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Das Neuroblastom ist der häufigste extrakraniell gelegene solide Tumor der pädiatrischen Onkologie. Der Verlauf der Erkrankung geht von spontaner Regression oder Differenzierung bis hin zu tödlich verlaufenden Erkrankungen. Die Mortalität von Patienten mit Tumoren in fortgeschrittenen Stadien ist immer noch sehr hoch. Die aggressivsten Tumoren sind die, die eine Amplifikation des Protoonkogens MYCN aufweisen. Eine Untergruppe dieser MYCN amplifizierten Tumoren weist eine Coamplifikation von DDX1 auf. Die Prognose dieser Patienten ist besser als die mit allein MYCN amplifizierten Tumoren, wenn auch immer noch schlechter als die von Patienten ohne MYCN Amplifikation. Das DDX1-Protein ist eine putative RNA-Helikase. Über seine genaue Funktion ist noch nicht viel bekannt. Ziel dieser Arbeit war es, potentielle Ziel-mRNAs von DDX1 zu identifizieren, um einen besseren Einblick in die Funktionen von DDX1 und mögliche Wege der Beeinflussung von Tumorverhalten und Prognose zu erhalten. Hierzu wurden eine DDX1 amplifizierte und eine nicht amplifizierte Zelllinie in Kultur genommen und eine Immunopräzipitation mit Zelllysaten der beiden Zelllinien durchgeführt – jeweils mit einem spezifischen Antikörper gegen DDX1 und einem unspezifischen Kontrollantikörper. Die Identifizierung der an DDX1 gebundenen mRNAs erfolgte mittels Microarray. Validiert wurden einige der im Microarray identifizierten RNAs mittels RT-PCR. CDK1, ATM und p18 ließen sich als spezifische Ziel-mRNAs von DDX1 identifizieren.

Regulation of N-myc oncogene expression is an important determinant of the biological behavior of neuroblastoma. The N-myc promoter contains several potential binding sites for transcription factors of the Sp1 family. Mutation of a CT-box motif contained within a 26 base pair region required for N-myc downregulation by retinoic acid decreased basal transcriptional activity and altered DNA-protein interactions of the promoter, while mutations flanking this motif did neither. On gel shift this region generated 3 specific DNA-protein complexes that were reliant on wild type sequence of the core CT element within it. Both Spl and Sp3 bound to the wild type probe as distinct complexes in specifically retarded bands, while neither protein was present on mutated sequences. Lysates from Drosophila S2 cells expressing exogenous Sp1 and Sp3 proteins were able to reproduce the gel shift complexes seen with neuroblastoma nuclear extract. Transient transfections of S2 cells showed that individually or together, Sp1 and Sp3 were able to trans-activate a N-myc CT-box-containing luciferase reporter construct in a dose-dependent manner. Conversely, transfection of CT-box oligonucleotide was able to decrease endogenous N-myc expression in neuroblastoma cells. Together these results suggest that the CT-box element serves a critical functional role, and in the basal state allows for N-myc transactivation by Spl and Sp3.

The suitability of neuroblastoma cell-based assays (Neuro-2a CBAs) was examined in order to characterize toxin profiles in ciguatera-related natural samples: microlagae (Gambierdiscus spp. and Prorocentrum rhathymum) and ciguatera-suspected fish samples from the Canary Islands. The elimination of the interferences of biological matrices and the specificity of the Neuro-2a CBA for ciguatoxins and maitotoxins were two aspects examined to favor the applicability of the Neuro-2a CBA for marine toxins detection. The results presented in this work have contributed to ciguatera risk assessment in the Canary Islands and support the use of CBAs as an alternative or complementary approach to the mouse bioassay for the determination of marine toxins. / La eficacia de los ensayos con células de neuroblastoma (Neuro-2a) ha sido examinada para la caracterización del perfil toxínico en muestras naturales asociadas a la ciguatera: microalgas (Gambierdiscus spp. y Prorocentrum rhathymum) y muestras de pescado potencialmente ciguatóxico procedentes de las Islas Canarias. La eliminación de las interferencias de las matrices biológicas y la especificidad de los ensayos con células Neuro-2a para ciguatoxinas y maitotoxinas han sido dos aspectos examinados para favorecer la aplicabilidad de los ensayos con células Neuro-2a a la detección de toxinas. Los resultados presentados en este trabajo han contribuido a la evaluación del riesgo de ciguatera en las Islas Canarias y apoyan el uso de los ensayos celulares como una aproximación alternativa o complementaria al bioensayo ratón para la determinación de toxinas marinas.

Prion diseases are neurodegenerative disorders that can affect humans and animals. The underlying event is a conformational change of the normal cellular prion protein (PrPC) into an aberrant isoform termed PrP-scrapie (PrPSc). PrPSc is thought to lead to neurodegeneration and activation of glial cells. Scrapie infection of neuroblastoma cells was shown to increase the expression of insulin receptor (IR). Additionally, a marked reduction of 125I-insulin binding sites was observed. Insulin stimulation showed alteration in both IR β-subunit tyrosine phosphorylation and extracellular signal regulated kinase-2 (ERK2) activity.  Furthermore, scrapie infection was shown to increase insulin-like growth factor-1(IGF-1) receptor (IGF-1R) expression, although the number of 125I-IGF-1-binding sites was reduced. Also binding affinity of 125I-IGF-1 to its receptor was reduced, and tyrosine phosphorylation of IGF-1R-β-subunit in response to IGF-1 was altered. The increased levels of neurotrophic receptors might represent a neuroprotective response to prion infection. However, scrapie infection instead leads to decreased function, decreased levels of functional receptors, or both, which could promote neurodegeneration in prion diseases, through attenuated neurotrophic support. In BV-2 microglial cells, LPS-induced iNOS (inducible nitric oxide synthase) expression and subsequent NO production were mainly mediated through c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) pathway. Antioxidant treatment indicates that oxidative suppressing mechanism(s) acts on JNK pathway possibly as a regulatory mechanism controlling the NO levels. The JNK pathway was also shown to play an important role in the survival of BV-2 cells. We show that BV-2 cells are protected from ongoing apoptosis by pro-survival activity mediated both by the JNK and p38 MAPK pathway during LPS-induced inflammation. This is very interesting findings since it is important for microglia to respond properly to a pathogen, without themselves being affected and undergo apoptosis.

Diss. (sammanfattning) Stockholm : Stockholms universitet, 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Thesis (Ph.D. )--University of Tennessee Health Science Center, 2007. / Title from title page screen (viewed on June 20, 2008 ). Research advisor: Clinton Stewart, Pharm.D. Document formatted into pages (xvi, 129 p. : ill.). Vita. Abstract. Includes bibliographical references (p. 112-129).

Neuroblastoma is an aggressive solid tumor that develops from immature cells of the nervous system and is almost exclusively diagnosed in infants and young children. Over the past decade a multitude of immune based therapies have been explored as therapeutic candidates for patients with neuroblastoma. The anti-GD2 monoclonal antibody, 3F8, and more recently, natural kill (NK) cell based therapies have been accepted as hopeful therapeutic options for patients with Neuroblastoma. These options however have many drawbacks including dose limiting pain, the development of tolerance, reliance on MHC mismatch and possible reliance on the invariant NK (iNK) cells population. Gamma Delta T cells, a subpopulation of T cells composed of a T cell receptor (TCR) with a gamma and a delta chain instead of an alpha and a beta; chain, have been shown to recruit a more robust immune response then both 3F8 and NK cells through their activation of antigen presenting cells (APCs) and non-reliance on MHC mismatch. Gamma Delta T cells are also able to recruit NK cells as well as other cytotoxic lymphocytes. For these reasons, it is believed that Gamma Delta T cell based treatment alone or in combination with an anti-GD2 monoclonal antibody may have a greater efficacy than either NK cells or an anti-GD2 monoclonal antibody alone. The intent of this thesis is to explore and evaluate the current state of Gamma Delta T cell based immunotherapy against the backdrop of NK cell based immunotherapy for neuroblastoma.

Tese (doutorado) - Pontifícia Universidade Católica do Paraná, Curitiba, 2011 / Bibliografia: f. 63-74 / Neuroblastoma é o tumor sólido maligno extracraniano mais comum da infância. Enquanto que em crianças muito pequenas podem regredir espontaneamente ou apresentar boa resposta ao tratamento, alterações genéticas que influenciam a apoptose, em alguns casos, / Neuroblastoma is the solid malignant extracranial tumor most common in childhood. While in very young children it may regress spontaneously or show good response to treatment, genetic changes in apoptosis pathway may result in resistance to chemotherapy o