O objetivo do estudo foi avaliar a importância da expressão das proteínas topoisomerase II alfa (topo II) e HER-2 como fatores preditvos da resposta à quimioterapia neoadjuvante e prognóstico em pacientes com câncer de mama nos estádio clínico II e III. Pacientes e métodos: 99 pacientes receberam quimioterapia neoadjuvante com docetaxel (75mg /m²) e epirrubicina (50 mg/m²) em infusão endovenosa no dia 1 a cada 3 semanas após terem sido submetidas a biópsia incisional. Foi complementado tratamento sistêmico com quimioterapia adjuvante com CMF ou FEC de acordo com o estado axilar avaliada após a cirurgia definitiva e/ou hormonioterapia de acordo com a avaliacãodos receptores hormonais. Avaliamos a taxa de resposta ao tratamento neoadjuvante e a influência da topo II alfa e do HER-2 na taxa de resposta à quimioterapia neoadjuvante bem comona sobrevida livre de doença e sobrevida global. Também foram avaliadas a expressão dos receptores hormonais. Resultados: a taxa de resposta clínica objetiva foi de 80,8 % com 9,1 % de resposta patológica completa. A expressão da topo II alfa nao apresentou significância nas taxas de resposta ou na sobrevida das pacietnes e nao houve correlação entre a expressão desta proteína e de HER-2. A superexpressão da proteína HER-2 foi associada com uma redução significante nas taxas de sobrevida livre de doença e sobrevida global (p= 0,04 e p= 0,004, respectivamente). Conclusão: a expressão da topo II alfa não demonstrou, em nosso estudo, ser fator preditivo ou prognóstico nas pácientes submetidas a quimioterapia neoadjuvante com docetaxel e epirrubicina. / The objective of this study is to evaluate the importance of the expression of the proteins topoisomerase II alpha (topo II) and HER-2 as predictive factors to response to neoadjuvant chemotherapy and the prognosis of patients diagnosed with clinical stage II and stage III breast cancer. Patients and methods: 99 patients have received neoadjuvant chemotherapy with docetaxel (75mg /m²) and epirrubicine (50 mg/m²) through intravenous infusion on D1 q3 weeks, after submitted to pathologic specimen harvest. Systemic treatment was then complemented with CMF or FEC according to the status of axilla involvement after surgical staging and/or hormone therapy according tohormone receptor status. We evaluated the response rate to neoadjuvant treatment and the influence of topo II alpha and HER-2 expression on the response rate and disease free survival and overall survival. The expression of hormone receptors was also evaluated. Results: Objective clinical response was 78,8%, with 8,2% of complete pathological response.Topo II alpha expression did not correlate to response to chemotherapy or survival and there was no correlation between topo II alpha expression and HER-2 expression. Superexpression of HER-2 protein was associated to a significant reduction in disease free survival and overall survival (p=0,04 and p=0,004, respectively). Conclusion: topo II alpha expression did not demonstrate, in our study, to be a predictive nor prognostic factor to the patientssubmitted to neoadjuvant with docetaxel and epirrubicin.

Human Epidermal Growth Factor Receptor 2 (HER-2/neu) is a breast tumor antigen (Ag) commonly overexpressed in 30% of breast cancer cases. Both HER-2/neu-targeted DNA-based and transgene modified dendritic cell (DC)-based vaccines are potent elements in eliciting HER-2/neu specific antitumor immune responses; however, there has been no side-by-side comparison of these two different immunization methods. We utilized an in vivo murine tumor model expressing the rat neu Ag to compare the immunization efficacy between DC transduced with replication-deficient adenovirus containing neu (AdVneu), to form DCneu, and plasmid DNA (pcDNA) vaccine. DCneu displayed an upregulation of immunologically important molecules and inflammatory cytokines expression such as IL-6 that partially mediated conversion of the regulatory T (Tr) cell suppression. Wildtype FVB/N mice immunized with DCneu induced stronger HER-2/neu-specific humoral and cellular immune responses compared to plasmid DNA immunized mice. Furthermore, mice immunized with DCneu remained completely protected from tumor challenge compared to partial or no protection observed in DNA immunized mice in two tumor animal models. In FVBneuN transgenic mice, which develop spontaneous breast tumors at 4-8 months of age, DCneu significantly delayed tumor onset when immunization conducted in mice at a younger age. Taken together, we demonstrated that a HER-2/neu-gene modified DC vaccine is more potent than a plasmid DNA vaccine in inducing neu specific immune responses resulting in greater protective and preventative effects in the tumor models examined. <p>In another study, we examined the use of a DC-based cancer vaccine involving the phagocytosis of apoptotic tumor cells expressing heat shock protein 70 (HSP70). The dual role of HSP70, as an antigenic peptide chaperone and danger signal, makes it especially important in DC-based vaccination. In this study, we investigated the impacts of apoptotic transgenic MCA/HSP tumor cells expressing HSP70 on DC maturation, T cell stimulation and overall vaccine efficacy. We found that DC with phagocytosis of MCA/HSP in the early phase of apoptosis expressed more peptide-major histocompatibility class (pMHC) I complexes, stimulated stronger cytotoxic T lymphocytes (CTL) responses and induced greater immune protection against MCA tumor cell challenge, compared to mice immunized with DC that phagocytosed MCA/HSP cells in the late phase of apoptosis. Taken together, our data demonstrated that HSP70 expression on apoptotic tumor cells stimulated DC maturation and DC with phagocytosis of apoptotic tumor cells expressing HSP70 in early phase of apoptosis more efficiently induced tumor-specific CTL responses and immunity than DC with phagocytosis of apoptotic tumor cells in late phase of apoptosis. <p>Overall, we have examined variations in designing DC-based cancer vaccines in two completely different model systems. Taken together, our results may have an important impact in designing DC-based antitumor vaccines.

Human Epidermal Growth Factor Receptor 2 (HER-2/neu) is a breast tumor antigen (Ag) commonly overexpressed in 30% of breast cancer cases. Both HER-2/neu-targeted DNA-based and transgene modified dendritic cell (DC)-based vaccines are potent elements in eliciting HER-2/neu specific antitumor immune responses; however, there has been no side-by-side comparison of these two different immunization methods. We utilized an in vivo murine tumor model expressing the rat neu Ag to compare the immunization efficacy between DC transduced with replication-deficient adenovirus containing neu (AdVneu), to form DCneu, and plasmid DNA (pcDNA) vaccine. DCneu displayed an upregulation of immunologically important molecules and inflammatory cytokines expression such as IL-6 that partially mediated conversion of the regulatory T (Tr) cell suppression. Wildtype FVB/N mice immunized with DCneu induced stronger HER-2/neu-specific humoral and cellular immune responses compared to plasmid DNA immunized mice. Furthermore, mice immunized with DCneu remained completely protected from tumor challenge compared to partial or no protection observed in DNA immunized mice in two tumor animal models. In FVBneuN transgenic mice, which develop spontaneous breast tumors at 4-8 months of age, DCneu significantly delayed tumor onset when immunization conducted in mice at a younger age. Taken together, we demonstrated that a HER-2/neu-gene modified DC vaccine is more potent than a plasmid DNA vaccine in inducing neu specific immune responses resulting in greater protective and preventative effects in the tumor models examined. <p>In another study, we examined the use of a DC-based cancer vaccine involving the phagocytosis of apoptotic tumor cells expressing heat shock protein 70 (HSP70). The dual role of HSP70, as an antigenic peptide chaperone and danger signal, makes it especially important in DC-based vaccination. In this study, we investigated the impacts of apoptotic transgenic MCA/HSP tumor cells expressing HSP70 on DC maturation, T cell stimulation and overall vaccine efficacy. We found that DC with phagocytosis of MCA/HSP in the early phase of apoptosis expressed more peptide-major histocompatibility class (pMHC) I complexes, stimulated stronger cytotoxic T lymphocytes (CTL) responses and induced greater immune protection against MCA tumor cell challenge, compared to mice immunized with DC that phagocytosed MCA/HSP cells in the late phase of apoptosis. Taken together, our data demonstrated that HSP70 expression on apoptotic tumor cells stimulated DC maturation and DC with phagocytosis of apoptotic tumor cells expressing HSP70 in early phase of apoptosis more efficiently induced tumor-specific CTL responses and immunity than DC with phagocytosis of apoptotic tumor cells in late phase of apoptosis. <p>Overall, we have examined variations in designing DC-based cancer vaccines in two completely different model systems. Taken together, our results may have an important impact in designing DC-based antitumor vaccines.

The aryl hydrocarbon receptor (AhR) binds synthetic and chemoprotective phytochemicals, and research in this laboratory has developed selective AhR modulators (SAhRMs) for treatment of breast cancer. Activation of the AhR through agonists such as TCDD inhibits hormone activation of several E2-responsive genes in breast cancer cell lines. In this study, inhibition of E2-induced proliferation and gene expression by TCDD has been investigated in the uterus of wildtype, ERKO and AhRKO mice. Cyclin D1, DNA polymerase ?, and VEGF mRNA levels are induced by E2 through ER? in the uterus as determined by in situ hybridization studies. TCDD down-regulated E2-induced cyclin D1 and DNA polymerase ? expression, but not E2-induced VEGF expression, in wild-type mice, but not AhRKO mice, confirming the role of the AhR. Furthermore, protein synthesis was not necessary for induction of cyclin D1 or DNA polymerase ?gene expression by E2 or inhibition of these responses by TCDD. Therefore, AhR-ER? crosstalk directly regulates the expression of genes involved in cell proliferation in vivo. AhR agonists induce down-regulation of ErbB family receptors in multiple tissues/organs suggesting possible inhibitory interactions with chemotherapeutic potential. Recently, it has been reported that the SAhRM 1,1??,2,2??-tetramethyldiindolylmethane inhibited DMBA-induced mammary tumor growth in rats and also inhibited MAPK and PI3-K pathways in human breast cancer cells. BT-474 and MDA-MB-453 cell lines are ErbB2-overexpressing breast cancer cells that express functional AhR and exhibit constitutive activation of MAPK and PI3-K pathways. Therefore, 1,1??,2,2??-tetramethyldiindolylmethane-induced inhibition of ErbB2 signaling was investigated in these cells lines and in the MMTV-c-neu mouse mammary tumor model, which overexpresses ErbB2 in the mammary gland. The growth of ErbB2 overexpressing cell lines and mammary tumors was inhibited by 1,1??,2,2??-tetramethyldiindolylmethane; however, modulation of MAPK or PI3-K pathways and cell cycle proteins nor induction of apoptosis by 1,1',2,2'-tetramethyldiindolylmethane was observed in the ErbB2overexpressing cell lines. Current studies are investigating mitochondrial effects of 1,1??,2,2??-tetramethyldiindolylmethane in the ErbB2-overexpressing cell lines, as well as continuing studies on gene expression profiles in the mammary glands of MMTV-c-neu mice to better understand and identify critical genes that are responsible for ErbB2-mediated transformation and growth of cancer cells/tumors.

In the era of personalized cancer treatment, adoptive T cell therapy (ACT) shows promise for the treatment of solid cancers. However, partial or mixed responses remain common clinical outcomes due to the heterogeneity of tumours. Indeed, in many patients it is typical to see a response to ACT in one tumour nodule, while others show little or no response. Thus, defining the tumour features that distinguish those that respond to ACT from those that do not would be a significant advance, allowing clinicians to identify patients that might benefit from this treatment approach. The first chapter of this thesis provides the necessary background to understand the principals behind and components of ACT. This chapter also offers selected historical advances contributing to the current state of the field. The second chapter introduces a novel murine model of breast cancer developed to investigate the tumour-specific mechanisms associated with immune evasion in an ACT setting. The third chapter describes the in vivo characterization of mammary tumour cell lines derived from our mouse model that reliably showed complete, partial or no response to ACT. Using these cell lines, we were able to characterize in vivo tumour-specific differences in cytotoxic T cell trafficking, infiltration, activation, and proliferation associated with response to ACT. In the fourth chapter, we used bioinformatics approaches to develop a preliminary predictive gene signature associated with response to ACT in our mammary tumour model. We used this signature to predict outcome and then test a number of murine mammary tumours in vivo, with promising results, wherein 50% of tumours responded to ACT as predicted based upon gene expression. Thus, using an innovative model for breast cancer, these results suggest that there are tumour-specific features that can be used a priori to predict how a tumour will respond to adoptive T cell therapy. Importantly, these findings might facilitate the design of immunotherapy trials for human breast cancer. / Graduate

Dendritic cells (DCs) are promising mediators of anti-tumour immune responses. Unfortunately, a major hindrance to the development of highly effective DC vaccines is their short lifespan. Tumour antigen presentation may also not be optimal. We hypothesize that the introduction of exogenous survival factors (SFs) would prolong DC longevity and that modulation of TAA glycosylation will improve antigen presentation. To this end, we have constructed bicistronic lentivectors (LVs) encoding the xeno Tumour-Associated-Antigen (TAA), rHER-2/neu, and one of five candidate SFs. We demonstrated that our LVs can effectively protect transduced DCs from apoptosis when subjected to apoptosis-inducing conditions. TAA glycosylation has been proposed to obstruct the processing and presentation of peptides on MHC molecules. To address this second issue, we have engineered a LV that encodes a partially deglycosylated rHER-2/neu. Overall, we have generated the tools to alter intrinsic DC properties, which we believe will be integral to improving DC vaccine efficacy.

Dendritic cells (DCs) are promising mediators of anti-tumour immune responses. Unfortunately, a major hindrance to the development of highly effective DC vaccines is their short lifespan. Tumour antigen presentation may also not be optimal. We hypothesize that the introduction of exogenous survival factors (SFs) would prolong DC longevity and that modulation of TAA glycosylation will improve antigen presentation. To this end, we have constructed bicistronic lentivectors (LVs) encoding the xeno Tumour-Associated-Antigen (TAA), rHER-2/neu, and one of five candidate SFs. We demonstrated that our LVs can effectively protect transduced DCs from apoptosis when subjected to apoptosis-inducing conditions. TAA glycosylation has been proposed to obstruct the processing and presentation of peptides on MHC molecules. To address this second issue, we have engineered a LV that encodes a partially deglycosylated rHER-2/neu. Overall, we have generated the tools to alter intrinsic DC properties, which we believe will be integral to improving DC vaccine efficacy.

The diploma thesis Forms of internal differentiation and their application in the textbook set Berliner Platz Neu 1 deals with internal differentiation in the chosen textbook set for German as a foreign language. The thesis is devided into a theoretical and a practical part. In the theoretical part, attention is given to the importance of internal differentiation as one of the possible solution to the heterogeneity in the classroom. The thesis focuses on four main forms of internal differentiation - the methodological, social, methodical and work- organizational differentiation. Each form is further divided into subcategories which are described in details. The practical part is devoted to the actual research. Firstly, it presents theoretically the textbook set Berliner Platz Neu 1, its authors and its parts. The textbook set consist of a textbook combined with an exercise book, an additional exercise book, a teśt book and a teacher's book. The textbook set is analysed according to given criteria for didactic and social differentiation. The thesis highlights these activities in the textbook that are suitable for differentiation and comments on the possibility of further differentiation. The aim of the thesis was to find out whether and how the selected textbook set Berliner Platz Neu 1 work with...

Obwohl Kinder- und Jugendbücher sowohl weltanschauliche als auch religiöse Inhalte und Normen widerspiegeln, sind sie von Seiten der (Religions-)Wissenschaft als Untersuchungsgegenstand bisher vernachlässigt worden. Während Theolog*innen, Literaturwissenschaftler*innen und Pädagog*innen mit Beginn des 21. Jahrhunderts allmählich damit begonnen haben, religionsaffine Kinder- und Jugendbücher auf ihre Aussagen zu Kategorien wie Geschlecht und Toleranz wie auch zu ihren Repräsentationen zu Religion(en) selbst zu untersuchen, sind religionswissenschaftliche Arbeiten zu dem Forschungsfeld weiterhin kaum vorhanden. Die vorliegende Publikation versteht sich als Beispiel für eine religionswissenschaftliche Herangehensweise an (neu-)heidnische Kinder- und Jugendliteratur. An dem Kinder- und Jugendbuch Die Geschichten von Yggdrasil. Das kleine Familienbuch der Nordischen Sagen von Luci van Org (2017) wird exemplarisch gezeigt , wie durch einen zweifachen methodischen Zugang aus Qualitativer Inhaltsanalyse und Narrativer Analyse die religiösen und weltanschaulichen Vor- und Darstellungen im Buch eruiert werden können.

Alzheimer’s disease (AD) is histopathologically characterized by neurodegeneration, the formation of intracellular neurofibrillary tangles and extracellular Aβ deposits that derive from proteolytic processing of the amyloid precursor protein (APP). As rodents do not normally develop Aβ pathology, various transgenic animal models of AD were designed to overexpress human APP with mutations favouring its amyloidogenic processing. However, these mouse models display tremendous differences in the spatial and temporal appearance of Aβ deposits, synaptic dysfunction, neurodegeneration and the manifestation of learning deficits which may be caused by age-related and brain region- specific differences in APP transgene levels. Consequentially, a comparative temporal and regional analysis of the pathological effects of Aβ in mouse brains is difficult complicating the validation of therapeutic AD treatment strategies in different mouse models. To date, no antibodies are available that properly discriminate endogenous rodent and transgenic human APP in brains of APP-transgenic animals. Here, we developed and characterized rat monoclonal antibodies by immunohistochemistry and Western blot that detect human but not murine APP in brains of three APP-transgenic mouse and one APP-transgenic rat model. We observed remarkable differences in expression levels and brain region-specific expression of human APP among the investigated transgenic mouse lines. This may explain the differences between APP-transgenic models mentioned above. Furthermore, we provide compelling evidence that our new antibodies specifically detect endogenous human APP in immunocytochemistry, FACS and immunoprecipitation. Hence, we propose these antibodies as standard tool for monitoring expression of endogenous or transfected APP in human cells and APP expression in transgenic animals.