Cell and Developmental Biology Commons^™

Systematics Of The African River Frog Genus Amietia (Anura: Pyxicephalidae) From Eastern Democratic Republic Of The Congo, Thornton Robert Larson

Open Access Theses & Dissertations

The African river frog genus Amietia is found near rivers and other lentic water sources throughout central, eastern and southern Africa. Because the genus includes multiple morphologically conservative species, taxonomic studies of river frogs have been limited. We sampled 49 individuals of Amietia from multiple localities in and near the Albertine Rift (AR) of Burundi, Democratic Republic of the Congo and Uganda. We utilized single-gene (16S) and concatenated (12S, 16S, cytochrome b and RAG1) gene-tree analyses and coalescent species-tree analyses to construct phylogenetic trees. Two divergence dating approaches were used in BEAST, including secondary calibration points with 12S, 16S and …

Phylogeny And Systematics Of Panaspis And Afroablepharus Skinks (Squamata: Scincidae) In The Savannas Of Sub-Saharan Africa, Maria Fernanda Medina

Open Access Theses & Dissertations

African snake-eyed skinks are relatively small lizards of the genera Panaspis and Afroablepharus. The allocation of these genera was frequently rearranged in the 20th century based on morphology, ecology and biogeography. Members of these genera occur primarily in savanna habitats throughout sub-Saharan Africa and include species that have highly conserved morphology, which poses a challenge for taxonomic studies. We sequenced two mitochondrial (16S and cyt b) and two nuclear genes (PDC and RAG1) from 91 Panaspis and Afroablepharus samples from various localities in eastern, central and southern Africa. Concatenated gene-tree and divergence dating analyses were conducted to infer phylogenies and …

Adp-Ribosylation Factor 6 (Arf6) Regulates Integrin Αiibβ3 Trafficking, Platelet Spreading, And Clot Retraction, Yunjie Huang

Theses and Dissertations--Molecular and Cellular Biochemistry

Endocytic trafficking of platelet surface receptors plays a role in the accumulation of granule cargo (i.e. fibrinogen and VEGF) and thus could contribute to hemostasis, angiogenesis, or inflammation. However, the mechanisms of platelet endocytosis are poorly understood. The small GTP-binding protein, ADP-ribosylation factor 6 (Arf6), regulates integrin trafficking in nucleated cells; therefore, we posited that Arf6 functions similarly in platelets. To address this, we generated platelet-specific, Arf6 knockout mice. Arf6^-/- platelets had a storage defect for fibrinogen but not other cargo, implying Arf6’s role in integrin αIIbβ3 trafficking. Additionally, platelets from Arf6^-/- mice injected with biotinylated-fibrinogen, showed …

Rheb Dynamics On Lysosomal Membranes Determines Mtorc1 Activity After Loss Of P53 Or Activation Of Ampk, Catherine M. Bell

Theses and Dissertations

The tumor suppressor TP53 is the most frequently altered gene in human cancers. The growth-promoting complex, mTORC1 plays a part of the oncogenic profile caused by dysfunctional p53. mTORC1 sits downstream of AMPK and other crucial tumor suppressors/oncogenes, PTEN, LKB1, and Akt. The antifolate pemetrexed was found by this laboratory to activate AMPK via the inhibition of the enzyme AICART in de novo purine synthesis. This work presents a mechanism of mTORC1 activation with p53 loss, as well as of mTORC1 inhibition by pemetrexed-induced AMPK. We have found that mTORC1 activity was substantially upregulated by the loss …

Physiological Function Of Fus: An Rna Binding Protein In Motor Neuron Disease, Liuqing Yang

Theses and Dissertations--Molecular and Cellular Biochemistry

FUS is an RNA binding protein implicated in the motor neuron disease— amyotrophic lateral sclerosis (ALS, also called Lou Gehrig’s disease). ALS is a fatal neurodegenerative disease characterized by progressive motor neuron death. Mutations in the FUS gene cause about 4% of familial ALS (FUS ALS). Mutated FUS protein mislocalizes from the motor neuron nucleus to the cytoplasm and forms inclusions in the cytoplasm. It is unclear how FUS mislocalization induces motor neuron dysfunction and degeneration. This dissertation research was designed to investigate the physiological functions of FUS in the nucleus, with a purpose to shed light on the pathogenesis …

Beryllium Nitrate Supports Fibroblast Migration As An Essential Component Of Skin And Limb Regeneration In Axolotls, Adam Boyd Cook

Theses and Dissertations--Biology

Tissue regeneration in salamanders is a robust process that is not easily interrupted or altered. Therefore, inhibiting regeneration provides a means to interrogate the underlying cellular and molecular mechanisms regulating this complex event. Here we show that application of a relatively low concentration of beryllium nitrate solution (100mM) causes a delay in skin regeneration and severely alters normal limb regeneration. We provide evidence showing a beryllium-induced reduction in dermal fibroblast migration in vivo and in vitro. We link this phenomenon to delayed regeneration of the skin and abnormal blastema formation resulting in limb patterning defects during regeneration. Though our …

Role Of Sox11 During Vertebrate Ocular Morphogenesis And Retinal Neurogenesis, Lakshmi Shashidharan Pillai

Theses and Dissertations--Biology

Microphthalmia, anophthalmia, and coloboma (MAC) are distinct abnormalities demonstrating a continuum of developmental eye defects that contribute to 15-20% of blindness and severe vision deficiencies in children worldwide. The genetic etiology of MAC is large, complex and encompasses the whole developmental biology of the eye. Understanding how the eye develops will aid in identifying genes and developmental pathways involved in MAC. Although investigation of the genetic architecture of congenital anomalies is growing exponentially, much work remains to be accomplished to understand the complex, genetically heterogeneous congenital anomalies, which significantly impact childhood vision.

With an interest in elucidating the mechanisms that …

Dual Functions For Insulinoma-Associated 1 In Retinal Development, Marie A. Forbes-Osborne

Theses and Dissertations--Biology

Proper visual system function requires tightly controlled proliferation of a pool of relatively homogeneous retinal progenitor cells, followed by the stepwise specification and differentiation of multiple distinct cell types. These retinal cells, both neuronal and glial, must be generated in the correct numbers, and the correct laminar location to permit the formation of synaptic connections between individual cell types. After synapses are made, constant signaling is required as part of normal retinal function, and to maintain cellular identity and connectivity. These processes rely on both extrinsic and intrinsic signaling, with regulation of gene expression by cascades of transcription factors having …

Characterization Of Cd8+ T Cell Differentiation Following Sivδnef Vaccination By Transcription Factor Expression Profiling, James M. Billingsley, Premeela A. Rajakumar, Michelle A. Connole, Nadine C. Salisch, Sama Adnan, Yury V. Kuzmichev, Henoch S. Hong, R. Keith Reeves, Hyung-Joo Kang, Wenjun Li, Qingsheng Li, Ashley T. Haase, R. Paul Johnson

Nebraska Center for Virology: Faculty Publications

The onset of protective immunity against pathogenic SIV challenge in SIVΔnef-vaccinated macaques is delayed for 15-20 weeks, a process that is related to qualitative changes in CD8⁺ T cell responses induced by SIVΔnef. As a novel approach to characterize cell differentiation following vaccination, we used multi-target qPCR to measure transcription factor expression in naïve and memory subsets of CD8+⁺ T cells, and in SIV-specific CD8⁺ T cells obtained from SIVΔnef-vaccinated or wild type SIVmac239-infected macaques. Unsupervised clustering of expression profiles organized naïve and memory CD8⁺ T cells into groups concordant with cell surface phenotype. Transcription factor …

Escherichia Coli Surface Display Of Single-Chain Antibody Vrc01 Against Hiv-1 Infection, Lin-Xu Wang, Michael Mellon, Dane Bowder, Meghan Quinn, Danielle Shea, Charles Wood, Shi-Hua Xiang

Nebraska Center for Virology: Faculty Publications

Human immunodeficiency virus type 1 (HIV-1) transmission and infection occur mainly via the mucosal surfaces. The commensal bacteria residing in these surfaces can potentially be employed as a vehicle for delivering inhibitors to prevent HIV-1 infection. In this study, we have employed a bacteria-based strategy to display a broadly neutralizing antibody VRC01, which could potentially be used to prevent HIV-1 infection. The VRC01 antibody mimics CD4-binding to gp120 and has broadly neutralization activities against HIV-1. We have designed a construct that can express the fusion peptide of the scFv-VRC01 antibody together with the autotransporter β-barrel domain of IgAP gene from …

Evidence For Placental Hpv Infection In Both Hiv Positive And Negative Women, Chrispin Chisanga, Dawn Eggert, Charles D. Mitchell, Charles Wood, Peter C. Angeletti

Nebraska Center for Virology: Faculty Publications

Human papillomaviruses (HPVs) have previously been reported to infect epithelial trophoblast cells of the placenta. To investigate this possibility, 200 placental samples from Zambian women were separated into HIV+ and HIV− groups and tested for HPV by redundant primer PCR, using GP5+/GP6+ and CPI/CPII primer sets. Three HPV genotypes (HPV6, 16 and 90) were detected in placental samples. Whereas, 20 different HPV genotypes were detected in vaginal sampling of the same patients, suggesting that compartment specific sub-populations of HPV may exist. The incidence of HPV16 in placental samples was almost 2-fold greater in HIV+ women compared to HIV− (p = …

The Hippo/Yap Pathway Interacts With Egfr Signaling And Hpv Oncoproteins To Regulate Cervical Cancer Progression, Chunbo He, Dagan Mao, Guohua Hua, Xiangmin Lv, Xingcheng Chen, Peter C. Angeletti, Jixin Dong, Steven W. Remmenga, Kerry J. Rodabaugh, Jin Zhou, Paul F. Lambert, Peixin Yang, John S. Davis, Cheng Wang

Nebraska Center for Virology: Faculty Publications

The Hippo signaling pathway controls organ size and tumorigenesis

through a kinase cascade that inactivates Yes-associated

protein (YAP). Here, we show that YAP plays a central role in

controlling the progression of cervical cancer. Our results suggest

that YAP expression is associated with a poor prognosis for cervical

cancer. TGF-α and amphiregulin (AREG), via EGFR, inhibit the Hippo

signaling pathway and activate YAP to induce cervical cancer cell

proliferation and migration. Activated YAP allows for up-regulation

of TGF-α, AREG, and EGFR, forming a positive signaling loop to

drive cervical cancer cell proliferation. HPV E6 protein, a major

etiological molecule of …

Kaposi’S Sarcoma At The University Teaching Hospital, Lusaka, Zambia In The Antiretroviral Therapy Era, Owen Ngalamika, Veenu Minhas, Charles Wood

Nebraska Center for Virology: Faculty Publications

With great interest, we read the recent publication “Kaposi’s sarcoma in HIV-infected patients in South Africa: Multicohort study in the antiretroviral therapy era” by Bohlius et al. We congratulate the authors for their contribution to this field. In this study the authors observed a decrease in incidence of Kaposi’s sarcoma (KS) in patients treated with anti-retroviral therapy (ART) when compared to patients who are not on ART. These results are encouraging because of the ongoing HIV epidemic in sub-Saharan Africa where KS is still one of the most prevalent cancers. Also, it is a relevant topic to study in …

Activation And Degradation Of Open Reading Frame 45 By The Replication And Transcription Activator Of Kaposi's Sarcoma-Associated Herpesvirus, Ying Wang, Kai Yu, Xiuzhi Pei, Tiancheng Zhang, Yuying Guo, Charles Wood, Jinzhong Wang

Nebraska Center for Virology: Faculty Publications

The open reading frame 45 (ORF45) of the Kaposi's sarcoma-associated herpesvirus (KSHV) is an immediate-early phosphorylated tegument protein critical for viral escape from host immune surveillance. Its expression is upregulated by the viral replication and transcription activator (RTA), a key protein that controls the switch from latency to lytic replication. We report here that ORF45 expression was not only upregulated by RTA, but ORF45 could also be degraded by RTA in a proteasome-dependent manner. The ORF45 was activated by RTA via activation of the ORF45 promoter, and the promoter region from nt 69 271 to nt 69 026 was involved. …

Single-Dose Cpg Immunization Protects Against A Heterosubtypic Challenge And Generates Antigen-Specific Memory T Cells, Alexander J. Vogel, Deborah M. Brown

Nebraska Center for Virology: Faculty Publications

Despite extensive research, influenza A virus (IAV) remains a major cause of morbidity, mortality, and healthcare expenditure. Emerging pandemics from highly pathogenic IAV strains, such as H5N1 and pandemic H1N1, highlight the need for universal, crossprotective vaccines. Current vaccine formulations generate strain-specific neutralizing antibodies primarily against the outer coat proteins, hemagglutinin and neuraminidase. In contrast to these highly mutable proteins, internal proteins of IAV are more conserved and are a favorable target for developing vaccines that induce strong T cell responses in addition to humoral immunity. Here, we found that intranasal administration with a single dose of CpG and inactivated …

Long-Acting Parenteral Combination Antiretroviral Loaded Nano-Drug Delivery System To Treat Chronic Hiv-1 Infection: A Humanized Mouse Model Study, Subhra Mandal, Guobin Kang, Pavan Kumar Prathipati, Wenjin Fan, Qingsheng Li, Christopher J. Destache

Nebraska Center for Virology: Faculty Publications

Human immunodeficiency virus (HIV) patients are often diagnosed in the chronic stage of HIV/AIDS. Combination antiretroviral therapy (cART) has improved quality of life for HIV-infected patients. Present study describes a novel long-acting parenteral formulation of combination antiretroviral (cARV) loaded nano-drugs for treating chronic HIV-1 (cHIV) in a humanized-BLT (hu-BLT) mice model. The cARV (elvitegravir + tenofovir alafenamide + emtricitabine; EVG + TAF + FTC) drugs (mimicking marketed Genvoy® one-pill for HIV-treatment) were encapsulated in poly (lactic-co-glycolic acid) nanoparticles (NPs). To establish cHIV, hu-BLT mice were intravaginally challenged with HIV-1 and maintained for 15 weeks. Plasma viral load (pVL) was monitored …

Mutations In The 5’ Ntr And The Non-Structural Protein 3a Of The Coxsackievirus B3 Selectively Attenuate Myocarditogenicity, Chandirasegaran Massilamany, Arunakumar Gangaplara, Rakesh H. Basavalingappa, Rajkumar A. Rajasekaran, Hiep Vu, Jean-Jack Riethoven, David Steffen, Asit K. Pattnaik, Jay Reddy

School of Veterinary and Biomedical Sciences: Faculty Publications

The 5’ non-translated region (NTR) is an important molecular determinant that controls replication and virulence of coxsackievirus B (CVB)3. Previous studies have reported many nucleotide (nt) sequence differences in the Nancy strain of the virus, including changes in the 5’ NTR with varying degrees of disease severity. In our studies of CVB3-induced myocarditis, we sought to generate an infectious clone of the virus for routine in vivo experimentation. By determining the viral nt sequence, we identified three new nt substitutions in the clone that differed from the parental virus strain: C97U in the 5’ NTR; a silent mutation, A4327G, in …

Confocal Fluorescence Microscopy: An Ultra-Sensitive Tool Used To Evaluate Intracellular Antiretroviral Nano-Drug Delivery In Hela Cells, Subhra Mandal, You Zhou, Annemarie Shibata, Christopher J. Destache

School of Veterinary and Biomedical Sciences: Faculty Publications

In the last decade, confocal fluorescence microscopy has emerged as an ultra-sensitive tool for real-time study of nanoparticles (NPs) fate at the cellular-level. According to WHO 2007 report, Human Immunodeficiency Virus/Acquired Immunodeficiency Syndrome (HIV/AIDS) is still one of the world’s major health threats by claiming approximately 7,000 new infections daily worldwide. Although combination antiretroviral drugs (cARV) therapy has improved the life-expectancy of HIV-infected patients, routine use of high doses of cARVhas serious health consequences and requires complete adherence to the regimen for success. Thus, our research goal is to fabricate long-acting novel cARV loaded poly(lactide-co-glycolic acid) (PLGA) nanoparticles (cARV-NPs) as …

Association Of Autophagy In The Cell Death Mediated By Dihydrotestosterone In Autoreactive T Cells Independent Of Antigenic Stimulation, Ting Jia, Annandurai Anandhan, Chandirasegaran Massilamany, Rajkumar A. Rajasekaran, Rodrigo Franco, Jay Reddy

School of Veterinary and Biomedical Sciences: Faculty Publications

Gender disparity is well documented in the mouse model of experimental autoimmune encephalomyelitis (EAE) induced with proteolipid protein (PLP) 139–151, in which female, but not male, SJL mice show a chronic relapsing-remitting paralysis. Furthermore, dihydrotestosterone (DHT) has been shown to ameliorate the severity of EAE, but the underlying mechanisms of its protective effects are unclear. Using major histocompatibility complex (MHC) class II dextramers for PLP 139-151, we tested the hypothesis that DHT selectively modulates the expansion and functionalities of antigen-specific T cells. Unexpectedly, we noted that DHT induced cell death in antigen-specific, autoreactive T cells, but the effects were not …

Evaluation Of An Interactive Workshop Designed To Teach Practical Welfare Techniques To Beef Cattle Caretakers And Decision Makers, Reneé Dewell, Christy Hanthorn, Jared Danielson, Rebecca Burzette, Johann Coetzee, D. Dee Griffin, Alejandro Ramirez, Grant Dewell

School of Veterinary and Biomedical Sciences: Faculty Publications

The purpose of the project was to evaluate the use of an interactive workshop designed to teach novel practical welfare techniques to beef cattle caretakers and decision makers. Following training, respondents reported being more likely to use or recommend use of local anesthesia for dehorning and castration and were more inclined to use meloxicam for pain management. Respondents also reported improvement in performing all skills taught in the workshop. These results suggest instructional techniques used to teach practical concepts/techniques relevant to beef cattle welfare led to improvements in both perceived competency and estimated likelihood that the material will be used. …