165 research outputs found

    Doctor of Philosophy

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    dissertationThermal ablation is widely used, first line local-regional therapy for unresectable hepatocellular carcinoma (HCC). Although high temperature delivered by thermal energy results in efficient coagulation necrosis in tumor cells, various factors including tumor size, shape, location, and cirrhosis can lead to un-uniform heat distribution and inefficient cell damage. As a result, the incomplete ablation causes high rates of tumor recurrence and poor survival for HCC patients. Cells that are not completely ablated can induce heat shock proteins (HSPs), which are cellular gatekeepers to protect tumor cells from thermal damage and prepare them for future neoplastic growth. Synchronous adjuvant chemotherapy targeting those cells can achieve more complete tumor abrogation and prevent future tumor recurrence. This dissertation describes a strategy to combat postablation recurrence by synchronous inhibition of heat shock protein 90 (HSP90) by thermo-responsive, elastin-like polypeptide (ELP)-based biopolymer conjugates. ELP copolymer carries high concentrations of a potent HSP90 inhibitor, geldanamycin (GA), which inhibit the induction of HSP90 and further destabilize numerous HSP90 client proteins critical for cell survival. It is hypothesized that combination of thermal ablation with concomitant inhibition of HSP90 via ELP-GA conjugates can achieve synergistic anticancer effect. Specifically, the ablation-created hyperthermia will sensitize tumor cells to be more vulnerable to the drug, which will be conjugated with high concentrations through thermally targeted, ELP-based biopolymer systems. The ELP conjugates, in turn, will reach and kill the remaining viable cells to prevent future recurrence. ELP-GA conjugates that ferry multiple GAs and rapidly respond to hyperthermia were synthesized, characterized, and evaluated for activity in HCC models. The cytotoxicity of ELP-GA conjugates was enhanced with hyperthermia treatment, and effective HSP90 inhibition was achieved in HCC cell lines. In a tumor-bearing mouse model, electrocautery-based thermal ablation offered effective destruction of tumor core and created a hyperthermia zone for targeted delivery and accumulation of ELP-GA conjugates. Results demonstrate that the combination of thermal ablation and targeted HSP90 inhibition can enhance the anticancer effect and cellular delivery of macromolecular chemotherapeutics to achieve safe, synergistic, and long-term anticancer effect with no tumor recurrence observed. The combination approach paves the way for developing molecular-targeted intervention to increase the efficacy of first-line local-regional therapies for HCC

    Foundational platform for mammalian synthetic biology

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    Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, February 2013Cataloged from PDF version of thesis.Includes bibliographical references (p. 116-129).The emergent field of synthetic biology is different from many other biological engineering efforts, in that its roots, design principles, and forward engineering perspective have been adopted from electrical engineering and computer science. Synthetic biology is uniquely poised to make great contributions to numerous fields such as bio-fuel, energy production, agriculture and eco-remediation, national defense, and biomedical and tissue engineering. Considerable progress has been made in engineering novel genetic circuits in many different organisms. However, not much progress has been made toward developing a formal methodology to engineer complex genetic systems in mammalian cells. One of the most promising areas of research is the study of embryonic and adult stem cells. Synthetic biology has the potential to greatly impact the progression and development of research in this area of study. A critical impediment to the development of stem cell engineering is the innate complexity, little to no characterization of parts, and limited compositional predictive capabilities. In this thesis, I discuss the strategies used for constructing and optimizing the performance of signaling pathways, the development of a large mammalian genetic part and circuit library, and the characterization and implementation of novel genetic parts and components aimed at developing a foundation for mammalian synthetic biology. I have designed and tested several orthogonal strategies aimed at cell-cell communication in mammalian cells. I have designed a characterization framework for the complete and proper characterization of genetic parts that allows for modular predictive composition of genetic circuits. With this characterization framework I have generated a small library of characterized parts and composite circuits that have well defined input-output relationships that can be used in novel genetic architectures. I also aided in the development of novel analysis and computational tools necessary for accurate predictive composition of these novel circuits. This work collectively provides a foundation for engineering complex intracellular transcriptional networks and intercellular signaling systems in mammalian cells.by Noah Davidsohn.Ph.D

    Somatic genome editing with the RCAS-TVA-CRISPR-Cas9 system for precision tumor modeling

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    To accurately recapitulate the heterogeneity of human diseases, animal models require to recreate multiple complex genetic alterations. Here, we combine the RCAS-TVA system with the CRISPR-Cas9 genome editing tools for precise modeling of human tumors. We show that somatic deletion in neural stem cells of a variety of known tumor suppressor genes (Trp53, Cdkn2a, and Pten) leads to high-grade glioma formation. Moreover, by simultaneous delivery of pairs of guide RNAs we generate different gene fusions with oncogenic potential, either by chromosomal deletion (Bcan-Ntrk1) or by chromosomal translocation (Myb-Qk). Lastly, using homology-directed-repair, we also produce tumors carrying the homologous mutation to human BRAF V600E, frequently identified in a variety of tumors, including different types of gliomas. In summary, we have developed an extremely versatile mouse model for in vivo somatic genome editing, that will elicit the generation of more accurate cancer models particularly appropriate for pre-clinical testing.A.C.-G is recipient of a Severo-Ochoa PhD fellowship. C.M. and V.M. are recipients of a "La Caixa "PhD fellowship. We thank A.J. Schuhmacher for the initial assistance with the intracranial injections in adult mice and C.S. Clemente-Troncone for the technical support. We thank Carmen Blanco, David Olmeda, and Marisol Soengas for sharing reagents and Orlando Dominguez for the help with the design of the BRAF high- throughput sequencing. We sincerely thank Dr. José Luis Rodríguez Peralto (Hospital U. 12 de Octubre Madrid) for the BRAF V600 IHCs staining. This research was supported by funds from the Acción Estratégica en Salud Spanish National Research and Development Plan, Instituto de Salud Carlos III (ISCIII), cofounder by FEDER (ERDF) (PI14/01884) to S.R.-P., by a 017 Leonardo Grant for Researchers and Cultural Creators from the BBVA Foundation and a grant from the Seve Ballesteros Foundation to M.S.S

    Topics on Cervical Cancer With an Advocacy for Prevention

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    Cervical Cancer is one of the leading cancers among women, especially in developing countries. Prevention and control are the most important public health strategies. Empowerment of women, education, "earlier" screening by affordable technologies like visual inspection, and treatment of precancers by cryotherapy/ LEEP are the most promising interventions to reduce the burden of cervical cancer.Dr Rajamanickam Rajkumar had the privilege of establishing a rural population based cancer registry in South India in 1996, as well as planning and implementing a large scale screening program for cervical cancer in 2000. The program was able to show a reduction in the incidence rate of cervical cancer by 25%, and reduction in mortality rate by 35%. This was the greatest inspiration for him to work on cerrvical cancer prevention, and he edited this book to inspire others to initiate such programs in developing countries. InTech - Open Access Publisher plays a major role in this crusade against cancer, and the authors have contributed to it very well

    Deciphering transcriptional regulation in cancer cells and development of a new method to identify key transcriptional regulators and their target genes

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    Cancer cells accumulate genetic changes during carcinogenesis. The dimension of these changes range from point mutations to large chromosomal aberrations. It has been widely accepted that essential genetic programs are thereby dysregulated that normally would prevent uncontrolled cellular division and growth. Transcription factors (TFs) are key proteins of gene regulation and are frequently associated with genetic pathologies, e.g. MYCN in neuroblastomas (NBs). Research on gene regulation -in general or condition-specific- thus is a central aspect in cancer research, and it is also the focus of my work. In a carcinogenesis model of NBs without MYCN-amplification, mutations of chromosome 11q (11q-CNA) are suspected to critically influence tumor development. We were able to refine this model by means of gene expression analysis on 11q-CNA in NBs with different clinical outcome. Gene expression profiles of NBs with unfavorable progression differed significantly between tumors with and without 11q-CNA, whereas 11q-CNA in NBs with favorable outcome is apparently compensated by a yet unknown mechanism. The TF-encoding gene CAMTA1 is located on the chromosomal region 1p, which is frequently deleted in NBs. In vitro experiments with ectopic induction of CAMTA1 yielded CAMTA1-regulated genes with different gene expression profiles that were functionally associated by enrichment analyses with cell cycle regulation and neuronal differentiation. The suggested role of CAMTA1 as a tumor suppressor gene was confirmed by additional in vivo experiments. Furthermore, we studied the effect of MYC and MYCN in NBs without MYCN-amplification and found that these TF also strongly regulate a large number of common target genes according to their own gene expression in these tumors. Promoter analyses and chromatin immunoprecipitation additionally supported the regulation of the determined target genes by MYC/MYCN. The genome-wide application of promoter and enrichment analyses on gene expression data from mouse models enabled us to predict target TFs of Rage signaling. E2f1 and E2f4 were validated experimentally as components of the Rage-dependent gene regulatory network. Finally, we used our experience from gene expression analysis to develop a novel machine learning method to precisely predict TF target gene relationships in human. We combined results from a genome-wide correlation meta-analysis on 4064 microarray gene expression profiles and promoter analyses on TF binding sites with known regulatory interactions between TFs and target genes in our approach. Our method outperformed other comparable methods in human, as we improved shortcomings of other algorithms specifically for higher eukaryotes, in particular the frequently (erroneously) assumed correlation between the mRNA expression of TFs and their target genes. We made our method freely available as a software package with multiple applications like the identification of key TFs in a multiplicity of cellular systems (e.g. cancer cells)

    Sox9 conditional knockdown reduces chondroitin sulphate proteoglycan expression, increases neuroplasticity, and improves motor function in a mouse model of spinal cord injury

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    This thesis investigates the effect of Sox9 knockdown on anti-regenerative scar gene expression, neuroplasticity, and hind limb functional recovery following mouse spinal cord injury. We hypothesized that Sox9 knockdown would reduce expression of anti-regenerative chondroitin sulfate proteoglycans both at the lesion site and at sites distant to the injury, thus providing an avenue for increased neuroplasticity and locomotor recovery after spinal cord injury. The first chapter provides a general introduction to the biological problem of spinal cord injury. The development of the glial scar and expression of the anti-regenerative chondroitin sulfate proteoglycan extracellular matrix is introduced, and Sox9 is identified as a transcription factor that may control expression of these anti-regenerative genes. The second chapter is a manuscript that describes the molecular changes and improved locomotor function seen when Sox9 knockdown is carried out just prior to spinal cord injury. The third chapter is more clinically relevant as it is a manuscript detailing the effects of Sox9 knockdown after spinal cord injury on the recovery of hind limb motor function. The fourth chapter is a manuscript investigating the neuro-anatomical mechanism of the improved functional recovery seen in Sox9 knockdown mice after spinal cord injury. The fifth chapter reflects on the findings presented herein, and suggests possible future plans of study. This dissertation demonstrates that inhibition of Sox9 leads to reduced CSPG expression, improved hind limb function, and increased total locomotion. It further provides compelling evidence that increased neuroplasticity as evidenced by increased reactive sprouting and increased expression of the presynaptic markers synaptophysin and VGLUT1 caudal to the injury site underlies the improved neurological recovery observed in spinal cord injured Sox9 conditional knockdown mice

    Tumor microenvironment models: ex vivo, in vitro and in silico approaches to address targeted therapies

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    "Cancer is the second leading cause of mortality worldwide, despite the extraordinary advances in the last two decades due to the development of targeted therapies. These target particular molecules required for cell growth and tumorigenesis; nonetheless, de novo or acquired resistance to therapy often lead to patient relapse and disease progression. There is cumulating evidence supporting the importance of tumor microenvironment (TME)-driven mechanisms in cancer progression and drug resistance. Therefore, there is a need for cancer models in which critical components of the TME, such as the non-malignant cell types and the extracellular matrix (ECM), are represented and tissue architecture is maintained. (...)"N/

    Mathematical approaches to the study of cellular heterogeneity, treatment design, and immune response in cancer

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    One of the complicating factors in treating cancer patients is the different levels of heterogeneity involved. In this thesis, we use a combination of mathematical methods (in silico experiments) and experimental data (in vitro and ex vivo experiments) to study cellular heterogeneity, treatment design, and immune response in cancer. This thesis demonstrates the importance and value of interdisciplinary communication and collaboration. In Chapter 2, we develop a framework that uses in vitro and in silico experiments to characterize cancer cell lines and investigate the cellular dynamics during early cancer development for specific cancer cell lines. We demonstrate this process with the breast cancer cell line, MCF-7, and present evidence that progenitor cells are the significant cancer subpopulation during early cancer development for MCF-7 cells. In Chapter 3, we modify and build on the agent-based model of Chapter 2 to characterize the effect of pressure on mammosphere formation with and without the presence of a chemotherapy drug. Our results suggest that pressure induces phenotypic plasticity. In Chapter 4, we identify the Hsp90 protein network as a means by which drug resistance can be overcome in a drug-tolerant cell. We construct a minimal in silico model of this network to design a treatment schedule for docetaxel and radicicol. In silico experiments are used to show that radicicol can overcome the development of drug resistance to docetaxel with the proper treatment sequence, which can be accomplished with a nanoparticle formulation. We present evidence that the intake rate and the decay rate of radicicol are drug formulation properties that will have the greatest impact on increasing the efficacy of the docetaxel-radicicol treatment sequence. In Chapter 5, we investigate the variability in immune system response to anti-PD-1 immunotherapy. In this work, we construct a systems biology model and use sensitivity analysis to identify potential biomarkers for a positive response to anti-PD-1 immunotherapy. We present evidence of the importance of two interaction networks with regards to response to anti-PD-1 immunotherapy: 1) the interaction between cancer cells and CD8+ cytotoxic Tc cells, and 2) the balance between CD4+ Th1 and Th2 helper cells. In each of the chapters, we investigate heterogeneity at a different level: cellular heterogeneity with and without the cell microenvironment, variability in protein expression, and variability in immune system response. By developing an in silico model to describe the biological phenomena, we can investigate the underlying mechanisms at work and provide potential biomarkers and potential improvements that can be tested further

    The Role of the CXCR3 Signaling Axes in Pancreatic Ductal Adenocarcinoma

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    Numerous cytokines promote pancreatic ductal adenocarcinoma (PDAC) progression and suppress anti-tumor immune response leading to poor prognosis in PDAC patients. Despite this, many cytokines, have not been investigated in PDAC. Bioinformatic analyses of PDAC microarray and RNA-Seq datasets were used to identify cytokines overexpressed in PDAC, confirm the expression of cognate receptors, determine the association of cytokines with patient survival and define key underlying molecular associations. Bioinformatic findings were validated using immunohistochemical (IHC) staining, comparative cytokine qPCR-array in KrasLSL-G12D:TP53LSL-R172H:Pdx1-Cre (KPC) and KrasLSL-G12D:Pdx1-Cre (KC) PDAC models and multicolor immunofluorescence staining. Tail-vein injections of PDAC cells with/without CXCR3 inhibition were used to study altered metastatic potential in vivo and functional assays were conducted to demonstrate causal relationships between CXCR3 activation and metastatic properties of PDAC cells. CXCR3 ligands CXCL9 and 10 were consistently overexpressed in PDAC datasets. CXCR3 was expressed in the majority of PDAC samples according to RNA-Seq, microarray and IHC analysis. CXCR3 ligands CXCL4, 9 and 10 were associated with poor patient survival and were overexpressed in the aggressive KPC murine model compared to KC mice. CXCR3 was associated with increased overall survival in humans. Pathway analysis showed that CXCR3 is associated with T-cell-related genes while CXCL9/10 were associated with T-cell and immunosuppressive genes. CIBERSORT, gene set enrichment and immunofluorescence analysis supported these findings. With respect to metastasis, inhibition of CXCR3 suppressed the number of cancer cells in the lungs following tail vein injection. Cancer cells treated with activated platelets and/or CXCL4 demonstrated increased ability to survive low attachment conditions and fluid shear stress and to adhere to endothelium, suggesting pleotropic roles in the metastatic process. Overall, CXCR3 ligands are overexpressed in PDAC and are associated with poor survival likely related to alterations in immune cell infiltrate/activity and augmented metastatic potential
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