This collection contains theses and dissertations submitted by graduate students under the Department of Cellular Biology and Anatomy for either a Master of Science degree or a Doctor of Philosophy degree.

Recent Submissions

  • Increased Membrane Thiol Oxidation in Sickle Erythrocytes

    Hill, Benjamin Albert; Department of Cell and Molecular Biology (1988-06)
  • IN VITRO AND IN VIVO STUDIES DEMONSTRATE A ROLE FOR SH3PX1 IN LAMELLIPODIA FORMATION.

    Hicks, Lawrence Joseph; Department of Cellular Biology and Anatomy (5/22/2018)
    Actin remodeling and endocytosis are essential functions for most cells. Defects in these processes present in a variety of diseases. Sorting nexins are known to contribute to endocytic uptake, cytokinesis, the retromer complex, and autophagy. Sorting nexin 9 (Snx9) interacts with major endocytic factors and proteins involved in regulation of actin cytoskeleton dynamics. Nonetheless, Snx9’s exact in vivo roles in these basic cellular processes and disease mechanisms are not known. By examining the roles of Sh3px1, we can better understand the mechanism by which this protein contributes to endocytosis and actin remodeling in vivo. Two additional paralogs, Snx18 and Snx33, complicate studies in mammalian models due to potential redundant mechanisms. Utilizing the single ortholog in Drosophila, sh3px1, this report describes the function of Sh3px1 in membrane organization and actin dynamics. Drosophila S2 cells that are depleted of Sh3px1 fail to form lamellipodia, a process that is also dependent on the actin nucleation factor, Scar. In addition, over-expression of Sh3px1 in S2 cells results in the formation of tubules and also long membrane protrusions, atypical of a classical BAR domain protein. An intact PX-BAR domain is required for these overexpression phenotypes. sh3px1 null flies are viable; however, mutant females have significantly compromised fertility. Female sh3px1 null egg chambers show many morphological defects. The age-dependent degeneration of the null egg chamber is not likely due to compromised endocytosis. Additionally, collective border cell migration is attenuated in the absence of Sh3px1. These cells are known for their reliance on endocytosis and modulation of actin dynamics for migration. We have found that Sh3px1 is essential in efficient lamellipodia production at the start of border cell migration. Our findings also suggest that Scar directly interacts with Sh3px1 and is upregulated in sh3px1 nulls. Mutation of Scar enhances many reproductive defects in sh3px1 nulls. Thus, our work reveals a main in vivo function of Sh3px1 in actin regulation for the production of structures such as lamellipodia.
  • DNA METHYLATION REGULATION IN ACUTE KIDNEY INJURY

    Dong, Zheng; Guo, Chunyuan; Department of Cellular Biology and Anatomy (4/26/2018)
    DNA methylation is a critical epigenetic mechanism, which is heritable during cell division, but does not involve the change of DNA sequence. It plays an essential role in regulating gene transcription in physiological and disease conditions. However, little is known about DNA methylation in renal diseases, especially in acute kidney injury (AKI). In this study, the role of DNA methylation in AKI was determined in both cell culture and mouse models. In cell culture, 5-aza-2’-deoxycytidine (5-aza), a pharmacological DNA methylation inhibitor, was used to inhibit DNA methylation. Interestingly, 5-aza increased both cisplatin- and hypoxia-induced apoptosis. These results suggest pharmacologic blockade of DNA methylation by 5-aza sensitizes renal tubular cells to apoptosis, supporting a cytoprotective role of DNA methylation in AKI. To determine the role of DNA methylation in vivo, we first successfully established conditional knockout mice that were deficient in DNMT1, DNMT3a or both exclusively in proximal tubules. In cisplatin-induced AKI, consistent with the effects of 5-aza in the cell culture, ablation of DNMT1 from proximal tubules exacerbated cisplatin-induced AKI in mice, and primary proximal tubular cells from PT-DNMT1-KO mice were more sensitive to cisplatin-induced apoptosis than wild-type cells. In sharp contrast, PT-DNMT1/3a-DK mice attenuated cisplatin-induced AKI, and primary proximal tubular cells from PT-DNMT1/3a-DK mice were more resistant to cisplatin-induced apoptosis. However, PT-DNMT3a-KO mice and PT-DNMT3a-WT mice showed similar AKI following cisplatin treatment. These results suggest different DNMTs play different roles in cisplatin-induced AKI. In ischemic AKI, none of the conditional knockout models showed differences in response to ischemia-reperfusion injury. Nevertheless, although ablation of both DNMT1 and DNMT3a in proximal tubular cells did not affect ischemia-reperfusion injury, it, indeed, suppressed renal fibroblast activation and ameliorated renal fibrosis. Furthermore, we found that Irf8 was regulated by DNA methylation during cisplatin treatment and knockdown of Irf8 in RPTC cells inhibited cisplatin-induced apoptosis, supporting a pro-death role of Irf8 in renal tubular cells. In ischemic AKI, although Bcl6 is hypermethylated and repressed in mice, overexpression of Bcl6 in RPTC cells had no impact on hypoxia-induced apoptosis. Collectively, these results suggest an important role of DNA methylation in AKI by regulating specific genes expression.
  • Targeting cyclic GMP signaling for the treatment of gastrointestinal diseases

    Sharman, Sarah Kristen; Department of Biochemistry and Molecular Biology / Cancer Center (1/25/2018)
    Continual renewal of the luminal epithelium in the gut is essential for the maintenance of a healthy intestine as it sustains the barrier that protects underlying tissue from infiltration of material passing through the lumen. Dysregulation of homeostatic processes involved in maintenance of the barrier have been implicated in numerous gastrointestinal diseases. The cGMP signaling axis has emerged as an important regulator of homeostasis in the intestinal mucosa, and has been implicated in the suppression of visceral pain, colitis, and colon cancer. While there is considerable interest in exploiting this pathway, until recently the approaches used to increase cGMP have been limited. The present study sought to test the hypothesis that elevation of cGMP in the intestinal epithelium using PDE5 inhibitors will alter epithelial homeostasis and be therapeutic for constipation and preventative for colon cancer. Healthy mice treated with the PDE5 inhibitor sildenafil or the GC-C agonist linaclotide exhibited reduced proliferation and apoptosis, and increased numbers of differentiated secretory cells in the intestinal epithelium. In addition to these homeostatic effects, both drugs normalized intestinal transit and fecal water content in two mouse models of constipation. Furthermore, administration of sildenafil to mice treated with dextran sulfate sodium tightened the disrupted epithelial barrier. Treatment of ApcMin/+ mice with sildenafil or linaclotide significantly reduced the number of polyps per mouse (67% and 50%, respectively). The effect of these cGMP-elevating agents was not on the polyps themselves but was rather on the pre-neoplastic tissue, which was less proliferative and more apoptotic in the presence of the drugs. Taken together, the results of this study demonstrate that increasing cGMP with a pediatric dose of PDE5 inhibitors could be a potential alternative to GC-C agonists for the treatment of gastrointestinal diseases.
  • Rapamycin, an evolving role in up-regulation of autophagy to improve stroke outcome and increase neuronal survival to stroke type injuries

    Buckley, Kathleen; Department of Cellular Biology and Anatomy (2015-10)
    Rapamycin was shown to reduce infarct size in a non-reperfusion and a slow reperfusion model of murine stroke; it also improved neurological score and survival in the slow-reperfusion model. The rapamycin improvement was 50 percent greater than that observed with chloroquine. In HT22 mouse hippocampal neurons, rapamycin was shown to improve survival to an oxidative/reperfusion injury with H2O2 and a hypoxic/ischemic injury with oxygen and glucose deprivation to a larger degree than chloroquine. Rapamycin treatment increased punctate microtubule light chain associated protein 3, LC3, in the HT22 neurons in an uninjured and oxygen and glucose deprivation injured HT22 neurons compared to untreated neurons. Finally, genetic knockdown of autophagy with shRNA to autophagy protein 5, ATG5, abrogated the rapamycin’s positive effect on survival to injury.
  • Role of microtubules and motor proteins in mRNA localization

    Sanghavi, Paulomi; Department of Cellular Biology and Anatomy (2015-08)
    Establishment of polarity is essential for many cell types to perform their functions. A common mechanism that is used to establish polarity is localization of mRNAs at specific sites. This results in spatial restriction of protein expression. mRNA localization is a widespread phenomenon, occurring in most species. However, the mechanism by which mRNAs are localized is poorly understood. Using Drosophila as the model system, we investigated the localization of one such localized transcript, oskar mRNA. Studying the mechanism by which oskar mRNA is localized is important because many factors involved in localizing this transcript also function in localizing mRNAs in mammalian neurons. oskar mRNA localizes at the posterior pole of the Drosophila oocyte. This results in the posterior restriction of Oskar protein, which is turn functions in establishment of polarity in the oocyte and the future embryo. Localization of oskar mRNA is microtubule-dependent. We, therefore, characterized the polarity of microtubules in the oocyte. Our findings suggest that the posterior region is highly enriched in microtubule plus ends. However, this polarization is not essential for oskar mRNA localization. Secondly, the posterior localization of oskar mRNA was shown to be mediated primarily by the Kinesin-1 motor. Our findings demonstrate the role of an additional motor, Dynein, in this pathway. We found that Dynein associates with oskar mRNA in vivo and depletion of Dynein caused a significant delocalization of oskar mRNA. Next, we examined the role of a Dynein adaptor, Egalitarian (Egl), in the oskar mRNA localization pathway. Egl has been shown to recruit localized mRNAs to the Dynein motor in Drosophila embryos. Our results suggest that Egl associates with oskar mRNA in vivo and is required for the posterior localization of this transcript. Interestingly, one of the mechanisms by which Egl affects the localization of oskar mRNA is by affecting the microtubule polarity in the oocyte. Additionally, depletion of Egl caused precocious translation of oskar mRNA in the oocyte. Thus, our findings revealed a novel function for Egl in organizing oocyte microtubules and in regulating the translation of a localized mRNA.
  • Androgenic Maintenance of Rat Penile Erection

    Reilly, Christopher M.; Department of Cellular Biology and Anatomy (1997-06)
    Prior studies from this laboratory, using untreated-castrated rats (CASTRATE) and testosterone-treated castrated rats (TESTO), have shown that the magnitude of the intracavemosal pressure increase during erection is androgen dependent. Studies from this and other laboratories have also presented evidence suggesting that penile erection is mediated principally by nitric oxide (NO). The present report was designed to confirm that androgens maintain the availability of cavemosal NO, and to determine if this androgenic action is exerted at the genomic level modulating the expression of the neuronal form of nitric oxide synthase (nNOS) gene. The results showed that administration of supplemental L-arginine failed to augment the erectile response in either group, suggesting that substrate availability is not a cause o f the reduced response in CASTRATE animals. Inhibition of NO synthesis with a nitro-arginine competitive inhibitor of NOS resulted in strong inhibition of erection in both TESTO and CASTRATE rats. When given in conjunction with ganglionic stimulation to induce erection, the NO releasing drug, sodium nitroprusside (SNP), increased intracavemosal pressure in CASTRATE but not in TESTO rats suggesting a deficiency of the available NO in CASTRATE animals. Finally, reverse transcription-polymerase chain reaction (RT-PCR) demonstrated that mRNA levels for the enzyme nNOS in the penis were greater in TESTO animals than in CASTRATE rats. These results support the hypothesis that androgens mediate the erectile response in the rat penis by stimulating the expression of the neuronal isoform of nitric oxide synthase, thus maintaining an adequate supply of NO.
  • Venous Contraction to Endothelin-1 in Congestive Hearth Failure

    Reddy, Vikram; Department of Cellular Biology and Anatomy (2003-04)
    Endothelin-1 (ET-1) is produced by endothelial cells and can stimulate either the ETa or the ETB receptors. The role o f ET-1 and the identity of the endothelin receptors involved in mediating tone in the mesenteric small veins of the Golden Syrian hamster are not known. ET-1 induces venoconstriction, thereby increasing the preload to the heart in congestive heart failure. However, mechanisms mediating contraction to ET-1 in the mesenteric small veins of the cardiomyopathic hamsters in the early and late stages of CHF are not known. Therefore, mechanisms mediating ET-1 induced contraction were determined in the mesenteric small veins o f the Golden Syrian and cardiomyopathic hamsters in the early and late stages of CHF. Baseline intraluminal diameter of small veins was measured before and after treatment with either ETa or ETB receptor antagonists. ET-1 induced contraction was higher in the early stage o f CHF, while it was decreased in the late stage of CHF. Blockade of the ETA receptor decreased ET-1 induced contraction in the mesenteric small veins from the control and cardiomyopathic hamsters in both the early and late stage o f CHF. ETB receptor blockade decreased the ET-1 induced contraction in the control and cardiomyopathic hamsters in the early, but not late, stage o f CHF. Therefore, ET-1 induced contraction in the mesenteric small veins is mediated by the ETa receptors alone in the late stage of CHF, while both the ETa and ETB receptors mediate vasoconstriction in the controls and in the early stage o f CHF. Stimulation of ET-1 receptors is associated with an increase in calcium levels within the vascular smooth muscle cells. It is not known whether the increase in reactivity to ET-1 in the early stage o f CHF or the decrease in reactivity to ET-1 in the late stage of CHF is due to problems with mobilization o f the intracellular calcium levels within the vascular smooth muscle cell. Following ET-1, calcium levels within the vascular smooth muscle cell were increased to a larger extent in the early stage o f CHF, than in the late stage of CHF, in agreement with the vascular reactivity data. Calcium levels were also measured before and after treatment with either ETa or ETb receptor antagonists. Blockade o f the ETa receptor inhibited the ET-1 induced increase in calcium levels in the mesenteric small veins from the control and cardiomyopathic hamsters in both the early and late stage of CHF. However, ETB receptor blockade inhibited the ET-1 induced increase in calcium levels in only the control and cardiomyopathic hamsters in the early stage o f CHF. These results indicate the absence of a functional responses mediated by the ETb receptor in the late stage of CHF. Studies have shown that NO can modulate the contraction to ET-1 in the vasculature. Baseline intraluminal diameter o f small veins were measured before and after treatment with N-nitro-L-arginine (LNA), a specific inhibitor o f nitric oxide synthase. LNA decreased the contraction to ET-1 in the early stage o f CHF, but increased contraction to ET-1 in the late stage of CHF. This indicates that NOS mediates a vasodilatory effect that counteracts contraction to ET-1 in the late stage, but contributes to the vasoconstrictor effect of ET-1 in the late stage o f CHF. NOS activity was measured to identify the NOS isoforms contributing to the modulation o f ET-1 induced vascular reactivity. Total NOS activity was significantly increased in the cytosolic fraction of small veins from hamsters in the late stage o f CHF and in the particulate fraction in hamsters in the early stage of CHF. In the late stage, the increase in NOS activity was inhibitable by 1400W, an iNOS selective inhibitor, suggesting that an increase in iNOS decreases the contraction to ET-1. In summary, in the early stage of CHF, there is an increase in the vascular reactivity to ET-1 associated with an increase in intracellular calcium levels and partially mediated by NOS. This may increase preload and impair myocardial function in CHF. There is an absence o f ETb receptor-mediated responses in the late stage of CHF, associated with very high plasma ET-1 levels and impaired intracellular calcium signaling. NOS activity is significantly enhanced in the mesenteric small veins from the cardiomyopathic hamsters in the late phase of CHF, and this increase in NOS activity is at least partially dependent on iNOS and may contribute to impaired venous contraction to ET-1 in cardiomyopathic hamsters. This may serve as a compensatory mechanism to decrease the preload to the failing heart.
  • Characterization of the Retinal Phenotype In Methylene Tetrahydrofolate Reductase (Mthfr) Deficient Mice, A Model Of Mild Hyperhomocysteinemia

    Markand, Shanu; Department of Cellular Biology and Anatomy (2015-05)
    (First Paragraph) The visual system is the primary source by which information is acquired by the human brain (Fernald, 1997). The eye is the organ responsible for vision. Blindness is a devastating condition as it affects the quality of life severely. In addition, there are financial consequences associated with blindness. Most untreatable disorders of vision that lead to blindness are due to disorders/degeneration of the retina. Retina is the photosensitive layer of the eye responsible for vision (Young et al, 2006). Several factors: genetic, environmental, systemic disease have been explored in the pathophysiology of various retinal disorders. One factor implicated in retinal diseases is excess levels of the amino acid homocysteine (hcy) (Selhub et al, 1999). The purpose of these studies was to analyze the expression of Cbs and Mthfr, key enzymes of hcy pathways in the mouse retina and to characterize the retinal phenotype in Mthfr deficient mice. To lay the foundation for this thesis, this introduction is organized in the following way: The eye, retina and retinal in-vivo diagnostics are described first. This is followed by description of hcy metabolism and its association with retinal diseases and mitochondrial dysfunction as a possible mechanism of hyperhomocysteinemia (Hhcy)-mediated retinal damage.
  • Impact of Genetic Predisposition and Environmental Stress on Measures of Preclinical Essential Hypertension

    Poole, Joseph C.; Department of Cellular Biology and Anatomy (2006-06)
    The main objective of this project was to determine the impact of genetic risk and chronic environmental stress on measures of preclinical essential hypertension (EH) (e.g., exaggerated cardiovascular reactivity, increased resting hemodynamics and increased left ventricular mass [LVM]). A secondary objective was to evaluate the moderating and interactive effects of ethnicity, gender, body mass index [BMI] and anger expression on EH risk indices. Two genes with relevance for blood pressure (BP) control (e.g., beta-2 adrenergic receptor [ADRB2] gene and serotonin transporter [5-HTT] gene) were used to define genetic risk. Chronic environmental stress was assessed by socioeconomic status (SES) and subjective social status (SSS). The project consisted of three sequential studies on a large, multiethnic cohort of young adults (N>500). The first two studies were cross-sectional and based on the analysis of cardiovascular reactivity, resting hemodynamics and LVM data collected at a single visit. The third study was longitudinal and involved the tracking of BP and LVM over a 15-year span from childhood to early adulthood. In the first study, ADRB2 haplotype significantly interacted with anger suppression in African Americans such that high anger suppressing carriers had the highest resting SBP (p<.05) and TPR reactivity to a cold pressor task (p<.01). In European Americans, ADRB2 haplotype significantly interacted with BMI to predict resting hemodynamics, such that carriers who were high in BMI showed the highest SBP (p<.05). In the second study, a significant interaction between the 5-HTT promoter region polymorphism (5-HTTLPR) and social status was found for cardiovascular reactivity, such that S allele homozygotes who were low in SES and high in SSS exhibited the greatest BP and TPR reactivity to the video game stressor (p-values<.05). No significant interaction was found between 5- HTTLPR and social status in the longitudinal study, however a significant 5- HTTLPR by BMI interaction was determined for LVM, such that obese LL homozygotes had the greatest LVM over time (p<.001). Results from this project expand what is currently known with regard to EH etiology and carry implications for the prevention of EH through the early consideration of genetic, environmental and demographic risk factors.
  • Role of NEK1 in VHL and Cell Cycle Regulation

    Patil, Mallikarjun; Department of Cellular Biology and Anatomy (2013)
    Nekl is the member of NIMA (Never in mitosis gene A) related protein kinase family that is widely expressed in mammals. Nekl is an essential protein because loss of function in Nekl gene causes polycystic kidney disease in mice, which is similar to ADPKD (Autosomal Dominant Polycystic Kidney Disease) in humans. In Humans Nekl mutations also cause short rib polydactyl syndrome characterized by renal cysts and other developmental defects. At the cellular level Nekl thought to regulate ciliogenesis, centrosome duplication and DNA damage response.Nekl mutations leading to PKD have long been attributed to its role in ciliogenesis. Interestingly, VHL (Von hippel lindau) protein a known tumor suppressor is also involved in ciliogenesis.VHL mutations cause cystic kidney disease and renal clear cell carcinoma. Since Nekl and VHL are involved in ciliogenesis and cystic kidney disease, my overall goal was to investigate if Nekl and VHL are part of common regulatory pathway and also to investigate the role of Nekl in cell cycle regulation. My results indicate that Nekl phosphorylates VHL and this has important role in cilia regulation. Nekl phosphorylates VHL on multiple sites and S168 of VHL a site phosphorylated by Nekl significantly affects its stability. Importantly renal cells expressing S168A VHL that cannot be phosphorylated by Nekl grow cilia that are resistant to serum stimulation and Nocodazole treatment. Surprisingly I also found that Nekl is an essential regulator of S phase. Nekl knockdown in HEK cells blocks cell cycle progression. Further characterization Nekl showed that Nekl is needed for S phase progression and DNA replication. Nekl deficient cells have replication stress and activate cell cycle check point. Nekl loads on to chromatin and this increases during replication stress. We have also identified that Nekl interacts with and affects Ku80 loading on to chromatin. These findings have provided novel insights into the Nekl functions, which help in understanding the pathophysiology and development of polycystic kidney disease in mice and short rib polydactyl syndrome mejawski in humans.
  • Bisphosphonate-Related Osteonecrosis of the Jaw: From Mechanism to Treatment

    Howie, Rebecca; Department of Cellular Biology and Anatomy (2015-04-20)
    With 55 million prescriptions issued each year, bisphosphonates are the second most prescribed class of drug in the United States. They are widely used to treat diseases with excessive osteoclastic resorption, including post-menopausal osteoporosis, Paget’s disease, and tumor metastasis to bone. Unfortunately, with long term intravenous administration of nitrogen-containing bisphosphonates some patients develop bisphosphonate-related osteonecrosis of the jaw (BRONJ). This debilitating disease has limited treatment options once it has manifested and no mechanism for its development has been elucidated. This dissertation explores the novel concept that bisphosphonates cause osteonecrosis of the jaw by impairing osteocyte-induced osteoclastogenesis and, through the physical accumulation of bisphosphonates in bone, impairing the ability of recruited osteoclasts to attach thereby arresting bone healing. Furthermore, it explores the possibility that chelating agents can be used for the removal of bisphosphonate attachment from bone systemically and locally during extractions, potentially leading to a future preventive treatment. It was found that 13 weeks of 80µg/kg intravenous tail vein injections of Zoledronate followed by two mandibular molar extractions caused the clinical presentation of BRONJ as analyzed by the gross, radiographic, and histological methods. Bone dynamic parameters and TRAP staining suggested an impaired ability for the bone to remodel and defective osteoclast attachment in treated groups that persisted eight weeks after the cessation of treatment. Additionally, it was found through the use of a fluorescently tagged bisphosphonate, that the decalcifying agents cadmium, EDTA, and citric acid all had the ability to cause the significant release of bound bisphosphonate from bone. Finally, this dissertation showed that the migration of monocytes treated with low doses of Zoledronate had increased migration, while their migration to conditioned media of osteocytes treated with Zoledronate was impaired. Collectively, these data suggest that invasive trauma by itself consistently precipitated massive bone necrosis in Zoledronate treated animals, possibly through a bisphosphonate driven alteration of monocyte migration and that the use of decalcifying agents could acutely remove bisphosphonate from bone both systemically and locally. This study establishes and effective rodent model for BRONJ and a possible preventive strategy for the side-effects of bisphosphonates during high-risk situations.
  • Characterization of Cardiac L-Type and T-Type Calcium Channels During Normal and Defective Chick Heart Development

    Nichols, Carol A.; Department of Cellular Biology and Anatomy (2000-03)
    (First Paragraph) The human heart is vital for survival from early in embryonic development throughout life. It begins developing around the third week of gestation from a pair of endocardial tubes that fuse to form a single primitive heart tube. The single-lumen heart tube develops a series of expanded areas and infoldings that divide it into four presumptive chambers. As the embryo grows, the heart begins looping. This looping process serves to bring the four presumptive chambers into the appropriate orientation for septation. The developing heart remodels itself into four separated chambers (two atria or holding chambers, two ventricles or pumping chambers) which provide for separate systemic and pulmonary circulation at birth. In most mammals, oxygenated blood enters the left atrium through four pulmonary veins. The blood is forced into the left ventricle when the left atrium contracts. When the left ventricle contracts, blood is pumped through the aorta and carried throughout the body. Deoxygenated blood returns to the right atrium via the superior and inferior vena cavae. Blood is forced into the right ventricle by contraction of the right atrium. Blood is then pumped through the pulmonary trunk and arteries to the lungs to be re-oxygenated. The four- chambered heart is formed by the eighth week o f gestation. (Larsen, 1997; de la Cruz & Markwald, 1998).
  • Regulation of Reduced-Folate Transporter-1 in Retinal Pigment Epithelium

    Naggar, Hany A.; Department of Cellular Biology and Anatomy (2003-04)
    (First Paragraph) The purpose of these studies was to analyze the regulation of the folate transport protein, reduced-folate transporter (RFT-1) in the retinal pigment epithelium (RPE) under conditions o f hyperglycemia, hyperhomocysteinemia and folate deficiency. A detailed description o f the retina, followed by information regarding folate and regulation o f RFT-1, is provided below.
  • The Effects of pp60v-src Expression on the Development of the Chicken Optic Tectum

    Mogan, John C.; Department of Biology and Anatomy (1999-03)
    The chicken optic tectum (OT) develops from the dorsal mesencephalon (midbrain) and processes crossed input from each retina. Previous experiments using a replicationdeficient retrovirus that contained the marker gene lacZ have demonstrated the normal pattern of development for the OT. Clonal cohorts derived from a single neuroepithelial stem cell migrate both radially and tangentially and differentiate into many types of neurons and at least three types of glia (radial glia and two types o f astrocytes). The goal of our laboratory is to identify important proteins involved in tectal development by: (1) directly altering expression of endogenous proteins through senseor antisense-containing retroviruses, or (2) indirectly altering endogenous protein expression or function by retroviral expression of an exogenous protein. These two approaches will allow us to determine which proteins are important in the normal and abnormal development of tectal clones. Many processes are involved in the development of the OT: proliferation, migration, differentiation, and synapse formation. Four non-receptor tyrosine kinases of the Src family (c-src, c-src+, fyn, and yes) are expressed in a spatially and temporally regulated manner in the nervous system. Their expression patterns in neural cells in vivo and in vitro have implicated these Src family members in all four of the major developmental processes mentioned above. Knockout mice of these three Src family members individually (c-src, fyn, or yes), however, show few or no overt neural developmental abnormalities. These unexpected results indicated that other members of the Src family can assume the roles of the missing kinase. Knockout mice for the major known negative regulator of Src family kinases, Csk (c-src kinase), however, show severe developmental abnormalities and defects in neural tube closure. These mice died around E9-E10 and showed elevated kinase activity for at least three Src family members (c-src, fyn, and Iyn). This fact makes it impossible to conclude that the overactivity of any one Src family tyrosine kinase is responsible for the developmental defects observed, and the early death of these embryos prevents the study of neural cell lineage, migration and differentiation in vivo. Given these results, the use of antisense to reduce expression of c-src would yield little information about the role of this kinase in neural development due to functional redundancy among Src family kinases. I decided to express in tectal clones an unregulated member of the Src family, pp60“'src, to determine how its expression alters normal tectal development. The v-src oncogene of Rous sarcoma virus was the first member of the Src family to be discovered, v-src encodes an activated tyrosine kinase (pp60,'"irc) which has lost a critical regulatory tyrosine present in the carboxy terminus of all other Src family members. Consequently, pp60'fcsrc expression affects the proliferation, migration and differentiation of many cell types in vitro and in vivo, but the effects of its expression on neural development in vivo are not well characterized. Expression of this kinase in tectal clones will provide an excellent system to study how a single unregulated Src kinase influences development of the nervous system. Expression o f a protein (pp60v'src) known to affect many different processes (proliferation, migration/cell adhesion and differentiation) in tectal clones will allow us to answer many questions of biological significance: (1) Is the proliferative potential of neuroepithelial stem cells restricted in vivo, or can stem cells generate clones of larger size?, (2) If multiple cell adhesion systems are presumptively inactivated in pp60*N,rc - expressing tectal neuroblasts, then how will clonal migration patterns differ from the norm?, (3) Is clonal differentiation in the OT controlled by only extracellular influence (e.g., growth factors, gradients) or can the developmental fate of stem cell progeny be altered by expression of pp60*’'src. In the first set of experiments I wanted to determine how wild-type pp60w'5rc expression alters the development of tectal clones in vivo. I used a replication-deficient retrovirus (LZIS), which efficiently coexpresses both LacZ and pp60*fc*rc, to determine the effects of pp60,,'*rc expression on several clonal parameters: cell number, migration pattern, and differentiation. In the next set of experiments I constructed and tested retroviral vectors which efficiently coexpress LacZ and mutated pp60w‘src proteins with deleted SH2 or SH3 domains (LZISASH2 and LZISASH3). These domains normally allow the pp60u'*rc tyrosine kinase to associate with certain cellular proteins which contain phosphotyrosines or a proline-rich stretch of amino acids, respectively. Mutation or deletion of these domains alters the biochemical and biological function of pp60^rc. I hoped to determine if the SIC or SID domains of pp60v'src are necessary for the wild-type pp60ltsrc phenotype, and to determine if they afford a unique but altered clonal phenotype compared to wild-type pp60v^rc. These experiments are novel in that they demonstrate that the overexpression of activated forms of Src family kinases influences development of the vertebrate brain. I conclude from my results that: (1) the proliferative potential of neuroepithelial stem cells in the OT is not restricted, (2) tangential migration of neuroblasts in the developing OT appears enhanced with pp60*fc*rc expression, and (3) the proper differentiation of radial glia is hindered but not prevented by pp60lHirc
  • The Effect of Blood Flow Rate on PMN Adherence and Protection Against Injury in the Isolated Blood Perfused Canine Lung Lobe Stimulated with PMA

    McCloud, Laryssa; Department of Cellular Biology and Anatomy (1998-05)
    In the lung neutrophil (PMN)-endothelial interactions contribute to the endothelial damage that occurs in many disease states, such as the adult respiratory distress syndrome (ARDS). Current literature states that PMN adherence is greater at low blood flow rates. How high blood flow rates affect PMN-mediated injury in the lung has not been investigated. This study was designed to determine the effects of increased blood flow on the ability of phorbol myristate acetate (PMA) to cause lung injury in the isolated canine lung lobe and on the ability of agents to protect against this injury. Injury was assessed by examining luminal endothelial bound angiotensin converting enzyme (ACE) activity, pulmonary vascular resistance (PVR), pulmonary artery pressure (Pa), double vascular occlusion pressure (Pdo), and the capillary filtration coefficient (Kf). PMN sequestration was measured using circulating white blood cell counts [WBC] and differentials and 51Cr labeled PMN retention by the lung. Lung lobes were perfused at low flow (LF, 0.599±0.001 L/min) or high flow (HF, 1.185±0.004 L/min) and divided into four groups. Group I, LF PMA, Group II, LF Control, Group III, HF PMA, and Group IV, HF Control. Groups I and III received PMA (10* M) while Groups II and IV were treated with the PMA vehicle. PMA decreased ACE activity and [WBC] at both flows while Pa, PVR and Kf were increased. PMA caused lung injury independent of blood flow rate. Isoproterenol (ISO) has been shown to protect against some forms of lung injury. To study the effect of flow rate on the ability of ISO (10*SM) to protect against PMAinduced injury, lobes were perfused at either 0.603±0.003 or 2.015±.0.064 L/min and were pretreated with either saline (Group I, LF Vehicle + PMA) and (Group II, HF Vehicle + PMA) or ISO (Group III, LF ISO + PMA) and (Group IV, HF ISO + PMA) for 20 min before PMA. After PMA Group I and II lobes showed significant decreases in ACE activity and increases in Pa and PVR. Kf measurements after injury could be completed in only three of the six lobes in Group II due to severe edema. Pa and PVR increased after injury in Group III lobes. In Group IV lobes ISO protected against the increases in Pa and PVR and decreases in ACE activity but caused an increase in Kf that was further increased after PMA. Thus, ISO protected against endothelial ectoenzyme dysfunction and partially protected against hemodynamic changes after PMA in lungs perfused at high blood flow rate. Lobes perfused at a low flow rate were not protected from the hemodynamic effects of PMA by ISO pretreatment. Pentoxifylline (PTX) is another agent reported to provide protection against various forms of lung injury. To study the ability of PTX (10'3M) to protect against PMA-induced injury, lobes were perfused at low flow (LF, 0.601±0.002 L/min) or high flow (HF, 1.170±0.005 L/min) and divided into four groups. Group I, LF PTX Control, Group II, LF PTX + PMA, Group III, HF PTX Control, and Group IV, HF PTX + PMA. Lobes were treated with PTX 30 min before PMA or vehicle. [WBC] and blood smear differentials were performed. PTX increased [WBC] in all groups but did not change any other measured parameters. In the presence of PTX, PMA resulted in no changes in ACE activity, Kf or hemodynamic parameters. PMA decreased [WBC] (P<0.05) in both th epresence and absence of PTX. PTX provided protection against PMA-induced lung injury at both flow rates. The injury to PMA was found to occur in lung lobes perfused at both high and low flow. PMA increased Pa, PVR and the Kf while decreasing circulating WBC counts, circulating PMN counts, A ^ /K ^ , and % metabolism of 3H-BPAP. Although the injury to PMA was found to occur independently of flow rate, the ability of ISO to protect against PMA-induced injury was found to be greatest during high flow perfusion. At high flow, ISO completely protected against increases in Pa, Pdo and PVR while attenuating the increase in the Kf. Plasma cAMP levels were also significantly increased by ISO pretreatment and were not altered by PMA in the high flow group. At low flow ISO did not prevent PMA-induced increases in Pa, Pdo or PVR. ISO did however protect against increases in the Kf and tended to increase plasma cAMP levels. Unlike ISO, PTX provided protection against PMA-induced lung injury independently of flow rate. During both high and low flow perfusion PTX protected against PMA-induced increases in Pa, PVR and the Kf while protecting against decreases in ACE enzyme activity. PTX caused the release of WBC from the lung significantly increasing both total WBC and PMN counts. PTX did not prevent the sequestration of PMN or the release of superoxide in response to PMA.
  • Use of Sigma Receptor Ligands to Prevent Retinal Ganglion Cell Apoptosis Characteristic of Diabetic Retinopathy

    Martin, Pamela M; Department of Cellular Biology and Anatomy (2003-04)
    (First Paragraph)Diabetic retinopathy is a major sight-threatening disease and is the leading cause of blindness among working-aged Americans, affecting approximately 10 to 12 million persons (Wu, 1995). Although retinal vasculature is particularly vulnerable to damage in diabetes, other retinal cells are at risk. Very recently, Barber et al. (1998) documented increased apoptosis of neural retinal cells in experimental diabetes in rats and diabetes mellitus in humans. Notably, retinal ganglion cells (RGCs) were found to be at particular risk. Ganglion cell death in diabetic retinopathy is thought to be mediated via overstimulation o f N-methyl-D-aspartate (NMDA) receptors by glutamate. oRl is a nonopiate and nonphencyclidine-binding site that has numerous pharmacological and physiological functions. In some studies, agonists for aR l have been shown to afford neuroprotection against overstimulation of the NMDA receptor. The purpose of these studies was to evaluate the potential use of aR ligands, particularly those that bind specifically to o R l, as neuroprotective agents in the treatment of RGC apoptosis characteristic of diabetic retinopathy. A detailed description of the retina, followed by information about diabetes and the mechanisms thought to be involved in the pathogenesis of diabetic retinopathy, particularly the apoptotic death of RGCs associated with diabetic retinopathy, is provided below.
  • Creating a Selective Advantage for Stem Cells: A Strategy for Gene Therapy

    Menezes, Kareena M.; Department of Cellular Biology and Anatomy (1999-10)
    (Statement of the Problem) Various approaches are used to treat the many known genetic diseases. The treatments are often incompletely effective, and they sometimes have undesirable side effects. Somatic cell gene therapy might provide truly effective permanent cures. Gene therapy, however, is still in the experimental stages, and much needs to be learned about stem cell biology before gene therapy becomes routine clinical practice. Moreover, inferences made from experiments in vitro do not necessarily model the in vitro setting. If treatments designed and tested in vitro can also be made workable and proven to be therapeutic in vivo, a major contribution to clinical gene therapy would be achieved. The described research, which attempts to encourage the stem cells to proliferate rather than divide down the hematopoietic cascade, could be significant in terms of increasing in number those hematopoietic cells that have been successfully modified by therapeutic vectors. The long-term goal of this research is to find a way to provide modified stem cells with a selective advantage in repopulating the marrow of a patient with a genetic disease. Ultimately it will be necessary to confer the selective advantage on somatic cells by introducing DNA into the patient’s defective bone marrow stem cells. However for purposes of preliminary laboratory analyses, a more reproducible system of testing a candidate genes’ potential for providing a selective advantage is necessary. In the present case, an Erythropoietin Receptor transgenic mouse line is used to provide stem cells, each of which already expresses the candidate selective-advantage gene.
  • Amyloid Peptide-a7 Nicotinic Acetylcholine Receptor Interactions: Implications For Cytoprotection In Vitro

    Li, Xinyu D.; Department of Cellular Biology and Anatomy (2006-11)
    Brain deposition of (3-amyloid peptide 1-42 (A(31 -42)-containing senile plaques has been a consistent finding in Alzheimer’s disease (AD). However, the link between Apl-42 and neuronal degeneration remains unclear. It has been reported that AP peptides bind with selectivity to a l nicotinic acetylcholine receptors (a7nAChRs), in both healthy and Alzheimer’s Diseased brain tissues. The goal of this study was to demonstrate the functional inhibition of oc7nAChRs induced by Api-42, both in systems in vitro and in vivo. Initially, differentiated PC-12 cells were preloaded with fura 2-AM and intracellular free Ca2+ levels were determined by fluorescent imaging. Nicotine-induced Ca2+ signals were inhibited by pretreatment with the a7nAChR-selective antagonists, abungarotoxin (BTX) and methyllycaconitine (MLA). Nicotine induced Ca2+ influx was also blocked by pretreatment with 100 nM Api-42. In the same model, nicotine produced a concentration-dependent increase in cell viability in differentiated PC-12 cells that underwent nerve growth factor (NGF) withdrawal for 24 hr. The cytoprotective action of nicotine was efficiently antagonized by co-treatment with a7nAChR antagonists. A concentration-dependent inhibition of the cytoprotective action of nicotine also was produced by co-treatment with Apl-42 (1-100 nM). Also in differentiated PC-12 cells, nicotine induced a concentration-dependent increase in cell surface Trk A receptor expression. This increase was almost completely reversed by a7receptor-selective antagonists, and by co-treatment with Api-42. In in vivo studies with rats, intracerebroventricular (icv) injection of choline, a selective a7nAChR agonist, produced transient, but dose-dependent pressor responses and prolonged decreases in heart rate. Icv pretreatment with BTX and MLA significantly inhibited the cardiovascular responses to subsequent injection of choline. Pretreatment with the Api-42 also significantly inhibited the choline-induced cardiovascular changes suggesting that the peptide can block an oc7nAChR-mediate response in vivo. Nicotine also was administered to rats by direct injection into a lateral cerebral ventricle. Estimation of Trk A expression in necropsied brain tissues revealed significant increases in hippocampus and entorhinal cortex. These increases were significantly inhibited in rats co-treated with a-bungarotoxin or with Api-42. The data derived from these in vitro and in vivo experiments support the hypothesis that low physiological concentrations of AP peptides inhibit the function of a7nAChRs, thereby contributing to the loss in neuronal viability that accompanies Alzheimer’s disease.
  • T-Type Calcium Current and Calcium-Induced Calcium-Release in Developing Chick Myocardium

    Kitchens, Susan A.; Department of Cellular Biology and Anatomy (2002-02)
    HYPOTHESES 1. The contribution of T-type calcium currents to the calcium transient are greater at young developmental ages, but decline with chick heart development. The decrease in contribution of T-type calcium current to the calcium transient mirrors the normal developmental reduction in magnitude of T-type current in the chick heart. 2. T-type calcium current plays a role in calcium-induced calcium-release during chick heart development. T-type current plays a significant role in the calcium-induced calcium-release process in younger embryos due to the greater magnitude of the current at earlier developmental stages. 3. More than one isoform of the T-type calcium channel is present in developing chick myocardium. The multiple isoforms will function concomitantly to provide sufficient T-type calcium current for proper development. 4. The expression of the T-type calcium channel in ventricle decreases with development. There is a concomitant decrease in T-type Ca2* current stimulation of CICR. SPECIFIC AIMS 1. To determine the contribution of T-type calcium current to the calcium transient during development in chick ventricular myocytes. The approach is to use a fluorescent calcium indicator to measure the transients from myocytes at embryonic day (ED) 5, EDI 1 andED15. 2. To determine the contribution of T-type calcium current to calcium-induced calciumrelease during chick heart development. The approach is to use pharmacological agents to quantify the contribution to the Ca3* transient from T-type Ca3* current stimulated CICR. 3. To determine which isoforms of the T-type calcium channel are likely to be present in chick myocardium. The approach is to use PCR methods to identify any T-type channel isoform mRNA expressed in chick ventricle. 4. To determine the level of expression of T-type calcium channel isoforms during the development of chick ventricle. The approach is to use molecular quantitation methods to examine the expression pattern of T-type channel isoforms in chick ventricle during development.

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