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The goal of the studies described in this thesis was to determine the changes in vascular density in the paraventricular nucleus (PVN) of the hypothalamus following prenatal exposure to the synthetic glucocorticoid hormone, dexamethasone (DEX). DEX is a synthetic glucocorticoid used clinically in women at risk for preterm delivery or

The goal of the studies described in this thesis was to determine the changes in vascular density in the paraventricular nucleus (PVN) of the hypothalamus following prenatal exposure to the synthetic glucocorticoid hormone, dexamethasone (DEX). DEX is a synthetic glucocorticoid used clinically in women at risk for preterm delivery or in preterm infants to promote proper pulmonary development in high-risk neonates. Prenatal exposure to glucocorticoids such as DEX may change the development of important brain regulatory centers such as the PVN, resulting in increased risk for diseases in adulthood.
Previous studies have demonstrated that the hypothalamus regulates neuroendocrine and autonomic function and behavior. Within the hypothalamus, the paraventricular nucleus (PVN) is an integratory node that contains neurons associated with the control of neuroendocrine and autonomic responses. The PVN also has one of the highest density of blood vessels within the brain. Alterations of normal PVN angiogenesis by dexamethasone could potentially result in long-term modifications of brain and endocrine functions.
Timed-pregnant Sprague Dawley female rats received DEX on gestational days 18-21 and the resulting progeny were sacrificed at Postnatal Day (PND) 0, 4, 14, and 21. A tomato lectin, Lycopersicon Esculentum labeled with DyLight594 was used to stain blood vessels in the PVN and scanning confocal microscopy was used to analyze the experimental brains for PVN blood vessel density
Analysis of data using a 3-way analysis of variance (ANOVA) with age, sex and treatment as main factors, showed a significant age effect in vascular density. Analysis of female data by 2-way ANOVA demonstrated a significant effect of age, but no treatment or interaction effects. Post-hoc analysis shows significant differences at PND 2, 4, 14, and 21 compared to PND0. A Student‘s t-test of a planned comparison on PND2 showed a significant reduction by DEX treatment (p < 0.05). Analysis of data from females, using 2-way ANOVA demonstrated a significant effect of age, but no treatment or interaction effects. Post-hoc analysis shows significant differences at PND 2, 4, 14, and 21 compared to PND0. A planned comparison at PND 2 using Student’s t-test indicated a significant reduction by dex treatment.
The results of these studies demonstrate that there is significant postnatal angiogenic programming and that the vascular density of the PVN is altered by prenatal dexamethasone administration at PND2. The time-course shows developmental fluctuations in vessel density that may prove to be physiologically significant for normal brain function and developmental programming of brain and behavior.
ContributorsWidener, Andrew John-Claude (Author) / Handa, Robert (Thesis director) / Orchinik, Miles (Committee member) / Mustard, Julie (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor)
Created2014-05
Description
A coincidence reporter construct, consisting of the p21-promoter and two luciferase genes (Firefly and Renilla), was constructed for the screening of drugs that might inhibit Olig2's tumorigenic role in glioblastoma. The reporter construct was tested using an Olig2 inhibitor, HSP990, as well as short hairpin RNA targeting Olig2. Further confirmatory

A coincidence reporter construct, consisting of the p21-promoter and two luciferase genes (Firefly and Renilla), was constructed for the screening of drugs that might inhibit Olig2's tumorigenic role in glioblastoma. The reporter construct was tested using an Olig2 inhibitor, HSP990, as well as short hairpin RNA targeting Olig2. Further confirmatory analysis is needed before the reporter cell line is ready for high-throughput screening at the NIH and lead compound selection.
ContributorsCusimano, Joseph Michael (Author) / LaBaer, Joshua (Thesis director) / Mangone, Marco (Committee member) / Mehta, Shwetal (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor)
Created2014-05
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Description
Diacylglycerol kinase catalyses the ATP-dependent conversion of diacylglycerol to phosphatidic acid in the plasma membrane of Escherichia coli. The small size of this integral membrane trimer, which has 121 residues per subunit, means that available protein must be used economically to craft three catalytic and substrate-binding sites centred about the

Diacylglycerol kinase catalyses the ATP-dependent conversion of diacylglycerol to phosphatidic acid in the plasma membrane of Escherichia coli. The small size of this integral membrane trimer, which has 121 residues per subunit, means that available protein must be used economically to craft three catalytic and substrate-binding sites centred about the membrane/cytosol interface. How nature has accomplished this extraordinary feat is revealed here in a crystal structure of the kinase captured as a ternary complex with bound lipid substrate and an ATP analogue. Residues, identified as essential for activity by mutagenesis, decorate the active site and are rationalized by the ternary structure. The γ-phosphate of the ATP analogue is positioned for direct transfer to the primary hydroxyl of the lipid whose acyl chain is in the membrane. A catalytic mechanism for this unique enzyme is proposed. The active site architecture shows clear evidence of having arisen by convergent evolution.
ContributorsLi, Dianfan (Author) / Stansfeld, Phillip J. (Author) / Sansom, Mark S. P. (Author) / Keogh, Aaron (Author) / Vogeley, Lutz (Author) / Howe, Nicole (Author) / Lyons, Joseph A. (Author) / Aragao, David (Author) / Fromme, Petra (Author) / Fromme, Raimund (Author) / Basu, Shibom (Author) / Grotjohann, Ingo (Author) / Kupitz, Christopher (Author) / Rendek, Kimberley (Author) / Weierstall, Uwe (Author) / Zatsepin, Nadia (Author) / Cherezov, Vadim (Author) / Liu, Wei (Author) / Bandaru, Sateesh (Author) / English, Niall J. (Author) / Gati, Cornelius (Author) / Barty, Anton (Author) / Yefanov, Oleksandr (Author) / Chapman, Henry N. (Author) / Diederichs, Kay (Author) / Messerschmidt, Marc (Author) / Boutet, Sebastien (Author) / Williams, Garth J. (Author) / Seibert, M. Marvin (Author) / Caffrey, Martin (Author) / College of Liberal Arts and Sciences (Contributor) / School of Molecular Sciences (Contributor) / Biodesign Institute (Contributor) / Applied Structural Discovery (Contributor) / Department of Physics (Contributor)
Created2015-12-17
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Description
Phytochromes are a family of photoreceptors that control light responses of plants, fungi and bacteria. A sequence of structural changes, which is not yet fully understood, leads to activation of an output domain. Time-resolved serial femtosecond crystallography (SFX) can potentially shine light on these conformational changes. Here we report the

Phytochromes are a family of photoreceptors that control light responses of plants, fungi and bacteria. A sequence of structural changes, which is not yet fully understood, leads to activation of an output domain. Time-resolved serial femtosecond crystallography (SFX) can potentially shine light on these conformational changes. Here we report the room temperature crystal structure of the chromophore-binding domains of the Deinococcus radiodurans phytochrome at 2.1 Å resolution. The structure was obtained by serial femtosecond X-ray crystallography from microcrystals at an X-ray free electron laser. We find overall good agreement compared to a crystal structure at 1.35 Å resolution derived from conventional crystallography at cryogenic temperatures, which we also report here. The thioether linkage between chromophore and protein is subject to positional ambiguity at the synchrotron, but is fully resolved with SFX. The study paves the way for time-resolved structural investigations of the phytochrome photocycle with time-resolved SFX.
ContributorsEdlund, Petra (Author) / Takala, Heikki (Author) / Claesson, Elin (Author) / Henry, Leocadie (Author) / Dods, Robert (Author) / Lehtivuori, Heli (Author) / Panman, Matthijs (Author) / Pande, Kanupriya (Author) / White, Thomas (Author) / Nakane, Takanori (Author) / Berntsson, Oskar (Author) / Gustavsson, Emil (Author) / Bath, Petra (Author) / Modi, Vaibhav (Author) / Roy Chowdhury, Shatabdi (Author) / Zook, James (Author) / Berntsen, Peter (Author) / Pandey, Suraj (Author) / Poudyal, Ishwor (Author) / Tenboer, Jason (Author) / Kupitz, Christopher (Author) / Barty, Anton (Author) / Fromme, Petra (Author) / Koralek, Jake D. (Author) / Tanaka, Tomoyuki (Author) / Spence, John (Author) / Liang, Mengning (Author) / Hunter, Mark S. (Author) / Boutet, Sebastien (Author) / Nango, Eriko (Author) / Moffat, Keith (Author) / Groenhof, Gerrit (Author) / Ihalainen, Janne (Author) / Stojkovic, Emina A. (Author) / Schmidt, Marius (Author) / Westenhoff, Sebastian (Author) / College of Liberal Arts and Sciences (Contributor) / School of Molecular Sciences (Contributor) / Biodesign Institute (Contributor) / Applied Structural Discovery (Contributor) / Department of Physics (Contributor)
Created2016-10-19
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Description
Antibodies are essential for structural determinations and functional studies of membrane proteins, but antibody generation is limited by the availability of properly-folded and purified antigen. We describe the first application of genetic immunization to a structurally diverse set of membrane proteins to show that immunization of mice with DNA alone

Antibodies are essential for structural determinations and functional studies of membrane proteins, but antibody generation is limited by the availability of properly-folded and purified antigen. We describe the first application of genetic immunization to a structurally diverse set of membrane proteins to show that immunization of mice with DNA alone produced antibodies against 71% (n = 17) of the bacterial and viral targets. Antibody production correlated with prior reports of target immunogenicity in host organisms, underscoring the efficiency of this DNA-gold micronanoplex approach. To generate each antigen for antibody characterization, we also developed a simple in vitro membrane protein expression and capture method. Antibody specificity was demonstrated upon identifying, for the first time, membrane-directed heterologous expression of the native sequences of the FopA and FTT1525 virulence determinants from the select agent Francisella tularensis SCHU S4. These approaches will accelerate future structural and functional investigations of therapeutically-relevant membrane proteins.
ContributorsHansen, Debra (Author) / Robida, Mark (Author) / Craciunescu, Felicia (Author) / Loskutov, Andrey (Author) / Dorner, Katerina (Author) / Rodenberry, John-Charles (Author) / Wang, Xiao (Author) / Olson, Tien (Author) / Patel, Hetal (Author) / Fromme, Petra (Author) / Sykes, Kathryn (Author) / Biodesign Institute (Contributor) / Innovations in Medicine (Contributor) / Applied Structural Discovery (Contributor) / College of Liberal Arts and Sciences (Contributor) / School of Molecular Sciences (Contributor)
Created2016-02-24
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Description
Serial femtosecond crystallography (SFX) using X-ray free-electron laser sources is an emerging method with considerable potential for time-resolved pump-probe experiments. Here we present a lipidic cubic phase SFX structure of the light-driven proton pump bacteriorhodopsin (bR) to 2.3 Å resolution and a method to investigate protein dynamics with modest sample requirement.

Serial femtosecond crystallography (SFX) using X-ray free-electron laser sources is an emerging method with considerable potential for time-resolved pump-probe experiments. Here we present a lipidic cubic phase SFX structure of the light-driven proton pump bacteriorhodopsin (bR) to 2.3 Å resolution and a method to investigate protein dynamics with modest sample requirement. Time-resolved SFX (TR-SFX) with a pump-probe delay of 1 ms yields difference Fourier maps compatible with the dark to M state transition of bR. Importantly, the method is very sample efficient and reduces sample consumption to about 1 mg per collected time point. Accumulation of M intermediate within the crystal lattice is confirmed by time-resolved visible absorption spectroscopy. This study provides an important step towards characterizing the complete photocycle dynamics of retinal proteins and demonstrates the feasibility of a sample efficient viscous medium jet for TR-SFX.
ContributorsNogly, Przemyslaw (Author) / Panneels, Valerie (Author) / Nelson, Garrett (Author) / Gati, Cornelius (Author) / Kimura, Tetsunari (Author) / Milne, Christopher (Author) / Milathianaki, Despina (Author) / Kubo, Minoru (Author) / Wu, Wenting (Author) / Conrad, Chelsie (Author) / Coe, Jesse (Author) / Bean, Richard (Author) / Zhao, Yun (Author) / Bath, Petra (Author) / Dods, Robert (Author) / Harimoorthy, Rajiv (Author) / Beyerlein, Kenneth R. (Author) / Rheinberger, Jan (Author) / James, Daniel (Author) / Deponte, Daniel (Author) / Li, Chufeng (Author) / Sala, Leonardo (Author) / Williams, Garth J. (Author) / Hunter, Mark S. (Author) / Koglin, Jason E. (Author) / Berntsen, Peter (Author) / Nango, Eriko (Author) / Iwata, So (Author) / Chapman, Henry N. (Author) / Fromme, Petra (Author) / Frank, Matthias (Author) / Abela, Rafael (Author) / Boutet, Sebastien (Author) / Barty, Anton (Author) / White, Thomas A. (Author) / Weierstall, Uwe (Author) / Spence, John (Author) / Neutze, Richard (Author) / Schertler, Gebhard (Author) / Standfuss, Jorg (Author) / College of Liberal Arts and Sciences (Contributor) / Department of Physics (Contributor) / Department of Chemistry and Biochemistry (Contributor) / Biodesign Institute (Contributor) / Applied Structural Discovery (Contributor) / School of Molecular Sciences (Contributor)
Created2016-08-22
Description

Affective computing allows computers to monitor and influence people’s affects, in other words emotions. Currently, there is a lot of research exploring what can be done with this technology. There are many fields, such as education, healthcare, and marketing, that this technology can transform. However, it is important to question

Affective computing allows computers to monitor and influence people’s affects, in other words emotions. Currently, there is a lot of research exploring what can be done with this technology. There are many fields, such as education, healthcare, and marketing, that this technology can transform. However, it is important to question what should be done. There are unique ethical considerations in regards to affective computing that haven't been explored. The purpose of this study is to understand the user’s perspective of affective computing in regards to the Association of Computing Machinery (ACM) Code of Ethics, to ultimately start developing a better understanding of these ethical concerns. For this study, participants were required to watch three different videos and answer a questionnaire, all while wearing an Emotiv EPOC+ EEG headset that measures their emotions. Using the information gathered, the study explores the ethics of affective computing through the user’s perspective.

ContributorsInjejikian, Angelica (Author) / Gonzalez-Sanchez, Javier (Thesis director) / Chavez-Echeagaray, Maria Elena (Committee member) / Computer Science and Engineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2021-05
Description

Cryptojacking is a process in which a program utilizes a user’s CPU to mine cryptocurrencies unknown to the user. Since cryptojacking is a relatively new problem and its impact is still limited, very little has been done to combat it. Multiple studies have been conducted where a cryptojacking detection system

Cryptojacking is a process in which a program utilizes a user’s CPU to mine cryptocurrencies unknown to the user. Since cryptojacking is a relatively new problem and its impact is still limited, very little has been done to combat it. Multiple studies have been conducted where a cryptojacking detection system is implemented, but none of these systems have truly solved the problem. This thesis surveys existing studies and provides a classification and evaluation of each detection system with the aim of determining their pros and cons. The result of the evaluation indicates that it might be possible to bypass detection of existing systems by modifying the cryptojacking code. In addition to this classification, I developed an automatic code instrumentation program that replaces specific instructions with functionally similar sequences as a way to show how easy it is to implement simple obfuscation to bypass detection by existing systems.

ContributorsLarson, Kent Merle (Author) / Bazzi, Rida (Thesis director) / Shoshitaishvili, Yan (Committee member) / Computer Science and Engineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2021-05
Description

This paper is centered on the use of generative adversarial networks (GANs) to convert or generate RGB images from grayscale ones. The primary goal is to create sensible and colorful versions of a set of grayscale images by training a discriminator to recognize failed or generated images and training a

This paper is centered on the use of generative adversarial networks (GANs) to convert or generate RGB images from grayscale ones. The primary goal is to create sensible and colorful versions of a set of grayscale images by training a discriminator to recognize failed or generated images and training a generator to attempt to satisfy the discriminator. The network design is described in further detail below; however there are several potential issues that arise including the averaging of a color for certain images such that small details in an image are not assigned unique colors leading to a neutral blend. We attempt to mitigate this issue as much as possible.

ContributorsMarkabawi, Jah (Co-author) / Masud, Abdullah (Co-author) / Lobo, Ian (Co-author) / Koleber, Keith (Co-author) / Yang, Yingzhen (Thesis director) / Wang, Yancheng (Committee member) / Computer Science and Engineering Program (Contributor, Contributor) / Barrett, The Honors College (Contributor)
Created2021-05
Description

This paper is centered on the use of generative adversarial networks (GANs) to convert or generate RGB images from grayscale ones. The primary goal is to create sensible and colorful versions of a set of grayscale images by training a discriminator to recognize failed or generated images and training a

This paper is centered on the use of generative adversarial networks (GANs) to convert or generate RGB images from grayscale ones. The primary goal is to create sensible and colorful versions of a set of grayscale images by training a discriminator to recognize failed or generated images and training a generator to attempt to satisfy the discriminator. The network design is described in further detail below; however there are several potential issues that arise including the averaging of a color for certain images such that small details in an image are not assigned unique colors leading to a neutral blend. We attempt to mitigate this issue as much as possible.

ContributorsMasud, Abdullah Bin (Co-author) / Koleber, Keith (Co-author) / Lobo, Ian (Co-author) / Markabawi, Jah (Co-author) / Yang, Yingzhen (Thesis director) / Wang, Yancheng (Committee member) / Computer Science and Engineering Program (Contributor, Contributor) / Barrett, The Honors College (Contributor)
Created2021-05