Chemotherapy has been highly successful in treating many forms of cancer; however there are increasing reports that this treatment causes cognitive declines in cancer survivors. These effects have been called “chemobrain” and while not affecting all patients, can persist for many years after the completion of treatment. The symptoms of chemobrain include a decline in concentration, memory and attention which are associated with a lower quality of life and in ability to return to work. Very little is known about the mechanisms behind these changes, or even the brain regions that are affected. Animal studies have found that systemic chemotherapy causes a decrease in the proliferation of neural stem cells (NSCs) in the subgranular zone (SGZ) neurogenic niche of the hippocampus and a decline in spatial memory. As hippocampal neurogenesis is required for a number of memory functions including the consolidation of long term memory, a decline in neurogenesis is likely to be one of the causes of the cognitive decline experienced by patients after chemotherapy. Previous animal work has shown that chronic treatment with the antidepressant fluoxetine prevents the decrease in neurogenesis and the associated cognitive decline. In the absence of fluoxetine chemotherapy spares dividing cells which are in contact with the surface of blood vessels in the SGZ of the hippocampus. This project used in vitro techniques to firstly look at whether fluoxetine has a direct effect on the sensitivity of neural cells to chemotherapy or whether treatment of astrocytes cells with fluoxetine produces an indirect effect on sensitivity. Secondly the effect of contact between NSCs and either astrocytes or endothelial cells was investigated to see if these cell types could provide protection to NSCs from chemotherapy. In the third part of the project, fluorescence activated cell sorting (FACS) and measurements of oxygen consumption after different drug treatments were used to investigate DNA damage, apoptosis and changes in the cell cycle and the metabolic response of different cell types to chemotherapy respectively. To do this we evaluated the relative sensitivity of primary NSCs, neural N2a cells, endothelial cells HBMEC, primary astrocytes, C6 astrocytes and 3T3 cells to the chemotherapy drug 5-fluorouracil (5-FU). NSCs were found to be more sensitive to 5-FU than other cell types. A concentration of 5 μM 5-FU was found to reduce NSC viability by 50% but largely to spare astrocytes and endothelial cells. FACS analysis showed that 5-FU was causing DNA damage and inducing apoptosis with some cells arresting in the G1 stage of the cell cycle. Measurements of oxygen consumption were not conclusive in explaining the differences in sensitivity between different cell types. Treatment with fluoxetine, either directly to neural cell cultures or via conditioned media from fluoxetine treated astrocytes or endothelial cells, was not found to be protective against 5-FU treatment. In contrast co-culture of NSCs with astrocytes or endothelial cells, protected NSCs from chemotherapy treatment. This effect was specific to cells in the neurogenic niche as co-culture with 3T3 fibroblasts did not provide protection. Direct cell contact between neural cells and astrocytes or endothelial cells was required for protection as neither conditioned media nor co-culture separated by a porous membrane was found to be effective. To investigate the mechanism behind astrocyte or endothelial contact dependent protection of neural cells, the gap junction protein Cx34 was stained for and found to be present on neural, astrocytic and endothelial cells. Fluorescent dye loading of marked cells showed that gap junctions were functional and dye could pass between them. Blocking gap junctions with the gap junction inhibitor carbenoxolone (CBX) abolished the protection provided by contact with astrocytes or endothelial cells. I hypothesise that the protection provided in vivo by fluoxetine may be mediated by its anti-inflammatory effect on the brain and is not a direct effect on cells in the stem cell niche. Contact with astrocytes or endothelial cells may provide Ca2+ buffering via gap junctions and in this way protect the more sensitive neural cells from the effects of chemotherapy.

Great progress has been made towards the understanding of the molecular mech- anisms driving factor induced somatic cell reprogramming to pluripotency since the discovery by Takahashi and Yamanaka. However this process remains highly stochastic and inef cient. More study is needed in order to achieve a more de- terministic cell fate conversion which could further improve the quality of stem cells generated, essential for prospective therapeutic applications. The work presented here was developed under the premise that natural embryonic devel- opment can serve as a guide to achieve more ef cient pluripotency induction. It was observed that the histone variant H2A.Z, which has a role in pluripotency in embryonic stem cells, is highly expressed in the oocyte and upon over-expression, together with Pou5f1, Sox2, Klf4 and Myc, was able to increase the ef ciency of somatic cell reprogramming to induced pluripotency. A gene co-expression network analysis of somatic cells being reprogrammed identi ed hub genes as- sociated with H2Af.z and chromatin remodelling related genes which could be tested for further improving the reprogramming ef ciency induced by H2Af.z over-expression. Moreover, the study of genetic networks from pre-implantation embryos identi ed preserved genetic circuits also present during the course of reprogramming. The most preserved network modules are associated with the nal stages of pluripotency induction. However the analysis also identi ed a genetic network associated with the zygotic genome activation in the totipotent embryo stage which is also found in a sub-population of pluripotent stem cells characterised by the expression of genes from the Zscan4 family, Tet1, Etv5 and Mga among other genes. This provocative observation led me to hypothesise that during the course of reprogramming the forced activation of hub genes from such network may help improve its ef ciency, possibly by recapitulating the natural embryonic processes which induces totipotency prior to pluripotency. The identi- cation of preserved network modules and its hub genes presented in this work may serve a platform for further reprogramming studies in a quest for improved cocktails of reprogramming factors capable of more ef ciently generating induced pluripotent stem cells.

Current culture methods to generate large quantities of cells destined for tissue engineering and regenerative medicine commonly use enzymatic digestion. However, this method is not desirable for subsequent cell transfer to the body due to the destruction of important cell-surface proteins and the risk of enzymatic contamination [1]. Therefore, research has led to the development of thermo-responsive surfaces for the continued culture of mammalian cells, with passaging achieved via a drop in the culture temperature. Recognising that the three-dimensional (3D) culture environment influences the cell phenotype, our aim was to generate a thermo-responsive 3D fibre-based scaffold, using electrospinning, to create an enzyme-free 3D culture surface for mammalian cell expansion that would be suitable for cells destined for the clinic. Thermo-responsive poly (poly (ethylene glycol) methacrylate), poly (PEGMA188), with lower critical solution temperature (LCST) of 26°C has been proposed for use within this thesis. It was used in combination with poly (lactic-co-glycolic acid) (PLGA) and poly (ethylene terephthalate) (PET) polymers in order to create 3D thermo-responsive non-woven electrospun fibrous scaffolds, on which different cell types could be cultured and passaged. Poly (PEGMA188) was prepared by free radical polymerization, and then incorporated with PLGA and PET polymers via four different methods: (i) surface adsorption, (ii) NaOH surface treatment, (iii) surface entrapment and (iv) blend-electrospinning. Blend-electrospinning was chosen over the other methods as it produced nano-PET and micro-PLGA bead-less fibres with responsive behaviour. The biocompatibility was assessed via the adhesion and proliferation of different mammalian cell types, including (i) red fluorescent protein (RFP)-expressing 3T3 fibroblasts, (ii) green fluorescent protein (GFP)-expressing primary immortalized human mesenchymal stem cells (ihMSCs), (iii) human colon adenocarcinoma cells (Caco2) and (iv) primary human corneal stromal stem cells (hCSSCs). The cell viability (Alamar Blue assay) was determined to measure the difference in cell populations adherent to the scaffolds while changing the culture temperature. These thermo-responsive scaffolds were able to support cell adhesion and proliferation at 37°C (hydrophobic surface). Furthermore, it was possible to detach the cells from the scaffolds by decreasing the temperature to 17°C (hydrophilic surface). Irrespective of the concentration of poly (PEGMA188) used, all scaffolds exhibited thermo-responsive proprieties; the cells were viable and proliferated in a similar manner to those cultured on control surfaces (PLGA or PET scaffolds). Finally, the effects of the thermo-responsive polymer and 3D culture environment on the hCSSC phenotype were assessed by quantitative reverse transcription-polymerase chain reaction (RT-qPCR) and immunocytochemistry. The application of 3D environments can promote the reversion of activated corneal stromal cells’ ‘fibroblastic phenotype’ to a desirable quiescent keratocyte phenotype. Therefore, seven thermal and enzymatic passages on responsive 3D scaffolds and 2D TCPS, respectively, were performed. Cell culture on the 3D scaffolds promoted the quiescent keratocyte phenotype, with the increased expression of the keratocyte markers, CD34 and ALDH, and decreased expression of the myofibroblast marker, ACTA2, when compared with cells cultured on the 2D culture flasks. In this thesis, the preparation and application of first generation, biocompatible thermo-responsive fibrous scaffolds are described. The combination of ease of preparation, positive cell response and the expansion of a desirable cell phenotype make the thermo-responsive fibres promising as a new class of materials for application in cell culture. The materials developed and studied in this thesis are believed to represent a significant contribution to the fields of biomaterials and tissue engineering.

Accurate chromosome segregation during meiosis is critical for generating genetic diversity and for producing gametes with the correct number of chromosomes. After meiotic S-phase, homologs pair, recombine, and then separate in meiosis I. Meanwhile sister chromatids remain cohesive until meiosis II. A tripartite cohesin ring made of Smc1, Smc3, and the klesin subunit (Rec8 in meiosis and Mcd1/Scc1 in mitosis) is thought to topologically entrap a pair of sister chromatids generating cohesion. The role of the fourth cohesin subunit, Scc3, remains elusive. To study the function of Scc3 in yeast meiosis, we constructed PCLB2SCC3, a meiosis-specific mutant in which the expression of Scc3 is activated in mitosis but is suppressed in meiosis. With the mutant, we first found that Scc3 is required for sister chromatid cohesion and nuclear division during meiosis in the budding yeast, Sacchromyces cerevisiae. Second, we found that Scc3 is also required for maintaining Rec8 protein level during meiosis, which is independent of the Scc3's function in sister chromatid cohesion and nuclear division. Therefore, we asked how Scc3 regulates Rec8 protein levels. Our results demonstrate that Scc3 regulates neither REC8 mRNA production nor Rec8 protein stability, which suggestes that Scc3 have a role in posttranscriptional regulation in REC8 expression. At last, Scc3 is essential for Rec8−chromosome binding, whereas Rec8 only partly regulates Scc3−chromosome binding. Our data provide molecular insights into the role Scc3 in accurate chromosome segregation during meiosis. / A Thesis submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Master of Science. / Summer Semester, 2010. / May 3, 2010. / cohesion, meiosis, yeast / Includes bibliographical references. / Hongguo Yu, Professor Directing Thesis; Yanchang Wang, Committee Member; George W. Bates, Committee Member; Hank W. Bass, Committee Member.

During the past 30 years, there have been several Flaviviridae threats. Among them, Hepatatis C virus (HCV) emerged in the Western hemisphere as the previously unidentified etiological agent of non-A non-B hepatitis in transfusion patients. Less than twenty-five years after the 1989 discovery of HCV, several high-efficacy direct acting antivirals (DAAs) boasting a >95% cure rate were approved for treatment of HCV infected patients. While the advent of these DAAs has revolutionized the prognosis for chronically infected HCV patients, a high level of HCV-induced disease burden remains due to low rate of diagnosis (% here) and the high cost of therapy. Without treatment, 80% of chronically infected hepatitis C individuals exhibit hepatic intracellular lipid accumulation, termed steatosis. This liver damage can eventually contribute to cirrhosis (20% of individuals) and hepatocellular carcinoma (5% of individuals), necessitating a liver transplant for patient survival. Hepatitis C virus (HCV) infection perturbs host lipid metabolism through both cellular and viral-induced mechanisms, with the viral core protein playing an important role in steatosis development. In the first three years of my Ph.D., I sought to identify mechanisms contributing to HCV-induced steatosis. This work was published and Spotlighted in Journal of Virology. In this study, we identified a liver protein, the cell death-inducing DFFA-like effector B (CIDEB), as a HCV entry host dependence factor that is downregulated by HCV infection in a cell culture model. We then further investigated the biological significance and molecular mechanism of this downregulation. Importantly, we validated our in vitro finding with an in vivo model system, and saw that HCV infection in a live mouse model downregulated CIDEB in the liver tissue. We also found that CIDEB gene knockout in a hepatoma cell line resulted in multiple aspects of lipid dysregulation that can contribute to hepatic steatosis, including reduced triglyceride secretion, lower lipidation of very low density lipoproteins, and increased lipid droplet (LD) stability. The potential link between CIDEB downregulation and steatosis was further supported by the requirement of HCV core and its LD localization for CIDEB downregulation, which utilized a proteolytic cleavage event that is independent of the cellular proteasomal degradation of CIDEB. In late 2015, the global scientific community became aware of the emergence and threat of another Flaviviridae virus called Zika virus (ZIKV) in Brazil. The previously obscure ZIKV, which had laid relatively dormant for the previous 70 years, began spreading rapidly through the Western hemisphere, thus prompting the World Health Organization (WHO) to declare a public health emergency in February 2016. In response to the global health emergency posed by the ZIKV outbreak and its link to microcephaly and other neurological conditions, we established a collaboration with Dr. Wei Zheng at the National Center for Advancing Translational Sciences (NCATS) at this time, with whom we together performed a drug repurposing screen of ~6,000 compounds that included approved drugs, clinical trial drug candidates and pharmacologically active compounds. We reported these results in Nature Medicine in August 2016. From this initial research, we identified 37 lead compounds that either inhibit ZIKV infection or suppress infection-induced caspase-3 activity in different neural cells. We found that emricasan, a pan-caspase inhibitor, inhibited ZIKV-induced increases in caspase-3 activity and protected human cortical neural progenitors in both monolayer and three-dimensional organoid cultures. Ten structurally unrelated inhibitors of cyclin-dependent kinases inhibited ZIKV replication. Niclosamide, a category B anthelmintic drug approved by the US Food and Drug Administration, also inhibited ZIKV replication. Finally, combination treatments using one compound from each category (neuroprotective and antiviral) further increased protection of human neural progenitors and astrocytes from ZIKV-induced cell death. We then continued our work to identify additional compounds by refining our high-throughput assay. We developed a high-throughput ZIKV-NS1 based FRET detection assay to rescreen all 6,000 compounds, and then validated 256 hits by a semi-automated viral titer assay we developed in our lab in collaboration with NCATS. From this combinatorial approach, we identified an additional 117 compounds for use in further antiviral development. Among these, we found a conserved role of proteasome inhibitors in inhibiting ZIKV infection, and identified additional lead compounds including emetine, an anti-protozoal small molecule compound. In collaboration with NCATS and Dr. Anil Mathew Tharappel who completed mouse studies, we found that emetine is effective in a live animal model at reducing Zika viral load and likely inhibits viral replication via a direct block on the ZIKV NS5 RNA dependent RNA polymerase. / A Dissertation submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Spring Semester 2018. / April 16, 2018. / Antiviral, Drug discovery, Flavivirus, Hepatitis C Virus, Steatosis, Zika virus / Includes bibliographical references. / Hengli Tang, Professor Directing Dissertation; Yi Ren, University Representative; Thomas C. Keller, Committee Member; Fanxiu Zhu, Committee Member; David Meckes, Committee Member.

Electron microscopy is an important technique for observing macromolecular structures, such as DNA and viruses, which would be too small to see under light microscopy. This type of microscopy utilizes electrons as its illumination source, produced by an electron gun, to generate an image of the specimen which is captured by either a charged coupled device (CCD) or direct electron detector (DED) camera. Specimens in electron microscopy can either be stained with heavy atom, embedded in plastic, or embedded in vitrified ice. Cryo-electron microscopy (cryo-EM) embeds specimens in a vitreous ice layer that resembles the specimen’s natural environment and increases the overall resolution of the specimen. In conjunction with cryo-electron tomography (cryo-ET), cryo-EM specimens can be tilted on a specimen holder to collect multiple 2D views in order to generate a 3D reconstruction through a weighted back-projection algorithm. The projections are first corrected to counter the effects of contrast transfer function (CTF), which can filter out high resolution information. The resulting tomogram undergoes cycles of image processing steps such as multivariate data analysis, classification, and subvolume averaging to bring out the features of the specimen. At the borders of the striated muscle sarcomere, there exists an electron dense structure called the Z-Disc. The arrangement of thin filaments in the Z-Disc differs between vertebrates and invertebrates. Z-Discs of vertebrates have a tetragonal lattice that contrasts with the hexagonal lattice seen in the A-Band, which might be caused by arrangement of an elastic protein named Titin from the A-Band to the Z-Disc. The tetragonal lattice in the vertebrate Z-Disc has two structural states, small-square and basket-weave, depending on the contraction state of the muscle. Invertebrate Z-Discs have a hexagonal lattice that contains five connecting densities that form large and small solvent channels. Z-Discs contain many proteins that are important for structural stability and signaling functions. Three Z-Disc proteins that are structurally important in invertebrate Z-Discs are α-actinin, an actin crosslinker, Kettin and Projectin, the latter being components of the elastic connecting filament. Alternative Z-Disc isolation methods were explored using Wild-type (WT) Drosophila and Sls-RNAi knockdown Drosophila for the possibility of using the specimens for cryo-EM. The insect flight muscle (IFM) was dissected from the thorax of WT Drosophila and the individual myofibrils were obtained through a homogenization and cleaning process. The Z-Discs were isolated from the myofibrils by exposing them to high salt buffers, to remove thick filaments, and gelsolin, to remove thin filaments. The isolated Z-Discs were negatively stained and observed under an electron microscope. The lattice arrangement of the thin filaments could not be seen due to the stain, but this method produced many Z-Discs on the EM grids. Cryo-EM samples of the isolated WT Drosophila Z-Discs could not be obtained due to problems pertaining to the plunge freezing method. Sls-RNAi Drosophila was obtained using the GAL4/UAS method to generate smaller Z-Discs and decrease the width of the myofibrils due to a decrease in the presence of Kettin. The IFM was extracted from the thorax, but the myofibrils were not exposed to high salt buffers and gelsolin. Under negative stain, the myofibrils observed produced Z-Discs about 500 nm in width, which is ideal for cryo-ET conditions. However, a width of 200-300 nm would produce higher resolution images for a 3D reconstruction. A cryo-EM 3D reconstruction was generated from isolated Lethocerus Z-Discs to confirm the structural features seen in plastic-embedded sections of Apis. Projections for cryo-ET were collected using a DED camera and underwent CTF correction. The tilt series images were coarse-aligned and went through cycles of refinement using an Appion-based database with Protomo. A 3D reconstruction was generated using a weighted back-projection algorithm, filtered to bring out structural features (subvolumes), and then the subvolumes were averaged through single- and multi-reference alignment. The results were visualized in CHIMERA which confirmed the hexagonal lattice arrangement of thin filaments as reported previously in Apis Z-Discs. The location of connecting densities, C1 and C2, were confirmed as forming apices and bases of the large solvent channel, while C3 and C5 were confirmed to be connecting thin filaments of opposite orientation at the tapered end of the small solvent channel. C4 connecting density/three-wheel spoke was seen linking the ends of three thin filaments in the same orientation that form the small solvent channel. C1 and C2 were proposed to contain α-actinin, especially in C2 where an atomic model of F-actin with CH1 domains closely interacted with an atomic model of α-actinin in the C2 density map. The results of this experiment confirmed what was currently known about the invertebrate Z-Disc structure, but the locations of Z-Disc proteins, Kettin and Projectin, are yet to be determined. / A Thesis submitted to the Department of Biological Science in partial fulfillment of the Master of Science. / Fall Semester 2016. / November 18, 2016. / Includes bibliographical references. / Kenneth A. Taylor, Professor Directing Thesis; M. Elizabeth Stroupe, Committee Member; P. Bryant Chase, Committee Member; Wu-Min Deng, Committee Member.

Thesis (Ph. D.)--University of Washington, 2008. / Vita. Includes bibliographical references (leaves 85-94).

Zhang, Lei. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 132-144). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

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