Charles E. Schmidt College of Medicine

The lens is responsible for focusing light into the retina. It accomplishes this through its maturation from an epithelial cell into a fiber cell. A large amount of research has been done on cellular differentiation. Nevertheless, we still lack knowledge on many different aspects of differentiation, including a complete theory on the mechanism behind differentiation. Due to the lens’ unique structure and cell types, this is an ideal model for studying differentiation. Our research has shown that αB crystallin, a small heat shock protein, is able to modulate cytochrome C levels and protect the mitochondria under oxidative stress. Also, cytochrome C release is often followed by caspase 3 activation. In addition, research has shown that low levels of caspase 3 activation is essential in driving differentiation. My work examined if αB crystallin could modulate cytochrome C to lower caspase 3 levels to allow for differentiation rather than apoptosis.

αB-crystallin is a small heat-shock chaperone protein (sHSP) required for the homeostasis of multiple tissues including eye lens, retina, heart and brain. Correspondingly, mutation or altered levels of αB-crystallin are associated with multiple degenerative diseases including cataract, retinal degeneration, cardiomyopathy and Lewy body disease. Based on its wide-ranging importance understanding the protective and homeostatic properties of α B-crystallin is critical for understanding degenerative diseases and could lead to the development of therapies to treat these diseases. αB-crystallin is localized to the mitochondria suggesting a direct effect on mitochondrial function. My thesis work has examined those molecular pathways required for translocation of αB-crystallin to the mitochondria and to identify the downstream pathways controlled by mitochondrial translocation of αB-crystallin that could be important for cellular protection and differentiation. My results point to a novel role of αB-crystallin in regulation of key apoptotic pathways that mediate the balance between cell survival and differentiation.

Alzheimer’s disease (AD) has been defined as a type of dementia that causes
problems with memory, thinking, and behavior. AD is characterized by tau tangles and
Aβ plaques in and around neurons, respectively. The impact this disease has on its
victims’ health, both physically and mentally, is unimaginable and the rate of progression
is not expected to decrease any time soon. This threat to our minds encourages the
importance of understanding AD. Amongst the theories as to what bio mechanisms cause
the brain to intertwine is the amyloid cascade hypothesis. The purpose of this thesis is to
review the amyloid cascade hypothesis and discuss treatments which utilize this model.
We also wish to examine social aspects such as loneliness and socioeconomic factors
which are associated with the progression of AD. Research presented provides evidence
that targeting the accumulation of Aβ in the brain will prevent further biochemical
responses to form neurodegenerative pathology. From the collected data, we observe that
therapies targeting the amyloidogenic pathway have received positive feedback in the
medical community. Amongst them, an Aβ synthetic peptide vaccine which made history
in vaccine development due to their responder rate. The impact of social factors such as
loneliness in the advancement of AD is also supported by research. While it is
acknowledged that any neurodegenerative disease is far too complex to narrow its cause
specifically, this thesis provides an association with multiple aspects that can be
understood and applied to future research in this field.

Drosophila melanogaster tolerates several hours of anoxia (the absence of
oxygen) by entering a protective coma. A burst of reactive oxygen species (ROS) is
produced when oxygen is reintroduced to the cells. ROS causes oxidative damage to
critical cellular molecules, which contribute to aging and development of certain agerelated
conditions. The amino acid, methionine, is susceptible to oxidation, although this
damage can be reversed by methionine sulfoxide reductases (Msr). This project
investigates the effect of Msr-deficiency on anoxia tolerance in Drosophila throughout
the lifespan of the animal. The data show that the time for recovery from the
protective comma as well as the survival of the animals lacking any Msr activity
depends on how quickly the coma is induced by the anoxic conditions. Insight into
the roles(s) of Msr genes under anoxic stress can lead us to a path of designing
therapeutic drugs around these genes in relation to stroke.

Alterations in activities of one family of proteases, the metzincins have been implicated
in an array of physiological and pathological processes. In the present study, metzincin
inhibitors were developed by utilizing topologically constrained peptides and
pseudopeptides. The endothelin-family framework was used to develop a disulfideconstrained
topology. This framework was chosen due to its three-dimensional similarity
with a family of endogenous metzincin inhibitors, the tissue inhibitors of
metalloproteases (TIMPs). The collagenous triple-helix was chosen as a second
framework, because only a subset of proteolytic enzymes have the capacity to bind and
hydrolyze a triple-helix. Both templates were successfully modified to generate an array
of inhibitors. These inhibitors displayed subnanomolar to micromolar apparent Ki values,
while being moderately selective metzincin inhibitors. In both cases the threedimensional
structure was determined to be important for activity. This work encourages
the further development of both frameworks as metzincin inhibitors.

The Mexican axolotl (Ambystoma mexicanum) carries a cardiac lethal mutation
resulting in mutant embryos with no heartbeat. This homozygous recessive gene results
in tropomyosin deficiency and absence of organized myofibrils. Co-culturing mutant
hearts with bioactive RNA, termed myofibril-inducing RNA (MIR), from normal axolotl
embryonic anterior endoderm causes the mutant hearts to beat. It is hypothesized that the
secondary structure of the MIR binds a specific protein(s) and this is required to
synthesize tropomyosin and form organized myofibrils. In this study mutant hearts are
co-cultured with human fetal and adult heart total RNA to assess rescue of the mutant
hearts. Results show that both human fetal and adult heart total RNA rescue the mutant
condition in a manner similar to the MIR. Thus, the MIR human functional homologs
induce events leading to normal heart differentiation and function. This finding may help
people with heart muscle damage regain normal heart function again.

tmRNA is a small stable RNA present in Eubacteria. Through a mechanism called
trans-translation, tmRNA mediates ribosome rescue and quality control of proteins and
mRNA. In this study, the Escherichia coli (E. coli) mutant lacking tmRNA was
demonstrated hypersensitive to oxidative stress. The role of tmRNA-mediated
surveillance mechanism in protecting E. coli cell under oxidative stress condition was
examined. The tmRNA-mediated tagged protein levels were elevated in cells under
oxidative stress condition, demonstrating the enhanced need for tmRNA under such
condition. Our results suggest that mRNA damage by oxidative stress may cause reduced
cell viability, and that tmRNA is required to rescue cells under such condition.
Furthermore, our observations showed that tmRNA is required for the optimal growth of
E. coli under normal aeration but not under anaerobic condition, suggesting that oxidation
ofmRNA is the major reason for requirement oftmRNA during normal aeration.

Troponin I is a contractile protein and plays an important role in cardiac function.
We have generated cTnl knockout and cTnI(R192H) transgenic mouse models. All of
cTnl knockout homozygous mice die at 17-18 days after birth. Some of cTnI(R192H)
transgenic mice die at early life stages, some mice develop heart failure at late stages.
High-resolution ultrasound imaging and Doppler echocardiography have been used to
evaluate cardiac function on cTnl deficient mice and cTnl(R192H) transgenic mice.
cTnI mice have damaged relaxation with gradually decreased E/A ratio(E/A<1). FS
and cardiac output dramatically decrease on 17-day-o1d cTnI mice indicating severe
cardiac dysfunction. We find that the damaged heart function is correspondent with the
Tnl expression level decline. 6-8 weeks transgenic mice have shown that the dimension
of left and right atria increase. In 15-month-old transgenic mice, the E/A ratio shows a
pseudonormal pattern indicating a diastolic dysfunction. This study demonstrate that
damaged heart function is tightly associated with Tnl levels in the heart.

Chitin Microparticles (CMPs, 1-10um), a special form of the ubiquitous and nontoxic
polysaccharide Chitin (GlcNAc), is capable of inducing a switch in macrophages
from the wound-healing M2 phenotype to the classically activated pro-inflammatory M1
phenotype; which has therapeutic implications in allergy and cancer. We hypothesized
that TLR2 forms a complex with CMPs and Chitin-Binding Proteins (CBPs) at the
surface of peritoneal macrophages and remains with that complex after internalization to
initiate downstream signaling events, leading to the production of the M1 cytokine, TNFalpha.
Our results from experiments performed in RAW 264.7 cells show that TLR2 and
TLR1, but not TLR6, are associated with the CMP binding fraction, and that both TLR1
and TLR2 might be important for M1 activation as a result of CMP phagocytosis. This
project sheds light on CMP as a potential therapeutic agent and provides more evidence
for a phagocytosis-dependent TLR2 signaling pathway.