OCT4 is one of four transcription factors known to induce pluripotency when expressed together in somatic cells. However, brief expression of these pluripotency inducing factors in somatic skin fibroblasts followed by treatment with lineage specific culture conditions results in direct conversion towards alternative lineage specific cell types. Our group has previously shown that expression of OCT4 alone in adult human fibroblasts followed by treatment with hematopoietic supportive conditions resulted in the generation of multi-potent blood progenitors without transitioning through a pluripotent intermediate. Despite never having been associated with transcriptional regulation within the hematopoietic compartment, expression of OCT4 induced expression of hematopoietic factors in skin fibroblasts. As such, I hypothesized that ectopic expression of OCT4 in human somatic cells can induce changes in transcription that bestow the potential to make cell fate choices in response to external stimuli. In direct support of this notion, we revealed that expression of OCT4 in adult human fibroblasts, followed by culturing in neural supportive conditions resulted in the generation of tri-potent neural progenitors, suggesting the effects of OCT4 were not specific or limited to activation of hematopoietic programs. In an effort to understand how OCT4 bestows the potential to make cell fate choices, we assessed the individual vs combined impact of OCT4 and the extracellular environment on transcription during direct conversion of fibroblasts to both blood and neural progenitors. In doing so, we have started to define an induced state of transcriptional activity that is distinct from cells transitioning to pluripotency, and instead characterized by expression related to lineage iii PhD Thesis – RR Mitchell McMaster University - Biochemistry development that is responsive to changes in the extracellular environment that we have termed OCT4 induced plasticity (OiP). Moreover, we revealed that OCT4 mediated direct conversion can facilitate the reprogramming of hematopoietic progenitor cells towards neural progenitor cells, suggesting that this cellular reprogramming approach is not limited to the use of differentiated fibroblasts. In summary, this thesis expands our current knowledge on both the use and understanding of OCT4 as a facilitator of cellular reprogramming in human somatic cells. / Thesis / Doctor of Philosophy (PhD)

Primary hepatocytes maintained in culture progressively down regulate liver-specific genes and lose their characteristic function and morphology. This process, termed dedifferentiation, is a hindrance to in vitro modelling of systems such as xenobiotic metabolism, liver disease and regeneration. However the results presented here demonstrate that dedifferentiated hapatocytes spontaneously induce expression of ductal genes, and therefore represent a useful model of cell reprogramming.

Over-the-air reprogramming is an important aspect of managing large wireless sensor networks. However, reprogramming deployed sensor networks poses significant challenges due to the energy, processing power and memory limitation of sensor nodes. For improved energy efficiency, a reprogramming mechanism should use less transmission and flash writing overhead. Past research has proposed different mechanisms for reprogramming deployed sensor networks. However, all of these mechanisms produce large patches if software modifications involve changing program layouts and shifting global variables. In addition, existing mechanisms use large amounts of external flash and rewrite entire internal flash. In this thesis, we present a differential reprogramming mechanism called QDiff that mitigates the effects of program layout modifications and retains maximum similarity between old and new software using a clone detection mechanism. Moreover, QDiff organizes the global variables in a novel way that eliminates the effect of variable shifting. Our experiments show that QDiff requires near-zero external flash, and significantly lower internal flash rewriting and transmission overhead than leading existing differential reprogramming mechanisms. / Thesis (Master, Computing) -- Queen's University, 2011-11-29 14:11:44.138

The demonstration that simply by introducing four selected proteins it is possible to change mammalian somatic cells from one phenotype to another is providing important new opportunities in medicine. However, this approach has several limitations. In contrast to other methods of changing cell fate such as cloning and cell fusion it is very slow, very inefficient and it is necessary to have identified the key transcription factors. In both cloning and fusion the nucleus is exposed to the cytoplasm of the recipient cell and it is this that changes nuclear function. With the hope of creating the same effect, extracts from cells of the desired cell type have been introduced into candidate cells. This induced some changes in cell function, but did not change cells from one phenotype to another. The aim of this project was to improve methods for the introduction of extract when human skin cells were exposed to extracts of mouse pluripotent stem cells. During early studies, it was noticed that external materials could enter cells spontaneously at a specific stage of cell cycle, metaphase. When cells in metaphase were exposed to cell extracts pluripotent cell-like colonies were formed. These cells expressed markers of pluripotency such as SSEA4 and Tra-1-60 and could form embryoid bodies that would further differentiate to all three germ layers. Unfortunately this protocol was found to be unrepeatable. In subsequent studies the temperature of exposure to extract was raised from 37oC to 40oC with the aim of promoting the fluidity of the cell membrane and so enhancing uptake of extract. A new treatment regime was introduced to increase the proportion of cells exposed to extract while in metaphase. A new protease inhibitor was introduced in order to promote persistence of the extract within the cells. Also, a new TLR3 agonist was introduced to enhance chromosome modification. After these modifications were made pluripotent cell-like colonies formed within 14 days of treatment with extract and these colonies were positive for alkaline phosphatase live staining. Further research is required to complete the development of a routine procedure.

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / Wireless Sensor Networks (WSN) is being used in various applications including environmental monitoring, site inspection and military. WSN is a distributed network of sensor devices that can be used to monitor temperature, humidity, light and other important metrics. The software that runs on the sensor devices define how the device should operate. In real world WSN deployment, device software update is required to maintain optimal operation. In this thesis, we propose a novel idea of updating the software of the sensor nodes using a mobile device running on Android Operating System. Our implementation builds upon Mobile Deluge with few enhancement which is a method of re-programming WSN with laptop computer. We have evaluated our application performance by lab experiments and in real world deployments of WSN and found the application stable and battery efficient.

acase@tulane.edu / Cancer’s response to mechanical vibration via high-intensity focused ultrasound and disruptive chemical agents (Mechanochemical Disruption) was examined in vitro and in vivo. We demonstrated that mechanochemical disruption of cellular structures induced phenotypic alterations in surviving tumor cells that prevented cancer progression. Mechanochemical disruption inhibited uncontrolled proliferation, tumorigenicity, metastatic development, and re-sensitized multiple cancer types to chemical treatment via alterations in protein expression and impediment of pro-survival signaling. Our study identified a novel curative therapeutic approach that can prevent the development of aggressive cancer phenotypes. / 1 / hakm murad

Direct in vivo cellular reprogramming offers the potential for local neural replacement to promote post-stroke neural regeneration and recovery. Pericytes are perivascular cells involved in blood-brain barrier maintenance under physiological conditions but are reprogrammed into multipotent induced neural progenitor cells (i-NPCs) in response to ischemia. The atypical protein kinase C (aPKC)-CREB binding protein (CBP) pathway regulates ischemia-activated pericyte (a-pericyte) reprogramming and neuronal differentiation. Our previous work showed that the pathway inhibitor compound C (CpdC) facilitated a- pericyte reprogramming into i-NPCs in culture, and that monoacylglycerol lipase (Mgll) inhibitor JZL184 was able to promote NPC differentiation to generate newborn neurons by mimicking pathway activation. In this regard, we propose to use acute CpdC treatment followed by chronic JZL184 treatment to enhance reprogramming of a-pericytes into i-NPCs and subsequently promote their neuronal differentiation, ultimately improving post-stroke functional recovery. Using the endothelin-1 (ET-1)/L-NAME ischemic stroke model in a pericyte lineage tracing transgenic mouse line, I characterized the ability of a-pericytes in the ischemic lesion site to generate neural (i-NPC, newborn neurons) and non-neural (microglia and fibroblasts) cell types. The CpdC+JZL184 treatment had early effects on enhancing a- pericyte reprogramming efficiency to produce i-NPCs at 7 days post-stroke and promoting their differentiation to generate neuroblasts at 14 days post-stroke. However, it did not affect stroke volume and produced minimal alterations to the pattern of post-stroke motor function recovery. Interestingly, I discovered a novel role of tamoxifen treatment prior to stroke in regulating reprogramming of a-pericytes and efficacy of compound C treatment. These studies inform the necessity of optimization of drug delivery for better control over the timing and duration to directly target in vivo i-NPC reprogramming and reveal a novel pharmacological paradigm to control the aPKC-CBP pathway.

Identification of therapeutically relevant molecules is necessary for the advancement of non-viral reprogramming of human cells for regenerative medicine. We have developed a novel non-viral model system that transforms primary human dermal fibroblasts into cells with induced regeneration competence (iRC). Low oxygen-mediated effects of fibroblast growth factor FGF2 lead to an increased cellular lifespan with a two fold increase in population doublings before senescence, remaining non-tumorigenic when injected into SCID mice while maintaining regeneration competence. This system allows us to study molecules that participate in increased cellular lifespan in a non-tumorigenic system. Analysis of chromatin modification enzymes by hybridization array, RT-PCR, and Western blots revealed upregulation of the arginine methyltransferase PRMT8 in iRC cells, challenging the paradigm that PRMT8 is solely expressed in brain tissue at the plasma membrane. Possibly leading to the erroneous conclusions that PRMT8 is brain specific at the plasma membrane is the fact that PRMT8 has several mRNA variants and protein isoforms. Here, I report expression of a novel PRMT8 variant in human dermal fibroblasts. Essential participation of PRMT8 in cellular proliferation was identified as a novel function for this enzyme through siRNA-mediated knockdown in both non-tumorigenic and tumorigenic cell lines. While other members of the PRMT family have known roles in cell cycle progression, I show for the first time that PRMT8 expression is reduced in both natural senescence and by premature induction of replicative senescence using sub-cytotoxic levels of hydrogen peroxide, implicating a correlation between PRMT8 expression and cell cycle progression. However, PRMT8 overexpression causes no significant change in the number of population doublings or the amount of time spent in culture prior to senescence, and does not alter the expression of key cell cycle regulatory genes. These results suggest that maintenance of PRMT8 expression is critical for cellular proliferation, but overexpression of PRMT8 alone is not sufficient to increase cellular lifespan. I determined that oxygen is the primary mediator of PRMT8 upregulation in the iRC system and therefore investigate histone occupancy of the PRMT8 promoter at hypoxia response elements. Through this analysis, I found bivalent occupancy regardless of culture conditions, indicating that PRMT8 maintains a state of poised readiness for transcriptional accessibility. The mechanism by which PRMT8 participates in cellular proliferation was investigated through binding partner identification. A binding partner of endogenous PRMT8 is identified here for the first time as FGF2 using co-IP and mass spectrometry. As iRC cells demonstrate a unique phenotype that uncouples the mechanisms of increased lifespan from tumorigenesis, I investigated the feasibility of PRMT8 as a cancer biomarker by mining publically available data in light of our own. I showed that PRMT8 is not only expressed in a variety of cancers, but that its expression is amplified. Moreover, PRMT8 expression significantly correlates to patient survival in specific cancers, strengthening the feasibility of this molecule as a biomarker. Aberrant expression of most PRMT family members has been described in various cancers, and specific PRMT variants are currently being used as prognostic markers. As such, I analyzed variant-specific PRMT8 expression in primary cancer cell lines and show that tumorigenic glioblastomas express PRMT8 mRNA variant 2. These data suggest that PRMT8 is a viable candidate for further study as a prognostic cancer biomarker, specifically for brain cancer.

In 2006, Kazutoshi Takahashi and Shinya Yamanaka demonstrated the ability of four transcription factors; Oct4, Sox2, Klf4 and c-Myc to 'reprogram' differentiated somatic cells to a pluripotent state. This technology holds huge potential in the field of regenerative medicine, but reprogramming also a model system by which to the common regulators of all forced cell identity changes, for example, transdifferentiation. Despite this, the mechanism underlying reprogramming remains poorly understood and the efficiency of induced pluripotent stem cell (iPSC) generation, inefficient. One powerful method for elucidating the gene components influencing a biological process, such as reprogramming, is screening for a phenotype of interest using genome-wide mutant libraries. Historically, large-scale knockout screens have been challenging to perform in diploid mammalian genomes, while other screening technologies such as RNAi can be disadvantaged by variable knockdown of target transcripts and off-target effects. Components of clustered regularly interspaced short palindromic repeats and associated Cas proteins (CRISPR-Cas) prokaryote adaptive immunity systems have recently been adapted to edit genomic sequences at high efficiency in mammalian systems. Furthermore, the application of CRISPR-Cas components to perform proofof- principle genome-wide KO screens has been successfully demonstrated. I have utilised the CRISPR-Cas9 system to perform genome-wide loss-of-function screening in the context of murine iPSC reprogramming, identifying 18 novel inhibitors of reprogramming, in addition to four known inhibitors, Trp53, Cdkn1a, Jun, Dot1l and Gtf2i. Understanding how these novel reprogramming roadblocks function to inhibit the reprogramming process will provide insight into the molecular mechanisms underpinning forced cell identity changes.