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Graduate Seminar (G.R.A.S.S.) – Yuxi Wang (Dimitrakopoulos) and Vishnu Raman (Forbes)

Date/Time: 

Tuesday, October 3, 2017 - 11:30am

Location: 

LGRT 201

Details: 

Vishnu Raman and Yuxi (Nancy) Wang will be presenting at the upcoming Chemical Engineering student seminar on Tuesday, October 3rd, 2017 at 11:30 a.m. in LGRT 201. 

See below presentation abstracts.

 

Vishnu Raman – Neil Forbes Lab

Intracellular Invasion of Salmonella into cancer cells is a driver of bacterial tumor colonization in vitro
Vishnu Raman and Neil Forbes

Salmonella colonize tumors at ratios greater than ten thousand to one over any other organ in the body. Unlike small molecule drugs, which disperse into tumors through passive diffusion, Salmonella can actively penetrate into tumors by several motility related mechanisms. The bacteria can also be genetically modified to produce DNA, RNA and peptide therapies. These characteristics make the bacteria well suited to deliver targeted and patient specific cancer therapies.

However, the mechanisms governing tumor colonization of Salmonella are not completely understood. While motility and chemotaxis influence tumor colonization, Salmonella also have the ability to intracellularly invade cancer cells. The dependence of intracellular invasion on tumor colonization has not yet been elucidated. In addition, bacterial motility and intracellular invasion have cross regulatory mechanisms that make each phenomenon codependent. Therefore, this study, aimed to test two hypotheses: (1) Overexpressing the master motility regulator, flhDC, in Salmonella increases colonization and intracellular invasion in tumor masses and (2) Intracellular invasion is the main driver of Salmonella tumor colonization in vitro. Salmonella utilize the transcription factor complex, flhDC, to upregulate flagellar synthesis and thus, motility. The transcriptional complex also increases type three secretion system (T3SS) synthesis, which, Salmonella use to intracellularly invade epithelial cells.

To determine if flagellar upregulation could increase intratumoral colonization and intracellular invasion of Salmonella, flhDC was overexpressed in the bacteria and perfused into a tumor-on-a-chip device. This motile Salmonella also expressed a red fluorescent protein (DsRed) constitutively and green fluorescent protein (GFP) selectively inside cancer cells. Time lapse fluorescence microscopy elucidated spatial and temporal dynamics of Salmonella colonization and intracellular invasion of the in vitro tumor masses. The motile Salmonella colonized tumor tissue and intracellularly invaded cancer cells five-fold and eight-fold more, respectively, than a Salmonella control. These results indicate that increasing motility in Salmonella improved both tumor colonization and intracellular invasion of the bacteria within in vitro tumor masses.

In order to isolate the dependence of bacterial motility on tumor colonization, flhDC was overexpressed in non-intracellularly invading Salmonella, and administered to the tumor-on-a-chip along with non-intracellularly invading Salmonella as a control. Both bacterial strains carried the DsRed and GFP reporter system for quantitative intratumoral and intracellular fluorescent detection, respectively. Increasing motility in non-intracellularly invading Salmonella did not improve tumor colonization as compared to control. These results demonstrate that tumor colonization of Salmonella is not solely dependent on bacterial motility.

In order to isolate intracellular invasion of Salmonella as a potential colonization driver, non-intracellularly invading Salmonella was transformed with the same DsRed/GFP reporter system and perfused into the tumor-on-a-chip model along with intracellular invasion competent Salmonella as a control. Intracellular invasion deficient Salmonella colonized tumor tissue five-fold less than the Salmonella control indicating that intracellular invasion is required for successful bacterial colonization of tumors in vitro. 

Since Salmonella are complex microbes with several colonization mechanisms, it is important to understand which of these most influences bacterial survival and growth within tumors in order to improve the effectiveness of bacterial cancer therapies. This study has demonstrated that bacterial motility by itself does not influence tumor colonization. However, motility strongly increases the ability of Salmonella to intracellularly invade tumor cells, which, drives tumor colonization in vitro. This study has provided a foundational framework to investigate if the same colonization mechanisms influence bacterial tumor colonization in vivo.

 

Yuxi (Nancy) Wang – Christos Dimitrakopoulos Lab

Plasma Induced Formation of Interlayer Covalent Bonds in Twisted Bilayer and Multilayer Graphene 
Yuxi (Nancy) Wang

Graphene is a promising material for electronic and mechanical applications due to its excellent charge carrier mobility, two-dimensional structure, and tensile strength, which is ten times higher than steel for the same thickness. Chemical Vapor Deposition (CVD) of graphene on metal surfaces is a relatively economical process for producing it. It requires moderate initial capital investment and produces high-quality, large-area, multi-domain graphene on metals that is usually transferred to another appropriate substrate, depending on the application. Pristine, monolayer graphene is the strongest material ever measured, and emergent mechanical applications will be readily implemented if strong, clean multilayer graphene sheets can be produced.  

In this talk, I will introduce methods of CVD graphene growth and monolayer graphene transfer from its copper foil growth substrate to a SiO2/Si wafer with nearly 100% graphene coverage and minimal content of defects in the final graphene film. We also stack multiple layers on top of one another to form multilayer graphene. 

Due to weak shear interactions between pristine graphene layers (limited to van der Waals interactions), the penetration resistance of graphene multilayers does not scale linearly with the number of stacked graphene sheets. Formation of covalent interlayer bonds is theoretically predicted to strengthen the shear strength in bilayer graphene (Maroudas et al.), and thus forming such bonds would enhance the mechanical properties of multilayer graphene. In addition, theoretical calculations (Maroudas et al.) predict the opening of an electronic band gap in graphene bilayers with interlayer covalent bonds, making it a semiconductor, which is important for electronics applications.

In this research project we investigate the effectiveness of various processing methods in forming such bond, and importantly identify techniques to detect their existence, and their distribution in the graphene plane. Until now, we have strong indications that plasma treatment and annealing in various atmospheres of bilayer or multilayer CVD graphene produce such interlayer covalent bonds. 

Here, CVD graphene grown on Cu foil is transferred to doped SiO2/Si wafers and annealed in various environments. Bottom-gate graphene field-effect transistors (GFETs) are fabricated for electrical characterization, and Raman spectroscopy is used for chemical bonding characterization. We need to corroborate the Raman results with ATR-FTIR to ensure that we indeed see interlayer C-C bonds in our bilayers and multilayers. We will also introduce the importance of controlling the twist angle in bilayer graphene in determining the density of interlayer bonds, and show how the method of transferring epitaxial single crystalline graphene from SiC wafers (Kim, …, Dimitrakopoulos Science, 2013) will enable accurate control of the twist angle.

 

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