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No description available.

No description available.

No description available.

No description available.

BCR-ABL is known as the most common translocation in the myeloproliferative (MPD) disorder chronic myelogenous leukemia (CML); it is the first leukemia to be described and associated with a consistent cytogenetic abnormality, termed the Philadelphia chromosome (Ph1). Ph1 is a shortened chromosome 22 that is the consequence of a reciprocal translocation between chromosomes 9 and 22, t(9;22)(q34;q11). BCR-ABL is known to display constitutively active tyrosine kinase activity that leads to the recruitment of downstream effectors of cell proliferation and survival, via several adapter proteins (e.g., GRB2, GAB2, CRKL.) and signaling pathways (e.g., RAS, PI3K, JAK STAT, PDk2-NFkB), all thought to contribute to the pathogenesis of CML. CML, essentially consists of 3 different phases based on disease severity; namely Chronic Phase (CP-AML), Accelerated Phase (AP-CML) and blast crisis (BC-CML). Imatinib, a small molecule ABL kinase inhibitor has been highly effective in treating chronic phase (CP) CML patients. However, a substantial number of patients undergo relapse due to development of resistance to imatinib therapy that leads to BC-CML, which is invariably fatal within weeks to months. Additional genetic aberrations assist in progression and identification of key players that are responsible for transformation is of utmost importance from a therapeutic point of view. Growth arrest DNA damage 45a (Gadd45a) gene, a member in the gadd45 family of genes including Gadd45b & Gadd45g, was identified as a myeloid differentiation primary response gene. There is evidence consistent with it's involvement in G2/M cell cycle arrest and apoptosis in response to multiple stressors, including genotoxic and oncogenic stress. Gadd45a has been shown to participate in cell cycle arrest, DNA repair, cell survival and apoptosis in response to environmental and physiological stress, via protein-protein interactions with key regulators such as PCNA, histones, cdk1, p21, MEKK4, MKK7 and p38. To investigate the effect of Gadd45a in the development of CML, we performed adaptive bone marrow transplantation experiments with either wild type or Gadd45a null myeloid progenitors expressing 210-kD BCR-ABL fusion oncoprotein. We showed that that loss of Gadd45a accelerated BCR-ABL driven CML and correlated with enlarged liver and spleen pointing to more aggressive leukemia. Additionally, we demonstrated that Gadd45a expression in presence of BCR-ABL was independent/distinct from well-known tumor suppressor p53, which suggests that Gadd45a could be considered as a prime and novel candidate for intervention in CML therapy. We also showed that transformed Gadd45a deficient progenitors in the presence of BCR-ABL, exhibited increased proliferation, increased survival and decreased apoptosis when compared to WT/BCR-ABL counterparts. Additionally, we demonstrate that recipients transplanted with Gadd45ako/BCR-ABL bone marrow exhibit increased number of Leukemic stem cells (LSC) harboring BCR-ABL which correlated with accelerated disease development in Gadd45a deficient background. Furthermore, we show that Gadd45ako/BCR-ABL progenitors exhibit increased self-renewal capabilities compared to WT/BCR-ABL progenitors through serial replating assays. Remarkably, we demonstrate that Gadd45ako/BCR-ABL bone marrow cells could be established as a factor independent cell-line and that this cell- line exhibited progenitor like properties; thereby confirming the status of Gadd45a as potent tumor suppressor factor. To shed more light into the mechanism of disease development, we demonstrate that the Gadd45ako/BCR-ABL bone marrow cells exhibit enhanced PI3K-AKT-4E-BP1 signaling and upregulated oncogenic p30C/EBPα expression along with hyperactivation of p38 and Stat5. Finally, to validate our observations in human CML population, we demonstrate that Gadd45a expression correlated with disease progression. We show that Gadd45a expression is upregulated in more indolent CP-CML samples and downregulated in aggressive AP-CML and BC-CML patient samples. Future studies to identify expression of downstream partners of Gadd45a in CML patients, gain of function experiments along with inhibitor studies to highlight mechanistic insights would shed more light into the tumor suppressor function of Gadd45a. Additionally, questions such as; do elevated levels of Gadd45a impede disease progression, does higher expression of Gadd45a indicate better response to Imatinib, still need to be answered in order to understand if Gadd45a agnostic and or combinatorial therapy in CML can be considered as a valid treatment option. The most common mutations of Ras are found in N-RAS (~30%), less frequently in K-RAS (~15%), and most rare in H-RAS in leukemia. Given the role of Gadd45a in modulating the response of hematopoietic cells to stress as well as its function in mediating oncogenic H-Ras carcinogenesis, we wanted to assess if and how Gadd45a loss modulates Nras driven leukemogenesis. By utilizing adaptive bone marrow strategy we show that loss of Gadd45a impeded activated Nras driven leukemia. This correlated with higher incidence of extramedullary hematopoiesis in the liver and spleen sections of WT/NrasD12 recipient mice compared to Gadd45ako/NrasD12 counterparts. Future studies to investigate the biological effects and shed more light on mechanistic insights in recipient mice is still underway. Taken together my work implies that also in the context of hematopoietic malignancies Gadd45a may function as suppressor or promoter of the leukemic phenotype dependent on the oncogenic stress. / Biochemistry

AML is a cancer of the blood and bone marrow characterized by the presence of highly proliferative and abnormally differentiated myeloblasts. Previous work from the Bhatia lab utilized the orthotopic xenograft model in order to isolate a population of leukemic regenerating cells (LRC) that exists prior to relapse. Affymatrix analysis of LRCs revealed up-regulation of 248 genes that can act as unique targets to prevent relapse. In order to screen compounds against all 248 targets, it is important to develop an in vitro model that is able to appropriately recapture the functional and molecular markers of LRCs. Primary AML samples were treated with 5-doses of 0.15 μM, 1 μM AraC, or DMSO control and several outcomes were measured. In vitro AraC treatment was not able to recapitulate the progenitor frequency curve and CD34 expression curve observed in vivo. Additionally, we were not able to see a consistent increase in select LRC targets DRD2, GLUT2, FUT3, and FASL via flow cytometry. Despite an increase in the mRNA levels of LRC genes 24h after treatment with 0.15 μM AraC, long term analysis could not be completed due to poor RNA quality and low expression of LRC-targets. Primary AML cells were co-culture with mouse MS-5 stromal cell line order to study the effects of mesenchymal stromal cells on AML response to AraC. Co-culture with MS-5 cells had different effects on select primary AML cells. AML 14939 showed an increase in CD34 and LRC targets DRD2 and FUT3 following AraC treatment when co-cultured with MS-5 cells; while A374 showed no differences between DMSO and AraC treated groups. Overall, these findings suggest the LRC signature is not induced by treatment with AraC alone. Complex interactions between AML cells and their bone marrow niche during AraC treatment plays an important role in the development of LRCs prior to AML relapse. / Thesis / Master of Science (MSc) / AML is a cancer of blood cells characterized by the presence of rapidly dividing cancer cells termed myeloblasts. AML has a high rate of disease relapse. The Bhatia lab modelled AML relapse in a mouse and discovered an unique population of cells that exist prior to relapse termed LRCs. LRCs express distinctive genes that can act as targets for the development of new therapies to prevent relapse. In order to screen potential relapse preventing compounds, we set out to recapture AML relapse using cells in a dish. AML cells from patients were treated with chemotherapy reagent AraC and the number of cancer progenitors and the expression of specific LRC proteins were measured. AraC did not increase the level of 3 out of 4 LRC proteins studied. We determined the LRCs were not caused by AraC treatment, and the physiology of the bone marrow environment plays an important role in inducing relapse.

Initial studies in this work explored the role of interleukin-6 (IL6) and leukemia inhibitory factor (LIF) in preimplantation bovine embryos. Neither cytokine affected the total percentage of embryos which developed to the blastocyst stage in vitro. However, supplementation of IL6 increased blastocyst inner cell mass (ICM) cell number without affecting trophectoderm (TE) cell number. Additionally, we found that IL6 activated signal transducer and activator of transcription 3 (STAT3) specifically within ICM cells. LIF, however, did not affect ICM cell number or activate STAT3 in ICM cells, and was not pursued further. This increase in ICM cell number by IL6 was largely comprised of hypoblast (GATA6+:NANOG-) cells, and most IL6-responsive cells in day 9 blastocysts were hypoblast cells (as measured by STAT3 activation). However, some epiblast (NANOG+) cells were also IL6-responsive, and IL6 appeared to initially slow epiblast differentiation. Finally, IL6-treated blastocysts also had increased transcripts of hypoblast/primitive endoderm (PE) markers. These results indicate that IL6 may improve pregnancy retention of IVP embryos by improving yolk sac development, but further work is needed to confirm this theory. Activation of STAT3 by IL6 could be blocked with a chemical Janus kinase 2 (JAK2) inhibitor (AZD1480). JAK2 inhibition from day 5 to 8 resulted in blastocyst ICMs with fewer than 10% the normal cell number, regardless of IL6 supplementation. This indicates that STAT3 is critical for bovine ICM development. Further analysis revealed that inhibition of JAK2/STAT did not prevent ICM formation but disrupted its maintenance. Additionally, we assessed the suitability of zinc sulfate and a bovine embryonic stem cell culture media (TeSR) for improving bovine embryo development in vitro. Zinc sulfate increased day 8 blastocyst total and ICM cell number. Therefore, zinc sulfate appears to improve blastocyst quality. The TeSR medium improved embryo development beyond day 8. In normal synthetic oviduct fluid, blastocysts degenerated after day 8, while blastocysts moved to TeSR had greatly increased cell numbers, and even exhibited PE migration out from the ICM, a phenomenon that has not been reported in vitro. This indicates that extended blastocyst culture is possible with TeSR media. / Doctor of Philosophy / Bovine embryos have been produced in vitro for the purpose of being transferred to recipient cattle to produce a calf since the 1980s. This practice allows cattle breeders to increase the number of offspring from their best females each year, and also allows for more rapid progress in generational genetic improvement. However, only approximately 10% of bovine oocytes survive and produce a calf. This poor efficiency of bovine in vitro embryo production negatively impacts the procedure's widespread use. A significant portion of these embryo losses are likely a result of inadequate in vitro culture conditions, particularly of the embryo culture media, the fluid in which embryos are grown. This media is often called "synthetic oviduct fluid," or SOF, because it is designed to mimic the fluid present in the cow's oviduct, where the embryo would normally reside. However, SOF is much simpler in nature than actual cow oviduct fluid, and this leads to reduced embryonic survival of in vitro produced embryos. Unfortunately, we know very little of what molecules control and promote bovine embryo development. Therefore, one major goal of bovine embryo research is to identify these factors and add them to SOF. The goal of this work was to examine the ability of three molecules, interleukin-6 (IL6), leukemia inhibitory factor (LIF), and zinc sulfate, to increase the number and quality of blastocysts produced through in vitro culture techniques. Additionally, I tested the replacement of SOF with a complex cell culture media, known as TeSR. This medium is more complex than SOF, and therefore should better promote embryo development. This work revealed that IL6, but not LIF, improves in vitro produced (IVP) bovine blastocyst quality. Unfortunately, neither IL6 nor LIF affected the percentage of embryos which survived to the blastocyst stage. However, IL6, but not LIF, increased the number of cells in the inner cell mass (ICM) of the blastocysts. ICM cells are the portion of the embryo which will produce the future calf. IVP bovine embryos are known to have fewer cells than normal, in vivo derived, blastocysts, and this issue is believed to cause some embryonic death after embryo transfer. Therefore, treatment with IL6 may increase the percentage of embryos which will survive after transfer and produce a calf. We also found the addition of zinc sulfate to SOF to benefit embryo quality. None of the concentrations of zinc significantly improved the percentage of embryos which survived to the blastocyst stage, but 2 µM zinc did increase ICM cell number. Like IL6, this may improve embryo survival after transfer. The use of the TeSR media as a replacement for SOF had some benefits. Unfortunately, this media is unusable for producing embryos for transfer to recipients, as we discovered early embryos could not survive in the media. However, blastocyst-stage embryos thrived in it, and could be cultured in vitro for a longer period of time as a result. Therefore, this media will be a useful tool for studying bovine embryo development in vitro, however it is unlikely to benefit calf production. In summary, this work provides evidence that zinc sulfate and IL6 are beneficial additions to SOF. However, future work is needed to determine if embryos produced with these factors are more able to produce a calf. Additionally, we discovered that TeSR is a superior extended blastocyst culture medium.

Corneal transplantation carries a high rate of success in the domestic cat and is an indicated treatment for specific corneal diseases in this species. The potential for iatrogenic transmission of viral diseases is a well-recognized problem in human corneal transplantation programs and screening donors for certain diseases is routine. Feline leukemia virus (FeLV) is a common agent of disease in domestic cats and available blood tests are highly effective in identification of infected individuals. This study investigates the presence of FeLV within corneal tissues of FeLV infected cats. Seventeen cats were identified to be positive for serum p27 antigen by enzyme-linked immunosorbent assay (ELISA). Twelve of these individuals were found to be positive on peripheral blood by immunofluorescent antibody (IFA) testing. Seventeen ELISA negative cats were identified to serve as negative controls. Full thickness corneal specimens were collected from all subjects and analyzed for the presence of FeLV proviral DNA and gp70 antigen by polymerase chain reaction (PCR) and immunohistochemical (IHC) testing, respectively. Eleven (64.7%) positive corneal PCR results were obtained from 17 ELISA positive cats. Of 12 cats which were both ELISA and IFA positive on peripheral blood, 10 (83.3%) had positive corneal PCR results. All corneal tissues from ELISA negative subjects were PCR negative. IHC staining of corneal sections revealed the presence of FeLV gp70 in corneal tissues of nine (52.9%) ELISA positive cats. Of the 12 cats which were both ELISA and IFA positive on peripheral blood, 8 (66.7%) had positive corneal IHC results. Positive IHC staining was localized to the corneal epithelium. Corneal tissues of all ELISA negative cats and all IFA negative cats were negative on IHC testing. This study reveals FeLV to be present within the corneal epithelium of some FeLV infected cats. Screening potential corneal donors for this virus is warranted. This work was funded by grants from the American College of Veterinary Ophthalmologists, the Virginia Veterinary Medical Association Pet Memorial Fund, and the DSACS Quick Response Fund. / Master of Science

Latent feline leukemia virus (FeLV) infections, in which proviral DNA is integrated into host DNA, but not actively transcribed, are suspected to be associated with many diseases. Bone marrow is the suspected site of the majority of latent infections. The purpose of this study was to determine if polymerase chain reaction (PCR) could detect FeLV proviral DNA in bone marrow and provide a method of detecting latent infections. Blood and bone marrow samples from fifty cats and bone marrow from one fetus were collected; sixteen had FeLV-associated diseases. Serum ELISA, blood and bone marrow immunofluorescent antibody test (IFA), and blood and bone marrow PCR were performed on each cat, and IFA and PCR on bone marrow of the fetus. Forty-one cats were FeLV negative. Five cats and one fetus were persistently infected with FeLV. Four cats were discordant; two ELISA positive with other tests negative, one bone marrow IFA negative with other tests positive, and one bone marrow IFA positive with other tests negative. No cats were positive on bone marrow PCR only. These results indicate that PCR can detect FeLV in bone marrow, but no cats in this study harbored FeLV only in the bone marrow. Not all cats with FeLV-associated diseases are persistently or latently infected with FeLV. / Master of Science