Biology

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Assessment of focal complexes in B35 Neuroblastoma growthcones following Lovastatin treatment
(1/1/2013) Deshmukh, Shweta Dilip; Hynds, DiAnna L.; Uphouse, Lynda L.; Beck, Brian W.
Proper development of the nervous system requires efficient and precise axon growth and guidance, growths requiring dynamic focal complex adhesion and de-adhesion in the neuronal growth cone. The formation of focal complexes is regulated in part by guanine triphosphatases (GTPases) of the Rho family, which signal bi-directionally to focal complex constituents. Rho GTPases are also regulated through binding of guanosine triphosphate (GTP), a process facilitated by membrane targeting through geranylgeranylation (a specific type of prenylation) at their C terminus. Prior work in our laboratory demonstrated that decreasing geranylgeranylation with the 3-hydroxy-3-methylglutaryl-coenzyme A (HMG CoA) reductase inhibitor, lovastatin, decreases neurite initiation and increases neurite branching and cell body rounding. These effects were reversed by co-treatment with geranylgeraniol, a precursor for geranylgeranylation. In the current work, we determined whether manipulating protein prenylation decreases the association of focal complex proteins (paxillin, talin or vinculin) with β1-integrin in B35 growth cones. B35 neuroblastoma cells were grown on uncoated glass coverslips, or coverslips coated with 25 μg/ml laminin or collagen. Cells were maintained in media were either not treated, or treated with lovastatin (LOV, 20 μM) alone or in combination with geranylgeraniol (GGOH, 10 μM). Focal complex formation in growth cones on the different substrata was assessed using co-immunoprecipitation and double-label immunocytochemistry. Culturing on different substrata did not alter the amount of paxillin or talin that associated with 1 integrin. However, vinculin co-localization with 1 integrin was decreased for cells plated on collagen or laminin; and co-localization with talin was increased in cells cultured on laminin, compared to cells plated on uncoated coverslips. For cells plated on collagen, GGOH decreased the amount of paxillin, but not talin or vinculin, that co-localized with β1 integrin compared to control cells maintained in reduced serum medium or treated with LOV. For cells cultured on their native, secreted substratum (undefined) or laminin, manipulating geranylgeranylation did not alter the association of focal complex proteins with 1 integrin in whole cell lysates, growth cones or cell bodies. However, treatment to alter protein geranylgeranylation (LOV, GGOH, LOV+GGOH) decreased the amount of co-localization of paxillin and talin, and GGOH treatment decreased the amount of vinculin co-localized with 1 integrin in growth cones, compared to cells in reduced serum medium (SCM) for cells cultured on collagen. Together, we interpret these data to indicate that inhibiting protein prenylation decreases growth cone focal adhesions, likely leading to decreased neurite initiation. Understanding the molecular mechanisms that regulate Rho GTPase control of neuronal process extension will facilitate our understanding of nervous system development, as well as identify potential therapeutic targets for treating developmental, traumatic and degenerative neural lesions.
Strain Differences in Fluoxetine-Induced Sexual Dysfunction
(1/1/2013) Miryala, Chandra Suma; Uphouse, Lynda; Hynds, DiAnna L.; Mills, Nathaniel; Beck, Brian; Fuchs, Janon
The present experiments were designed to examine strain differences in sexual dysfunction after acute fluoxetine treatment. Female Fischer and Sprague-Dawley rats were used. Three major experiments were performed. (1) Strain differences were examined in regularly cycling female rats and in ovariectomized rats hormonally primed with 0.067 µg/g estradiol benzoate and 3.333 µg/g progesterone following treatment with 5, 10, 15, 20 or 30 mg/kg fluoxetine. (2) The effects of the 5-HT2 receptor antagonist, ketanserin 0.416, 0.5, 1, 2, 5 or 10 mg/kg, were compared in the two strains. (3) The combination of 10 mg/kg fluoxetine and l mg/kg ketanserin was examined. The major outcomes of this study were: (i) consistent with prior studies, fluoxetine reduced female rat sexual behavior in both hormonally-primed, ovariectomized and in naturally cycling rats; (ii) hormonally primed, ovariectomized rats were more sensitive to the lordosis inhibiting effects of fluoxetine than the intact, naturally cycling females; (iii) in both hormonally-primed and naturally cycling rats, Sprague-Dawley females were less sensitive to the lordosis-inhibiting effects of fluoxetine than Fischer females; (iv) a 5-HT2A/2C receptor antagonist, ketanserin, reduced lordosis behavior in both strains with a slightly greater effect in Sprague-Dawley females, but the difference was modest in comparison to the strain differences in response to either fluoxetine, and (v) the combination of fluoxetine and ketanserin did not amplify negative effects on lordosis behavior relative to the individual drugs alone. From these experiments, it was concluded that Fischer female rats are more sensitive than Sprague-Dawley females to the lordosis inhibiting effects of fluoxetine and that the 5-HT2 receptor, may not be involved. Potential mechanisms responsible for strain differences in the receptor activation are discussed.
Sunflowers and Honeybees: A Study of the Mutualistic Relationship from a Biochemical and Morpho-Anatomical Perspective
(1/1/2013) Wojtaszek, Jennie W.; Maier, Camelia; Conrad-Webb, Heather; Beck, Brian; McIntire, Sarah
The mutualistic relationship between Helianthus annuus (Asteraceae) and Apis mellifera is reflected in their co-evolutionary adaptations. The corolla morphology and pigmentation of sunflowers help form a target pattern under UV, recognizable by bees. While collecting rewards, bees cross-pollinate the disk florets. Morpho-anatomical co-adaptations of the sunflower and honeybee were studied with LM, SEM, and CLSM. This study reports for the first time the presence of one to three rows of transitional papillae on stigma, which may function in protection of the receptive stigma from self-pollination. A model of the cross-pollination of sunflower inflorescence by honeybees is presented. The chemical characterization of flavonoid pigments in disk florets, known to contribute to the target pattern of the inflorescence, accomplished with chromatographic and MS techniques, revealed the presence of luteolin and pelargonidin pigments. This is the first report on the presence of luteolin and pelargonidin in sunflower disk florets. Results of this study will contribute to the metabolomics of the phenylpropanoid pathway in H. annuus in addition to enhancing understanding of mutualism and biosemiotic relationships between flowering plants and pollinators.
Regulation of Expression of the M143 Promoter of Mouse Cytomegalovirus
(1/1/2013) Eluru, Seneetha Reddy; Hanson, Laura K.; Conrad-Webb, Heather; Mills, Nathaniel C.
The objective of this research is to examine the regulation of an essential gene (m143) in MCMV that has sequence and functional homology to HCMV genes IRS1/TRS1. All of these prevent the shutdown of protein synthesis that is an antiviral defense. Little is known about the regulation of these genes at either the transcriptional or posttranscriptional levels. Sequential deletion mutant analysis of the m143 promoter was done in the context of an SEAP reporter plasmid. The results indicated that viral proteins are required for activation and the presence of repressor-binding sites and activation-binding sites. EMSA shows that SP1 may be important for the activation. Two important viral transcriptional regulators IE1 and IE3, each alone weakly activates the m143 promoter, but together synergize to efficiently activate this promoter. The absence of repression with co-transfection experiments support repression by other viral proteins. Such studies may lead to novel treatments for cytomegalovirus infection.
Calcium-dependent global chromatin compaction protects DNA from UV inflicted damage
(1/13/2020) Abbas, Mohammad M; Bergel, Michael
Eukaryotic genomes are packaged into chromatin, which is the physiological substrate for all DNA-mediated functions, including DNA damage repair. At the DNA damage site, chromatin organization undergoes critical rearrangements during the repair process. These rearrangements around the lesion sites accommodate at least three steps: providing access to the repair factors, repair, and restoring the DNA’s pre-lesion. chromatin architecture. However, the global changes to chromatin after UV-irradiation were less explored and understood. To investigate the relationship between chromatin condensation and UV irradiation, HeLa-S3 cells were irradiated and subjected to micrococcal nuclease digestion analysis. The results showed that chromatin globally commenced compaction five minutes after UV-irradiation. Twenty-four hours after irradiation chromatin returned to the pre-UV steady-state. Southwestern blots showed that cells were irradiated twice at 15 J/m2 with a five minutes break had a significantly lower cyclobutane pyrimidine dimers (CPD) and DNA (6-4) photoproduct (6-4PP) rate in comparison to cells subjected to 30 J/m2, and had no significant difference from cells irradiated with a single dose of 15 J/m2. Western blot analysis demonstrated a post-UV core histone deacetylation wave which followed the chromatin condensation. Western blots analysis of caspase-3, which is activated in apoptotic cells both by extrinsic and intrinsic pathways, showed no caspase-3 activation after five and ten minutes post UV-irradiation. Here, we demonstrate that an environmental genotoxic agent, UV radiation, causes immediate and global chromatin compaction in HeLa cells and this compaction results in a robust reduction in the newly formed lesions. Our data suggest an influx of calcium cations after UV irradiation is directly involved in inducing chromatin compaction.
Irradiation effects on gonad development to 65 days of age in male rats irradiated on the first day of postnatal life
(1/21/1970) Thomas, Sarah Nell; Hupp, Eugene; Aboul-Ela, Mohamed; Burkett, Ray; Fry, Kenneth; Fuerst, Robert
Functional analysis of M139, a pathogenesis factor of mouse cytomegalovirus
(1/28/2021) Pathak, Sushila; Hanson, Laura K.
Cytomegalovirus (CMV) is a common herpes virus that infects almost 60% of the adult population in the United States. It is transmitted via body fluids: saliva, tears, urine, stool, semen, and breast milk. Once a person is infected, it can be retained in the body for life with rarely any symptoms. CMV may cause several complications like retinitis or hepatitis in immunocompromised people and hearing loss or mental retardation in neonates. CMV is host specific meaning that human cytomegalovirus (HCMV) can only infect humans. For this reason, mouse cytomegalovirus (MCMV) is often studied in the mouse model for understanding the function of conserved and pathogenically important gene families. The M139, M140 and M141 genes in MCMV belong to the US22 gene family, members of which play important roles in pathogenesis. It has been shown that the deletion of any one of the M139, M140, and M141 genes leads to replication impairment of MCMV in macrophages. While the M140 protein is required for stable viral gene encapsidation in macrophages, the role of the M139 protein in virus replication is still unknown. Dr. Clive Sweet’s lab showed that a mutant virus with a C-terminal truncation of 79 amino acids in M139 replicates normally at 37°C in both fibroblasts and macrophages but the replication is impaired at 40°C in fibroblasts. We have determined that this mutant virus is also temperature sensitive in a neuroblastoma cells indicating that this defect is not restricted to fibroblast cells. The combination of lack of a temperature sensitive defect in ΔM140 and ΔM141, the coprecipitation of pM140 with the pM139trun, and that the defect occurs after capsid assembly, unlike ΔM140, supports that the pM139 has a function separable from the complex with pM140-pM141. Although the stability of the MutM139 infectious particle is not altered, higher number of non-infectious MutM139 particles are produced from infected cells at 40˚C than at 37˚C. The most likely possibility for the production of large number of non-infectious MutM139 particles would be a defect in the incorporation of one or more tegument proteins, possibly including pM139, into the virus particles.
Evaluating protein-protein interactions of CpsA, a virulence factor in Group B Streptococcus
(1/8/2019) Steffey, Danielle Renea; Hanson, Laura K.
Streptococcus agalactiae, Group B Streptococcus (GBS), is an important human pathogen, causing life-threatening sepsis and meningitis in newborns. The virulence of GBS in part can be attributed to its ability to produce a polysaccharide capsule. CpsA has been recognized as an integral part of capsule synthesis as well as having a role in transcriptional regulation. We hypothesize that protein-protein interactions with CpsA play a role in virulence and may be required for CpsA function. Elucidating CpsA protein-protein interactions may provide a better understanding of CpsA function in the capsule synthesis pathway. Using a BACTH system we have confirmed that CpsA interacts with the CpsC protein. Interactions between CpsA and predicted partner CpsE were not supported while interactions between CpsA and CpsY may be transient. Our pull down did not find any of these proteins however, we have identified multiple additional proteins of interest, including FtsZ and CodY. Understanding protein-protein interactions of CpsA will help in new antimicrobial therapies targeting capsule synthesis.
The role of upstream activating factor in suppressing Pol II rRNA transcription in Saccharomyces cerevisiae
(1/8/2020) Bhatt, Kushal; Conrad-Webb, Heather
Ribosome synthesis is the most resource and energy – intensive process in all eukaryotic cells and is tightly coupled with growth rate. In addition, defects in synthesis and assembly of ribosomal RNA (rRNA) and ribosomal proteins result in G1 arrest and cell death (Bernstein & Baserga, 2004). As the rate limiting step in ribosome synthesis, rRNA transcription is tightly regulated on many levels. RNA polymerase (Pol I) transcribes the ribosomal DNA (rDNA) to generate a 35S ribosomal RNA (rRNA) precursor which is post-transcriptionally modified to mature 18S, 5.8S, 28S rRNAs (Warner, 1999). However, under chronic stress conditions when Pol I transcription is repressed, rRNA can also be synthesized by RNA polymerase II (Pol II) using a cryptic promoter overlapping the Pol I promoter. This phenomenon of rRNA synthesis by Pol II is termed as ‘polymerase switch’ (Conrad-Webb & Butow, 1995). Since this process is conserved throughout eukaryotes including humans and plants, this phenomenon may play a universal role in the regulation of rRNA. Because the Pol I transcription factor, upstream activating factor (UAF), is known to generate rDNA chromatin inhibitory to Pol II during non-stress conditions, we hypothesized that UAF inhibited the polymerase switch during normal nitrogen conditions and that this inhibition is released during nitrogen deprivation, facilitating the switch. During nitrogen deprivation, UAF steady state levels decreased 2-fold and UAF binding to the rDNA promoter also decreased. Consistent with our hypothesis, UAF subunits H3 and H4 are differentially modified upon nitrogen deprivation with an increase in H3K4 and H3K36 methylation and a decrease in acetylation at H4K5. Contributing to the inhibitory chromatin structure in non-stress conditions, Pol I interacting protein Hmo1 represses polymerase switch as determined by reporter gene assays; whereas, Sir2 does not influence the polymerase switch. Furthermore, transcriptional repressors binding to the Pol II rDNA promoter recruit Ssn6-Tup1 to further repress the Pol II mediated transcription. Thus, during non-stress conditions, UAF triggers the assembly of Pol II inhibitory chromatin and recruitment of HmoI. This inhibitory chromatin is enhanced by the recruitment of the Ssn6-Tup1 repressor. This work has enhanced our understanding of the Pol I regulation during stress conditions and the role Pol II rRNA synthesis plays in overall regulation of ribosome synthesis upon stress.
RhoA and Rac1 prenylation: Effects on serine/threonine signaling for actin polymerization
(1/9/2020) Raut, Namrata; Hynds, DiAnna L.
RhoA and Rac1 are small guanosine triphosphates (GTPases) that regulate cytoskeletal rearrangements, cell polarity and axon guidance and signaling pathways. Alteration of the subcellular localization or activation of Rho GTPases is implicated in several neurological conditions. Rho GTPases are activated by binding to guanosine triphosphate (GTP) and is thought to require translocation to the plasma membrane by the addition of an isoprenoid moiety (geranylgeranyl) to the protein. However, previous experiments indicate that RhoA or Rac1 can be activated in the cytosol, without translocation to the plasma membrane. Based on this and evidence the Rho GTPases are centrally located in signaling pathways regulating actin dynamics, it was hypothesized that overexpressing non-geranylgeranylatable RhoA or Rac1 would decrease the actin filament content in neuronal growth cones by altering the location of actin regulating molecules. In particular, it was hypothesized that non-geranylgeranylatable RhoA or Rac1 would decrease the activity of actin filament promoters (i.e association of WAVE with the ARP2/3 complex and activation of JNK and ERK), and inactivation of the actin-depolymerizing protein cofilin. Overexpressing non-geranylgeranylatable (EmGFP-RhoAC190A), but not wild-type RhoA (EmGFP-RhoA), increased activation of ERK in the cytosol and increased association of WAVE with the ARP2/3 complex at the membrane, compared to cells overexpressing only the empty vector (EmGFP). However, overexpressing neither RhoA construct affected actin dynamics. Overexpressing wild-type Rac1 (EmGFP-Rac1), but not non-geranylgeranylatable Rac1 (EmGFP-Rac1C189A), increased the actin filaments content in growth cones compared to neurons expressing only EmGFP, concomitant with an increase in JNK activation. Overexpression of EmGFP-RacC189A decreased JNK activation and increased WAVE/ARP2/3 complexing, compared to cells expressing wild-type Rac1 or EmGFP alone. Studies with signaling molecule inhibitors indicated significant cross-talk between signaling pathways, which is altered by overexpressing wild-type or non-geranylgeranylatable forms of RhoA or Rac1. The results suggest that altering the subcellular localization of RhoA or Rac1 changes the activation of signaling molecules that regulate actin dynamics in neuronal growth cones. Elucidating the signaling cascades of the active GTPases may identify the distinct functions of these GTPases in the cytosol and can be used as novel targets to facilitate axon regeneration in neurodegenerative and neurological conditions.
Drug release kinetics and blood-brain barrier crossing efficacy of polymer encapsulated magnetic nanocarriers
(1/9/2020) Sebastian, Sumod; Hynds, DiAnna L.
Spinal cord injury (SCI) causes neuronal death and leads to permanent loss of motor and sensory functions. Treatment of SCI is challenging as axon regeneration from damaged neurons is limited by their intrinsic inability to regenerate as well as by the presence of extrinsic growth inhibitory molecules at the injury site. Moreover, targeting therapeutics to damaged CNS neurons is difficult due to the presence of the blood brain or blood spinal cord barrier (BBB/BSCB). This increases the demand for developing new treatment strategies for SCI. Unique properties of nanomaterials make them promising for targeted drug delivery to CNS neurons. We have developed a polymer encapsulated magnetic nanocarrier (PE-MNC) system for targeted and controlled drug delivery across the BBB. PE-MNCs were synthesized by precipitation polymerization method and tested for their biocompatibility in neurons. Drug loaded nanocarriers were tested for their release kinetics and targeted drug delivery to neurons in culture. We have also tested their size dependent BBB crossing efficacy in the presence and absence of an external magnetic field as well as induced hypoxia. We found that the physicochemical properties of PE-MNCs are ideal for their in vivo applications. Specifically, PE-MNCs possess superparamagnetic behavior in the presence of an external magnetic field and a higher LCST value. Increasing doses of PE-MNC treatment for up to 96 h did not detrimentally affect the neuron morphology. These nanocarriers exhibited a 50-70 nm volumetric reduction at physiological temperatures and released 80% of the imbibed drugs in a controlled manner. Moreover, they could induce neurite outgrowth on inhibitory substratum by delivering outgrowth promoting drugs to cortical neurons. We have used two different sizes of PE-MNCs for transport studies across bovine brain microvascular endothelial cell in vitro BBB model. In the presence of an external magnetic field, irrespective of their size, PE-MNCs were transported across a hypoxic in vitro BBB model. We conclude that PE-MNCs have the potential to deliver therapeutics to severed neurons across BBB following SCI.
Anti-proliferative activity of novel amidoximes in human and murine malignant cell lines and in mouse mammary carcinoma
(10/25/2019) Gekombe, Amon; Bergel, Michael; Mirsaleh-Kohan, Nasrin
Cancer incidence and mortality is on the rise worldwide, which necessitates newer and more potent therapies to combat cancer. In this study, four novel amidoximes, JJMB5, JJMB6, JJMB7 and JJMB9, were analyzed for their ability to inhibit proliferation of human and murine malignant cell lines, and in mouse mammary carcinoma. We established that JJMB5 and JJMB6 induced inhibition of acetylation of core histones H3K9 and H4K5 prior to apoptosis. Moreover, JJMB5, JJMB6 and JJMB9 inhibited acetylation of core histone H3K27, but JJMB7 did not. Additionally, JJMB5, JJMB7 and JJMB9 inhibited the proliferation of murine mammary malignant cell lines in culture but did not inhibit mouse embryonic fibroblasts, as established by the MTS colorimetric assay. When human and murine malignant cell lines were treated individualy, or in combination of various amidoximes or amidoximes with cisplatin, several combination treatments resulted in significantly lower growth inhibitory concentration (GI50) compared to individual treatments. Once the MTDs for the amidoximes were established, tumor studies were carried out. Results on tumor volume reduction were mixed. For example, mice treated with JJMB7 demonstrated significant tumor volume reduction in the first experiment but not in subsequent experiments. On the other hand, mice treated with JJMB9 had the lowest tumor volumes in all experiments. In The second experiment, tumor volumes were significant up to day 21. There were no differences in lung metastasis area between the treatment groups. Acetylation studies of 4T1 cells from tumors revealed that JJMB9 induced significant inhibition of acetylation of H4K5. Also, JJMB9 was able to induce drug resistance in MCF-7 cells, and this resistance was reversed by cisplatin. The amidoximes have potential if certain changes can be made on the protocol such as decreasing 4T1 cells implanted into the mammary fat pad, amidoxime dose escalation, and using alternate routes of administration.
The chemical analysis of Euphorbia bicolor (Euphorbiaceae) latex and its analgesic and antiproliferative properties
(10/29/2019) Basu, Paramita; Maier, Camelia
People with intense pain arising from disease or traumatic injuries are most commonly treated with opioids. Despite substantial advances in pain research and treatment, the negative side effects of opioids, such as addiction, physical dependency, and tolerance, remain a significant challenge to long-term pain management. On the other hand, globally breast cancer is one of the most frequently diagnosed cancers in women and is the second leading cause of cancer-related deaths in the USA. Both pain therapeutics and breast cancer treatments are accompanied by debilitating side effects. For that reason, the search for plant-derived natural compounds that could be used as pain and breast cancer therapeutics with better efficacy and lesser side effects has been enhanced. The present research investigated the pain relieving and antiproliferative properties of Snow-on-the-prairie, Euphorbia bicolor (Euphorbiaceae), a plant native to the south-central United States. Analgesic activity of the latex extract was tested in two distinct somatosensory systems of rat models of inflammatory and pain. Estrogenic and antiestrogenic activities were evaluated by employing a steroid-regulated yeast system and antiproliferative activity was assessed on estrogen receptor (ER) positive and negative breast carcinomas. Eleven bioactive latex phytochemicals were identified by UPLC-ESI-MS/MS. Injection of latex extract in rat hindpaw or orofacial region significantly induced long-lasting non-opioid analgesia in both male and female rats, in part via transient receptor potential V1 ion channels (TRPV1), pain-generating ion channels present in the peripheral nervous system. Furthermore, TRPV1-mediated analgesia occurred in part via modulating cytokines/chemokines, as well as downregulating the oxidative stress markers advanced oxidation protein products (AOPP), NADPH oxidase 4 protein (NOX4), and reactive oxygen species (ROS). E. bicolor latex extract and its phytochemicals induced different degrees of estrogenic and antiestrogenic activities in the steroid-regulated yeast system and the extract and its phytochemicals resiniferatoxin and rutin significantly reduced the growth of ER-positive (MCF-7 and T47D) and ER-negative (MDA-MB-231 and MDA-MB-468) breast carcinomas. This is the first study on the phytochemical analyses and biological properties of E. bicolor latex extract. Overall, the results indicate that latex phytochemicals could contribute to the development of novel, non-opioid pain relieving and antiproliferative drugs.
Role of progesterone in sexual receptivity and orofacial pain
(11/8/2019) Hornung, Rebecca Sharon; Averitt, Dayna L.; Uphouse, Lynda
Two understudied disorders that are challenging to treat, sexual dysfunction and orofacial pain, are more prevalent in women and can be impacted by the gonadal hormone’s estrogen and progesterone. Estrogen’s effects on women’s health are well-known, but much less is known about progesterone. The data in this dissertation indicates progesterone may be beneficial in alleviating these two major issues in women. The major drug prescribed for depression increases brain serotonin levels resulting in sexual inhibition, which likely occurs via the serotonin 1A receptor (5-HT1A) as agonists inhibit sexual behavior. Interestingly, progesterone protects against sexual inhibition by an unknown mechanism that may involve the intracellular progesterone receptor (iPR). Here we hypothesized that progesterone’s attenuation of 5-HT1A receptor-induced sexual inhibition involves the iPR. Also, progesterone may be beneficial for alleviating temporomandibular joint disorder (TMD) pain, which is 3-4x more common in women. TMD pain dissipates during pregnancy and after menopause but reemerges for some post-menopausal women prescribed estrogen replacement therapy. Here we hypothesized that therapeutic doses of progesterone in female rats undergoing estrogen replacement would attenuate inflammatory pain behaviors of the temporomandibular joint (TMJ). Ovariectomized, estradiol-primed female rats injected with an iPR antagonist before or after progesterone, then injected with a 5-HT1A receptor agonist had sexual receptivity parameters measured to determine if progesterone’s attenuation of 5-HT1A receptor-induced sexual inhibition involves the iPR. To determine progesterone’s effect on TMD pain, female rats were tested for basal sensory thresholds at the TMJ then injected with complete Freund’s adjuvant (CFA) to trigger inflammation in the TMJ. Mechanical allodynia (developed touch sensitivity) was confirmed then rats were ovariectomized and reassessed for allodynia following various estrogen and progesterone treatment paradigms. This dissertation reports that progesterone acting at the iPR can attenuate 5-HT1A receptor-induced sexual inhibition (Chapter II), progesterone can rapidly attenuate estrogen-evoked TMD pain behaviors (Chapter III), and orofacial sensory neurons contain progesterone-metabolizing enzymes and receptors (Chapter IV). Overall, adjunctive progesterone treatment may provide beneficial gender-based therapies for women facing serotonin-induced sexual dysfunction or post-menopausal women experiencing a return in TMD pain following estrogen treatment.
Assessing university biology students’ critical thinking skills resulting from team-based learning with case studies in the classroom
(12/15/2017) Collier, Jayme E.; Westmoreland, Sandra L.; Maier, Camelia; Gumienny, Tina L.
Research was conducted to measure the effectiveness of new course assessments to increase post-semester exam scores and critical thinking abilities of undergraduate students enrolled in a large lecture Principles of Biology course. New complex problem scenarios were designed to reinforce biological topics studied with Team-Based Learning (TBL) and to promote development of higher order thinking skills. The new problem scenarios were introduced in unit exams following a unit of study with TBL. Research results showed significant increases in student post-semester test scores for specific TBL-aligned questions when compared to previous semester student scores. This result validated the hypothesis that student content knowledge scores would increase due to new TBL-aligned problem scenario assessments. Comparisons between students’ successive problem scenario scores showed significant differences. Comparisons of student scores with different science backgrounds were not significant for post-test scores and problem scenario essays.
Effects of RhoA and Rac1 prenylation on Alzheimer's disease proteins
(12/2/2019) Chabayta, Rawand Khaled
Alzheimer’s disease (AD) is a progressive neurodegenerative disease characterized by brain extracellular amyloid plaques and intracellular neurofibrillary tangles.  amyloid (Aβ) plaques are produced from the cleavage of amyloid precursor protein (APP) by β-amyloid cleavage enzyme (BACE1) and -secretase. Intracellular neurofibrillary tangles are formed from the hyperphosphorylation of tau accomplished by increased kinase and/or decreased phosphatase activity. Clinically, the cholesterol-depleting drugs, statins, which also decrease the isoprenoid production, are associated with decreased incidence of AD. Geranylgeranyltransferase I activity adds a 20-carbon geranylgeranyl group (a type of prenylation) to proteins and its activity decreases with age or AD. This enzyme prenylates Rho guanosine triphosphatases (GTPases), including RhoA and Rac1. Some evidence indicates that dysregulation of Rho GTPase or activation or prenylation is associated with altered AD pathological markers A or tau. Here, we investigated how altered prenylation of Rac1 or RhoA affects AD pathological makers. We hypothesized that overexpressing Rac1 or RhoA that cannot be prenylated would increase APP and tau by increasing their processing secretases and kinases. To address this, we overexpressed emerald green fluorescently protein (EmGFP)-tagged prenylatable (wild-type) Rac1 or RhoA or non-prenylatable Rac1 or RhoA and measured APP, A, BACE1, -secretase, tau, phosphorylated tau, and levels of one kinase that phohsphorylates tau, glycogen synthesis kinase 3 (GSK3β), and activation of BACE1. In whole cell lysates of B35 neuroblastoma cells, overexpressing prenylatable or non-prenylatable Rac1 did not alter APP, A BACE1, presenilin 2 (the active subunit of  secretase), or GSK3β levels. However, APP levels in membranes were decreased by overexpressing either prenylatable or non-prenylatable Rac1, with the latter decreasing membrane-associated APP less than overexpressed wild-type Rac1. Cells overexpressing prenylatable or non-prenylatable RhoA increased total levels of tau, but only overexpression of wild-type RhoA increased levels of tau phosphorylation. We interpret results showing increased Rac1 and decreased RhoA prenylation increases tau phosphorylation to indicate a possible contribution to neurofibrillary tangle formation. However, since altering Rac1 or RhoA prenylation did not affect Aβ formation and evidence indicates that Rho GTPases function both upstream and downstream of AD marker production, RhoA GTPase prenylation, regardless of activation, may result from AD marker production instead of synthesis regulation.
Polyethylene glycol copolymer nanocarriers: Biocompatibility, uptake and intracellular trafficking in neurons
(12/20/2017) Veettil, Remya; Uphouse, Lynda; Hanson, Laura K.; Brower, Christopher; Ghosh, Santaneel
Spinal cord injury (SCI) causes neuronal death and leads to persistent loss of motor and sensory functions. Treatment of SCI is challenging as axon regeneration from damaged neurons is largely inhibited in the central nervous system (CNS). Moreover, targeting therapeutics to damaged CNS neurons is difficult due to barriers, including the blood brain barrier and injury-induced inhibitors of regeneration. This prompts the exploration of new treatment strategies for SCI. Due to their unique properties, nanomaterial-based drug delivery systems (nanocarriers) are promising excipients for targeted drug delivery to neurons. We have developed four types of nanocarriers and performed experiments to further optimize their potential use in SCI treatment. One of them is a polymer (PEG copolymer) encapsulated magnetic nanocarrier (PE-MNC) and was tested for its biocompatibility in a neuron model (PC12 cells), as well as in chick dorsal root ganglion (DRG) sensory neurons, cells that are damaged during SCI. We have performed time- and dose-dependent studies showing that treatment of up to 150 µg/mL PE-MNC for 72 to 96 hours does not affect the morphology and neurite outgrowth in DRG sensory neurons and neuronal cell line, as assessed using immunocytochemistry and confocal microscopy. The other three nanomaterial systems are surface functionalized nanocarriers (SFNCs) made of fluorescently-labeled PEG copolymers without the magnetic core. One system is derivatized by covalently-attached amino groups, producing SFNCs of 150 nm diameter (N150). The other two systems are functionalized with covalently-attached carboxyl groups, producing SFNCs with diameters of 150 (C150) and 750 (C750) nm. We have used these SFNCs to study the effect of nanocarrier size and charge in their clathrin-mediated endocytosis (CME), intracellular trafficking and uptake efficiency in neurons and glia, assessed using immunocytochemistry, confocal microscopy and live cell imaging. We have observed that, irrespective of size and charge, a portion of all SFNCs are internalized by CME in neurons, where they follow endo-lysosomal trafficking in B35 cells, but not in PC12 cells. Moreover, the efficiency by which SFNCs are taken up into cortical neurons is higher than that of glia, significantly for the uptake of C750 system. We conclude that modifying PE-MNC with C750 and/or N150 properties provides a potential nanocarrier for drug delivery to SCI-damaged neurons in vivo.
Characterization and prediction of residues forming functional protein-protein interfaces using network analysis
(12/21/2017) Mehta, Isha D.; Hynds, DiAnna L.; Beck, Brian W.; Uphouse, Lynda; Hanson, Laura K.; Brower, Christopher
Characterization of protein-protein interfaces can provide understanding of the fundamental properties of their functionality. Additionally, such understanding aids functional annotation of the interfaces and prediction to help in drug design. In this work, we classified protein interfaces broadly into two functional categories: Functionally Linked Interfaces of Proteins (FLIPs) and Functionally uncorrelated Contacts (FunCs). We used a method, based on network analysis followed by statistical analysis, to classify protein interfaces based on their functionality. Additionally, the method used for classification of protein interfaces characterizes each subunit in the complex individually allowing to expand the use of our method towards prediction of functionally linked interfaces. We assessed two different types of network constructs, as well as a method combining the best features from both the network constructs, for their classification accuracy and ability to predict functional interfaces. In the first network construct, Residue Interaction Networks (RINs), amino acid interactions are represented by spatial distance cut-offs of 8 Å between two Cα atoms of the residues. This simple approach classified FLIPs from FunCs with a consistent accuracy of 72%. It also identified dissimilarities among FLIP and FunC organizations. The positive correlation of FLIPs and network organization features suggest that FLIPs have a surface rich in concavities, while FunCs have smooth surface. The second network construct, Protein Energy Network, identified interactions among the amino acid residues using PyRosetta generated interaction energy scores. This construct was designed to assimilate chemical information in addition to geometrical information based RIN construct to study energy influenced organization of interfaces. A 70% classification accuracy was achieved with this method; however, the compromise in overall accuracy is balanced by accuracies of categorization of a few functional subcategories such as structural interfaces were classified with 100% accuracy. Finally we combined the identified spatial and energetic organization features from both the constructs and analyzed the classification potential of the combined model. It achieved an accuracy of 73% in classifying FLIPs from FunCs. Analysis of organization features from both the constructs, and also, the combination using Receiver Operating Characteristic (ROC) Curves identified that the classification abilities of combined organization features can be translated to a better prediction method among the three. Predictions of functionally linked interfaces of protein showed that 60% of the observed interfaces were correctly predicted, along with identification at least one connected triplet in 87% of the FLIPs. However, a 75% over prediction rate was observed. Analysis of FunCs on the other hand showed that we identified a connected triplet in 70% of the proteins, but rarely predicted functionally uncorrelated interface residues. Overall, our work suggests that network analysis can successfully characterize, classify and predict functionally linked interfaces of proteins.
Implementing team-based learning in principles of biology to determine effects on students' content knowledge, thinking skills, and attitudes about teamwork
(12/30/2015) Agogo, Angela; Westmoreland, Sandra; Conrad-Webb, Heather; Maier, Camelia
Respiratory viruses promote glycolysis the critical role of metabolism in human plasmacytoid dendritic cell antiviral responses
(12/30/2015) Bajwa, Gagan; Hanson, Laura K.; Gill, Michelle A.; Conrad-Webb, Heather; Mills, Nathaniel

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