Thesis (M.S.)--Florida State University, 2005. / Advisor: Dr. W. Ross Ellington, Florida State University, College of Arts and Sciences, Dept. of Biological Science. Title and description from dissertation home page (viewed June 15, 2005). Document formatted into pages; contains x, 51 pages. Includes bibliographical references.

Pathways and mechanisms for human memory T cell differentiation and maintenance have largely been inferred from studies of peripheral blood, though the majority of T cells are found in lymphoid and mucosal sites. We present here a novel, multidimensional, quantitative analysis of human T cell compartmentalization and maintenance over six decades of life in blood, lymphoid and mucosal tissues obtained from 56 individual organ donors. Our results reveal that the distribution and tissue residence of naïve, central and effector memory, and terminal effector subsets is contingent on both differentiation state and tissue localization. Moreover, T cell homeostasis driven by cytokine or TCR-mediated signals is dependent on CD4+ or CD8+ T cell lineage, subset differentiation and tissue localization, and cannot be inferred from blood. Our data provide an unprecedented spatial and temporal map of human T cell compartmentalization and maintenance, supporting new pathways for human T cell fate determination and homeostasis. Memory T cells can remain in tissues as non-circulating, resident memory populations, which provide optimal protection against infection at barrier surfaces. Lung-resident memory T cells (TRM) mediate in situ protection to respiratory pathogens, though mechanisms for their maintenance and retention are unknown. Through whole transcriptome profiling, we identify a cohesive network of genes enriched in lung CD4+ TRM, including Itgad (CD11d), Cd69, and IFN-associated responders. We find that upregulation of CD11d enhances CD69 expression through type I IFN signaling downstream of homotypic cell adhesion, and is required for optimal T cell differentiation and lung retention. Moreover, blockade of IFNαR1 reduces CD11d expression and retention of influenza-generated lung TRM, suggesting that CD11d-dependent type I IFN signaling promotes TRM establishment. Our results implicate CD11d and type I IFN in retaining lung CD4+ TRM cells, and identify potential targets for modulating tissue immunity.

Thesis (Ph. D.)--University of Missouri--Columbia, 2004. / Typescript. Vita. Includes bibliographical references (leaves 166-179). Also issued on the Internet.

This dissertation presents the development of a multielectrode microcompartment platform for understanding signal transduction in the nervous system. The design and fabrication of the system and the characterization of the system for pharmacological and electrophysiological measurements of cultured neurons is presented in this work. The electrophysiological activity of cultured dorsal root ganglion (DRG) neurons and cortical neurons is shown on the MEA substrate. These recordings were measured and tied to the toxicological effects of the chemotherapeutic drug vincristine on DRGs. Conventional electrophysiological recordings (via a patch micropipette) are made routinely to record action potentials and ion channel activity in neurons. Moreover, Campenot chambers (traditional compartmented culture systems) have been used for the last thirty years to study the selective application of drugs to neurons. Both of these techniques are useful and well established; however they have their limitations. For instance, Campenot chambers cannot be used very well for small processs-producing neurons, since the barriers are difficult to tranverse. Moreover, conventional patch recordings are labor-intensive, especially when more than one microelectrode needs to be positioned. The developed system is composed of a two compartment divider, each compartment capable of housing axons or cell bodies. Underneath the divider, the substrate has 60 electrodes, arranged in several lines to accommodate several different neurite tracks. Neurons can be stimulated and their activity can be recorded in both of the compartments. The neurotoxin and chemotherapeutic drug vincristine was tested in the system on the DRGs. The drug caused length-dependent axonal degeneration in the DRGs when applied locally. Moreover, electrophysiological activity in both compartments showed that only the activity in the axonal compartment was affected, leading us to believe that the mechanism behind the degeneration is localized to the distal axon.

Thesis (Ph. D.)--University of Missouri--Columbia, 2004. / "May 2004." Typescript. Vita. Includes bibliographical references (leaves 166-179). Also issued on the Internet.

INTRODUÇÃO: Os carcinomas papilífero e folicular são neoplasias malignas primárias da glândula tireóide. Em conjunto, recebem o nome de carcinoma bem diferenciado. Determinar o risco individual da ocorrência de metástase nesses casos auxilia na seleção da terapêutica que é atualmente baseada na classificação de acordo com fatores prognósticos, aos quais pode ser associada a pesquisa de marcadores biológicos. Dentre eles, destaca-se a galectina-3, cujas funções exercidas nos compartimentos celulares foram descritas em uma variedade de neoplasias. Entretanto, seu papel no carcinoma tireóideo permanece controverso. Com o intuito de investigar se a galectina-3 pode auxiliar na predição do risco individual da ocorrência de metástase e se está associada aos critérios de malignidade do carcinoma bem diferenciado, a presente pesquisa objetivou verificar as seguintes hipóteses: 1) se há diferença da imunoexpressão da galectina-3 nos compartimentos do tireócito maligno entre os doentes com e sem metástase e se é possível predizer o risco de metástase em função da quantificação digital desse marcador; 2) se há diferença da imunoexpressão da galectina-3 entre o tecido tireóideo maligno e o não neoplásico; conforme a presença de invasão tecidual; e conforme a sobrevivência; 3) se há indício do envolvimento da galectina-3 com apoptose, indução da proliferação celular e angiogênese. MÉTODO: Trata-se de estudo retrospectivo de caso-controle que envolveu 109 doentes operados por carcinoma bem diferenciado da tireóide e seguidos por mais de cinco anos, distribuídos em dois grupos equivalentes: com e sem metástase. Foram feitos coleta de dados clínicos, avaliação anátomo-patológica e análise imunohistoquímica digital dos biomarcadores galectina-3, Ki-67, caspase-3 e CD-34. RESULTADOS: 1) A média do índice de positividade nucleolar da galectina-3 foi maior no grupo de doentes com metástase linfática cervical (1,78 ± 0,41 nucléolos/CGA contra 0,35 ± 0,13, p=0,004). A expressão nucleolar da galectina-3 apresentou especificidade de 75% para identificação da ocorrência de metástase e foi fator independente associado à ocorrência metástase linfática (p=0,01). A equação logística obtida permitiu calcular o risco individual de desenvolvimento de metástase linfática cervical que é próximo a 100% quando a galectina-3 está imunoexpressa em quatro ou mais nucléolos por campo microscópico de grande aumento. 2) não houve expressão da galectina-3 no tireócito não neoplásico; o índice de expressão citoplasmático foi fator independente associado à presença de invasão linfática (p=0,013) e a média desse índice foi maior nos casos com extensão extratireóidea (52,7 ± 3,9 uo/µm2 contra 41,0 ± 4,0, p=0,037); não houve associação dos índices de imunoexpressão da galectina-3 e sobrevivência; 3) no grupo de doentes com metástase, a expressão nucleoplasmática da galectina-3 correlacionou-se de forma positiva com o índice de positividade do Ki-67 e, nos dois grupos, a expressão citoplasmática com o índice de expressão da caspase-3. CONCLUSÕES: Foi possível predizer o risco individual da ocorrência de metástase linfática cervical em função da quantificação digital da imunoexpressão nucleolar da galectina-3. O presente estudo sugere que alta expressão citoplasmática está associada com algumas características de invasão local. Houve indícios do envolvimento da galectina-3 com indução da proliferação celular e apoptose no grupo de doentes com metástase. / INTRODUCTON: Papillary and follicular carcinomas are primary malignant neoplasias of the thyroid gland and are classified as well-differentiated carcinoma. In these cases, determination of individual risk of metastasis allows offering an adequate treatment. Nowadays therapy is chosen based on classification according to prognostic factors and biomarkers can be associated with them. Galectin-3 is one of these markers and has been thoroughly studied. A wide range of functions that it carries out in the subcellular compartments have been described in several neoplasms. However, its role in thyroid carcinomas remains controversial. In order to investigate if galectin-3 can be used to predict the individual risk of metastasis and if this marker is associated with malignant criteria of well-differentiated carcinoma, this study was proposed to verify the following hypotheses: 1) if galectin-3 immunostaining in subcellular compartments of the malignant thyrocyte is different when comparing patients with and without metastasis and if it is possible to predict the individual risk of metastasis based on digital quantification of the galectin-3 immunostaining; 2) if galectin-3 immunoexpression is different from malignant and benign thyroid tissue; according to tissue invasion and survival; 3) if there are indications that galectin-3 plays a role in apoptosis and cell proliferation or angiogenesis induction. METHODS: It was performed a retrospective case-control study involving 109 patients treated for well-differentiated thyroid carcinoma and followed up for more than five years. They were divided into two equivalent groups: with and without metastasis. The search of clinical data, morphological evaluation and digital immunohistochemical analysis with galectin-3, Ki-67, caspase-3 and CD-34 antibodies were done. RESULTS: 1) the average of the nucleolar galectin-3 positive index was higher in lymph node metastasis group (1.78 ± 0.41 nucleoli/HPF versus 0.35 ± 0.13, P=.004). Nucleolar staining was an independent factor associated with lymph node metastasis (P=.01) and its specificity to identify metastasis was 75%. The logistic model allowed predicting the individual risk of cervical lymph node metastasis. It was almost 100% for carcinomas displaying more than four galectin-3 immunostained nucleoli by microscopic high power field. 2) There was no galectin-3 immunostaining in non-neoplasic thyrocyte; the cytoplasmic galectin-3 expression index was an independent factor associated with lymphatic invasion (P=.013) and these index average was higher in cases with extrathyroidal extension (52.7 ± 3.9 uo/µm2 versus 41.0 ± 4.0, P=.037); there was no association of galectin-3 immunostaining indexes with survival; 3) in the metastasis group, there was positive correlation between nucleoplasmic staining of galectin-3 and Ki-67 positive index; there was positive correlation between cytoplasmic staining of galectin-3 and caspase-3 positive index in both groups. CONCLUSION: It was possible to predict the individual risk of cervical lymph node metastasis based on digital quantification of the nucleolar galectin-3 immunostaining. This study suggests that there is association of high cytoplasmic expression with local tissue invasion. In the metastasis group there were indications that galectin-3 plays a role in cell proliferation and apoptosis induction.

INTRODUÇÃO: Os carcinomas papilífero e folicular são neoplasias malignas primárias da glândula tireóide. Em conjunto, recebem o nome de carcinoma bem diferenciado. Determinar o risco individual da ocorrência de metástase nesses casos auxilia na seleção da terapêutica que é atualmente baseada na classificação de acordo com fatores prognósticos, aos quais pode ser associada a pesquisa de marcadores biológicos. Dentre eles, destaca-se a galectina-3, cujas funções exercidas nos compartimentos celulares foram descritas em uma variedade de neoplasias. Entretanto, seu papel no carcinoma tireóideo permanece controverso. Com o intuito de investigar se a galectina-3 pode auxiliar na predição do risco individual da ocorrência de metástase e se está associada aos critérios de malignidade do carcinoma bem diferenciado, a presente pesquisa objetivou verificar as seguintes hipóteses: 1) se há diferença da imunoexpressão da galectina-3 nos compartimentos do tireócito maligno entre os doentes com e sem metástase e se é possível predizer o risco de metástase em função da quantificação digital desse marcador; 2) se há diferença da imunoexpressão da galectina-3 entre o tecido tireóideo maligno e o não neoplásico; conforme a presença de invasão tecidual; e conforme a sobrevivência; 3) se há indício do envolvimento da galectina-3 com apoptose, indução da proliferação celular e angiogênese. MÉTODO: Trata-se de estudo retrospectivo de caso-controle que envolveu 109 doentes operados por carcinoma bem diferenciado da tireóide e seguidos por mais de cinco anos, distribuídos em dois grupos equivalentes: com e sem metástase. Foram feitos coleta de dados clínicos, avaliação anátomo-patológica e análise imunohistoquímica digital dos biomarcadores galectina-3, Ki-67, caspase-3 e CD-34. RESULTADOS: 1) A média do índice de positividade nucleolar da galectina-3 foi maior no grupo de doentes com metástase linfática cervical (1,78 ± 0,41 nucléolos/CGA contra 0,35 ± 0,13, p=0,004). A expressão nucleolar da galectina-3 apresentou especificidade de 75% para identificação da ocorrência de metástase e foi fator independente associado à ocorrência metástase linfática (p=0,01). A equação logística obtida permitiu calcular o risco individual de desenvolvimento de metástase linfática cervical que é próximo a 100% quando a galectina-3 está imunoexpressa em quatro ou mais nucléolos por campo microscópico de grande aumento. 2) não houve expressão da galectina-3 no tireócito não neoplásico; o índice de expressão citoplasmático foi fator independente associado à presença de invasão linfática (p=0,013) e a média desse índice foi maior nos casos com extensão extratireóidea (52,7 ± 3,9 uo/µm2 contra 41,0 ± 4,0, p=0,037); não houve associação dos índices de imunoexpressão da galectina-3 e sobrevivência; 3) no grupo de doentes com metástase, a expressão nucleoplasmática da galectina-3 correlacionou-se de forma positiva com o índice de positividade do Ki-67 e, nos dois grupos, a expressão citoplasmática com o índice de expressão da caspase-3. CONCLUSÕES: Foi possível predizer o risco individual da ocorrência de metástase linfática cervical em função da quantificação digital da imunoexpressão nucleolar da galectina-3. O presente estudo sugere que alta expressão citoplasmática está associada com algumas características de invasão local. Houve indícios do envolvimento da galectina-3 com indução da proliferação celular e apoptose no grupo de doentes com metástase. / INTRODUCTON: Papillary and follicular carcinomas are primary malignant neoplasias of the thyroid gland and are classified as well-differentiated carcinoma. In these cases, determination of individual risk of metastasis allows offering an adequate treatment. Nowadays therapy is chosen based on classification according to prognostic factors and biomarkers can be associated with them. Galectin-3 is one of these markers and has been thoroughly studied. A wide range of functions that it carries out in the subcellular compartments have been described in several neoplasms. However, its role in thyroid carcinomas remains controversial. In order to investigate if galectin-3 can be used to predict the individual risk of metastasis and if this marker is associated with malignant criteria of well-differentiated carcinoma, this study was proposed to verify the following hypotheses: 1) if galectin-3 immunostaining in subcellular compartments of the malignant thyrocyte is different when comparing patients with and without metastasis and if it is possible to predict the individual risk of metastasis based on digital quantification of the galectin-3 immunostaining; 2) if galectin-3 immunoexpression is different from malignant and benign thyroid tissue; according to tissue invasion and survival; 3) if there are indications that galectin-3 plays a role in apoptosis and cell proliferation or angiogenesis induction. METHODS: It was performed a retrospective case-control study involving 109 patients treated for well-differentiated thyroid carcinoma and followed up for more than five years. They were divided into two equivalent groups: with and without metastasis. The search of clinical data, morphological evaluation and digital immunohistochemical analysis with galectin-3, Ki-67, caspase-3 and CD-34 antibodies were done. RESULTS: 1) the average of the nucleolar galectin-3 positive index was higher in lymph node metastasis group (1.78 ± 0.41 nucleoli/HPF versus 0.35 ± 0.13, P=.004). Nucleolar staining was an independent factor associated with lymph node metastasis (P=.01) and its specificity to identify metastasis was 75%. The logistic model allowed predicting the individual risk of cervical lymph node metastasis. It was almost 100% for carcinomas displaying more than four galectin-3 immunostained nucleoli by microscopic high power field. 2) There was no galectin-3 immunostaining in non-neoplasic thyrocyte; the cytoplasmic galectin-3 expression index was an independent factor associated with lymphatic invasion (P=.013) and these index average was higher in cases with extrathyroidal extension (52.7 ± 3.9 uo/µm2 versus 41.0 ± 4.0, P=.037); there was no association of galectin-3 immunostaining indexes with survival; 3) in the metastasis group, there was positive correlation between nucleoplasmic staining of galectin-3 and Ki-67 positive index; there was positive correlation between cytoplasmic staining of galectin-3 and caspase-3 positive index in both groups. CONCLUSION: It was possible to predict the individual risk of cervical lymph node metastasis based on digital quantification of the nucleolar galectin-3 immunostaining. This study suggests that there is association of high cytoplasmic expression with local tissue invasion. In the metastasis group there were indications that galectin-3 plays a role in cell proliferation and apoptosis induction.