M.S. - Developmental and Reproductive Biology

Permanent URI for this collectionhttps://hdl.handle.net/10125/36909

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Investigating exocyst inhibition for reducing neuronal pathophysiologies associated with Alzheimer’s disease
(University of Hawai'i at Manoa, 2025) Aou, Shion; Fogelgren, Ben; Developmental & Reproductive Biology
Alzheimer’s disease (AD) is a progressive neurodegenerative disorder and the leading cause of dementia among older adults. It is characterized by memory loss, cognitive decline, and hallmark features such as amyloid beta (Aβ) plaques, tau tangles, and metabolic dysfunction. While Aβ accumulation results from amyloidogenic cleavage of the amyloid precursor protein (APP), the upstream trafficking mechanisms that regulate this process remain unclear. The exocyst complex, an evolutionarily conserved eight-protein vesicle tethering complex, has been proposed to guide APP trafficking and support neuronal homeostasis. This study investigates the role of the exocyst in modulating APP processing and AD-related phenotypes in differentiated SH-SY5Y neurons expressing familial APP mutations (Swedish and Indiana). Using genetic knockdown of Exoc5 and pharmacological inhibition with Endosidin2 (ES2), we examined effects on endosomal morphology, Aβ production, tau phosphorylation, metabolic function, and gene expression. Our findings demonstrate that exocyst disruption did not rescue APP-induced dysfunction but instead exacerbated endosomal defects, mitochondrial impairment, and expression of genes associated with apoptosis and inflammation. These results suggest that the exocyst complex is essential for neuronal integrity and that its inhibition may worsen rather than alleviate AD-related cellular pathologies.
Morphometric analysis of the fossa ovalis in human cadaveric hearts
(University of Hawai'i at Manoa, 2025) Garcia, Chloe Nicole G.; Alarcón, Vernadeth B.; Developmental & Reproductive Biology
Coronary artery disease (CAD) is highly prevalent in the United States, leading to an increased need for interventions such as mitral valve replacement via transseptal puncture (TSP). TSP utilizes the fossa ovalis (FO), a remnant of the foramen ovale located between the right and left atria. However, detailed normative data describing FO anatomy remain limited. Inadequate understanding of FO morphology during TSP may result in serious complications, including cardiac tamponade caused by misdirected puncture. Accurate measurement of the FO is therefore essential for clinical decision-making during left-sided cardiac interventions. This study aimed to develop a comprehensive understanding of FO anatomy by investigating its morphological features in relation to surrounding cardiac structures and donor demographics. Thirty-six hearts from donors in the Willed Body Program at the John A. Burns School of Medicine were examined using a standardized method to measure and quantify FO morphology. Measurements of FO area, circularity, and aspect ratio were analyzed in relation to donor demographics, including age, sex, height, body weight, body mass index, body surface area, heart weight, and heart circumference. Additional anatomical characteristics, such as the presence of a patent foramen ovale, quality of the limbus, presence of the right septal pouch, and presence of the valve of the foramen ovale, were also evaluated. Analysis revealed significant correlations between FO morphology, heart weight, heart circumference, and donor demographics. Notably, sex-based differences were observed, with females demonstrating a significantly larger FO area when normalized using the heart weight and circumference. These findings enhance the current knowledge of FO anatomy and may contribute to improving the safety and efficacy of TSP during cardiac interventions.
The exocyst regulates exocytosis of the insulin-sensitive GLUT4 channel in neurons
(University of Hawai'i at Manoa, 2025) Kumasaka, Hanna; Fogelgren, Ben; Developmental & Reproductive Biology
Alzheimer’s Disease (AD) is the leading cause of dementia, impacting an estimated 6.7 million Americans, and the prevalence is projected to double in the next 25 years. This irreversible neurodegenerative disease manifests as severe memory loss and cognitive decline due to a progressive loss of neurons in the brain. Meta-analytic data demonstrate a substantially increased risk of AD among individuals with type 2 diabetes mellitus (T2DM), and up to 81% of people living with AD are estimated to have T2DM. Racial and ethnic groups suffering from high rates of T2DM, such as African Americans and Native Hawaiians and other Pacific Islanders, also have disproportionately higher rates of AD and show an earlier onset of cognitive decline. Despite the breadth and rigor of clinical data, we still have a poor understanding of the mechanistic relationship between insulin levels in the brain and the etiology of AD. Although the causes of AD are not yet fully understood, a classic pathological hallmark of the AD brain is the accumulation of amyloid beta (Aβ) plaques. The generation of these plaques involve the improper neuronal cleavage of the transmembrane amyloid precursor protein (APP), yielding small Aβ fragments that aggregate in the extracellular space. We have discovered that the exocyst protein complex may be an insulin-sensitive regulator of APP trafficking and processing in neurons, which would represent a new molecular mechanism directly linking insulin signaling and AD. Using both mouse hippocampal neurons and human SH-SY5Y neuroblastoma cells differentiated into neurons, we have shown that exocyst subunits are co-localized with APP on intracellular transport vesicles, however this APP-exocyst interaction is dramatically decreased within minutes of insulin treatment. In these SH-SY5Y neuronal cells expressing an APP transgene with familial AD mutations, RNAi silencing of exocyst genes led to substantial decreases in Aβ production. In this thesis work, we have engineered SH-SY5Y neurons to express combinations of fluorescently-labeled APP and exocyst proteins for live-cell total internal reflection fluorescence (TIRF) microscopy measurement of coordinated movement of APP and exocyst subunits. To further study these phenomena, we have engineered SH-SY5Y neurons to express both a pH-sensitive fluorescent glucose transporter 4 (GLUT4) with an mScarlet tag measuring total GLUT4. Through this tool, we have found evidence that the exocyst regulates GLUT4 translocation to the plasma membrane of neurons in response to insulin, which has only been previously shown in adipocytes and muscle cells. The anticipated impact of this research is the identification of a novel molecular pathway that directly links insulin signaling in neurons with early AD pathogenesis, providing a better understanding of how insulin dysregulation increases the risk of AD.
Using in vitro gastrulation models to assess the teratogenicity of antiviral drugs: dolutegravir and remdesivir
(University of Hawai'i at Manoa, 2021) Kirkwood-Johnson, Lauren; Marikawa, Yusuke; Developmental & Reproductive Biology
About 1 in every 33 babies born in the U.S. each year has a major structural birth defect, which is one of the leading causes of infant death. Due to the prevalence of and severe risk associated with birth defects, further investigation into their causes is needed. One of the potential causes of birth defects are teratogens, which include infectious agents such as viruses, as well as the medications that may be prescribed for the treatment of infectious agents. If a woman contracts a virus during pregnancy, it can negatively affect both her health as well as that of the fetus, so proper treatment is imperative. However, the medications may also negatively impact the developing embryo. It is this concept that formed the basis for this study, where the teratogenicity of important antiviral medications (one used to treat HIV and another to treat COVID-19) was examined using in vitro gastrulation models. The in vitro gastrulation models consist of aggregates of mouse or human pluripotent stem cells which undergo axial elongation mimicking the process of gastrulation. As gastrulation is an embryological event critical to generating the basic body plan, a disruption to this process may result in birth defects. Therefore, adverse effects on the growth, elongation, and gene expression in the gastrulation model serve as an indication that exposure to the drugs may be teratogenic. The results of this study demonstrated that both dolutegravir, the anti-HIV medication, and remdesivir, the only FDA-approved treatment for COVID-19, caused significant morphological and molecular changes in the gastrulation model when cell aggregates were exposed to therapeutically relevant concentrations of the drugs. These results suggest that dolutegravir and remdesivir may have teratogenic effects due to the impairment of key developmental processes. This supports the need to seek alternative therapeutic options with a lesser risk to fetal development. The findings also provide insight for further investigations to reveal the mechanisms of their teratogenicity.
An assessment of the reproductive toxicity of the anti-COVID-19 drug molnupiravir using stem cell-based embryo models
(University of Hawaii at Manoa, 2023) Huntsman, Margaret Carrell; Marikawa, Yusuke; Developmental & Reproductive Biology
It is important that women of reproductive potential know what risks the medications they are consuming pose to reproductive health. However, reproductive risk information is particularly limited for new drugs, such as those to treat COVID-19. Because the FDA no longer requires in vivo animal testing during preclinical tests, the information obtained through suitable in vitro models are particularly crucial. The overall objective of this study was to evaluate the embryotoxic effects of molnupiravir, an anti-COVID-19 drug, using in vitro embryo models made of pluripotent stem cells. In Specific Aim 1, the efficacy of morphological assay using mouse P19C5 stem cell model, or embryoid bodies (EBs), was assessed in reference to known embryotoxic drugs. The ICH recently released a list of drugs with their in vivo concentrations known to cause embryotoxic effects. This ICH list can be used to validate the sensitivity and specificity of an in vitro assay to identify embryotoxicity. Adverse effects of drugs on the P19C5 EB model were determined from disruptions in morphology. The in vitro results with P19C5 EBs closely reflected those of the ICH’s in vivo results for many of the drugs examined, validating the efficacy of the assay. In Specific Aim 2, the adverse effects of molnupiravir, and its metabolite N-hydroxycytidine (NHC) were examined using in vitro embryo models made of P19C5 cells and also of human embryonic stem cells. Morphology-based analyses indicated that molnupiravir had an adverse effect at concentrations much higher than the therapeutic level, whereas NHC had a dose-dependent adverse effects at therapeutically relevant concentrations. Molecular studies using P19C5 EBs further demonstrated that NHC altered the expression patterns of several embryo-patterning genes, providing mechanistic insights into the embryotoxic action of molnupiravir. These results emphasize the need for further studies into the effectiveness of the in vitro embryo models and the embryotoxic effects of the COVID-19 antiviral drugs.
Optimizing The Targeting Of The Piggybac And T2022 Transposon System
(University of Hawaii at Manoa, 2022) Xie, Kristal; Owens, Jesse B.; Developmental & Reproductive Biology
Gene therapy, which involves correcting pre-existing genetic interruptions, could provide a long-term solution for those suffering from genetic diseases such as cystic fibrosis (CF), a monogenic, autosomal recessive disease. Typical methods, however, for introducing DNA into cells have limitations such as random insertion, limited cargo capacity, and immune response. Thus, this thesis aims to optimize DNA binding domain (DBD)-directed transposition to develop a novel approach that would result in a reliable and effective non-viral method of transporting a gene of interest to a particular location in the genome in non-dividing cells. We hypothesize that the piggyBac (pB) and t2022 transposon systems can be optimized to target and integrate at a specific TTAA site through (a) introducing mutations in the pB and the t2022 transposases to reduce integration at off-target TTAA sites and (b) pairing the pB and the t2022 transposon systems to programmable targetable nucleases that contain a DNA binding domain capable of binding to a DNA sequence near a TTAA site of interest. In Chapter 2, we generate mutations in the dimerization interface and the DNA binding domain of the pB and the t2022 transposases to reduce their natural abilities to bind and integrate at various TTAA sites in the genome. In Chapter 3, we fuse the modified pB and the t2022 transposases to a CRISPR-associated protein 9 (Cas9) and synthesize user-defined sgRNAs. that bind to our region of interest. We also fuse the modified pB and t2022 transposases to custom-built transcription activator-like effectors (TALEs) that can direct integration to specific TTAA sites in the genome. The sgRNAs and TALEs are designed to target a specific TTAA site in the cystic fibrosis transmembrane conductance regulator (CFTR) gene as well as a specific TTAA site in the genomic safe harbors: CCR5, hROSA26, and AAVS1. Overall, the optimization of the pB and t2022 transposon systems will lead to a safer and more efficient approach to delivering therapeutic genes into the genome that have the potential to reverse various genetic diseases.
Investigations Of The Effects Of Selected Y-chromosome Encoded Gene Deletion And Viral Infection On Testicular Function In A Mouse Model
(University of Hawaii at Manoa, 2022) Bakse, Jackson Elias; Ward, Monika A.; Developmental & Reproductive Biology
Murine and human testes are affected by both Y-chromosome encoded gene loss and the direct or indirect effects of some viral infections. Primary focus into the impact of Y-chromosome gene loss and viral infection on male fertility has been primarily focused on the Y-chromosome encoded testis determining factor, Sry, and viruses that transmit vertically, like ZIKA virus (ZIKV). Zinc finger Y encoded genes (Zfy), termed Zfy1 and Zfy2 in mouse and ZFY in humans, were once suspected to be the testes determining factor (TDF). This was proven incorrect when the true TDF was identified as another Y-chromosome gene, SRY (sex-determining region Y encoded). Subsequent research using mouse models with Zfy deficiencies and Zfy transgenic additions have indicated significant contributions from these genes to other aspects of male fertility in the mouse. However, complete knockout (KO) of Zfy genes has not been conducted. Existing data from testicular biopsies obtained from men who succumbed to SARS-CoV2 (COVID-19) infection, many with underlying conditions, revealed extensive testicular damage in the absence of active viral infection of the testes, despite the presence of the viral cellular receptor, angiotensin converting enzyme 2 (ACE2). However, the effect of SARS-CoV-2 infection on testicular function has not been researched in a well-controlled mouse model. The projects herein are based on the hypothesis that complete Zfy gene loss and SARSCoV-2 viral infection have profound effects on testicular function in the mouse. To assess effect of Zfy gene loss, select spermiogenic phenotypes were assessed in mice with complete Zfy1 (Zfy1 KO), Zfy2 (Zfy2 KO), and both Zfy homologs (Zfy DKO) knockout developed with CRISPR/Cas9 technology. Assessment of the testicular effect of COVID-19 disease was performed using ii transgenic K18-hACE mice expressing human ACE in epithelia, infected with SARS-CoV-2, and assessed 3-, 5-, 8-, and 14-days post infection (DPI). The analyses of sperm and testes Zfy KO mice using silver-stained sperm spreads and light microscopy and transmission electron microscopy (TEM) revealed defects in sperm morphogenesis in Zfy2 KO and Zfy DKO males, but not Zfy1 KO males. The analyses of testicular PAS-stained testicular sections from Zfy DKO males revealed seminiferous tubule abnormalities. Histopathological assessment of testes from K18-hACE mice at 5 different DPI revealed significant increase in seminiferous tubule abnormalities and increase in germ cell apoptosis males with DPI of at least 5. This data shows that the testes are highly susceptible to loss of Zfy1 and Zfy2, and to SARSCoV-2 infection, illustrating the fragility of male fertility. The novel mouse models utilized to examine the effect of Zfy deletion and COVID-19 disease allow for the assessment of attributed solely to intended manipulations (gene knockout or viral infection). This provides insight for further investigations to elucidate the mechanisms of Zfy gene contribution and COVID-19’s lasting impact on mice and by extension possibly mankind.
Three-dimensional (3-D) ovarian tissue culture system supported by synthetic polyvinyl alcohol (PVA) hydrogel
(University of Hawaii at Manoa, 2022) Miyagi, Marissa; Yamazaki, Yukiko; Developmental & Reproductive Biology
Cancer therapies such as radiation and chemotherapy may damage ovarian function and result in infertility. The development of assisted reproductive technologies (ARTs) supports fertility preservation and has become a significant area of study to improve the quality of life for cancer patients undergoing chemoradiotherapy. In vitro follicle culture is a promising ART to preserve the fertility of cancer patients. Generally, conventional two-dimensional (2-D) ovarian tissue culture is unable to maintain the normal architecture of the ovarian tissues, and suppresses the transition from secondary to antral follicle development. To overcome this problem, the Yamazaki lab previously developed a three-dimensional (3-D) ovarian tissue culture system supported by Matrigel. As a scaffold, Matrigel significantly enhanced antral follicle development and oocyte quality. However, Matrigel is an animal-derived natural hydrogel and is therefore not clinically applicable.In this study, we tried to develop a clinically applicable 3-D ovarian tissue culture system using the mouse ovary as a model. For this purpose, we replaced animal-derived Matrigel with synthetic polyvinyl alcohol (PVA) hydrogel. PVA hydrogel is non-animal derived and synthetically engineered. PVA hydrogel has a defined composition, no batch-to-batch variability, and can be applied clinically. To establish a 3-D ovarian tissue culture system supported by PVA hydrogel, we first set the appropriate mechanical condition of PVA hydrogel to support follicle development (Specific Aim 1: To determine whether PVA hydrogel supports follicle growth in the 3-D culture system). Next, we examined ECM-mimetic bioactive modification in PVA hydrogel (Specific Aim 2: To determine the effect of extracellular matrix (ECM)-derived cell-adhesive peptides on follicle growth and oocyte development). To examine the effect of three ECM-derived cell-adhesive peptides (collagen-derived Gly-Phe-Hyp-Gly-Glu-Arg (GFOGER), laminin-derived Tyr-Ile-Gly-Ser-Arg (YIGSR), and fibronectin-derived Arg-Gly-Asp (RGD)) on follicle growth and oocyte development, the ovarian tissues were cultured in peptide-modified PVA hydrogels. We found that (1) PVA hydrogel successfully supported follicle growth as a scaffold, (2) RGD-modified PVA hydrogel significantly promoted antral follicle development and oocyte maturation, and (3) the combination of three peptides in PVA hydrogel synergistically enhanced folliculogenesis in vitro. In conclusion, our 3-D system supported by synthetic PVA hydrogel can be a promising technology to preserve the fertility of cancer patients, as well as an excellent model to define bioactive molecules essential for in vitro folliculogenesis.
The Role of Deptor in Intrauterine Growth Restriction
(University of Hawaii at Manoa, 2021) Nunes, Lance Gregory A.; Urschitz, Johann; Developmental & Reproductive Biology
Maternal obesity or insufficient nutrient supply during pregnancy can result in fetal overgrowth and fetal growth restriction, respectively, and cause serious health problems for both the mother and the fetus. Dysregulated placental nutrient transport is believed to be a mediator for both conditions. The mammalian target of rapamycin (mTOR) is thought to be the nutrient sensor of the placenta. mTOR is comprised of two complexes, mTORC1 and mTORC2. Both mTORC1 and mTORC2 have downstream targets that regulate cell growth, proliferation, ion transport, cytoskeletal remodeling, and more. Intrauterine Growth Restriction (IUGR) is associated with decreased nutrient transport and restricted nutrient availability for the fetus. It has been shown that mTOR is downregulated in placentas of pregnancies complicated by IUGR. The DEP-domain containing mTOR interacting protein (DEPTOR) has recently been shown to be an endogenous inhibitor of mTOR. We hypothesized that placenta-specific DEPTOR knockdown (KD) results in restoration of mTOTRC1/2 signaling, normalizing placental amino acid transport capacity and preventing IUGR. In the first part of this thesis, we generated a transgenic mouse model that expressed a placenta-specific DEPTOR KD cassette to study the in vivo effects of placental DEPTOR KD. Results showed that trophoblastic DEPTOR inhibition leads to increased expression of mTORC1/2 signaling, increased activity of the two main placental amino acid transport systems, and a marked increase in birth weight. In the second part of this thesis, we aimed to optimize parameters for placental sonoporation, a minimally invasive in vivo gene delivery technique that utilizes ultrasound and DNA conjugated lipid microbubbles. We observed successful placental transgene expression after sonoporation following intravenous injection of DNA conjugated lipid microbubbles. Taken together, this thesis has elucidated the role of placental DEPTOR in the regulation of mTORC1/2 signaling and fetal nutrient transport, providing insight into the molecular mechanisms that govern fetal growth and, ultimately, health. Furthermore, the successful sonoporation trials illustrate the possibility for this technique to be used as an alternative to transgenics for studying in vivo gene modulation, as well as a potential adaptable technique for the treatment of complications due to placental dysregulation.
The Role of N-WASP in ORC4 Mediated Polar Body Extrusion
(University of Hawaii at Manoa, 2020) Elento, Dominique Chanel Olita; Ward, W. Steve; Developmental & Reproductive Biology
Oocyte meiosis is completed though two asymmetric cellular divisions, where the oocyte extrudes half of its DNA two times, into two smaller daughter cells called polar bodies. N-WASP is an important factor in the actin polarization pathway. We investigated whether N-WASP is required for the extrusion of both polar bodies during mouse female meiosis. Previous work in our laboratory demonstrated that the DNA replication protein origin recognition complex 4 (ORC4) forms a cage around the chromosomes that will be extruded during polar body extrusion (PBE), but not the chromosomes that remain in the oocyte. My hypothesis is that N-WASP’s involvement in action polarization may be important for PBE, and related to ORC4 cage formation. We first localized N-WASP during oocyte progression through meiosis and found that it co-localizes with ORC4, except that N-WASP was not present in the initial stage, GV oocytes, while ORC4 was. This finding suggested the possibility that N-WASP was synthesized during meiosis. We tested this by injecting siRNA into GV oocytes upon collection, then allowing them to mature. We found that only 23.2% + 2.5% (MEAN+SD) of oocytes injected with siRNA directed towards N-WASP progressed to MII (the normal end point for in vitro GV maturation), while 92.9% + 10.1% of oocytes injected with control siRNA progressed to MII. These data suggest that (1) N-WASP may function in the same process that regulates PBE that ORC4 does, (2) that N-WASP is synthesized during meiosis, and (3) that N-WASP is required for progression to MII.
Identification of Quantitative Trait Loci (QTL) for Vibration Attraction, Locomotor, and Sleep Behaviors in Astyanax mexicanus
(University of Hawaii at Manoa, 2020) Lactaoen, Kimberly Diego; Yoshizawa, Masato; Developmental & Reproductive Biology
Rapid evolutionary shifts of multiple behavioral traits are frequently observed in the animal kingdom. Certain genetic mechanisms of these behavioral traits are thought to parallel psychiatric conditions. What puzzles geneticists and evolutionary biologists is that many behavioral traits are multigenic, which conflicts with rapid evolution. One possible explanation for this was shown through human genetics studies on Autism Spectrum Disorder (ASD), in which a high-burden of common risk variants induce many behavioral shifts in an individual. A core assumption is that many risk variants should be pleiotropic—one gene influences multiple behaviors. To test this assumption, we used Astyanax mexicanus, a teleost species constituting two morphs, a surface morph and a cave morph. The cave morph, evolved from its conspecific, exhibits many distinct behavioral phenotypes derived from cave-type mutations in the genome. In this thesis, F2 progeny generated from a pair of a cavefish and a surface fish (N=580), addressed the hypothesis that the evolution of a set of polygenic behaviors are influenced by pleiotropic loci. In Chapter 1, four diversified behaviors (vibration attraction, locomotor activity, sleep, and turning) were measured in cavefish (N=8), surface fish (N=8), and F2 hybrids (N=580). Behavioral correlation analyses among F2 hybrids indicated a tight link between night activity and night sleep, suggesting shared (pleiotropic) genetic factors among these behaviors. In Chapter 2, a genetic linkage map comprising of 27 linkage groups with 1,445 markers (40,268 cM) was generated through the genotyping-by-sequencing data of genomic DNA extracted from the F2 individuals in Chapter 1. On this linkage map, 16 quantitative trait loci (QTL) associated with vibration attraction, locomotor, and sleep behaviors were detected. Also, 11 of 16 QTLs were associated with two or more behavioral measurements, supporting the hypothesis that rapid multi-behavioral shifts are underlain by pleiotropic genetic factors. Interestingly, newly detected QTLs for daytime and nighttime sleep were distinct, suggesting separate pathways for regulating diurnality. In summary, shared genetic factors can provide the framework for studying rapid evolution and can inform potential target pathways in the study of psychiatric conditions.
Juvenile Shank3b Deficient Mice Present With Behavioral Phenotype Consistent With Autism Spectrum Disorder
(University of Hawaii at Manoa, 2017-12) Balaan, Chantell; Developmental & Reproductive Biology
Autism spectrum disorder (ASD) is a pervasive, multifactorial neurodevelopmental disorder diagnosed according to deficits in three behavioral domains: communication, social interaction, and stereotyped/repetitive behaviors. Mutations in Shank genes account for ~1% of clinical ASD cases with Shank3 being the most common gene variant. In addition to maintaining synapses and facilitating dendritic maturation, Shank genes encode master scaffolding proteins that build core complexes in the postsynaptic densities of glutamatergic synapses. Male mice with a deletion of the PDZ domain of Shank3 (Shank3B KO) were previously shown to display ASD-like behavioral phenotypes with reported self-injurious repetitive grooming and aberrant social interactions. Our goal was to extend these previous findings and use a comprehensive battery of highly detailed ASD-relevant behavioral assays including an assessment of mouse ultrasonic communication carried out on key developmental days in male and female Shank3B KO mice. We demonstrate that ASD-related behaviors, atypical reciprocal social interaction and indiscriminate repetitive grooming, are apparent in juvenile stages of development of Shank3B KO mice. Our findings underscore the importance of utilizing Shank mutant models to understand the impact of this gene in ASD etiology, which may enable future studies focusing on etiological gene-environment interactions in ASD.
Exocyst Dysfunction Impairs Urothelial Development Resulting in Congenital Ureter Obstruction
(University of Hawaii at Manoa, 2016-08) Napoli, Josephine
Hydronephrosis is the most commonly detected abnormality in prenatal ultrasounds. The most Prevalent cause of prenatal hydronephrosis is congenital obstructive nephropathy, with obstructions at the ureteropelvic junction (UPJ) region as the most common site for obstruction. The cause and developmental process of obstruction formation at the UPJ is poorly understood. We developed a Sec10 conditional knockout (Sec10-CKO) mouse model where Sec10, a critical subunit of the exocyst complex, has been knocked out in the urothelium that lines the ureter. Sec10-CKO embryos develop complete bilateral UPJ obstructions and hydronephrosis by day 18.5 of gestation (E18.5). Using this model, we were able to study the development of prenatal hydronephrosis. We determined that the urothelium lining the ureters of Sec10-CKO mice fails to differentiate, with the earliest evidence of this starting at E16.5. Loss of key terminal differentiation markers, including uroplakins, weakens the urothelial barrier that prevents urine from permeating into ureter tissues. Our data suggests that a luminal wound healing response at the UPJ starts at E17.5, which would correspond with the time point urine first starts to flow. We measured three characteristics consistent with a fibrotic wound healing response: (1) increased expression of TGFβ1, a key regulator of wound healing and fibrosis, (2) gene expression profiles consistent with myofibroblast activation, with increased proliferation at the site of obstruction, and (3) evidence of stromal remodeling. Our findings support a model where prenatal UPJ obstructions may be caused by an impaired urothelial barrier that allows urine to permeate and damage tissues lining the lumen, inducing a fibrotic wound healing response.
Cell Survival Characterization of Human Amnion Mesenchymal Cells Grown in Novel 3D Culture System and Gene Expression Comparison to 2D Culture
(University of Hawaii at Manoa, 2015-08) Foca, Kimberly
The Teratogenic Effects of Anti-Diabetic Drugs: In Vitro Assays Using Embryoid Body Morphogenesis
(University of Hawaii at Manoa, 2015-05) Kim, Iris
Many adults take some form of medication to treat chronic conditions, however little is known about their effects on embryogenesis and pregnancy. With the increase in fertility problems society is facing and the increase in use of medications, it is important to understand what effects pharmaceuticals have on the developing embryo to ensure that future generations are not adversely affected by teratogenic compounds that can lead to birth defects. In this study, teratogenicity of anti-diabetic compounds was examined using the in vitro P19C5 embryoid body (EB) elongation morphogenesis model. As the EB model recapitulates key embryological events crucial for body patterning and axis formation, adverse impact of drugs on EB growth and elongation implicates their potential teratogenicity. Of the 31 anti-diabetic compounds examined, 27 decreased EB size and 11 either decreased or increased EB elongation at a given concentration. Morphogenetic impact of four specific anti-diabetic drugs, dapagliflozin, phenformin HCl, manidipine 2HCl, and resveratrol, was further examined by investigating expression patterns of key developmental regulators. Each drug exhibited a unique effect on gene expression patterns. Notably, dapagliflozin significantly reduced the level of the Wnt3 gene, a crucial regulator of the primitive streak formation. The Wnt-inhibiting effect of dapagliflozin was also confirmed by the TOPFLASH reporter assay. Furthermore, it was revealed that the drug impact on EB size was likely mediated by adverse effect on cell proliferation. These results suggest that anti-diabetic compounds impact EB morphogenesis by perturbing key developmental regulators, cellular signaling, and proliferation. The present research should lay the foundation for further investigations, including animal and human studies, to determine teratogenicity of specific anti-diabetes medications.
Double-Tagging Ligation for Library Construction of Double-Strand Break Regions in Sperm Chromatin Fragmentation
(University of Hawaii at Manoa, 2015-05) Jin, Xiao
Sperm chromatin fragmentation (SCF) (Shaman et al. 2006) is a chemical induction of endogenous DNA fragmentation including both double-strand and single-strand breaks in mouse spermatozoa. The reaction can be activated by Mn (II) alone or in concert with Ca (II). The resultant double-strand breaks (DSBs) occur at a regular 25 kb interval throughout the mouse sperm genome. It has been postulated that the SCF-induced DSB regions (SCFRs) may be associated with the nuclear matrix attachment regions (MARs) in the mouse spermatozoa (Ward 2010). Such proposition demands a technical solution in sequencing of the SCFRs and subsequent mapping of the reads to the mouse genome. In this study, We have developed a double-tagging ligation protocol for sequencing the SCFRs. We first purified the SCF fragments and labeled the SCFRs. We then enclosed the SCFRs into the final sequencing construt containing sequencing adaptors on each end. The library preparation for SFCR break site analysis that we have performed in this study is a technical quest necessary for understanding and characterization of these structurally unique DNA elements.
The Role of the Human Amnion in Amniotic Fluid Volume Regulation
(University of Hawaii at Manoa, 2015-05) Gardiner, Juliet
Dose-dependent functions of FGF9 in male primordial germ cell differentiation
(University of Hawaii at Manoa, 2013-08) Ulu, Ferhat
Primordial Germ Cells (PGCs) are the precursors of gametes. For PGC male differentiation, (i) inhibition of meiosis and (ii) male-inducing factor(s) are essential. It was recently revealed that Fibroblast Growth Factor 9 (FGF9) is one of male-inducing factors. Using isolated PGC culture system, we examined the function of FGF9 in PGC differentiation. We found low FGF9 treatment (0.2 ng/ml) induced PGC male differentiation. Conversely, high FGF9 treatment drastically stimulated PGC proliferation (40%) compared with the control and low FGF9 groups (5~10%). As a result, high FGF9 treatment (25 ng/ml) prevented PGCs to enter the male pathway. Also, we demonstrated that high FGF9 induced ERK1/2 signaling activation for stimulating PGC proliferation while low FGF9 enhanced phosphorylation of p38 signaling to push PGCs into the male pathway.
Cardiovascular disease training for community health workers serving native Hawaiians and pacific people
(University of Hawaii at Manoa, 2013-08) Moleta, Chace Donovyne Ikaika
It has been well documented that Native Hawaiians and other Pacific Peoples (NHPP) are disproportionately affected by cardiovascular disease (CVD) in the United States, and particularly in Hawaiʻi. Nationally, community health workers (CHW) have been shown to be effective in delivering CVD self-management education. They are utilized by clinics and health agencies for individual and group education, as well as in case management and outreach; however, they are often hired with no formal health education and with limited clinical experience. In Hawaiʻi, CHW are used extensively in community health centers (CHC) and throughout the Native Hawaiian Healthcare System (NHHS). A 2008 needs assessment of 19 CHC and the NHHS identified the training of CHW in CVD as a top priority. The focus of this thesis was to further develop and evaluate a CVD training program tailored specifically for CHW serving NHPP. A community-based participatory research (CBPR) framework was utilized to both identify this priority and guide the development of a training program. Our efforts were motivated by the hypothesis that CHW knowledge in CVD could be increased, and subsequently retained, through training that is both culturally competent and interactive. Specific aspects of culture-based education (CBE) were systematically incorporated throughout the development of the training. The resulting product, "Heart 101," is a 5-hour long training seminar that is taught by a multidisciplinary team and balances a PowerPoint guided lecture with interactive class games, group discussions, and role-play scenarios. To date, Heart 101 has been delivered nine times, reaching 162 individuals, primarily CHW. For our evaluations we examined participants from three training seminars held during 2010. Identical pre-, post-, and 6-month post-training CVD knowledge tests were administered to seminar participants to assess gain and retention of CVD knowledge. Additionally, participants were also asked to complete a satisfaction survey. The results of our analyses of changes in mean test scores revealed both significant gains in CVD knowledge from pre-to post-seminar, as well as retention of that knowledge measured at 6-months post-training. Participant feedback about their experience in Heart 101 was overwhelmingly positive. A subsequent analysis to investigate knowledge change by CVD subtopics compared the frequency of correct answers by question from pre-to post-seminar. Although there were clear increases in knowledge for all subtopics reviewed, we also found baseline understanding among participants to be stronger in clinical knowledge as opposed to that of the basic sciences. This finding provides us with a basis to strengthen the Heart 101 curriculum and may serve as a guide for the development of future CHW training programs. The demonstrated success of Heart 101 has positive implications for the standardization of CHW education and their professional development. Our findings show that a CHW training program like Heart 101 can be effective in providing the necessary tools for the further development of this important and growing segment of the healthcare workforce. As the utilization of CHW in efforts to ameliorate health disparities increases, so will the importance of their ability to assist in the delivery of chronic disease management directives.
The molecular cloning of a hyperactive PiggyBac transposase and its transfection into HEK293T cells for the purpose of protein purification
(University of Hawaii at Manoa, 2013-08) Macias, Jessie Lin
Traditionally, a virus with genes of interest is generated to integrate its cargo into the host genome. More recently however, has been the advancement of harnessing the ability of naturally occurring transposases and constructed plasmids to cut and paste genes of interest into the host genome, also creating genetically modified organisms. A transposase is a protein that has specific recognition sequences that it binds to; cuts out all DNA (to be the transposon) in between these two flanking recognition sequences and then inserts it into the host chromosomal DNA. The goal of this work was to create a new method of gene delivery in embryos, by injecting purified hyperactive piggyBac transposase (HyPBase) into embryos, instead of the DNA that codes for it, thereby excluding the time needed for transcription and translation to occur. We first created a HyPBase vector with a histidine tag to aid in the transposase protein detection by antibody. Then we produced a stable HyPBase expression cell line by transfecting human embryonic kidney (HEK) cells with our cloned vector. The resulting protein was then purified using streptavidin resins according to a tandem affinity purification (TAP) protocol. The purified HyPBase protein was then injected into mice embryos by pronuclear microinjection, cytoplasmic microinjection and intracytoplasmic sperm injection methods. The 2-cell stage embryos were transferred to surrogate mothers and 2 of 17 live pups were transgenic. This will result in more rapid gene delivery, and lower the percentage of mosaicism in transgenic animals.

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