Thesis (Ph. D.)--University of Missouri-Columbia, 2006. / Title from title screen of research.pdf file (viewed on December 22, 2006) The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. "May 2006" Includes bibliographical references.

Undergraduate honors paper--Mount Holyoke College, 2006. Dept. of Biological Sciences. / Includes bibliographical references (leaves 66-68).

Thesis (Ph. D.)--University of Chicago, Division of the Biological Sciences and the Pritzker School of Medicine, June 1999. / Includes bibliographical references. Also available on the Internet.

Thesis (M. Med. Sc.)--University of Hong Kong, 2005.

Undergraduate honors paper--Mount Holyoke College, 2007. Dept. of Biological Sciences. / Includes bibliographical references (leaves 69-70).

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 46-69).

BACKGROUND: In their lifetime, 42% of men and 38% of women will be diagnosed with cancer. One of the most prominent cancers in women is breast cancer, which represents 14.8% of newly diagnosed cancers. Research has demonstrated the importance of the lysyl oxidase (LOX) gene, more specifically the lysyl oxidase propeptide (LOX-PP), in the attenuation of breast cancer and in mouse xenograft models, making it an important and ongoing area of investigation. In recent, unpublished experiments, mice with a variant of LOX-PP were treated with 7,12-Dimethylbenz[a]anthracene (DMBA) to induce breast cancer. However, some of the treated mice developed poor body conditions without any signs of having developed breast cancer. Published studies have demonstrated that, following treatment with DMBA, some mice generate leukemia or other lymphoproliferative diseases. Research has shown that identification of these diseases is possible via histological analyses or immunohistochemistry. Objective: To determine the cause of poor body conditions in mice treated with DMBA that did not develop mammary tumors by histological analyses and immunohistochemistry. Additionally, to determine the difference between a variant of LOX-PP (LOX-PPv) knock-in (Ki) and wild type (WT) mice in the generation of poor body conditions or leukemia/lymphoproliferative disease. METHODS: A total of 48 mice, 24 LOX-PPv Ki and 24 WT, were treated with DMBA and observed for mammary tumor formation. Body conditions of the mice were observed and noted, and liver samples from these mice were obtained and analyzed via histological and immunohistochemistry interventions. In Hematoxylin & Eosin (H&E) assessments of mice livers, mice were classified as normal or as having possible lymphocyte infiltration, mild lymphocyte infiltration, or massive lymphocyte infiltration. Staining with the Ki-67 antibody in immunohistochemistry allowed for classification of mouse livers as normal or as having slightly positive or massively positive expression of the Ki-67 antigen. The degree to which each liver sample was positive for both readings was compared and further analyzed. RESULTS: A total of 9 mice (30.4% of Ki mice and 8.7% of WT mice) displayed poor body conditions following DMBA treatment. Of the 9 (7 Ki and 2 WT) mice having poor body condition, 2 Ki mice suffered from severe dermatitis. For the 7 remaining mice, no cause for such poor body conditions was obvious. 6 of these mice (85.7%) were diagnosed with leukemia. In all other DMBA-treated mice, 52.6% of Ki and 23.8% of WT displayed some degree of abnormal lymphocyte infiltration in the liver, while a total of 39.1% of the K¬I mice and 69.6% of the WT mice had normal liver histologies. 60.9% of Ki and 45.5% of WT mice displayed some degree of positivity for expression of Ki-67. CONCLUSIONS: Mice not diagnosed with breast cancer but that were suffering from poor body conditions likely had developed leukemia or some other lymphoproliferative disease. In the mice not definitively diagnosed with leukemia by a pathologist, H&E analyses and immunohistochemistry of the livers showed lymphocyte infiltration and cellular proliferation possibly indicative of leukemia., but further tests are necessary to confirm this. Furthermore, LOX-PPv Ki mice appeared to have a greater likelihood of generating poor body conditions or some observable sign of abnormal lymphocyte infiltration in the liver versus their WT counterparts when treated with DMBA. / 2018-06-16T00:00:00Z

Sequencing B and T cell receptor genes allows for detailed characterisation of the genetic diversity underlying adaptive immune responses in health and disease. In the context of infectious diseases this can act as a powerful tool for identification of pathogen-specific immune signatures and genetic determinants of immune memory, protection and response to re-exposure. As part of my PhD I developed and optimised a method for high-resolution profiling of B cell receptor (BCR) immune repertoires based on the barcoded sequencing of the human immunoglobulin genes. The use of molecular barcodes allowed for reduction of technical noise, which can lead to erroneous assignment of lymphocyte function. I applied this methodology to the study of natural infection with measles virus in unvaccinated children. Childhood measles causes a profound immune suppression, which can last for weeks to months post infection, with large reductions in numbers of circulating B cells. Interestingly, long-term consequences of measles immune suppression result in increased incidence of secondary infections up to 3 years after resolution of measles. Vaccination against measles virus with the MMR vaccine has been a major factor in reducing direct and secondary childhood morbidity and mortality. The maintenance of sufficient global vaccine coverage, however, has been challenging due to the refusal of vaccination, mainly in religious communities, resulting in increasing number of outbreaks worldwide. In addition to the overall drop in measles virus herd immunity, measles-induced immune suppression can compromise immunity to other infectious pathogens, thus complicating global vaccination and surveillance efforts. The exact mechanisms underlying the prolonged immune-suppression associated with measles remain elusive and have not been investigated in humans. I applied BCR sequencing to characterise the long-term immunological effects of natural measles virus infection in a cohort of unvaccinated children. Specifically, I addressed the restructuring of immune memory and the possible loss of immunity to non-measles pathogens. My work provided evidence for previously hypothesised depletion of B cell memory pools, referred to as ‘immunological amnesia’. Loss of clonally expanded B memory populations lead to immune re-setting and convergence in repertoire diversity between measles-infected and control groups. In addition to the loss of individual-specific variation in immune memory, a subset of measles-infected individuals exhibited dramatic collapse in the diversity of their naïve B cell compartment, despite the recovery of normal B naïve cell counts. An effect of measles on serological immunity was also demonstrated in a ferret model of measles, where lymphotropic challenge lead to significant loss of vaccine-acquired immunity to influenza virus. The work presented in this dissertation demonstrates the utility of BCR sequencing for understanding adaptive immune responses in the context of infectious diseases and highlights the potential of this approach to uncover novel mechanisms of immune (dys)function.

Orientador: Sara Teresinha Olalla Saad / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-16T01:25:11Z (GMT). No. of bitstreams: 1 Rodrigues_PatriciaCristina_D.pdf: 2756759 bytes, checksum: c32a31ef7786358790e9e69edaf2c2db (MD5) Previous issue date: 2010 / Resumo: A proteína humana ANKHD1 (Ankyrin Repeat Single KH Domain containing 1) foi inicialmente descrita em células de adenocarcinoma de próstata humano (LNCaP), e sua expressão também é observada em diversas linhagens leucêmicas. ANKHD1 é homóloga a proteína Mask (Multiple Ankyrin repeat and single KH domain) de Drosophila melanogaster, relacionada com diferenciação, sobrevivência e proliferação de fotorreceptores dos olhos de drosófila. Interações diretas entre ANKHD1 e SHP2, proteína que exerce um papel crítico na hematopoiese, foram descritas nas linhagens mielóide K562, de adenocarcinoma de próstata, LNCaP, e em linhagem de mieloma múltiplo RPM1 8226. Porém pouco se sabe sobre a função de ANKDH1 e seu papel na leucemogênese. O objetivo do presente estudo foi investigar o envolvimento da ANKHD1 em processos biológicos relacionados com a leucemogênese. Para tal utilizamos duas abordagens metodológicas principais: inibição e hiperexpressão de ANKHD1. A inibição de ANKHD1 em linhagem LNCaP foi seguida de rastreamento das demais alterações gênicas associadas através de microarray. O microarray realizado mostrou 706 genes cuja expressão foi aumentada e 159 genes cuja expressão foi diminuída. Os genes foram agrupados em 26 classes funcionais, sendo que diversos genes conhecidos por regularem ciclo celular e apoptose tiveram seu perfil de expressão alterada. Importante ressaltar que a inibição de ANKHD1 resultou em aumento significativo da expressão de um grande número de Histonas, sugerindo seu papel no controle epigenético. Dentre as modulações validadas, algumas foram verificadas também em amostras provenientes de portadores de leucemia. Neste estudo, foi demonstrada a baixa expressão gênica de ANKHD1 em amostras de 38 pacientes com diagnóstico de leucemia aguda, quando comparadas às células de medula óssea normal. Uma vez que esta amostra leucêmica se mostrou como um modelo natural de inibição de ANKHD1, a expressão de outros genes modulados no array foi testada, e o gene BMF, que se mostrou aumentado no array, se mostrou também mais expresso em amostras de leucemias, confirmando a oposição de expressão destes dois genes. Simultaneamente, células de leucemia aguda e mielodisplasia foram submetidas à ação de diversas drogas usadas no tratamento dessas doenças mostrando aumento de expressão de ANKHD1. Estes dados em conjunto com os observados no microarray sugerem a participação da ANKHD1 na via de JAK/STAT1 e vias de controle epigenético. A segunda abordagem utilizada neste trabalho foi a hiperexpressão ANKHD1, isoformas 1 e 2, em linhagens tumorais, seguidas da detecção de expressão protéica de ANKHD1 em cotransfecção com os genes Siva e Vpr. As proteínas Siva e Vpr foram recentemente descritas como capazes de interagir fisicamente com as isoformas 1 e 2 de ANKHD1 respectivamente. Tanto a proteína a Siva quanto a Vpr foram atribuídos papéis de indução de apoptose em linfócitos T. Observou-se que ao cotransfectarmos Siva ou Vpr com ANKHD1 houve diminuição de expressão de ANKHD1 tanto para a isoforma 1 quanto para isoforma 2 (western blotting) e também se observou que a isoforma 1 de ANKHD1 é citoplasmática assim como ocorre com a Mask de drosófila, porém a isoforma 2 se mostrou predominantemente nuclear em microscopia de fluorescência. A análise funcional da hiperexpressão de ANKHD1 seguida de análise por FACS sugere diminuição de apoptose em células leucêmicas. Em conclusão, o presente estudo identificou diversos possíveis participantes da via de sinalização de ANKHD1 por análise microarray, relatou a diminuição da sua expressão em amostras de pacientes portadores de leucemia, bem como o aumento de sua expressão em linhagens leucêmicas sob a ação de diversos tratamentos correntemente utilizados no combate de desordens hematopoiéticas. Descrevemos também o aumento de expressão da expressão do gene BMF nas mesmas amostras em que há diminuição de ANKHD1, corroborando os dados encontrados no microarray da inibição de ANKHD1. Descrevemos também pela primeira vez a localização nuclear da isoforma 2 de ANKHD1, confirmando todas as previsões computacionais de localização de ANKHD1. Os resultados aqui descritos sugerem que ANKHD1 esteja envolvida com o fenótipo anormal da célula leucêmica e permitirão direcionar novos estudos com o objetivo de melhor elucidar as funções específicas de ANKHD1 na hematopoiese / Abstract: The human protein ANKHD1 (Ankyrin Repeat Single KH Domain containing 1) was initially described in Human prostate adenocarcinoma cells (LNCaP), and can also be expressed in many different leukemic cell lines. ANKHD1 is homologous to the Drosophila melanogaster Mask protein (Multiple Ankyrin repeat and single KH domain), related to differentiation, proliferation and survival in photoreceptors of Drosophila eyes. Direct interactions between ANKHD1 and SHP2, a protein that plays a critical role in hematopoiesis, have been described in the myeloid cell line K562, in prostate adenocarcinome, LNCaP, and in multiple myeloma cell line RPM1 8226. Nevertheless, little is known of the function of ANKDH1 and its role in leukemogenesis. The purpose of this work was to investigate the involvement of ANKHD1 in biological processes related to leukemogenesis. For this purpose, we used two main methodological approaches: ANKHD1 inhibition and overexpression. The inhibition of ANKHD1 in LNCaP cell lines was followed by the tracking through microarray of the remaining associated genetic alterations. Microarray revealed 706 genes with upregulated expression and 159 genes with downregulated expression; the genes were gathered in 26 functional groups and many genes, known for regulating cell cycle and apoptosis, had alterations in their expression profile. Importantly, inhibition of ANKHD1 resulted in significant increase in the expression of a large number of Histones, suggesting its role in epigenetic control. Among the validated modulations, some were also observed in the samples collected from leukemia patients. In this study, the downregulation of ANKHD1 was demonstrated in samples collected from 38 acute leukemia patients when compared to normal bone marrow cells. As this leukemic sample represented a natural model of inhibition of ANKHD1, the expression of other genes processed through array were tested, and the BMF gene, which was upregulated in the array, was also more elevated in leukemia samples. Simultaneously, cells of acute leukemia and myelodysplasia were subjected to the action of various drugs used to treat these diseases showing increased expression of ANKHD1. These data together with those observed in the microarray suggest the participation of ANKHD1 towards JAK/STAT1 and pathways of epigenetic control. The second approach used in this work was the ANKHD1overexpression, isoforms1 and 2, in tumor lines, followed by the detection of protein expression in cotransfection with Siva and Vpr genes. The proteins Siva and Vpr have been recently described as being capable of physical interaction with the isoforms 1 and 2 of ANKHD1, respectively. The role of inducing apoptosis in T lynphocytes was attributed to both Siva and Vpr. By cotrasfecting Siva and Vpr with ANKHD1 a reduction of ANKHD1 expression to isoform 1 as well as to isoform 2 (western blotting) was obtained and isoform 1 of ANKHD1 was observed to be citoplasmatic, such as occurs with drosophila Mask, although isoform 2 happened to be predominantly nuclear in the fluorescence microscope. Functional analysis of overexpression of ANKHD1 followed by FACS analysis suggests reduction of apoptosis in leukemic cells. Finally, this study identified many possible participants in the signaling pathway of ANKHD1 by microarray analysis, and related the reduction of its expression in leukemia patients, as well as the increasing of its expression in leukemic cell lines under the effect of many different treatments currently used against hematopoietic disorders. We also described the increasing of BMF expression in the same samples where we found a reduction of ANKHD1, corroborating all the data we obtained from the ANKHD1 inhibition microarray. In addition, we described, for the first time, the nuclear location of ANKHD1's isoform 2, confirming all the computer-aided predictions regarding the location of ANKHD1. The results herein described suggest that ANKHD1 could be involved with the abnormal phenotype of leukemic cells and these results could guide further studies towards elucidating the specific functions of ANKHD1 in hematopoiesis / Doutorado / Biologia Estrutural, Celular, Molecular e do Desenvolvimento / Doutor em Fisiopatologia Medica

Class of 2014 Abstract / Specific Aims: Evaluate serum posaconazole concentrations following dose adjustment in response to subtherapeutic serum concentrations. Determine optimal dose adjustment schema and identify toxicity with doses above 600 mg daily (e.g.: 200 mg per os three times daily). Methods: The health records were reviewed for 29 patients ≥ 18 years with acute myeloid leukemia over a four-year period. Participants initially received posaconazole 200 mg per os three times daily as prophylaxis and required at least one dose adjustment secondary to a subtherapeutic posaconazole serum concentration. Patients were stratified by posaconazole dosing following dose adjustment (A=200mg QID, B=300mg TID, C=400 mg TID, D=400 QID). Main Results: There was a statistically significant increase in posaconazole serum concentration in each group compared to baseline serum concentration, aside from group C (group A and B P<0.001, group C P=0.236, and group D P=0.0076). The majority of participants in 3 of the 4 groups reached therapeutic serum concentration (A=0.87, B=0.76, D=0.80) whereas group C had a serum posaconazole concentration on average below therapeutic range (0.51). There was no significant difference between the four groups in regards to renal function (p=0.35) or hepatic function (AST p=0.676, ALT p=0.877, total bilirubin p=0.097). Conclusion: A dose increase led to an increase in posaconazole serum concentration except for the dosing regimen of 400 mg three times daily. However, the study is limited by a small patient population, an unequal number of patients in each group, and potentially by poor absorption of posaconazole suspension. Further research is required to expand on these findings.