Zugl.: Hamburg-Harburg, Techn. Universiẗat, Diss., 2000.

A Thesis submitted to The University of Arizona College of Medicine - Phoenix in partial fulfillment of the requirements for the Degree of Doctor of Medicine. / Recent advances in cancer immunology, highlighted by immune checkpoint inhibitors, have demonstrated that immunotherapy is a viable option in the oncologist’s armamentarium. Despite these advances, many patients are nonresponders. Preliminary studies have suggested that non-responders lack a de-novo anti-tumor antigen immune response that can be unmasked by checkpoint blockade; thus, strategies to induce anti-tumor immune responses are needed. We hypothesized that many tumor associated antigens (Ag) are readily susceptible to immune attack, but only in the context of identifying the tumor antigen epitopes that can reliably initiate an immune response, regardless of individual patient human leukocyte antigen (HLA) haplotype restrictions. We further hypothesized that epitope prediction strategies which seek to identify pan- or highly promiscuous-HLA binding epitopes would reduce the number of potential candidates and be more likely to accurately identify high-priority tumor Ag epitopes. Utilizing known HLA-serotype frequencies and setting a threshold of ninety percent of population coverage, regardless of race or ethnicity, twenty-nine different HLA-DRB1 haplotypes were chosen for antigen prediction utilizing the open source epitope prediction algorithm netMHCIIpan. Predictions were also performed for HLA-A serotypes utilizing the open source algorithm netMHCpan. Predicted epitopes were synthesized in the form of synthetic long peptides and tested in immune system sensitization assays involving unfractionated peripheral blood mononuclear cells (PBMC). Briefly, PBMC were subjected to a two-step culture, first synchronizing their exposure to the long peptides with aggressive surrogate activation of innate immunity, followed by IL-7-modulated T-cell hyperexpansion. Predictions resulted in identification of highly promiscuous-HLA binding epitopes. Unexpectedly, these epitopes clustered together forming high priority regions: unique “hot spots” with high densities of promiscuous HLA-binding epitopes from the widely expressed oncoproteins MUC1, HER2/neu and CMV-pp65 (p<0.0001, for predicted HLA-DRB1 binding affinities, compared to non-hot spot regions). Added synthetic long peptides (>20aa) derived from “hot spot” regions of MUC1, HER2/neu, and CMVpp65 reliably produced selective and sustained expansion of both CD4+ and CD8+ peptide-specific, interferon-γ (IFNγ)-producing Tcells when synchronized with step 2 exposure to exogenous IL-7 (p<0.0001 and p=0.0048, for CD4+ and CD8+ Ag-specific T-cells, respectively, compared to T-cells directed against peptides from non-hot spot regions). “Hot spot” peptide Ag-specific T-cells preferentially recognized endogenous tumor derived MUC1, either in MUC1 expressing tumor cell killing assays (p=0.038, compared to non-peptide Ag-specific T-cells) or as MUC1 tumor lysate when pulsed onto restimulatory PBMC (p=0.022 and 0.025, for CD4+ and CD8+ T-cells, respectively, compared to T-cells directed against peptides from non-hot spot regions). This mechanistically rational antigen selection sequence, effective even for unvaccinated donors, regardless of HLA-haplotype, enables rapid identification of tumor protein regions relevant for cancer immunology, including adoptive immunotherapy, vaccines, and even identification of tumor neo-antigens unique to each patient.

Proton beam radiotherapy is an emerging treatment tool for cancer. Its basic principle is to use a high-energy proton beam to deposit energy in a tumor to kill the cancer cells while sparing the surrounding healthy tissues. The therapeutic proton beam can be either a broad beam or a narrow beam. In this research, we mainly focused on the design and simulation of the broad beam produced by a passive double-scattering system in a treatment nozzle. The NEU codes package is a specialized design tool for a passive double-scattering system in proton beam radiotherapy. MCNPX is a general-purpose Monte Carlo radiation transport code. In this research, we used the NEU codes package to design a passive double-scattering system, and we used MCNPX to simulate the transport of protons in the nozzle and a water phantom. We used "mcnp_pstudy" script to create successive input files for different steps in a range modulation wheel for SOBP to overcome the difficulty that MCNPX cannot be used to simulate dynamic geometries. We used "merge_mctal" script, "gridconv" code, and "VB script embedded in Excel" to process the simulation results. We also invoked the plot command of MCNPX to draw the fluence and dose distributions in the water phantom in the form of two-dimensional curves or color contour plots. The simulation results, such as SOBP and transverse dose distribution from MCNPX are basically consistent with the expected results fulfilling the design aim. We concluded that NEU is a powerful design tool for a double-scattering system in a proton therapy nozzle, and MCNPX can be applied successfully in the field of proton beam radiotherapy.

Made available in DSpace on 2014-06-12T23:04:27Z (GMT). No. of bitstreams: 2 arquivo8877_1.pdf: 1460374 bytes, checksum: c08f2c8b72d50467c3068b2ce9762c97 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2007 / A proteína cerbB-2 tem sido bastante estudada não só por sua importância como fator prognóstico dos carcinomas da mama, mas sobretudo por ser um indicador para terapias com esquemas quimioterápicos. Para eliminar a subjetividade da interpretação do método imunohistoquímico convencional, este estudo tem como objetivo quantificar, morfometricamente, a imunomarcação da proteína cerbB-2 expressa em tumores de mama. Fragmentos de tecido mamário normal (n=10) e com doença tumoral (carcinoma ductal invasivo, CDI, n=51; fibroadenoma, n=11) foram fixados em formalina, submetidos à rotina histológica para inclusão em parafina. Cortes histológicos (4mm), corados em hematoxilina e eosina foram examinados para confirmar o diagnóstico. Os cortes foram incubados com solução de anticorpos por uma por uma hora em temperatura ambiente. A marcação foi visualizada após incubação com diaminobenzidina (DAB) e peróxido de hidrogênio. A análise morfométrica foi realizada utilizando uma estação de análise digital de imagens através do software de análise OPTIMAS®. A partir dos resultados obtidos pode-se concluir que a superexpressão do cerbB-2 em casos de CDI é um fenômeno condizente com o estágio de proliferação das células neoplásicas e quando analisados os casos de fibroadenoma, este marcador não exibiu qualquer correlação ou padrão específico, ao contrário apresentaram resultados semelhantes ao tecido mamário normal. Não houve diferenças significativas entre os diferentes scores qualitativos e a análise morfométrica digital, o que no mínimo demonstra a necessidade de estudos mais acurados a fim de resolver esta dificuldade de interpretação

<p> DER, the Drosophila Epidermal Growth Factor Receptor (DEgfr) is the only known fly orthologue of vertebrate Neu/ErbB2 receptor tyrosine kinase family. Receptor Tyrosine Kinases (RTKs) like DER and ErbB2 play an important role in regulating cell differentiation, cell proliferation and cell survival in metazoan animals. Neu/ErbB2 is over-expressed in 20-30% human breast cancers, which correlates with poor clinical prognosis in cancer patients. </p> <p> Our previous studies showed that rat-NeriJErbB2 could successfully signal in vivo using Drosophila adaptor and second messenger molecules. Here we regenerated the transgenic fly lines with various neu add-back alleles. We further re-established mis-expression phenotypes in various adult structures such as wings and eyes, the tissues known to require DEgfr signaling. By using genetic approach, we have demonstrated that the tyrosine residue at the 1028 site (NeuYA), might have an inhibitory role in RTK signaling. In addition we have already generated a number of double add-back neu alleles where tyrosine site at the 1028 site (neuYA) was added back to another Neu allele and made neuYAB, neuYAc neuYAD and neuYAE. Transgenic flies with these alleles will be generated to further study the inhibitory role of Neu^YA. </p> <p> Finally, our on going large-scale genetic screening is likely to reveal the component(s) of NeuYE (Y1253) pathway that does not utilize the function of Ras. </p> / Thesis / Master of Science (MSc)

The Neu/ErbB2 receptor tyrosine kinase has been implicated in the induction of mammary tumourigenesis. Both Ras-dependent and independent signalling downstream of activated Neu is believed to mediate cellular signalling that contributes cellular transformation in oncogenic Neu. Signalling from five 𝘣𝘰𝘯𝘢 𝘧𝘪𝘥𝘦 autophosphorylation sites (termed sites A through E) in the carboxyl tail of the receptor mediate these signals to the cytosol. Of the four positive regulatory sites (B, C, D, E), only three (B, C, D,) signal through known signalling molecules. Site E, (Y1253) in Neu, though known to interact with DOKR is essentially an orphan site. The discovery of a 34kD protein capable of associating to peptides corresponding to site E prompted investigations into the nature of site E signalling. Mass spectrometric analyses revealed that the 34kD protein is 2,4-dienoyl-CoA reductase (DECR1), a lipid metabolism protein typically localized to the mitochondria. Investigations into this protein reveal that DECR1 is capable of associating with Neu site E in a tyrosine phosphorylation and sequence specific manner. Furthermore, analyses with site E second-site mutants (YE[APEY], YE[DPEY], YE[NAEY] and YE[NDEY]) show that DECR1 associates in a manner consistent to a PTB domain-containing protein. Analyses of amino acid sequence demonstrate the presence of a putative Bcl-domain in DECR1 suggestive of a role in apoptosis. Experiments into the apoptotic activity of DECR1 proved inconclusive. The examination of sub-cellular localization of Neu and DECR1 showed that the two proteins are found in both the mitochondrial and plasma membrane fractions. These results demonstrate that DECR1 may be a veritable binding partner for Neu Y1253. / Thesis / Master of Science (MSc)

The Drosophila Epidermal Growth Factor Receptor (DEgfr) is the only fly orthologue of the vertebrate Neu/ErbB2 receptor tyrosine kinase (RTK) family. In Drosophila, DEgfr signaling is required in the developing wing discs, and for the determination and differentiation of wing veins. Expression of constitutively active rat Neu/ErbB2 transgenes, each having a single phosphotyrosine (pTyr) residue in the adaptor domain, generates cellular responses in Drosophila consistent with the activation of signaling cascades employed by intrinsic DEgfr. We have performed extensive genetic screening to identify adaptor and second messenger pathways that are activated by individually reconstituted pTyr, by determining which signaling gene mutants alter neu wing phenotypes. In addition, we have screened for genetic deletions on the second and third chromosome that enhance or suppress the wing phenotypes generated by the neu add-back alleles. This approach has enabled us to identify several signaling proteins that differentially affect the Neu signaling pathway via association with specific pTyr residues. We have also identified 41 genomic regions in Drosophila, which modify signals from either individual or multiple neu add-back alleles. As Neu signaling appears to function in a manner similar to the DEgfr, we sought to determine whether these receptors were capable of heterodimerizing. In order to examine this we co-expressed a kinase inactive version of the neu^YD allele with activated DEgfr. Co-expression of these receptors suggested that the DEgfr was unable to dimerize with, and transphosphorylate, the YD pTyr on Neu, as no potent anti-apoptotic phenotype was detected. Additionally, co-expression of activated neu and the kinase inactive version of neu^YD resulted in a decrease in glial cell numbers, in relation to mis-expression of the activated neu allele alone. These findings suggest that Neu does not interact with the DEgfr, but rather functions via homodimerization of it's receptor subunits. / Thesis / Master of Science (MS)

The ets gene family of transcription factors has been widely implicated in a variety of human tumors. PEA3, the founding member of the PEA3 subfamily of ets genes is overexpressed in a mouse model of mammary tumorigenesis as well as in primary human breast tumors. PEA3 deregulation in these tumors is thought to increase the metastatic potential of mammary tumors by increasing the expression of various matrix metalloproteinases. The identification of other ets gene's expression using a mouse model of HER2/Neu-induced mammary tumorigenesis would provide insight into the mechanisms behind these mammary tumors. Degenerate RT-PCR analysis was used to screen for expression of all known ets genes in these tumors. A large spectrum of ets genes was identified as being expressed in these tumors. Quantitative analyses including semi-quantitative RT-PCR and ribonuclease protection assays, indicate that the PEA3 subfamily of ets genes, including PEA3, ERM and ER81, as being overexpressed in these tumors, while other ets genes, Ets-1, Ets-2 and GABP(alpha) were not. These results imply a specific role for the PEA3 subfamily in this model of mammary tumorigenesis and isolate this subfamily of ets genes as a possible therapeutic target. / Thesis / Master of Science (MS)

Made available in DSpace on 2016-08-10T10:38:33Z (GMT). No. of bitstreams: 1 FLAVIA ALEIXO FERREIRA.pdf: 856261 bytes, checksum: 437e20f50936aac2d69cf7dcc15d95ed (MD5) Previous issue date: 2011-07-01 / Breast cancer is the second most frequent type of cancer worldwide and more common among women, accounting for 22% of new cases each year. These tumors appear as heterogeneous evolution and response to different treatment available whose prognostic and predictive factors guiding the therapeutic approach being used. The detection of oncogenic protein c-erbB-2 by immunohistochemistry is a prognostic factor for diagnostic, and in most cases, is associated with a worse prognosis. This study aimed to raise clinicopathological data of patients with breast cancer, as well as evaluating the expression c-erbB-2 in tumor tissue paraffin samples of these patients. Medical records of 286 female patients with breast carcinoma treated at Hospital Araújo Jorge (Association of Cancer Combat of Goiás), between 1979 and 2002 were collected and tabulated together with data from immunohistochemical detection of c-erbB protein -2. In our series the positive immunodetection of c-erbB-2 in tumor cells showed no association with conventional parameters clinicopathologics. The immunodetection of the protein c-erbB-2 did not significantly affect the survival of patients analyzed in our series, consistent with data obtained by other studies. We conclude that molecular methods should become more sensitive. / O câncer de mama é o segundo tipo mais frequente de câncer no mundo e o mais comum entre as mulheres, corresponde por 22% dos casos novos a cada ano. Esses tumores apresentam-se heterogêneos quanto a evolução e resposta às diferentes opções terapêuticas disponíveis, cujos fatores prognósticos e preditivos norteiam a conduta terapêutica a ser utilizada. A detecção da proteína oncogênica c-erbB-2 por imuno- histoquímica é um fator prognóstico auxiliar para avaliação diagnóstica e, na maioria dos casos, associa-se a um pior prognóstico. O presente estudo teve como objetivo levantar os dados clínicopatológicos de pacientes com câncer de mama, bem como avaliar a expresssão de c-erbB-2 nas amostras de tecido tumoral parafinadas dessas pacientes. Prontuários de 286 pacientes do sexo feminino com carcinoma de mama atendidas no Hospital Araújo Jorge (Associação de Combate ao Câncer em Goiás), entre 1979 e 2002, foram coletados e tabulados juntamente com os dados de imuno- histoquímica para detecção da proteína c-erbB-2. Em nossa casuística a imunodetecção positiva de c-erbB-2 nas células tumorais não demonstrou associação com os parâmetros clinicopatológicos convencionais. A imunodetecção da proteína c-erbB-2 não influenciou significativamente a sobrevida das pacientes analisadas em nossa série, condizendo com dados obtidos por outros estudos. Assim concluímos que os métodos moleculares devem se tornar mais sensíveis

The dissertation addresses the trajectory of the Reading Society Hermann Faulhaber, that has its origins in the Library founded in 1903, in the old Colony of Neu-Württemberg, current municipality of Panambi/RS. The research focuses on the creation, constitution and maintenance of the Reading Society, as an institution dedicated to the preservation of culture among immigrants/descendants. Thus, it analyzes the Library, in the initial years of formation in the Colony, by the initiative of its owner Hermann Meyer, under the management of Marie and Hermann Faulhaber, and its transformation in the Reading Society in 1927, as well as its characteristics before the collection was confiscated in 1942, due to the nationalization policies adopted by the Estado Novo. The research also discusses the decision of not to close activities of the Reading Society, after the seizure of the books, strategies for recovery of the collection, as well as the restructuring actions, with emphasis on the National Institute of the Book (INL). Finally, the dissertation addresses the role played by Marie Faulhaber in the formation of readers in the Colony, highlighting her trajectory, her studies in Germany and her work in the community. The dissertation allows us to understand the role of the Reading Society, associated with the Faulhaber family, especially in bond with the German culture, through the reading practices of the former Colony of Neu-Württemberg. / A dissertação aborda a trajetória da Sociedade de Leitura Hermann Faulhaber, que tem suas origens na Biblioteca fundada em 1903, na antiga Colônia de Neu-Württemberg, atual município de Panambi/RS. A pesquisa está centrada na criação, constituição e manutenção da Sociedade de Leitura, enquanto instituição voltada à preservação da cultura alemã entre os imigrantes/descendentes. Assim, analisa a fundação da Biblioteca, nos anos iniciais de formação da Colônia, por iniciativa do seu proprietário Hermann Meyer, sob administração do casal Marie e Hermann Faulhaber, e a sua transformação na Sociedade de Leitura em 1927, bem como suas características antes do acervo ser confiscado em 1942, devido às políticas de nacionalização adotadas pelo Estado Novo. A pesquisa também discorre sobre a decisão de não encerrar as atividades da Sociedade de Leitura, depois da apreensão dos livros, as estratégias para recuperação do acervo, assim como as ações de reestruturação, com destaque a vinculação ao Instituto Nacional do Livro (INL). Por fim, a dissertação aborda o papel exercido por Marie Faulhaber na formação de leitores na Colônia, destacando a sua trajetória individual, seus estudos na Alemanha e a sua atuação na comunidade. O conjunto da dissertação permite compreender o papel da Sociedade de Leitura, associada ao protagonismo da família Faulhaber, sobretudo no que diz respeito à manutenção dos vínculos com a cultura alemã, através das práticas de leitura da antiga Colônia de New-Württemberg.