R-3 Repository :: Browsing by Author "Lwigale, Peter"

Browsing by Author "Lwigale, Peter"

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Amniotic Fluid-derived Stem Cell Isolation, Maintenance, and Differentiation for Cardiac Tissue Engineering
(2014-12-05) Connell, Jennifer Petsche; Jacot, Jeffrey G; Fraser, Charles D; Lwigale, Peter; Grande-Allen, K. Jane
Cardiac tissue engineering is limited by the lack of a clinically relevant cell source. Amniotic fluid-derived stem cells (AFSC) are broadly multipotent and proliferate rapidly, making them a promising cell source for tissue engineering applications. AFSC can also be utilized autologously for congenital heart defects, the most severe of which are identified in utero, allowing for ample time to isolate and expand the cells to prepare a patch for implantation shortly after birth. This thesis focused on the characterization of AFSC and their potential to differentiate towards a cardiac lineage. For characterization studies, stem cells from amniotic fluid were sorted for c-kit protein expression at the first passage or left unfractionated and then expanded in 5 different media. Protein and gene expression of markers common to pluripotent stem cells were analyzed from passages 2 through 6, and differentiation capacity of the stem cells towards osteogenic, endothelial, and neurogenic lineages were compared at passages 5 and 6. The unfractionated AFSC maintained higher expression of stem cell markers but displayed a significant decrease in those markers at passage 6. Correspondingly, indicators of the lineages of interest were higher following differentiation at passage 5 compared to passage 6. To investigate the cardiac tissue engineering potential of AFSC, cells were differentiated in indirect co- cultures with neonatal rat ventricular myocytes (NRVM) and under a small molecule- based directed differentiation regime. NRVM induce AFSC to form functional gap junctions following indirect co-culture. AFSC undergoing directed differentiation also localized gap junctions to cell membranes and additionally demonstrated an up regulation in cardiac transcription factors and sarcomere proteins. In both co-culture and small molecule-based differentiation methods, however, no organized sarcomeres or spontaneously beating cells were observed. While AFSC hold great potential for regenerative medicine applications, particularly in congenital defect repair, functional cardiomyocytes have not yet been obtained, and it is likely that additional cues beyond chemical signaling and growth factors will be required. Overall, these studies led to a greater understanding of the cardiac potential of AFSC and the effect of sorting and culture conditions on maintenance of stem cell properties in AFSC.
An investigation of the role of the Drosophila gene jim lovell in endopolyploid growth
(2018-07-13) Zhou, Fanli; Beckingham, Kathleen M.; Lwigale, Peter
The Drosophila gene jim lovell (lov) encodes a putative transcription factor of the BTB/POZ (Bric-a-Brac, Tramtrack, Broad/Pox virus and Zinc finger) domain class that is expressed in many elements of the developing larval nervous system. Lov has roles in innate behaviors such as larval locomotion and adult courtship. I have identified new roles of lov in controlling endopolyploid growth. Endopolyploid growth occurs when cells repeatedly duplicate the genomic DNA without cell division. This type of growth is a normal feature of growth in some plants and animal tissues and is also used by cancer cells for survival. Most of the rapid growth of the Drosophila larva is achieved by this mechanism. I discovered that decreasing lov expression in endopolyploid larval tissues such as the tracheae and the salivary gland, reduces their cell size and endoploidy levels. dMyc is a well-known regulator of both mitotic and endopolyploid growth in Drosophila, acting mainly on nucleolar functions. I have shown that Lov is a nucleolar protein and that decreasing Lov levels can partially suppress the effects of dMyc on endopolyploid growth. I have also established that Lov interacts with another BTB/POZ domain protein called Ribbon (Rib) in controlling endopolyploid growth. Lov also regulates tracheal growth by affecting transcription of the uninflatable (uif) gene, which encodes an apical plasma membrane protein. Together, my findings indicate that Lov’s role in endopolyploid growth involves interactions with dMyc, Rib, and Uif.
Interplay of Perlecan and MMP-7/Matrilysin Regulates Metastatic Prostate Cancer Cell Behavior: Basic and Clinical Implications
(2015-04-23) Grindel, Brian John; Farach-Carson, Mary C; Lwigale, Peter; Segatori, Laura
Perlecan/HSPG2 is a large extracellular heparan sulfate proteoglycan concentrated at tissue borders and separating epithelium and stroma. Along with its proteolytic consumers, the matrix metalloproteinases (MMPs), perlecan helps orchestrate development and homeostasis in nearly all studied multicellular organisms. However, both molecule classes can be coopted by prostate cancer (PCa) to advance the disease to its most deadly metastatic form. This work aimed to understand that relationship both at the basic and clinical level. Perlecan with its HS chains and tight domain structure is generally resistant to proteolysis, but a PCa cell must produce an associated enzyme to cleave the border proteoglycan in order to metastasize. This work was the first to identify an active protease produced by PCa cells that can completely digest intact perlecan. Following in silico proteolytic analysis, matrilysin/MMP-7, was identified as a likely candidate for in vitro assays. MMP-7, unlike other enzymes tested, cleaved perlecan when presented in multiple contexts. Perlecan and a subdomain, domain IV-3 (Dm IV-3), but not other subdomains (Dm I, IV-1 and IV-2), induced a striking clustering phenotype. MMP-7 incubation completely reversed this effect to favor cell dispersion and adhesion. Proteomic signaling arrays point towards global Src kinase activation as a major influence of perlecan DmIV-3 effects. To determine if this perlecan/MMP-7 relationship exists in PCa subjects, I performed a tissue microarray, along with β2-microglobulin (β2M), a GF that binds perlecan and induces MMP secretion. Besides increased levels of the two proteins within the patients (cancer/normal), MMP-7 and perlecan levels statistically correlated in multiple grades and localized at tissue interfaces. Additionally, I developed a new assay probing the perlecan fragment signature in the same PCa subjects’ serum. Perlecan fragments were largely increased in PCa and some of the fragments were associated with MMP-7 expression in the subjects. Overall, this work demonstrates a unique interplay between perlecan and its efficient proteolyzer, MMP-7, a relationship that is relevant from the cell and tissue to the clinic and which is likely to contribute to PCa progression to metastatic lethal disease.
Neural Crest and the Cornea: Transformation of the transcriptome and the role of Nephronectin during ocular development
(2020-09-15) Ma, Justin; Lwigale, Peter; Carson, Daniel
Corneal development is an intricate process that involves signaling and interactions between the periocular neural crest cells (pNC)s, presumptive corneal epithelium, lens, and the extracellular matrix. Cross-talk between these divisions are critical in regulating the migration, proliferation, and differentiation of the periocular neural crest to form the cornea. Major signaling pathways (retinoic acid, TGFβ, and Wnt) and cell-matrix interactions are essential for corneal formation, but the mechanisms of how they influence pNC behavior are not well understood. In this study, I use RNA-sequencing to establish the transformation in the transcriptomic profile from the pNC to the formation of the cornea. With this data, I establish the pNC identity and analyze how differential regulation of the major signaling pathways regulate pNC behavior and contribute to the corneal cell fate. In addition, I take note of the expression of key extracellular matrix, matrix-remodeling, and cell-matrix receptor genes and link their differential regulation to essential corneal developmental events. Within this niche, I focused on studying the role of a particular strongly expressed matrix protein, nephronectin (Npnt). Npnt has been linked to many embryonic developmental roles including the kidney, bone, heart, tooth, and lungs. However, a role for it has yet to be described in the cornea. Using both the chick and mouse as my model organisms, I found that both Npnt and its cellular receptor integrin α8β1 were strongly expressed in the migrating pNC. This led to the hypothesis that Npnt/Itgα8β1 has a role in regulating pNC behavior during corneal development. Using functional studies by knocking down or overexpressing Npnt in the chick, I show that loss or gain of Npnt correlates to a decrease or increase of corneal thickness and cell counts, respectively. This result was further confirmed by knocking down Itga8 and see corresponding decrease in corneal thickness, suggesting that Npnt/Itgα8β1 has a role in cell migration and/or proliferation. In clarifying respective assays. I found that cell proliferation was not increased by overexpressing Npnt and that Npnt-mediated signaling, which is a stronger inducer of periocular neural crest migration in vitro, is abrogated by inhibition of Itgα8β1. Altogether, in this thesis, I analyzed the transcriptomic profile in the developing cornea and characterize a previously unknown ECM-receptor interaction (Npnt/Itgα8β1), which may be a novel regulator of pNC migration and corneal development. Identifying a functional role for Npnt also validates the potential of the transcriptomic study as a valuable resource to identify novel genes essential for corneal development and potential targets for corneal therapies.
Primary cilia deficiency in neural crest cells models anterior segment dysgenesis in mouse
(eLife Sciences Publications, Ltd, 2019) Portal, Céline; Rompolas, Panteleimos; Lwigale, Peter; Iomini, Carlo
Defects affecting tissues of the anterior segment (AS) of the eye lead to a group of highly debilitating disorders called Anterior Segment Dysgenesis (ASD). Despite the identification of some causative genes, the pathogenesis of ASD remains unclear. Interestingly, several ciliopathies display conditions of the AS. Using conditional targeting of Ift88 with Wnt1-Cre, we show that primary cilia of neural crest cells (NCC), precursors of most AS structures, are indispensable for normal AS development and their ablation leads to ASD conditions including abnormal corneal dimensions, defective iridocorneal angle, reduced anterior chamber volume and corneal neovascularization. Mechanistically, NCC cilia ablation abolishes hedgehog (Hh) signaling in the periocular mesenchyme (POM) canonically activated by choroid-secreted Indian Hh, reduces proliferation of POM cells surrounding the retinal pigment epithelium and decreases the expression of Foxc1 and Pitx2, two transcription factors identified as major ASD causative genes. Thus, we uncovered a signaling axis linking cilia and ASD.
Structural and functional studies of the envelope protein and viral polymerase in Hepatitis B Virus
(2021-10-18) Lin, Zihan; McNew, James A.; Shamoo, Yousif; Lwigale, Peter; Suh, Junghae
Hepatitis B Virus (HBV), a member of the Hepadnaviridae family, is a serious pathogen that causes chronic Hepatitis B and affects over 350 million people globally. Though a vaccine has been available since 1982, it cannot cure established infections and their effectiveness in preventing blood-borne transmission from an infected mother to her infant is only 90%. In order to better understand human HBV with the aim of finding effective treatments, much work has been dedicated toward investigating HBV virally encoded proteins. Unfortunately, few of these proteins have been able to be expressed or purified in vitro. As a result, current efforts seek to determine candidate HBV surface antigens as a possible immunotherapeutic target. Particularly promising candidates include those with identical function as HBV envelope L protein, which interacts with host proteins such as sodium-taurocholate cotransporters polypeptide (NTCP) and possibly epidermal growth factor receptor (EGFR) for entry. Meanwhile, current FDA-approved drugs to control human HBV target the viral polymerase. In order to develop more effective drugs against HBV infection, a better understanding of the HBV polymerase structure and function is necessary. Such information would not only inform us about the enzymatic mechanism of the viral polymerase, but also provide insights into the mechanism of current and mutation-mediated drug resistance. Here, we report the expression, purification, and analysis of the HBV LM envelope protein – a promising surface antigen. Lone LM was found to be an intrinsically disordered monomeric protein capable of interaction with host protein NTCP - and possibly EGFR. Addition of single-chain variable fragment 2H5 to LM formed a much more stable complex while conserving biological activity. Our results overall suggest that LM and the 2H5-LM complex exhibit favorable physical and biochemical properties for future in vitro study, in addition to serving a key role in promoting HBV entry and infection. We also demonstrate the expression and purification of the RNA dependent RNA polymerase domain and RNase H viral polymerase domains from different HBV species.
Visualization of RNA virus infection in a marine protist with a universal biomarker
(Springer Nature, 2023) Coy, Samantha R.; Utama, Budi; Spurlin, James W.; Kim, Julia G.; Deshmukh, Harshavardhan; Lwigale, Peter; Nagasaki, Keizo; Correa, Adrienne M. S.
Half of the marine virosphere is hypothesized to be RNA viruses (kingdom Orthornavirae) that infect abundant micro-eukaryotic hosts (e.g. protists). To test this, quantitative approaches that broadly track infections in situ are needed. Here, we describe a technique—dsRNA-Immunofluorescence (dsRIF)—that uses a double-stranded RNA (dsRNA) targeting monoclonal antibody to assess host infection status based on the presence of dsRNA, a replicative intermediate of all Orthornavirae infections. We show that the dinoflagellate Heterocapsa circularisquama produces dsRIF signal ~ 1000 times above background autofluorescence when infected by the + ssRNA virus HcRNAV. dsRNA-positive virocells were detected across > 50% of the 48-h infection cycle and accumulated to represent at least 63% of the population. Photosynthetic and chromosomal integrity remained intact during peak replication, indicating HcRNAV infection does not interrupt these processes. This work validates the use of dsRIF on marine RNA viruses and their hosts, setting the stage for quantitative environmental applications that will accelerate understanding of virus-driven ecosystem impacts.

Browsing by Author "Lwigale, Peter"

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