Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2016-07-12T13:09:04Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertacao_MitalleMatos_PPGG.pdf: 3448234 bytes, checksum: fe598459348fe967a8f1c2e5067f7d99 (MD5) / Made available in DSpace on 2016-07-12T13:09:04Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertacao_MitalleMatos_PPGG.pdf: 3448234 bytes, checksum: fe598459348fe967a8f1c2e5067f7d99 (MD5) Previous issue date: 2015-03-02 / CNPq / Danos causados por doenças virais estão entre os principais fatores limítrofes da produtividade do feijão-caupi. Em condições de estresse os fatores de transcrição (TFs) participam ativamente das etapas iniciais do processo de detecção e sinalização, regulando a expressão de vários grupos gênicos. Neste sentido, objetivou-se caracterizar TFs da família MYB e avaliar sua expressão diferencial frente à infecção viral, bem como determinar genes de referência (RGs) para normalização dos dados em RT-qPCR sob diferentes condições de estresse e controles. Por meio de análises in silico no banco NordEST, identificamos no transcriptoma do feijão-caupi um total de 86 candidatos a TF MYB, classificados em três subfamílias. A análise dos componentes estruturais do domínio R2R3-MYB permitiu observar a conservação dos aminoácidos característicos desta classe protéica em feijão-caupi. Por sua vez, o padrão de distribuição em pseudocromossomos de Phaseolus vulgaris indicou que genes MYB sofreram duplicações em tandem e intercromossomais, contribuindo para sua expansão no feijão-caupi. A análise filogenética formou 18 subclados, apoiados pela estrutura dos motivos funcionais da região C-terminal das proteínas. Das tags SuperSAGE diferencialmente expressas sob infecção viral, três foram reguladas positivamente, indicando a participação de candidatos MYB na resposta ao estresse viral. Dos sete RGs avaliados em três conjuntos experimentais, β-tubulina, Skip16 e Act2/7 + Skip16 foram as melhores combinações para seca, salinidade e vírus, respectivamente, podendo ser recomendados como normalizadores para estudos de expressão diferencial em feijão-caupi. Neste estudo identificamos a maior família de TFs em plantas observando sua participação ativa na resposta de defesa contra estresses em feijão-caupi. / Damages caused by viral diseases are among the main factors affecting the cowpea productivity. Under stress conditions, transcription factors (TFs) actively participate in the initial stages of the detection and signaling process by regulating the expression of various gene groups. In this sense, the objective of the present work was to characterize members of the MYB TF-family and evaluate their differential expression under viral infection, also determining reference genes (RGs) for data normalization in RT-qPCR under different stress and control conditions. Using in silico approaches to analyze the NordEST databank, a total of 86 MYB TF-candidates could be identified, being classified into three subfamilies. An analysis of the structural components of the R2R3-MYB domain allowed the identification of conserved amino acid residues of this protein class in cowpea. In turn, the MYB distribution pattern in the pseudochromosomes of Phaseolus vulgaris indicated that MYB members suffered in tandem and interchromosomal duplications, contributing to their expansion in cowpea. Phylogenetic analysis formed 18 subclades, supported by structural features of motifs in the C-terminal region of the protein. Of differentially expressed SuperSAGE tags under viral infection, three were upregulated, indicating the involvement of MYB candidates in response to viral stress. Considering the seven tested RGs under three experimental conditions, β-tubulin, Skip16 and Act2/7 + Skip16 were the best combinations for drought, salinity and viruses, respectively, recommended as normalizer genes in studies of differential expression in cowpea. In the present work we identified members of the largest family of TFs in plants observing their active participation in defense against stress response in cowpea.

O linfoma de células do manto é associado à translocação t(11;14)(q13;q32) e à hiperexpressão da ciclina D1. Os pacientes com linfoma de células do manto apresentam-se com doença avançada ao diagnóstico e a fase leucêmica da doença é observada em cerca de um terço dos casos. Os linfócitos B virgens pré-centro germinativo, que ocupam a zona do manto dos folículos linfóides secundários, constituem a origem celular do linfoma de células do manto. A hiperexpressão da ciclina D1, por si só, não é suficiente para a patogênese da neoplasia, e a elucidação das alterações moleculares adicionais poderá fundamentar novas estratégias terapêuticas. Nesse contexto, os métodos de estudo do perfil de expressão gênica em larga escala têm potencial para auxiliar na descoberta dessas alterações moleculares adicionais. Todavia, nos estudos que empregaram esses métodos até o momento, o material genético foi obtido de amostras de gânglios acometidos pelo tumor, que contêm uma proporção variável de células normais do estroma do tecido linfóide. Por isso, ainda não se sabe quais dos genes identificados como alterados no linfoma de células do manto são específicos das células linfomatosas, e quais são dependentes das células normais que perfazem o estroma do gânglio. Com o objetivo de elucidar as alterações moleculares do linfoma de células do manto em nível celular, realizamos um estudo comparativo entre o perfil de expressão gênica de células linfomatosas purificadas e de linfócitos B virgens, utilizando microarrays de oligonucleotídeos. Células linfomatosas e linfócitos B virgens (IgD+CD38±CD27-) foram purificados em colunas magnéticas a partir do sangue periférico de pacientes com linfoma de células do manto em fase leucêmica e de amígdalas de indivíduos normais, respectivamente (pureza > 95%). Três indivíduos foram selecionados em cada grupo e os experimentos foram realizados em duplicatas usando microarrays comerciais modelo CodeLink Human UniSet I, com 10.000 genes. Foram identificados 106 genes com variação de expressão maior do que três vezes, 63 deles induzidos e 43 reprimidos no linfoma de células do manto em relação aos linfócitos B virgens. Dez genes (seis induzidos e quatro reprimidos) foram selecionados para quantificação, por RT-PCR em tempo real, em amostras de sangue periférico de 21 pacientes com linfoma de células do manto em fase leucêmica, além de 14 pacientes com outras doenças linfoproliferativas crônicas e de sete indivíduos sem doenças neoplásicas. Os resultados dos experimentos com microarrays foram confirmados pela quantificação por RT-PCR em tempo real em todos os 10 genes selecionados e os valores de expressão do gene TLR1 obtidos por esse método demonstraram correlação significativa com a sobrevida dos pacientes com linfoma de células do manto. Além de identificar vários genes modulados no linfoma de células do manto em nível celular, este estudo também revelou a expressão aberrante de diversos genes relacionados à apoptose e às vias de sinalização PI3K/AKT, WNT e TGFβ. Esses resultados adicionam informações originais à patogênese molecular do linfoma de células do manto e apontam para vias específicas de sinalização molecular como potenciais alvos para novas abordagens terapêuticas. / Mantle cell lymphoma is associated with the translocation t(11;14)(q13;32) and overexpression of cyclin D1. Mantle cell lymphoma is predominantly disseminated at diagnosis and a frank leukemic phase is detected in one third of patients. The pre-germinal-center naive B-cells, which populate the mantle zone of the secondary lymphoid follicles, are the cells of origin of mantle cell lymphoma. Overexpression of cyclin D1 is not sufficient by itself to cause lymphoma, and a better understanding of the additional molecular lesions may provide insights toward new therapeutic approaches. In this context, large scale gene expression studies may be useful in the investigation of such additional molecular lesions. However, the great majority of mantle cell lymphoma cases studied by these methods to date had the genetic material harvested from lymph nodes, which have a variable proportion of normal cells from the lymphoid stroma. It is therefore not known how many genes identified as differentially expressed in mantle cell lymphoma by tumor versus normal experiments are cell-autonomous rather than dependent on the tumor microenvironment. To address this issue, we compared the gene expression profile of mantle cell lymphoma cells and normal naive B-cells using oligonucleotide microarrays. Lymphoma cells and naive B-cells (IgD+CD38±CD27-) were highly purified, by magnetic activated cell sorting, from the peripheral blood of patients with mantle cell lymphoma in the leukemic phase and from tonsils of normal individuals, respectively (purity > 95% in all samples). Three individuals were selected for each group and experiments were performed in replicates using the Amersham CodeLink Human UniSet I Bioarrays, with 10,000 genes. We identified 106 genes differentially expressed with a fold change of at least three-fold, 63 induced and 43 repressed in mantle cell lymphoma in comparison with naive B-cells. Ten genes were selected (six induced and four repressed in lymphoma cells) for quantification by real-time RT-PCR in non-purified peripheral blood samples from 21 patients with mantle cell lymphoma in the leukemic phase, as well as in 14 patients with other chronic lymphoproliferative diseases and seven normal individuals. Microarray results were confirmed by real-time RT-PCR for all selected genes and expression values of the TLR1 gene as quantified by this method showed significant correlation with patient survival in mantle cell lymphoma. In addition to the identification of several modulated genes in mantle cell lymphoma at cellular level, this study revealed that some genes functionally connected through apoptosis or the PI3K/AKT, WNT and TGFβ signaling pathways are aberrantly expressed in mantle cell lymphoma. These results add original data to the molecular pathogenesis of mantle cell lymphoma and point to specific molecular signaling pathways related to inhibition of apoptosis as potential targets for new therapeutic approaches.

Doctor of Philosophy / Department of Plant Pathology / Barbara S. Valent / Rice blast is a major fungal disease on rice, caused by the hemibiotrophic filamentous ascomycete fungus, Magnaporthe oryzae. This disease accounts for 157 million tons of grain loss annually. The fungus produces a specialized cell called appressorium to penetrate the host surface barrier and enter inside. It produces intracellular Invasive Hyphae (IH) that grow form cell to cell to colonize the host. The mechanisms of appressorium formation and host penetration have been studied in detail but the host colonization strategies remain largely unknown. We applied live-cell imaging to characterize spatial and temporal development of IH and plant responses inside successively-invaded rice cells. Early loading experiments with the endocytotic tracker, FM4-64, showed dynamic plant membranes around IH. These hyphae showed remarkable plasticity and recruited plant cell components. IH exhibited pseudohyphal growth and were sealed in plant membrane, termed the Extra-Invasive Hyphal Membrane (EIHM). The fungus spent up to 12 hours in the first cell, often tightly packing it with IH. IH that moved into neighboring cells were biotrophic, although they were initially thinner and grew more rapidly. IH in neighboring cells were wrapped in EIHM with distinct membrane caps at the hyphal tips. Time-lapse imaging showed IH scanning plant cell walls before crossing them, and transmission electron microscopy showed crossing occurring at pit fields. This and additional evidence strongly suggest that IH co-opt plasmodesmata for cell-to-cell movement. Our studies have revealed insights into a novel hemibiotrophic strategy employed by the blast fungus. Few genes have been previously characterized that impact the biotrophic IH. To understand the molecular basis of the biotrophic infection strategy we employed Laser Microdissection (LM) technology to isolate and purify the IH at this early growth stage. We compared the gene expression of these samples with axenically-grown mycelium using M. oryzae whole genome microarrays. We identified several hundreds of infection specific genes. We have shown that LM technology can be used to isolate homogenous cells from the infected rice tissues to study the underlying molecular mechanisms of signaling during disease formation. These studies will be very critical to understand the host-pathogen interactions to eventually develop durable management strategies.

Adult hippocampal neurogenesis is regulated at various levels and by various factors. Genetic influence is an important key determinant of adult neurogenesis and exerts its effects at all levels. In vivo studies have suggested that adult hippocampal neurogenesis is highly variable and heritable among different laboratory strains of mice. To dissect the genetic effect from other contributing factors, it is necessary to study adult neurogenesis under highly controlled environment conditions. We extracted adult hippocampal precursor cells (AHPCs) from 20 strains of the BXD set of recombinant inbred mice, cultured them and studied the effect of genetic background on neurogenesis. The BXD panel consists of mouse lines derived from an intercross between inbred parentals C57BL/6J and DBA/2J. Both of the parentals are fully sequenced and all the strains are well characterized in terms of genotypic and phenotypic characteristics. This allows us to use advanced genetic techniques to identify novel genomic loci and gene-gene interactions important in adult neurogenesis. Comparison of the AHPCs from 20 BXD strains, with respect to cell proliferation and neuronal and astrocytic differentiation in vitro, revealed a large variation for these traits across the strains. Proliferation, as measured by BrdU incorporation, showed over two- fold differences between the extremes. Similar differences were observed for neurogenic (4-fold) and astrogenic differentiation (2-fold). These three traits all showed strong heritability values indicating that the differences were mainly attributed to the genetic component. QTL mapping, with these phenotypic data, revealed that there was no major contribution from single loci controlling these traits. Instead, we found many loci with smaller effects associated with these traits. Gene expression profiling using RNA samples from proliferating cultures of the 20 BXD mice strains yielded two cis eQTL candidates that directly regulated proliferation, LRP6 and Chchd8. LRP6 is well known as a co-receptor of Wnt signaling, but the function of Chchd8 is not known. Further experimentation, using over expression and gene silencing demonstrated that LRP6 negatively regulates AHPCs proliferation. Thus, from this study using a system genetics approach, we were able to identify, LRP6 as a novel regulator of adult hippocampal neurogenesis.

Colorectal cancer is the third most common cancer and the leading cause of cancer deaths in Western industrialised countries. Despite recent advances in the screening, diagnosis, and treatment of colorectal cancer, an estimated 608,000 people die every year due to colon cancer. Our current knowledge of colorectal carcinogenesis indicates a multifactorial and multi-step process that involves various genetic alterations and several biological pathways. The identification of molecular markers with early diagnostic and precise clinical outcome in colon cancer is a challenging task because of tumour heterogeneity. This Ph.D.-thesis presents the molecular and cellular mechanisms leading to colorectal cancer. A systematical review of the literature is conducted on Microarray Gene expression profiling, gene ontology enrichment analysis, microRNA and system Biology and various bioinformatics tools. We aimed this study to stratify a colon tumour into molecular distinct subtypes, identification of novel diagnostic targets and prediction of reliable prognostic signatures for clinical practice using microarray expression datasets. We performed an integrated analysis of gene expression data based on genetic, epigenetic and extensive clinical information using unsupervised learning, correlation and functional network analysis. As results, we identified 267-gene and 124-gene signatures that can distinguish normal, primary and metastatic tissues, and also involved in important regulatory functions such as immune-response, lipid metabolism and peroxisome proliferator-activated receptors (PPARs) signalling pathways. For the first time, we also identify miRNAs that can differentiate between primary colon from metastatic and a prognostic signature of grade and stage levels, which can be a major contributor to complex transcriptional phenotypes in a colon tumour.

<p>Based on breast cancer clinical trial data accumulated over the last several decades it is obvious that standard breast cancer therapeutics extend survival in breast cancer patients. However, only a minority of patients within these trials derive benefit from treatment. In a population of breast cancer patients treated with adjuvant therapy after surgery, many patients are over-treated, as they would never experience relapse even without receiving adjuvant therapies. Among the remaining patients, some achieve durable remission from therapy, whereas others relapse despite therapy. Hence, there is an obvious need to develop biomarkers that can serve to identify these three populations of patients, such that only patients who are likely to benefit from available therapies are treated with these therapies, as well as to develop new therapies for the treatment of patients who aren’t afforded durable remission by approved treatments. Here, we present the identification of biomarkers to identify low risk breast cancer patients who experience excellent long-term survival even without adjuvant therapy. Conversely, high risk patients represent those patients most likely to benefit from intervention with aggressive treatment regimens. We also report on the identification of biomarkers which can predict the likelihood of response to approved chemotherapy regimens, which could be used to further stratify high risk patients into responders and non-responders. Finally, for high risk patients unlikely to be afforded durable remission from available therapies, we report on the identification of agents that target breast tumor-initiating cells, and may be effective for the treatment of these patients.</p> / Doctor of Philosophy (PhD)

Der JAK/STAT-Signalübertragungsweg ist im Tierreich evolutionär konserviert und spielt eine Rolle in der Entwicklung eines Organismus, sowie beim Erhalt von Stammzellen. Desweiteren verursacht eine erhöhte Signalaktivität in Blutzellen Leukämie. Mit Hilfe der genetischen und molekularen Methoden, die in der Taufliege Drosophila melanogaster verfügbar sind, wurden die Hauptkomponenten der JAK/STAT-Signalkaskade identifiziert.Ziel dieser Arbeit war die Identifikation von Effektoren, die durch die JAK/STAT-Signalkaskade in Drosophila angeschaltet werden und die Bildung von Bluttumoren verursachen. Die Untersuchung des Geneexpressionsmusters ergab, dass 1197 Gen-Loci direkt oder indirekt durch den Liganden der Signalkaskade namens UPD zu unterschiedlichen Zeitenpunkten reguliert werden. Mit Hilfe von bioinformatischen Methoden konnten diese 1197 Gene zu immunologisch relevanten Kategorien (auch Gene Ontology genannt) zugewiesen werden, welche ebenfalls eine zeitlich dynamische Verteilung aufweisen. Weiterhin identifizierte die Promotoranalyse von hoch-regulierten Genen DNA-Bindungsstellen, an die der Transkriptionsfaktor der JAK/STAT-Signalkaskade mit hoher oder niedriger Affinität bindet. Die Rolle von zehn der 1197 Gene wurde in der Taufliege bezüglich der Tumorentwicklung untersucht. Darunter befinden sich Gene, die für die Zellpolarität verantwortlich sind; eine Funktion, die kürzlich mit der Aktivierung der JAK/STAT-Signalkaskade im Epithelgewebe in Verbindung gebracht wurde.

Un défi de la transcriptomique est d'explorer l'intégralité du répertoire des transcrits normaux et anormaux. Les analyses de GEP (Gene Expression Profiling) basées sur la technologie des puces à ADN sont largement exploitées en cancérologie depuis plusieurs années. Parallèlement, les nouvelles méthodes basées sur le séquençage à haut débit offrent désormais la possibilité de réaliser des analyses précises et sensibles nécessaires à l'étude des cellules normales et cancéreuses. Quelle que soit la méthode, la caractérisation de l'ensemble des transcrits codants et non-codants représente un réel défi biologique pour la recherche de nouveaux marqueurs de diagnostic et de pronostic, et pour la bonne prise en charge des patients. Dans ce travail, j'ai eu l'occasion de traiter deux aspects différents de la biologie qui convergent vers l'identification de transcrits exprimés de manière aberrante dans les leucémies myéloïdes. Le premier aspect a consisté à proposer une sélection de biomarqueurs moléculaires pour la caractérisation de la leucémie myélomonocytaire chronique (LMMC). A partir de l'expression de ces gènes, nous avons développé un score de pronostic qui a permis de définir deux groupes de patients cliniquement distincts. Nous avons ensuite complété notre étude par une caractérisation phénotypique par cytométrie en flux des sous-populations cellulaires aberrantes constituant les lignages mono- et granulocytaires. Une partie de ce travail a été étendue aux leucémies aiguës myéloïdes (LAM) à caryotype normal (CN). L'autre aspect a consisté à participer à la mise en place d'une approche computationnelle intégrée pour caractériser de nouveaux ARNs non annotés et fort probablement non-codants. En explorant des données de Digital Gene Expression (DGE), nous avons quantifié et caractérisé la fraction de ces transcrits dans les régions intergéniques. Nous avons vérifié l'expression de ces nouveaux transcrits dans les tissus normaux et cancéreux en croisant avec d'autres données d'expression, telles que le RNA-Sequencing, et regarder leur conservation et leur expression dans d'autres espèces. / A challenge of transcriptomics is to explore the full repertoire of normal and abnormal transcripts. Gene expression profiling analyses based on microarray technology are widely used in cancer research since many years. Meanwhile, new methods based on high-throughput sequencing methods offers henceforth the possibility to undergo accurate and sensitive analyses necessary for studying normal and cancer cells. Despite the method, the characterization of all coding and non-coding transcripts is a real biological challenge in identifying novel diagnostic and prognostic markers, and for the proper care of patients. In the present work, I had the opportunity to address two different aspects of biology, both convergent toward the identification of aberrantly expressed transcripts in myeloid leukemia. The first aspect was to provide a selection of molecular biomarkers for the characterization of chronic myelomonocytic leukemia (CMML). We developed a gene expression-based prognostic score which identified two clinically distinct groups of patients. We then completed our study with a phenotypic characterization by flow cytometry of aberrant subpopulations constituting the myeloid and granulocytic lineages. A part of this work has been extended to acute myeloid leukemia (AML) patients with normal karyotype. The other aspect was to participate in the implementation of an integrated computational approach in order to characterize novel non annotated RNAs, more likely non-coding. We quantified and characterized the proportion of these transcripts in intergenic regions by exploring Digital Gene Expression (DGE) data. We checked their expression in normal and cancer tissues by crossing with RNA-Seq data, and their conservation and expression in other species.

Aplicando tecnologia de microarray, objetivamos traçar o perfil do programa de maturação das Mo-DC pulsadas com HIV autólogo inativado por AT-2, a fim de identificar marcadores específicos de ativação funcional e sugerir um perfil de expressão de genes úteis na identificação de respostas ao modelo in vitro das Mo-vacina DC. Essas informações podem ajudar a estabelecer assinaturas moleculares das funções celulares mais relevante para a melhoria das vacinas terapêuticas. O perfil transcricional foi analisado com base das vias celulares moduladas das Mo-DCs no estado imaturo, transitório e maduro. O HIV-1 inativado por AT-2 induz ativação de genes associados à apresentação de antígenos. Os conjuntos de genes do citoesqueleto podem influenciar a mudança de comportamento migratório das Mo-DCs ativadas. O aumento na expressão dos receptores celulares contribuem para o recrutamento de monócitos, DCs e macrófagos para o local da infecção. Além disso, modulam a resposta imune inata e adaptativa, incluindo a polarização das células Th e sub-regulação da resposta inflamatória, que pode interferir significativamente com a resposta imune. Coletivamente, o perfil transcricional das Mo-DCs induzido pelo HIV-1 inativado com AT-2 reflete uma significativa reprogramação imunológica e celular das células envolvidas na resposta imune do hospedeiro. Os resultados deste estudo focaram na interpretação de genes específicos dos perfis de transcrição das Mo-DCs como modelo terapêutico utilizado na vacina anti-HIV. As análises de assinaturas gene associado e sua correlação as respostas funcionais simplificam a identificação de indivíduos susceptíveis a vacina e a compreensão de eventuais falhas em ensaios clínicos. Microarray permitiu a análise quantitativa e simultânea da expressão de um elevado número de genes. Os estudos do perfil de expressão foram extremamente úteis para identificar os eventos moleculares e vias envolvidas nas funções de celular induzida por estímulos específicos. Em particular, os resultados sobre o padrão global da expressão dos genes subjacentes as modificações induzidas pelo HIV-1 inativado por AT-2, na fase inicial da administração do antígeno, pôde ser extremamente útil para a identificarmos marcadores de ativação e avaliar os efeitos biológicos que poderiam estar envolvidos para modificação e otimização de estratégias vacinação com Mo-DC / Applying microarray technology, we intend to profile the program to mature Mo-DC pulsed with autologous inactivated HIV by AT-2, in order to identify specific markers of functional activation and suggest a profile of expression of specific genes, useful identification of responders to in vitro model of Mo-DC vaccine. Such information may help to establish detailed molecular signatures of cellular functions most relevant to improving the therapeutic vaccines. The transcriptional profile was analyzed on the basis of the cellular pathways modulated in immature MoDC, transitional MoDC and mature MoDC. The AT-2-inactivated HIV-1 induction of MoDC results in the activation of genes associated with antigen presentation functions. A set of cytoskeletal genes that may potentially mediate shape change and migratory behavior of activated MoDC is also observed. The increase in the expression of immune receptors contribute to the recruitment of monocytes, DCs, and macrophages to the site of infection. Moreover, they modulate both innate and adaptive immune response, including the polarization of Th cells, and the down-regulation of the inflammatory response, which may significantly interfere with the immune response. Collectively, the transcriptional profile induced by AT-2-inactivated HIV-1 in MoDc reflects a significant cellular and immunological reprogramming of cells directly involved in the host immune response. The results of this study focused on the interpretation of specific genes of transcription profile of MoDC used in therapeutic HIV vaccine model. Supplementing the analyses with examination of associated gene signatures and their correlation to functional responses will simplify the identification of responsive vaccine individuals and the understanding of eventual failures in individuals enrolled in clinical trials. Microarray approach allows quantitative and simultaneous analysis of gene expression of a large amount of genes and the systematic studies of expression patterns are extremely useful for identify molecular events and key pathways involved in cellular functions induced by specific stimuli. In particular, data on the global pattern of gene expression underlying the modifications induced by AT-2-inactivated HIV-1 in MoDC, at early stages of antigen administration, may be extremely helpful for the identification of exclusive activation markers to trace the biological effects of modifications/optimizations of the MoDc vaccination strategy

Nach der Eradikation der humanen Pockenerkrankung stellen zoonotische Orthopockenvirus-(OPV-)Infektionen heute eine mögliche Bedrohung der öffentlichen Gesundheit dar. Hierbei sind insbesondere Kuhpocken-(CPXV), Affenpocken-(MPXV) und Vaccinia Viren (VACV) von Bedeutung. In dieser Arbeit wurde das Genexpressionsprofil humaner (HeLa) Zellen nach Infektion mit CPXV, MPXV oder VACV untersucht. Es wurden sowohl zelluläre Gene identifiziert, welche generell von allen verwendeten Viren reguliert wurden, als auch Gene, die eine Virus-spezifische Regulation durch individuelle OPV aufwiesen. Gemeinsamkeiten zeigten sich insbesondere zwischen CPXV und MPXV, welche, im Gegensatz zu VACV, die Expression zahlreicher Cytokine und Chemokine induzierten. Insbesondere für Interleukin-6, -8 und CXCL1 konnte auch auf Proteinebene eine gesteigerte Sekretion durch CPXV-infizierte Zellen nachgewiesen werden. Vermutlich aufgrund dieser Induktion, trat in vitro eine verstärkte Rekrutierung von Monozyten und Makrophagen in Folge einer CPXV-, nicht aber einer VACV-Infektion auf. Makrophagen spielen eine kontroverse Rolle im Rahmen einer OPV-Infektion und sind sowohl für deren Bekämpfung, als auch, im infizierten Zustand, für die Ausbreitung der Viren im Organismus von Bedeutung. Daher wurde die Replikationsfähigkeit von CPXV und VACV in Makrophagen charakterisiert. Der Virulenzfaktor p28, welcher von den meisten VACV Stämmen nicht kodiert wird, konnte als essentiell für die Replikation von CPXV in einer murinen Makrophagen-Zelllinie, primären peritonealen Makrophagen der Ratte und in Makrophagen aus primären humanen PBMCs identifiziert werden. In Anbetracht der Bedeutung der Replikationsfähigkeit in Makrophagen für die Ausbreitung einer OPV-Infektion im Wirtsorganismus, deuten diese Ergebnisse darauf hin, dass CPXV, im Fall einer weiteren Adaption an den Menschen, ein höheres Bedrohungspotential im Vergleich zu VACV aufweisen könnten. / Today, following the eradication of human smallpox, zoonotic infections caused by orthopoxviruses (OPV) are emerging as a potential human health threat. Especially cowpox viruses (CPXV), vaccinia viruses (VACV), and monkeypox viruses (MPXV) are gaining importance as a cause of infectious disease of man and livestock. This study aimed to analyse and compare the gene expression profile of human (HeLa) cells following infection with CPXV, MPXV or VACV. Cellular genes were identified which were either commonly modulated by infection with any of the three viruses, or which were specifically modulated by one individual OPV. Particularly similar effects on cellular gene expression were observed in the case of CPXV and MPXV infection, which both induced the expression of several cytokine and chemokine genes. Especially interleukin-6, -8, and CXCL1 were strongly secreted by CPXV-infected cells but not by VACV-infected cells. Consequently, CPXV infection also induced a strong chemotactic recruitment of monocytes and macrophages in vitro in contrast to VACV infection. Especially macrophages are known to play a controversial role during OPV infection. On the one hand, macrophages are of importance for the control of the infection. On the other hand, infected macrophages also facilitate virus spread across the organism. Therefore, the capability of CPXV and VACV to replicate in macrophages was analysed. Thereby, the poxviral virulence factor p28, which is absent from most strains of VACV, was identified as an essential factor, allowing CPXV replication in a murine macrophage cell line, primary peritoneal rat macrophages and in human PBMC-derived macrophages. Concerning the importance of productively infected macrophages for OPV spread, these results suggest that CPXV, if further adapted to human beings as host species, may harbor a greater threat to human health when compared to VACV.