31 |
Cellular reprogramming of human acute myeloid leukemia patient somatic cellsSalci, Kyle 15 December 2015 (has links)
Acute myeloid leukemia (AML) is a fatal cancer of the human hematopoietic system characterized by the rapid accumulation of non-functional, immature hematopoietic cells in the bone marrow (BM) and peripheral blood (PB) of affected patients. Limited sources of safe hematopoietic stem/progenitor cells (HSPCs) for transplantation and incomplete mechanistic understandings of disease initiation, progression and maintenance have impeded advances in therapy required for improvement of long-term AML patient survival rates. Toward addressing these unmet clinical needs, the ability to generate induced pluripotent stem cells (iPSCs) from human somatic cells may provide platforms from which to develop patient-specific (autologous) cell-based therapies and disease models. However, the ability to generate iPSCs from human AML patient somatic cells had not been investigated prior to this dissertation. Accordingly, I hypothesized that cellular reprogramming of human AML patient somatic cells to iPSCs is possible and will enable derivation of autologous sources of normal and dysfunctional hematopoietic progenitor cells (HPCs).
I first postulated that reprogramming AML patient fibroblasts (AML Fibs) to pluripotency would provide a novel source of normal autologous HPCs. Our findings revealed that AML patient-specific iPSCs devoid of leukemia-associated aberrations found in the matched bone marrow (BM) could be generated from AML Fibs, and demonstrated that this cellular platform allowed for the derivation of healthy HPCs capable of normal differentiation to mature myeloid lineages in vitro. During the tenure of these experiments we also redefined conventional reprogramming methods by discovering that OCT4 transcription factor delivery combined with culture in pluripotent-supportive media was minimally sufficient to induce pluripotency in AML and normal Fibs.
Toward capturing and modeling the molecular heterogeneity observed across human AML samples in vitro, we next asked whether reprogramming of AML patient leukemic cells would enable generation of iPSCs and derivative HPCs that recapitulated dysfunctional differentiation features of primary disease. Our results demonstrated that conventional reprogramming conditions were insufficient to induce pluripotency in leukemic cells, but that generation of AML iPSCs could be reproducibly achieved in one AML sample when reprogramming conditions were modified. These AML iPSCs and their derivative HPCs harboured and expressed the leukemia-associated aberration found in the BM leukemic cells and similarly possessed dysfunctional differentiation capacities.
Together, this body of works provides the proof of principle that cellular reprogramming can be applied on a personalized basis to generate normal and dysfunctional HPCs from AML patient somatic cells. These foundational findings should motivate additional studies aimed at developing iPSC-based cell therapies and disease models toward improving AML patient quality of life and long-term survival rates. / Thesis / Doctor of Philosophy (PhD)
|
32 |
Testing Mice at Risk of Pancreatic Cancer for Altered Protein Pathways Found in DiabetesCheung, Henley 01 January 2017 (has links)
Pancreatic cancer is nearly asymptomatic, which can result in extensive grow and even metastasis to other organs before detection. When diagnosed at a late stage, the survival rate is 3%. Early detection is therefore the key to treating pancreatic cancer. Diabetes was identified as a risk factor for the development of pancreatic cancer, but the mechanism remains unknown. In this project, the objective was to delineate a link between diabetes and pancreatic cancer by examining their shared protein signaling pathways. In a previous study, hyper-activation of AKT1 resulted in a pre-diabetic phenotype and also increased upregulation of downstream phosphorylated mTOR and phosphorylated p70S6 kinase. More recently, mice with mutations that hyper-activated AKT1 and KRAS showed a significantly higher blood glucose level compared to littermate matched wild-type, mutant AKT1, or mutant KRAS mice. Interestingly, mice with a combination of mutations that hyper-activated AKT1 and KRAS also showed faster development of pancreatic cancer compared to these other groups of littermate mice.
Toward determining a molecular basis for the crosstalk between AKT1 and KRAS, pancreas and liver tissues were collected from all four groups of mice including wild-type, mutant AKT1, mutant KRAS, and mice with dual AKT1/KRAS hyper-activation. One strategy was to examine expression and/or phosphorylation of downstream protein signaling crosstalk by analysis of p70S6K using Western Blots. Erk 1/2 proteins were also tested as downstream proteins of KRAS to provide a molecular view of the individual and cooperative roles of AKT1 and KRAS in the mouse models. A potential feedback mechanism to affect insulin receptor signaling in the pancreas was examined using enzyme-linked immunosorbent assays (ELISA). A significant decrease in insulin receptor phosphorylation, possibly contributing to insulin resistance, was found when mice had mutant hyper-activated KRAS. Contrary to the original expectations, mice with combined mutations of AKT1 and KRAS may contribute to the accentuated diabetic phenotype by targeting two different points in the AKT and KRAS protein signaling pathways. The information can help understand the relationship between glucose metabolism, diabetes, and pancreatic cancer development. By thoroughly studying the interactions between targets in the AKT1/KRAS signaling pathways, key molecular events that induce metabolic changes and potentially early biomarkers may lead to an improved understanding of risk and/or detection of pancreatic cancer.
|
33 |
Simple derivation of skeletal muscle from human pluripotent stem cells using temperature-sensitive Sendai virus vector / 温度感受性センダイウイルスベクターを用いてヒト多能性幹細胞から骨格筋細胞を簡便に作製する方法TAN, GHEE WAN 23 March 2022 (has links)
京都大学 / 新制・課程博士 / 博士(医学) / 甲第23812号 / 医博第4858号 / 新制||医||1059(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 金子 新, 教授 山下 潤, 教授 朝長 啓造 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM
|
34 |
Canine chronic enteropathy—Current state-of-the-art and emerging conceptsJergens, Albert E., Heilmann, Romy M. 25 July 2024 (has links)
Over the last decade, chronic inflammatory enteropathies (CIE) in dogs have received great attention in the basic and clinical research arena. The 2010 ACVIM Consensus Statement, including guidelines for the diagnostic criteria for canine and feline CIE, was an important milestone to a more standardized approach to patients suspected of a CIE diagnosis. Great strides have been made since understanding the pathogenesis and classification of CIE in dogs, and novel diagnostic and treatment options have evolved. New concepts in the microbiome-host-interaction, metabolic pathways, crosstalk within the mucosal immune system, and extension to the gut-brain axis have emerged. Novel diagnostics have been developed, the clinical utility of which remains to be critically evaluated in the next coming years. New directions are also expected to lead to a larger spectrum of treatment options tailored to the individual patient. This review offers insights into emerging concepts and future directions proposed for further CIE research in dogs for the next decade to come.
|
35 |
DIET-INDUCED OBESITY: DOPAMINERGIC AND BEHAVIORAL MECHANISMS AS OUTCOMES AND PREDICTORSNarayanaswami, Vidya 01 January 2013 (has links)
Obesity and drug abuse share common neural circuitries including the mesocoticolimbic and striatal dopamine reward system. In the current study, a rat model of diet-induced obesity (DIO) was used to determine striatal dopamine function, impulsivity and motivation as neurobehavioral outcomes and predictors of obesity. For the outcome study, rats were randomly assigned a high-fat (HF) or a low-fat (LF) diet for 8 wk. Following the 8-wk HF-diet exposure, rats were segregated into obesity-prone and obesity-resistant groups based on maximum and minimum body weight gain, respectively, and neurobehavioral outcomes were evaluated. For the predictor study, neurobehavioral antecedents were evaluated prior to an 8-wk high-fat diet exposure and were correlated with subsequent body weight gain. Striatal D2 receptor density was determined by in vitro kinetic analysis of [3H]raclopride binding. DAT function was determined using in vitro kinetic analysis of [3H]dopamine uptake, methamphetamine-evoked [3H]dopamine overflow and no net flux in vivo microdialysis. DAT cell-surface expression was determined using biotinylation and Western blotting. Impulsivity and food-motivated behavior were determined using a delay discounting task and progressive ratio schedule for food-reinforcers, respectively. Relative to obesity-resistant, obesity-prone rats exhibited 18% greater body weight, 42% lower striatal D2 receptor density, 30% lower total DAT expression, 40% lower in vitro and in vivo DAT function, 45% greater extracellular dopamine concentration, and 2-fold greater methamphetamine-evoked [3H]dopamine overflow. Obesity-prone rats exhibited higher motivation for food, but were less impulsive relative to obesity-resistant rats. Neurobehavioral antecedents of DIO included greater motivation for high-fat reinforcers in rats subsequently shown to be obesity-prone relative to obesity-resistant. Impulsivity, DAT function and extracellular dopamine concentration did not predict the DIO-phenotype. Thus, motivation for food is linked to both initiation and maintenance of obesity. Importantly, obesity results in decreased striatal DAT function, which may underlie the maintenance of compulsive food intake in obesity.
|
36 |
A MECHANISTIC STUDY OF AN iPSC MODEL FOR LEIGH’S DISEASE CAUSED BY MtDNA MUTATAION (8993 T>G)Galdun, John P 01 January 2016 (has links)
Mitochondrial diseases encompass a broad range of devastating disorders that typically affect tissues with high-energy requirements. These disorders have been difficult to diagnose and research because of the complexity of mitochondrial genetics, and the large variability seen among patient populations. We have devised and carried out a mechanistic study to generate a cell based model for Leigh’s disease caused by mitochondrial DNA mutation 8993 T>G. Leigh’s disease is a multi-organ system disorder that depends heavily on the mutation burden seen within various tissues. Using new reprogramming and sequencing technologies, we were able to show that Leigh’s disease patient fibroblasts reprogrammed to induced pluripotent stem cells maintain the same level of mutation burden seen in the original patient cell line. Mutation burden was maintained through several passages and spontaneous differentiation. This cell based model could be useful for future pathogenesis studies, or therapeutic drug screenings in a patient and tissue specific manner.
|
37 |
FUNCTIONAL CHARACTERIZATION OF SCAFFOLD PROTEIN SHOC2Jang, HyeIn 01 January 2018 (has links)
Signaling scaffolds are critical for the correct spatial organization of enzymes within the ERK1/2 signaling pathway and proper transmission of intracellular information. However, mechanisms that control molecular dynamics within scaffolding complexes, as well as biological activities regulated by the specific assemblies, remain unclear.
The scaffold protein Shoc2 is critical for transmission of the ERK1/2 pathway signals. Shoc2 accelerates ERK1/2 signaling by integrating Ras and RAF-1 enzymes into a multi-protein complex. Germ-line mutations in shoc2 cause Noonan-like RASopathy, a disorder with a wide spectrum of developmental deficiencies. However, the physiological role of Shoc2, the nature of ERK1/2 signals transduced through this complex or mechanisms regulating the function of Shoc2 remain largely unknown. My dissertation addresses the mechanisms by which Shoc2 accelerates ERK1/2 signal transmission and the biological outputs of the Shoc2-guided signals.
To delineate Shoc2-mediated ERK1/2 signals, I have utilized a vertebrate zebrafish model. I demonstrated that loss of Shoc2 protein expression leads to early embryonic lethality resulting from a significant reduction in the number of circulating erythropoietic and myelopoietic blood cells, underdeveloped neurocranial and pharyngeal cartilages, and a profound delay in calcification of bone structures. Together, this data demonstrates that the Shoc2 scaffolding module transmits ERK1/2 signals in neural crest development and blood cell differentiation.
This dissertation also addresses the mechanistic basis of how allosteric ubiquitination of Shoc2 and RAF-1 is controlled. I have characterized a molecular interaction of Shoc2 with its previously unknown binding partner Valosin-Containing Protein (VCP/p97). These studies demonstrated that hexametric ATPase VCP modulates ubiquitination of Shoc2 and RAF-1 through the remodeling of the scaffolding complex in a spatial-restricted manner. Experiments utilizing fluorescence microscopy and biochemical methods show that VCP/p97 sequesters the E3 ligase HUWE1 from the Shoc2 module, thereby altering the ubiquitination of Shoc2 and RAF-1 as well as the amplitude of ERK1/2 signals. These studies also show that the levels of Shoc2 ubiquitination and ERK1/2 phosphorylation are imbalanced in fibroblasts isolated from Inclusion Body Myopathy with Paget’s disease of bone and Frontotemporal Dementia (IBMPFD) patients harboring VCP germline mutations. This data also suggests that ERK1/2 pathway deregulation is part of IBMPFD pathogenesis.
In summary, these studies make a significant advance in our understanding of the mechanisms by which the Shoc2 scaffold regulates specificity and the dynamics of the ERK1/2 signaling networks. They also make important insights into our understanding of biological activities and targets of Shoc2-mediated ERK1/2 signals at the early stages of embryonic development and disease.
|
38 |
Examination of Strain-Dependent Differences in S. sanguinis Virulence and GrowthBaker, Shannon 01 January 2019 (has links)
Streptococcus sanguinis, an abundant and benign inhabitant of the oral cavity, is an important etiologic agent of infective endocarditis, particularly in people with pre-disposing cardiac valvular damage. Although commonly isolated from patients with IE, little is known about the factors that make any particular S. sanguinis isolate more virulent than another or, indeed, whether significant differences in virulence exist among isolates. To investigate the virulence of multiple isolates, a variation of the Bar-seq (barcode sequencing) method was employed. A conserved chromosomal site was identified for subsequent insertion of a barcode identifier, unique for each strain. Barcode insertion did not affect growth in vitro or in a rabbit model of endocarditis. Pooling of these strains and inoculation into rabbits demonstrated that all strains were capable of causing disease; however, virulence varied widely among strains. Genomic comparisons of the more virulent strains versus less virulent strains failed to conclusively identify any single gene responsible for virulence. Given this result, we continued our examination of the manganese transport system SsaACB, which is present in every strain of S. sanguinis examined. Although its contribution to virulence has not been confirmed in any strain other than SK36, it has been shown to be required for virulence in multiple species of streptococci, making it a candidate for emerging targeted therapies. In S. sanguinis strain SK36, previous studies have confirmed that loss of the manganese transport protein SsaB is tantamount to loss of virulence. Moreover, ssaB-deficient mutants are deficient for serum growth—a phenotype we have previously found to be associated with virulence. Our in vitro studies of manganese transporter-deficient strain SK36 supported this, but also revealed the emergence of suppressor mutants. In each suppressor mutant that was isolated, mutations were identified that mapped to a common gene, SSA_0696. Deletion of SSA_0696 resulted in restored in vitro growth in the ssaACB-deficient background, unearthing a novel mechanism for bacterial growth under manganese limitation. Fortunately, the suppressor mutant phenotype was not maintained in vivo; however, the combined results of these experiments suggest the efficacy of future therapeutics may require consideration of virulence at the species level and the incorporation of multiple targets.
|
39 |
Coral Disease Epizootiology in the Florida Keys (U.S.A.) and Cayman Islands (British West Indies), and the Development of the Simulation of Infected Corals ModelBrandt, Marilyn Elizabeth 11 December 2007 (has links)
Understanding coral disease dynamics within the heterogeneous populations in which they act is critical for predicting how the structure of reefs may change as a result of enzootic or epizootic levels of these important sources of mortality. This work focused on combining field studies and the development and testing of a spatially-explicit, individual-based epizootiological computer model with the aim of gaining a greater understanding of the dynamics and impact of white plague, a significant source of mortality on reef-building corals in the Caribbean region. Field studies focused on the incidence and distribution of all sources of coral mortality, including suspect white plague in situ, at two locations; the Florida Keys (United States of America) and Little Cayman Island (Cayman Islands, British West Indies). Results indicated that in both regions disease was the most significant source of mortality during the monitoring time periods, and that suspect white plague type II in Cayman is likely contributing to major structural changes. In Florida, observations made during a mass bleaching event indicated that a significant relationship exists between bleaching severity and disease incidence, and that mortality during the event was largely the result of disease and not bleaching. The simulation model was developed using a long-term data set from Little Cayman, and results of calibration indicated that suspect white plague type II on these reefs is transmissible between colonies within a limited field and require a yearly input from an outside source, and that host susceptibility to infection is low and likely not variable among species. Parameters describing the distribution and composition of the coral population were varied, and results indicated a significant effect of colony density, aggregation, and mean size on the impact of disease. Scenario testing of various disease management strategies indicated that should local prevention measures be developed in the future, it is they, and not treatment, that will likely be the most effective in limiting the impact of disease.
|
40 |
Estimating Seasonal Drivers in Childhood Infectious Diseases with Continuous Time ModelsAbbott, George H. 2010 May 1900 (has links)
Many important factors affect the spread of childhood infectious disease. To
understand better the fundamental drivers of infectious disease spread, several researchers
have estimated seasonal transmission coefficients using discrete-time models.
This research addresses several shortcomings of the discrete-time approaches,
including removing the need for the reporting interval to match the serial interval
of the disease using infectious disease data from three major cities: New York City,
London, and Bangkok. Using a simultaneous approach for optimization of differential
equation systems with a Radau collocation discretization scheme and total variation
regularization for the transmission parameter profile, this research demonstrates that
seasonal transmission parameters can be effectively estimated using continuous-time
models. This research further correlates school holiday schedules with the transmission
parameter for New York City and London where previous work has already been
done, and demonstrates similar results for a relatively unstudied city in childhood
infectious disease research, Bangkok, Thailand.
|
Page generated in 0.0667 seconds