Made available in DSpace on 2014-10-09T12:49:29Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:02:34Z (GMT). No. of bitstreams: 1 10385.pdf: 5520353 bytes, checksum: 8afc34c986ae698ddacb9c2d3093ea7a (MD5) / Dissertacao (Mestrado) / IPEN/D / Intituto de Pesquisas Energeticas e Nucleares, IPEN/CNEN-SP

In the normal adult human, hemopoiesis appears to be maintained by the simultaneous activity of many stem cell-derived clones. Conversely, most examples of human myeloid malignancies have been shown to represent clonal populations arising as a result of the unregulated expansion of a single transformed hemopoietic stem cell. The limits of the proliferative capacity of normal hemopoietic stem cells in humans and their persistence in hemopoietic malignancies have, however, not been extensively Investigated. One of the most likely reasons for this is the lack, until very recently, of a widely applicable method to analyze the clonality status of human cell populations. Methylation analysis of two polymorphic genes. HPRT and PGK, now allows such studies to be performed in approximately 50 % of females. The possibility that normal human hemopoietic stem cells might have the capacity to mimic the behaviour of some transformed stem cells by generating clones of progeny that could dominate the entire hemopoietic system was then examined. Such a phenomenon has been well documented in animal models of marrow cell transplantation. I therefore undertook an analysis of all allogeneic marrow transplants performed over a 1 to 1-1/2 year period where the genotype of the donor made clonality analysis using the HPRT or PGK systems possible. Using this approach, I obtained evidence in two patients suggesting that a single or, at most, a very small number of normal primitive hemopoietic stem cells were able to reconstitute the hemopoietic system. In one case the data suggested that such reconstitution was likely to have derived from a stem cell with both lymphopoietic and myelopoietic potential. However, in all other cases hemopoiesis in the transplant recipient was found to be polyclonal. Such findings indicate that clonal dominance in the hemopoietic system is not sufficient to infer that a genetically determined neoplastic change has occurred. In addition, these findings have implications for the design of future gene therapy protocols. The same methodology was also applied to investigate the clonality of different hemopoietic cell populations in patients with chronic myelogenous leukemia (CML) and essential thrombocytosis (ET). In both of these myeloproliferative disorders, the neoplastic clone produces terminally differentiated progeny that appear minimally different from normal. Data from the CML studies confirmed the non-clonal nature of the cells emerging in long-term CML marrow cultures. Similarly, patients transplanted with cultured autologous marrow were shown to undergo polyclonal and bcr-negative reconstitution of their hemopoietic system. Analysis of a series of patients with a clinical diagnosis of ET showed that polyclonal hemopoiesis in the presence of an amplified neoplastic clone is not a rare event in this disorder, and that clonality results do not always correlate with other neoplastic markers associated with myeloproliferative diseases in general. Another example of polyclonal hemopoiesis in the presence of an amplified neoplastic clone was demonstrated in a patient with Ph¹-positive ALL whose disease appeared to have originated in a lymphoid-restricted stem cell. The studies described in this thesis reveal a level of complexity of normal and neoplastic stem cell dynamics not previously documented. They highlight the need for more precise information about the molecular basis of regulatory mechanisms that govern hemopoietic cell proliferation and survival at every level of differentiation. Finally they support the accumulating evidence that acquisition of full malignant potential requires several additive genetic changes first postulated many years ago as the somatic mutation theory of carcinogenesis. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

No description available.

As a first step in characterizing the genes involved in O-side chain synthesis in <i>Brucella abortus</i> strain 2308, a portion of the genomic DNA was cloned from a rough mutant created by Tn5 (KnR) mutagenesis. This mutant was rough based on the lack of reactivity by either whole cells or extracted LPS to an O-side chain monoclonal antibody (BRU-38). A 30 kb <i>Xba</i>I genomic fragment (including Tn5) from the rough strain was subcloned into a sequencing vector to create pJM6. When <i>B. abortus</i> 2308 was electroporated with pJM6, KnR clones were unable to react with BRU-38; a Southern analysis of these clones revealed Tn5 in the 30 kb <i>Xba</i>I genomic fragment. Various regions of the 30kb fragment were subcloned and tested for their ability to complement specific <i>rfa</i> and <i>rfb</i> mutants of <i>Escherichia coli</i> and <i>Salmonella typhimurium</i>. One particular DNA fragment complemented an <i>rfbD</i> mutation in <i>E. coli</i> as judged by agglutination with <i>E. coli</i> anti-O (0:85) serum. The same DNA fragment failed to cause <i>E. coli rfbD</i> to react with either BRU-38 or <i>B. abortus</i> anti-O polyclonal antisera. The <i>B. abortus</i> 30 kb <i>Xba</i>I fragment contains a gene which has been identified by comple-mentation as containing the equivalent of the <i>rfbD</i> gene encoding dTDP-rhamnose synthetase in <i>E. coli</i>. Since <i>Brucella</i> is not known to have rhamnose in its core this enzyme may have a different function in <i>Brucella</i> LPS synthesis. / Master of Science

T cells can fight cancer, but an immunosuppressive tumor microenvironment (TME) disallows them from carrying out their function over time. Upregulation of inhibitory checkpoint molecules such as programmed cell death protein 1 (PD1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA4) can lead to such an immunosuppressive TME. Despite their widespread use, immune checkpoint blockade (ICB) antibodies targeting checkpoint molecules remain ineffective in most cancer patients. We do not understand why some patients respond to ICB better than others. To understand the heterogeneity of ICB response, we must understand the heterogeneity of the T cell subsets acted upon by such therapies. Here, we ask how T cell subsets change in the presence and absence of ICB. We track T cell clones through their T cell receptor sequences and link phenotypes with T cell receptor specificities. Through multiplexed single cell TCR sequencing, single cell RNA sequencing, and the use of cell- surface CITE-seq antibodies, coupled with surgical biopsy, we longitudinally tracked the fate of individual T cell clones within tumors at baseline and in response to ICB in an immunogenic mouse tumor model. Furthermore, computational clustering of T cells solely based on their gene expression profiles may ignore upstream regulatory mechanisms that control T cell gene expression. Hence, we employed Virtual Inference of Protein-activity by Enriched Regulon (VIPER) analysis to cluster CD8+ and CD4+ T cell phenotypes. VIPER leverages inference of gene regulatory networks to allow full quantitative characterization of protein activity for transcription factors, co-factors, and signaling molecules by assessing the enrichment of their transcriptional targets cell-by-cell among expressed genes. This gave us a window into the transcriptional states and their inferred protein activity. We next developed a computational analysis toolkit to study TCR clonality incorporating sub-sampling of TCR clonotypes, forward and back tracing of shared clones between timepoints, and in turn, inferred shared clonal evolution. We employed the above workflow to MC38 tumor-infiltrating and tumor-draining lymph node-derived CD8+ and CD4+ T cells. We found that T cell phenotypes are highly dynamic within tumors at baseline, in the absence of ICB, particularly within the window that they are responsive to therapy. In the absence of ICB, effector phenotype of CD8+ T cells diminished, while the exhaustion phenotype was enhanced as tumors progressed. Within the CD4+ population, a heterogenous subset of regulatory CD4+ T cells (Tregs) changed phenotype over time, and CD4+ Th1 like effectors, along with stem like progenitor CD4+ showed distinct dynamism. Next, by analyzing responses to therapy within his context, we found that both anti-PD1 and anti-CTLA4 act through distinct mechanisms on CD8+ and CD4+ T cells. Anti-PD1 acted upon intra-tumoral effector CD8+ T cells to slow their progression to terminally differentiated exhausted cells, i.e., increased their persistence within tumors. Anti-CTLA4 therapy increased recruitment of novel effector CD8+ T cell clones to tumors from lymph nodes while diminishing tumor-infiltrating Tregs. ICB also potentiated CD4+ Th1 like phenotype. These results uncovered a behavior pattern of CD8+ and CD4+ T cells within tumors at baseline tumor progression, and then in the presence of ICB. We believe these findings have added to our understanding of the subtleties of T cell phenotypes in tumors, specifically in response to ICB. This will provide a practical framework for designing and validating novel checkpoint blockade therapies in the future.

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Made available in DSpace on 2014-10-09T12:43:42Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T13:57:43Z (GMT). No. of bitstreams: 1 06555.pdf: 4601344 bytes, checksum: d54da5711d592aac4fbdb3cbb1ac8e1a (MD5) / Tese (Doutoramento) / IPEN/T / Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP