapie eine anhaltende funktionelle Verbesserung des sensomotorischen Defizites auf. Während die zusätzliche frühzeitige Zelltransplantation (6h) keinen weiteren Therapieeffekt zeigte, führte die Zelltransplantation nach 48h zu einer Aufhebung des protektiven G-CSF Effektes. Die G-CSF-Therapie bewirkte erwartungsgemäß einen deutlichen Anstieg der zirkulierenden Leukozyten. Interessanterweise wurde der Granulozytengehalt im Blut und in der Milz durch die einmalige Zelltherapie nach 48h signifikant erhöht. Ein Großteil der transplantierten BM MNC (48h) konnte in der Milz nachgewiesen werden und führte dort vermutlich zu einer kompetitiven Hemmung des Granulozytenabbaus. Dies hatte sowohl den Anstieg der zirkulierenden Granulozyten als auch deren vermehrte Infiltration in das ischämische Hirngewebe zur Folge und könnte schließlich den negativen Einfluss auf die funktionelle Verbesserung erklären. Die beobachteten Interaktionsmechanismen werfen ein interessantes Licht auf die mögliche Wirkungsweise von Zelltherapien und unterstreichen die entscheidende Rolle des Immunsystems in der Pathophysiologie des Schlaganfalls.

Mucosal immune system comprises not only the major compartment of the immune system but also important interface with the outer environment. It is responsible in maintaining an intricate balance with the danger and non-danger stimuli of the outer world by employing specific anatomical features and unique functional mechanisms. Mucosal immune system has been long understudied, perhaps due to the limited accessibility, and its biological importance is thus still underevaluated. However, it has become evident that it is important to study mucosal immune system not only in local mucosal affections but also when uncovering pathogenic mechanisms and novel prevention strategies of organ specific autoimmune diseases such as type 1 diabetes. Thus, the first, more clinically oriented part of this thesis is focused on mucosal immune system of the upper respiratory tract in disease conditions - in nasal polyposis (NP). Because there is a substantial accumulation of eosinophils and neutrophils in the most frequent type of NP, we investigated and described increased expression of chemokine receptors CCR1 and CCR3 in NP versus nasal mucosa. Both innate immune mechanisms as well as homeostasis of epithelial cells may participate in NP. We have documented increased numbers of iNOS-positive and insulin-like growth...

Cancer development and progression vary depending on tumor type, localization, invasion, immunogenicity and the ability of immune system to become activated. There are frequent interactions between tumor cells and immune cells, occuring locally at the site of primary tumor or distally through paracrine signalling of various mediators and cytokines. The main subject of this PhD thesis is to study key factors and aspects of immune response in cancer patients. In the first part, we analyzed immune cells infiltrating tumor tissues of ovarian cancer patients at different stages of disease. We focused on the dynamics of immune response, primarily on frequency of individual T lymphocyte populations in peripheral blood and tumor infiltrating T lymphocytes in tumors of early and advanced stages of ovarian cancer. We found that during disease progression there is a gradual decrease of proinflammatory Th17 and Th1 immune responses and a specific recruitment of regulatory T cells to the tumor site, which results in a significant immune suppression in the tumor microenvironment. In the second part, we demonstrated that the character of immune response in HPV-positive head and neck cancer patients is very different from the patients with tumors not associated with HPV infection. In HPV-positive patients, significantly...

Over the last few years, significant immune system changes have been observed in tumor patients exposed to different treatments. Moreover, evaluation and prediction of the responses of treatments given to these patients can become a key component. Therefore, it is critical to develop a pipeline for a systematic approach where complete monitoring of the immune system and interpretation of data from different assaysare involved in predicting treatment effects. Most of the children in the study involved were diagnosed with neuroblastoma, osteosarcoma, wilms tumor, and brain tumor. In this study,whole blood samples from children with solid tumors were used. The analysis was done on cells, proteins, and genes to gather comprehensive information on the immune system's composition and function. This study extracts cell-specific signatures with modern mass cytometry technology, proteins with a proximity extension assay, and mRNA whole blood sequencing. These assays were analyzed with different statistical models such as multivariate, mixed effect model, and multi-omics factor analysis. These assay signatures provided information about the contribution of specific immune cells, plasma proteins, and genes associated with a tumor. The use of these signatures in diagnosing and treating solid tumors has been discussed in the form of a shared trajectory of recovery among tumor patients / <p>Presentation was approved with Instructions of additional information in the report.</p>

HIV-1 protease is a very important drug target for AIDS therapy. Nine protease inhibitors have been proved by FDA and used in AIDS treatment. Due to the high replication rate and the lack of fidelity of the HIV-1 reverse transcriptase, HIV-1 virus developed various drug-resistant variants. Although experimental methods such as crystallography and isothermal titration calorimetry provide structural and thermodynamic data on drug-resistant variants, they are unable to discern the mechanism by which the mutations confer resistance to inhibitors. Understanding the drug-resistance mechanism is crucial for developing new inhibitors more tolerant to the drug-resistant mutations. Computational methods such as free energy calculations and molecular dynamic simulations can provide insights to the drug resistance mechanism at an atomic level. In this thesis, I have focused on the elucidation of the energetic and dynamics of key drug-resistant variants of HIV-1 protease. Two multi-drug resistant variants, in comparison with wild-type HIV-1 protease were used for the comparisons: Flap+ (L10I, G48V, I54V, and V82A) which contains a combination of flap and active site mutations and ACT (V82T, I84V) that only contains active site mutations. In Chapter II, I applied free energy simulations and decomposition methods to study the differential mechanism of resistance to the two variants, Flap+ and ACT, to the recently FDA-approved protease inhibitor darunavir (DRV). In this study, the absolute and relative binding free energies of DRV with wild-type protease and the two protease variants were calculated with MM-PB/GBSA and thermodynamic integration methods, respectively. And the predicted results are in good agreement with the ITC experimental results. Free energy decomposition elucidates the mutations alter not only its own interaction with DRV but also other residues by changing the geometry of binding pocket. And the VdW interactions between the bis-THF group of DRV is predominant even in the drug-resistant variants. At the end of this chapter, I offer suggestions on developing new inhibitors that are based on DRV but might be less susceptible to drug-resistant mutations. In Chapter III, 20-ns MD simulations of the apo wildtype protease and the apo drug-resistant protease variant Flap+ are analyzed and compared. In these studies, these mutations have been found to decrease the protease flexibility in the apo form but increase the mobility when the protease is binding with inhibitor. In Chapter IV, more details of the free energy simulation and decomposition are discussed. NMR relaxation experiments were set up as a control for the MD simulation study of the dynamics of the Flap+ variant. The difficulty of finishing the NMR experiment is discussed and the solution and some preliminary results are shown. In summary, the scope of this thesis was to use computational methods to study drug-resistant protease variants’ thermodynamic and dynamic properties to illuminate the mechanism of protease drug resistance. This knowledge will contribute to rational design of new protease inhibitors which bind more tightly to the protease and hinder the development of drug-resistant mutations.

Gain of function NOTCH1 mutations are common in both patients with T-ALL and in mouse models of the disease. Inhibiting the Notch pathway in T-ALL cell lines results in growth arrest and/or apoptosis in vitro, suggesting a requirement for Notch signaling in T-ALL. Therefore, we sought to examine the role of Notch1 signaling in both premalignancy and in the maintenance of leukemic growth. Using a murine model of T-ALL, in which expression of the Tal1 and Lmo2 oncogenes arrests thymocyte development, our preleukemic studies reveal that Notch1 mutations are early events that contribute to the clonal expansion of DN3 and DN4 progenitors. We also demonstrate that progenitors are maintained within the tumor and are enriched in leukemia-initiating cell (L-IC) activity, suggesting Notch1 may contribute to L-IC self-renewal. By studying the effects of Notch signaling in murine T-ALL cell lines, we also demonstrate that Notch1 promotes the proliferation and survival of leukemic blasts through regulation of Lef1 and the Akt/mTOR pathways. Given that T-ALL cell lines are dependent on Notch signaling in vitro, we investigated the effects of Notch inhibition in vivo. We provide evidence that Notch1 can be successfully targeted in vivo and that Notch inhibition, with γ-secretase inhibitors (GSIs), significantly extends the survival of leukemic mice. We also demonstrate that administration of GSIs in combination with rapamycin inhibits human T-ALL growth and extends survival in a mouse xenograft model. Given that NOTCH1 may be required to maintain both L-IC and bulk leukemic growth, targeting NOTCH1 may prove to be an efficacious targeted therapy for T-ALL patients with aberrant NOTCH1 activation.

A constant flow of various pathogens enters the respiratory system on daily basis through the involuntary mechanism of breathing. Respiratory viral infections are common yet can be fatal in vulnerable populations. Respiratory syncytial virus (RSV) is one of the first and most common viruses that the human population acquire in the first two years of life. Despite the ability of most infants to recover from a RSV infection, many require hospitalization and, in few cases, die from such an infection. The pattern of seasonality of respiratory viruses also applies to RSV. In this work the temperature dependence of infectivity was studied in Hep-2 cells infected with RSV that had been incubated with bronchoalveolar lavage (BAL) fluid. The results indicate a temperature dependence of infectivity. Inhibition of the viral infectivity was observed at three different temperatures 37 ̊C, 40 ̊C and 42 ̊C. The inhibition appears to be linked to the appearance of large agglutinates that appear to reduce the infectivity of RSV. Such a study found that viral neutralization is dependent on a temperature-dependent agglutination reaction. The causality of agglutination formation requires further investigation in order to conclusively confirm the immunological component(s) of this reaction, and how temperature is contributing to this reaction.

Unsolicited e-mails generally present a major problem within the e-mail communication nowadays. There exist several methods that can detect spam and distinguish it from the requested messages. The theoretical part of the masters thesis introduces the ways of detecting unsolicited messages by using artificial immune systems. It presents and subsequently analyses several methods of the artificial immune systems that can assist in the fight against spam. The practical part of the masters thesis deals with the implementation of a spam filter on the basis of the artificial immune systems. The project ends with comparison of effectiveness of the newly designed spam filter and the one which uses common methods for spam detection.

Huge amount of medical data has been generated in practical experiments which makes data analysis a challenging problem. This requires novel techniques to be developed. The improvements in computational power suggest to use computerbased modeling approaches to process a large set of data. One of the important systems in the human body to be investigated is the immune system. The previous studies of medical scientists and ongoing experiments at Karolinska Institute provide information about the human immune system. This information includes attributes of human immune system’s blood cells and the interactions between these cells. This interactions are provided as ‘if-then’ logical rules. Each rule verifies a condition on the attribute of one cell and it may initiate interaction processes to modify the attributes of other cells. A specific temporal value is associated to each process to quantify the speed of that process in the body (i.e., slow, medium, fast). We propose an agent-based model (ABM) to study human immune system cells and their interactions. The ABM is selected to overcome the complexity of large amount of data and find emergent properties and behavior patterns of the cells. Immune system cells are modeled as autonomous agents which have interactions with each other. Different values of a cell attributes define possible states of the cell and the collection of states of all cells constructs the state of the whole agent-based model. In order to consider the state transitions of the cells, we used a finite state machine (FSM). The first state is constructed from the input initial values for the cells and considering the logical time of 1. In each step, the program goes one time unit further and computes next state by applying the changes based on the cells’ interactions rules. This evolution of states in time is similar to game of life (GOL) automaton. The final model based on three modeling approaches of ABM, FSM and GOL are used to test medical hypothesis related to human immune system. This model provides a useful framework for medical scientists to do experiments on the cells’ attributes and their interaction rules. Considering a set of cells and their interactions, the proposed framework shows emergent properties and behavior patterns of the human immune system.