Targeting PI3Kδ and PI3Kγ signalling disrupts human AML survival and bone marrow stromal cell mediated protection

Phosphoinositide-3-kinase (PI3K) is an enzyme group, known to regulate key survival pathways in acute myeloid leukaemia (AML). It generates phosphatidylinositol-3,4,5-triphosphate, which provides a membrane docking site for protein kinaseB activation. PI3K catalytic p110 subunits are divided into 4 isoforms; α,β,δ and γ. The PI3Kδ isoform is always expressed in AML cells, whereas the frequency of PI3Kγ expression is highly variable. The functions of these individual catalytic enzymes have not been fully resolved in AML, therefore using the PI3K p110δ and p110γ-targeted inhibitor IPI-145 (duvelisib) and specific p110δ and p110γ shRNA, we analysed the role of these two p110 subunits in human AML blast survival. The results show that PI3Kδ and PI3Kγ inhibition with IPI-145 has anti-proliferative activity in primary AML cells by inhibiting the activity of AKT and MAPK. Pre-treatment of AML cells with IPI-145 inhibits both adhesion and migration of AML blasts to bone marrow stromal cells. Using shRNA targeted to the individual isoforms we demonstrated that p110δ-knockdown had a more significant anti-proliferative effect on AML cells, whereas targeting p110γ-knockdown significantly inhibited AML migration. The results demonstrate that targeting both PI3Kδ and PI3Kγ to inhibit AML-BMSC interactions provides a biologic rationale for the pre-clinical evaluation of IPI-145 in AML

Identification of Bruton's tyrosine kinase as a therapeutic target in acute myeloid leukemia

Bruton's tyrosine kinase (BTK) is a cytoplasmic protein found in all hematopoietic cell lineages except for T cells. BTK mediates signalling downstream of a number of receptors. Pharmacological targeting of BTK using ibrutinib (previously PCI-32765) has recently shown encouraging clinical activity in a range of lymphoid malignancies. This study reports for the first time that ibrutinib inhibits blast proliferation from human acute myeloid leukaemia (AML) and that treatment with ibrutinib significantly augmented cytotoxic activities of standard AML chemotherapy cytarabine or daunorubicin. Here we describe that BTK is constitutively phosphorylated in the majority of AML samples tested, with BTK phosphorylation correlating highly with the cell's cytotoxic sensitivity towards ibrutinib. BTK targeted RNAi knock-down reduced colony forming capacity of primary AML blasts and proliferation of AML cell lines. We showed ibrutinib binds at nanomolar range to BTK. Furthermore, we also showed ibrutinib's anti-proliferative effects in AML are mediated via an inhibitory effect on downstream nuclear factor-κB (NF-κB) survival pathways. Moreover, ibrutinib inhibited AML cell adhesion to bone marrow stroma. Furthermore, these effects of ibrutinib in AML were seen at comparable concentrations efficacious in chronic lymphocytic leukemia (CLL). These results provide a biologic rationale for clinical evaluation of BTK inhibition in AML patients

Prehistoric human impact on tree island lifecycles in the Florida Everglades

The current study provides a fine-grained analysis of evidence for sustained pre-Columbian human occupation and socio-ecological interaction within Everglades National Park. Utilizing archaeological data on dietary and cultural patterns recovered from recent excavations at a prehistoric tree island site, we argue the role of ancient human populations in the formation or augmentation of tree islands should be incorporated into environmental models of the tree island lifecycle. High phosphorus levels in human waste, especially the largely organic waste of prehistoric populations, as well as other anthropogenic factors have not been adequately factored into current environmental models of tree island formation or the ecological evolution of the Everglades. More broadly, while socio-ecological modeling is at the core of current scholarly and restoration paradigms, expanded collaboration between environmental scientists and archaeologists will lead to more accurate identification of anthropogenic environmental impacts over time

Prehistoric human impact on tree island lifecycles in the Florida Everglades

The current study provides a fine-grained analysis of evidence for sustained pre-Columbian human occupation and socio-ecological interaction within Everglades National Park. Utilizing archaeological data on dietary and cultural patterns recovered from recent excavations at a prehistoric tree island site, we argue the role of ancient human populations in the formation or augmentation of tree islands should be incorporated into environmental models of the tree island lifecycle. High phosphorus levels in human waste, especially the largely organic waste of prehistoric populations, as well as other anthropogenic factors have not been adequately factored into current environmental models of tree island formation or the ecological evolution of the Everglades. More broadly, while socio-ecological modeling is at the core of current scholarly and restoration paradigms, expanded collaboration between environmental scientists and archaeologists will lead to more accurate identification of anthropogenic environmental impacts over time

Doctor of Philosophy

dissertationIt is well known that corrosion and simultaneous cyclic loading have a detrimental impact in the integrity of devices or structures. Understating these mechanisms is critical to ensure safety of aircraft. This work presents an extensive literature review on issues of corrosion mechanisms including pitting, exfoliation and intergranular attack. Moreover, models for phases of life and pitting corrosion are presented. Relevant definitions related to these failure modes are presented. The nucleation of fatigue cracks from corrosion pits was investigated by evaluating the effects of two variables on the fatigue life of dog-bone specimens of aluminum alloys 7075-T6 and 2024-T3. The specimens were exposed to different levels of corrosion in an acidified saline solution of 3.5% NaCl. In addition, the specimens were exposed to concomitant fatigue and corrosion until failure by fracture occurred. SEM analysis indicated that fatigue cracks formed/nucleated from each pit, and subsurface mechanisms of degradation were identified associated with the pitting nucleation sites including subsurface pitting, cracking, tunneling and intergranular attack. Failure data were analyzed by ANOVA methods and three transformations were evaluated to minimize the variance, including natural log, inverse square root and power with a lambda of 1/3. Contour and surface plots were developed to show how these variables impact the response of cycles to failure for the conditions evaluated. The effects of stress are more detrimental than corrosion time on the fatigue life of the specimens for the values previously defined by the DOE matrix. The research reported herein presents a methodology for accelerated corrosion fatigue of high strength aluminum alloys in an acidified saline environment. Subsequently a statistical based methodology to assess the impact of multiple variables into the fatigue life of specimens is presented. Statistical models are developed to assess the effect of two variables, stress and corrosion time into the fatigue life of the specimens. Stress levels were chosen to simulate conditions of current aircraft, such as fuselage bulkheads in the F-16. Development of statistical models to predict the behavior of materials will increase our ability to predict and prevent catastrophic structural failures thereby increasing the safety of our aircraft structures

Blastic plasmacytoid dendritic cell neoplasm: Genomics mark epigenetic dysregulation as a primary therapeutic target

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and aggressive hematologic malignancy for which there is still no effective B therapy. In order to identify genetic alterations useful for a new treatment design, we used whole-exome sequencing to analyze 14 BPDCN patients and the patient-derived CAL-1 cell line. The functional enrichment analysis of mutational data reported the epigenetic regulatory program to be the most significantly undermined (P<0.0001). In particular, twenty-five epigenetic modifiers were found mutated (e.g. ASXL1, TET2, SUZ12, ARID1A, PHF2, CHD8); ASXL1 was the most frequently affected (28.6% of cases). To evaluate the impact of the identified epigenetic mutations at the gene-expression and Histone H3 lysine 27 trimethylation/acetylation levels, we performed additional RNA and pathology tissue-chromatin immunoprecipitation sequencing experiments. The patients displayed enrichment in gene signatures regulated by methylation and modifiable by decitabine administration, shared common H3K27-acetylated regions, and had a set of cell-cycle genes aberrantly up-regulated and marked by promoter acetylation. Collectively, the integration of sequencing data showed the potential of a therapy based on epigenetic agents. Through the adoption of a preclinical BPDCN mouse model, established by CAL-1 cell line xenografting, we demonstrated the efficacy of the combination of the epigenetic drugs 5’-azacytidine and decitabine in controlling disease progression in vivo

Ultrassonografia anal e transperineal : nova abordagem para o diagnóstico anorretal em cães

Orientadora : Profª. Drª. Tilde Rodrigues FroesDissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências Agrárias, Programa de Pós-Graduação em Ciências Veterinárias. Defesa: Curitiba, 28/03/2017Inclui referências : f. 98-105Resumo: A presente pesquisa teve como objetivo mostrar achados ultrassonográficos da região anal de cães como uma nova abordagem no diagnóstico por imagem veterinário, a fim de contribuir com clínicos e cirurgiões na tomada de decisões terapêuticas nesses animais. Para isso, o trabalho foi dividido em três capítulos que se complementam. O primeiro capítulo revisa conceitos anatômicos e semiológicos do canal anal de cães, incluindo aspectos radiográficos, além de apresentar as doenças dos sacos anais nessa espécie. Essa revisão deixa claro que a avaliação anorretal em cães ainda é carente de diagnóstico complementar ao clínico. Assim, o segundo capítulo apresenta uma nova forma de avaliação do canal anal: a ultrassonografia anal e transperineal com transdutor linear, um método não invasivo que permite analisar as estruturas dessa região com facilidade e baixo custo, mostrando ao ultrassonografista que é possível examinar a área do ânus na sequência da avaliação abdominal se houver indicação clínica, e encontrar informações interessantes. Esses achados foram estudados em cães saudáveis de diferentes idades, portes, escores corporais e sexos, para assim termos um parâmetro de normalidade como referência para os cães com doenças anorretais apresentados no terceiro capítulo. No último capítulo foram descritos os achados desse mesmo exame em cães com doenças dos sacos anais, neoplasia perianal ou perineal, hérnia perineal, disquesia e tenesmo não relacionados à doença anorretal, incontinentes fecais, e furunculose anal revelando características ultrassonográficas interessantes em cada uma dessas enfermidades. Concluímos que a ultrassonografia anal e transperineal pode auxiliar diferentes especialistas veterinários de pequenos animais, como cirurgiões, dermatologistas, oncologistas e gastrenterologistas na avaliação clínica anorretal de forma eficaz, indolor, rápida e de fácil execução, além de baixo custo. Portanto, seguem dois estudos inéditos na medicina veterinária e uma revisão de literatura. Palavras-chave: canino, ânus, ultrassom, sacos anais, esfíncter, doenças perianais e perineaisAbstract: The present research aimed to show ultrasonographic findings of dog's anal region as a new approach in veterinary imaging diagnosis in order to contribute with clinicians and surgeons in the therapeutic decision making in these animals. For this, the study was divided into three chapters that complement each other. The first chapter reviews anatomical and semiological concepts of dogs' anal canal, including radiographic aspects, besides presenting the diseases of the anal sacs in this species. This review makes it clear that anorectal evaluation in dogs is still lacking in complementary diagnosis to the clinician. Thus, the second chapter presents a new form of anal canal evaluation: anal and transperineal ultrasonography with linear transducer, a non-invasive method that allows to analyze the structures of this region with ease and low cost, showing to the sonographer that it is possible to examine the anus area following abdominal evaluation if there is clinical indication, and find interesting information. These findings were studied in healthy dogs of different ages, sizes, body scores and sexes, in order to have normality parameters as reference for dogs with anorectal diseases presented in the third chapter. In the last chapter were described the findings of this same examination in dogs with anal sacs diseases, perianal or perineal neoplasia, perineal hernia, dyschezia and tenesmus not related to anorectal disease, fecal incontinents, and anal furunculosis revealing interesting sonographic characteristics in each of these diseases. We conclude that anal and transperineal ultrasound can assist different veterinary specialists of small animals, such as surgeons, dermatologists, oncologists and gastroenterologists in the anorectal clinical evaluation an effective, painless, quick and easy to perform, and low cost way. Therefore, there are two unpublished studies in veterinary medicine and a review of the literature. Keywords: canine, anus, ultrasound, anal sacs, sphincter, perianal and perineal disease

MUC1-C drives myeloid leukaemogenesis and resistance to treatment by a survivin-mediated mechanism

Acute myeloid leukaemia (AML) is an aggressive haematological malignancy with an unmet need for improved therapies. Responses to standard cytotoxic therapy in AML are often transient because of the emergence of chemotherapy-resistant disease. The MUC1-C oncoprotein governs critical pathways of tumorigenesis, including self-renewal and survival, and is aberrantly expressed in AML blasts and leukaemia stem cells (LSCs). However, a role for MUC1-C in linking leukaemogenesis and resistance to treatment has not been described. In this study, we demonstrate that MUC1-C overexpression is associated with increased leukaemia initiating capacity in an NSG mouse model. In concert with those results, MUC1-C silencing in multiple AML cell lines significantly reduced the establishment of AML in vivo. In addition, targeting MUC1-C with silencing or pharmacologic inhibition with GO-203 led to a decrease in active β-catenin levels and, in-turn, down-regulation of survivin, a critical mediator of leukaemia cell survival. Targeting MUC1-C was also associated with increased sensitivity of AML cells to Cytarabine (Ara-C) treatment by a survivin-dependent mechanism. Notably, low MUC1 and survivin gene expression were associated with better clinical outcomes in patients with AML. These findings emphasize the importance of MUC1-C to myeloid leukaemogenesis and resistance to treatment by driving survivin expression. Our findings also highlight the potential translational relevance of combining GO-203 with Ara-C for the treatment of patients with AML