Cellular signal transduction

The central premise of this dissertation is that mitochondrial antioxidant enzymes
are essential to lens cell viability by preserving lens cell mitochondria and protecting
and/or repairing lens cell proteins, and two mitochondrial-specific antioxidant enzymes,
Peroxiredoxin 3 (PRDX3) and Methionine sulfoxide reductase A (MsrA), are explored.
In this dissertation, we will examine the expression ofPRDX3 in the human lens, its colocalization
to the lens cell mitochondria, its ability to be induced by H20 2-oxidative
stress, and speculate how PRDX3 function/sf could affect the lens. We will also examine
the reduced levels of MsrA by targeted gene silencing and its effect on reactive oxygen
species production and mitochondrial membrane potential in human lens cells to
determine its role in mitochondrial function in the lens. Lastly, we will examine the
ability of MsrA to repair and restore function to a critical mitochondrial protein,
Cytochrome c. The collective evidence strongly indicates that the loss of mitochondrial-specific enzymes, such as PRDX3 and MsrA, are responsible for increased reactive
oxygen species levels, decreased mitochondrial membrane potential, protein aggregation
and lens cell death, and further indicates that mitochondrial repair, protective, and
reducing systems play key roles in the progression of age-related cataract and other agerelated
diseases.

Taurine, an endogenous ammo acid and neuromodulator, has been found to be
neuroprotective against numerous forms of neurotoxicity including glutamate-induced
excitotoxicity. Previously we have shown that taurine inhibits glutamate-induced
calcium influx through VGCCs and NMDA receptors. Although the neuroprotective
effects of taurine against excitotoxicity have been attributed to its intracellular Ca2+
regulatory functions, the complete mechanism underling taurine neuroprotection has
remained unclear. Using primary rat cortical neuronal cell cultures, we have determined
key cytosolic components to the mechanism of taurine neuroprotection. In this study we
have found that taurine inhibits excitotoxicity by suppressing glutamate-induced
elevations in [Ca2+]i, preventing calpain activation, and inhibiting reductions in Bel-
2:Bax ratios and consequently activation of the intrinsic pathway.

Huntington's disease (HD) is an inherited neurological disorder characterized by a
selective loss of neurons in the striatum and cortex leading to involuntary movement,
dementia and eventually cell death. HD is caused by an expanded polyglutamine (PolyQ)
repeat in Huntingtin (Htt) protein. It is well known that misfolded mutant Htt could form
intracellular aggregates, trigger ER stress and ultimately lead to apoptosis. However, the
molecular link between ER stress and apoptosis in mitochondria is poorly understood. In
the present study, we identified Bim (Bcl-2 interacting mediator of cell death) as the
essential protein. We first established a cellular model of HD by over expressing the Nterminus
of wild type and mutant Htt into HEK293 cell lines. We showed that the
accumulation and aggregation of misfolded mutant Htt protein triggers ER stress and
apoptosis. The Bim protein expression level was greatly increased in mutant Htt
transfected cells and this increase was partially due to up-regulation of Bim mRNA as analyzed using quantitative RT-PCR. We further showed that Bim phosphorylation
also played an important role in regulating Bim expression. Moreover, up-regulation of
Bim facilitates the translocation of Bax to mitochondrial membrane, which lead to
cytochrome c release and apoptosis. We also silenced Bim using siRNA to further
investigate the essential role of Bim in mutant Htt induced ER stress and apoptosis.
Identifying the Bim pathway that is altered in response to the mutant Htt protein is
important for understanding the cellular processes impacted by the disease.

Cell penetrating peptides (CPPs) are short sequences of amino acids that excel in
crossing the cellular membrane without inducing cytotoxicity Interest in these peptides
stem from their ability to be attached, and grant their penetrating properties to, a variety
of cargo In this work we have combined the application of Confocal Raman Microscopy
(CRM) and Atomic Force Microscopy for the first time to examine the interactions of
unlabeled Transportan (TP), one of the most well studied CPPs, with mammalian cells
CRM’s capability to discriminate control and treated cell groups was verified by principal
component analysis (PCA) and linear discriminant analysis (LDA) and was 93-100%
accurate We’ve determined that at a concentration of 20 μM TP enters cells through a
non-endocytotic mechanism, has a high affinity for the cytoplasm and membranes, and
results in a significant increase in cellular stiffness Our work provides the first direct
evidence of this cell-stiffening phenomenon SFTI-1, the smallest member of a bicyclic, cysteine rich class of CPPs, was
examined by CRM to determine the potential role of cyclic structure on cellular uptake
The peptide, along with monocyclic and linear analogs was heavy isotope labeled and
incubated with mammalian cells at numerous concentrations and timespans Our work is
the first SFTI-1 uptake study forgoing the use of fluorophore conjugates, which have
been linked to artificial cellular uptake We demonstrate herein the absence of any CRM
detectable uptake, providing the first evidence that SFTI-1 may not be a CPP
Finally, CRM was applied to the discrimination of normal and basal cell
carcinoma cells obtained from the same donor The use of patient matched cells avoids
the normal biochemical variations that exist among individuals, ensuring that
discrimination is based solely on the cell’s diseased state CRM spectra, analyzed by
PCA and LDA, were capable of spectral discrimination with 100% accuracy Major
differences in the cancerous cells were an increase in lipids and nucleic acids, and an
overall decrease in protein We also demonstrate an enhancement in Raman signal
through the use of an aluminum foil substrate, providing a practical approach for
measuring cells with thin morphologies

Solid tumors can hijack many of the same programs used in neurogenesis
to enhance tumor growth and metastasis, thereby generating a plethora of
neurogenesis-related molecules including semaphorins Among them, we have
identified Semaphorin7A (SEMA7A) in breast cancer We first used to the DA-3
mammary tumor model to determine the effect of tumor-derived SEMA7A on
immune cells We found that tumor-derived SEMA7A can modulate the
production of proangiogenic chemokines CXCL2/MIP-2 and CXCL 1, and prometastatic
MMP-9 in macrophages We next aimed to determine the expression
and function of SEMA7A in mammary tumor cells We found that SEMA7A is
highly expressed in both metastatic human and murine breast cancer cells We
show that both TGF-β and hypoxia elicits the production of SEMA 7 A in mammary
cells SEMA7 A shRNA silencing in 4T1 cells resulted in decreased mesenchymal
markers MMP-3, MMP-13, Vimentin and TGF-β) SEMA7A silenced cells show increased stiffness with reduced migratory and proliferative potential In vivo,
SEMA7A silenced 4T1 tumor bearing mice showed decreased tumor growth and
metastasis Genetic ablation of host-derived SEMA7A synergized to further
decrease the growth and metastasis of 4T1 cells Our findings suggest novel
functional roles for SEMA7A in breast cancer and that SEMA7A could be a novel
therapeutic target to limit tumor growth and metastasis

This study determined the impact of vitamin C dose on genistein-induced apoptosis in LNCaP cancer cells at various treatment regimens in vitro. Although the linear regression of viability assay (MTT) indicated a p-value = 0.11; NBT assay reveal a declining SOD activity during cell death. Apoptosis induction was the main mode of treatment induced cell death. The overall data showed the trend of treatment efficacy as;(Gen 10uM + Vit C 40uM) > (Gen 30uM + Vit C 40uM) > (Gen 70uM + Vit C 40uM) > 10uM genistein > 70uM genistein. The chi-square test for comparing necrosis, apoptosis and life cells showed that Vitamin C could impact genistein-induced apoptosis in LNCaP cells (p = 0.0003). This study forms the basis for in vivo studies of the impact of vitamin C on genistein-induced apoptosis in LNCaP prostate cancer cells.

Small conductance Ca2+-activated K+ (SK) channels have been shown to alter the encoding of spatial and non-spatial memory in the hippocampus by shaping glutamatergic postsynaptic potentials and modulating NMDA receptor-dependent synaptic plasticity. When activated, dendritic SK channels reduce hippocampal neuronal excitability and LTP. Similar SK channel properties have been demonstrated in lateral amygdala (LA) pyramidal neurons. Additionally, induction of synaptic plasticity and beta-adrenoreceptor activation in LA pyramidal neurons causes PKA-mediated internalization of SK channels from the postsynaptic density. Chronic activation of the amygdala through repetitive stressful stimuli can lead to excitatory synaptic strengthening that may create permanent hyper-excitability in its circuitry. This mechanism may contribute to a number of mood and anxiety disorders. The selective influence of SK channels in the LA on anxiety and fear conditioning are not known. The thesis project outlined herein examined whether SK channel blockade by bee venom peptide, apamin, during a repetitive acute fear conditioning paradigm was sufficient to alter fear memory encoding and the resulting behavioral outcome. Following the final fear memory test session, mice were tested in the open field immediately after the second fear conditioning test session. The findings indicate that intracranial LA microinfusions of apamin did not affect memory encoding or subsequent anxiety.

Transcription and translation of proteins are required for the consolidation of episodic memory. Arc, an effector immediate early gene, has been linked to synaptic plasticity following learning and memory. It is well established that the rodent hippocampus is essential for processing spatial memory, but its role in processing object memory is a point of contention. Using immunohistochemical techniques, hippocampal sections were stained for arc proteins in the CA1 region of the dorsal hippocampus in mice following two variations of the novel object recognition (NOR) task. Results suggest mice that acquired strong object memory showed significant hippocampal activation. In mice that acquired weak object memory, hippocampal activation was not significantly different from controls. Arc expression was also examined in other hippocampal sub-regions, as well as in the perirhinal cortex. These results suggest that the mice must acquire a threshold amount of object information before the hippocampal CA1 region is engaged.

Oxidative stress is considered a major factor in the etiology of age related diseases and the aging process itself. Organisms have developed mechanisms to protect against oxidative damage resulting from increased production of reactive oxygen species during aging. One of the major antioxidant systems is the methionine sulfoxide reductase (Msr) enzyme family. The two major Msr enzymes, MsrA and MsrB, can stereospecifically reduce the S and R epimers, respectively, of methionine sulfoxide in proteins back to methionine. This study, using Drosophila melanogaster, decribes the first animal system lacking both MsrA and MsrB. The loss of either MsrA or MsrB had no effect on lifespan in Drosophila, but loss of MsrB results in a slight decrease in locomotor activity from middle age onward. Double mutants lacking both forms of Msr have a significantly decreased lifespan and decreased locomotor activity at all ages examined. The double Msr mutants had no detectable increase in protein oxidation or decrease in mitochondrial function and were not more sensitive to oxidative stress. These results suggested that other cellular antioxidant systems were protecting the flies against oxidative damage and the decreased life span observed in the double knockouts was not due to widespread oxidative damage. However, one cannot exclude limited oxidative damage to a specific locus or cell type. In this regard, it was observed that older animals, lacking both MsrA and MsrB, have significantly reduced levels of dopamine, suggesting there might be oxidative damage to the dopaminergic neurons. Preliminary results also suggest that the ratio of F to G actin is skewed towards G actin in all mutants. The present results could have relevance to the loss of dopaminergic neurons in Parkinson’s disease.

The purpose of this study was to examine the acute and temporal response of CK- MM and FGF-21 to 3-day/wk. different repetition-range, volume-equated resistance training programs over 8-weeks in previously trained males. Sixteen trained, college- aged males were counterbalanced into high (DUP-HR) or low (DUP-LR) repetition groups. Subjects performed the squat and bench press 3x/wk. for 8 weeks. Blood samples were collected at various intervals throughout the study. Trained individuals did not elicit significant acute or chronic changes in CK-MM or FGF-21 following training and the lack of change was present in both groups. Additionally, neither biomarker correlated with changes in 1RM strength. There was a very strong correlation between acute mean (r=0.95) and acute percentage change (r=0.97) increase from pre training to post training in week #1. Additionally, a moderate correlation in percentage change was observed (r=0.59) of both biomarkers from pre training to 48 hours post training in week #2.