Cellular control mechanisms.

The primary purpose of this study was to examine the impact of acute highintensity
interval Exercise (HIIE) on plasma cfDNA and IL-6 responses in obese and
normal-weight subjects. Fifteen subjects (8 obese and 7 normal-weight) were recruited to
participate in an acute HIIE protocol. Our results demonstrated a significant elevation
across time in plasma cfDNA and IL-6 immediately following acute HIIE, with no
difference between obese and normal-weight subjects. Furthermore, cfDNA was not
correlated with IL-6 in response to acute HIIE in either group. These findings indicate
that the obese state does not further exacerbate the release of acute HIIE-induced
inflammatory mediators (cfDNA and IL-6), which suggests that HIIE training may serve
as a time-effective exercise strategy to improve obesity-associated inflammation.

In the developing CNS, presynaptic neurons often have exuberant overgrowth and
form excess (and overlapping) postsynaptic connections. Importantly, these excess
connections are refined during circuit maturation so that only the appropriate connections
remain. This synaptic rearrangement phenomenon has been studied extensively in
vertebrates but many of those models involve complex neuronal circuits with multiple
presynaptic inputs and postsynaptic outputs. Using a simple escape circuit in Drosophila
melanogaster (the giant fiber circuit), we developed tools that enabled us to study the
molecular development of this circuit; which consists of a bilaterally symmetrical pair of
presynaptic interneurons and postsynaptic motorneurons. In the adult circuit, each
presynaptic interneuron (giant fiber) forms a single connection with the ipsilateral,
postsynaptic motorneuron (TTMn). Using new tools that we developed we labeled both
giant fibers throughout their development and saw that these neurons overgrew their targets and formed overlapping connections. As the circuit matured, giant fibers pruned
their terminals and refined their connectivity such that only a single postsynaptic
connection remained with the ipsilateral target. Furthermore, if we ablated one of the two
giant fibers during development in wildtype animals, the remaining giant fiber often
retained excess connections with the contralateral target that persisted into adulthood.
After demonstrating that the giant fiber circuit was suitable to study synaptic
rearrangement, we investigated two proteins that might mediate this process. First, we
were able to prevent giant fibers from refining their connectivity by knocking out
highwire, a ubiquitin ligase that prevented pruning. Second, we investigated whether
overexpressing Netrin (or Frazzled), part of a canonical axon guidance system, would
affect the refinement of giant fiber connectivity. We found that overexpressing Netrin (or
Frazzled) pre- & postsynaptically resulted in some giant fibers forming or retaining
excess connections, while exclusively presynaptic (or postsynaptic) expression of either
protein had no effect. We further showed that by simultaneously reducing (Slit-Robo)
midline repulsion and elevating Netrin (or Frazzled) pre- & postsynaptically, we
significantly enhanced the proportion of giant fibers that formed excess connections. Our
findings suggest that Netrin-Frazzled and Slit-Robo signaling play a significant role in
refining synaptic circuits and shaping giant fiber circuit connectivity.

The vertebrate eye lens functions to focus light onto the retina to produce vision.
The lens is composed of an anterior monolayer of cuboidal epithelial cells that overlie a
core of organelle free fiber cells. The lens develops and grows throughout life by the
successive layering of lens fiber cells via their differentiation from lens epithelial cells.
Lens developmental defect and damage to the lens are associated with cataract formation,
an opacity of the lens that is a leading cause of visual impairment worldwide. The only
treatment to date for cataract is by surgery. Elucidating those molecules and mechanisms
that regulate the development and lifelong protection of the lens is critical toward the
development of future therapies to prevent or treat cataract. To determine those
molecules and mechanisms that may be important for these lens requirements we
employed high-throughput RNA sequencing of microdissected differentiation statespecific
lens cells to identify an extensive range of transcripts encoding proteins expressed by these functionally distinct cell types. Using this data, we identified
differentiation state-specific molecules that regulate mitochondrial populations between
lens epithelial cells that require the maintenance of a functional population of
mitochondria and lens fiber cells that must eliminate their mitochondria for their
maturation. In addition, we discovered a novel mechanism for how lens epithelial cells
clear apoptotic cell debris that could arise from damage to the lens and found that UVlight
likely compromises this system. Moreover, the data herein provide a framework to
determine novel lens cell differentiation state-specific mechanisms. Future studies are
required to determine the requirements of the identified molecules and mechanisms
during lens development, lens defense against damage, and cataract formation.

Establishing appropriate animal models for the study of human memory is
paramount to the development of memory disorder treatments. Damage to the
hippocampus, a medial temporal lobe brain structure, has been implicated in the memory
loss associated with Alzheimer’s disease and other dementias. In humans, the role of the
hippocampus is largely defined; yet, its role in rodents is much less clear due to
conflicting findings. To investigate these discrepancies, an extensive review of the rodent
literature was conducted, with a focus on studies that used the Novel Object Recognition
(NOR) paradigm for testing. The total amount of time the objects were explored during
training and the delay imposed between training and testing seemed to determine
hippocampal recruitment in rodents. Male C57BL/6J mice were implanted with bilateral
dorsal CA1 guide cannulae to allow for the inactivation of the hippocampus at discrete
time points in the task. The results suggest that the rodent hippocampus is crucial to the
encoding, consolidation and retrieval of object memory. Next, it was determined that there is a delay-dependent involvement of the hippocampus in object memory, implying
that other structures may be supporting the memory prior to the recruitment of
hippocampus. In addition, when the context memory and object memory could be further
dissociated, by altering the task design, the results imply a necessary role for the
hippocampus in the object memory, irrespective of context. Also, making the task more
perceptually demanding, by requiring the mice to perform a two-dimensional to three-dimensional
association between stimuli, engaged the hippocampus. Then, in the
traditional NOR task, long and short training exploration times were imposed to
determine brain region activity for weak and strong object memory. The inactivation and
immunohistochemistry findings imply weak object memory is perirhinal cortex
dependent, while strong object memory is hippocampal-dependent. Taken together, the
findings suggest that mice, like humans, process object memory on a continuum from
weak to strong, recruiting the hippocampus conditionally for strong familiarity.
Confirming this functional similarity between the rodent and human object memory
systems could be beneficial for future studies investigating memory disorders.

The lens is a crystallin tissue of the anterior part of the eye that focuses light onto the
retina. Aged-related cataract, which is the result of loss of lens transparency, is the most
common cause of blindness in the world. Being constantly exposed to UV-light, lens is
significantly affected by its UVA spectrum. UV-light exposure has been shown to result
in apoptosis of lens cells which can lead to cataract formation. This suggests the need for
molecular mechanisms to remove apoptotic debris from the lens. In the set of
experiments it was proven that integrin αvβ5-mediated pathway is involved in
phagocytosis of apoptotic cell debris in the ocular lens, thus contributing to its
homeostasis. Additionally, it was shown that exposure to UV-light plays role in cataract
formation by influencing integrin αvβ5-mediated phagocytosis function.

Changes in synaptic strength underlie synaptic plasticity, the cellular substrate for learning and memory. Disruptions in the mechanisms that regulate synaptic strength closely link to many developmental, neurodegenerative and neurological disorders. Release site probability (PAZ) and active zone number (N) are two important presynaptic determinants of synaptic strength; yet, little is known about the processes that establish the balance between N and PAZ at any synapse. Furthermore, it is not known how PAZ and N are rebalanced during synaptic homeostasis to accomplish circuit stability. To address this knowledge gap, we adapted a neurophysiological experimental system consisting of two functionally differentiated glutamatergic motor neurons (MNs) innervating the same target. Average PAZ varied between nerve terminals, motivating us to explore benefits for high and low PAZ, respectively. We speculated that high PAZ confers high-energy efficiency. To test the hypothesis, electrophysiological and ultrastructural measurements were made. The terminal with the highest PAZ released more neurotransmitter but it did so with the least total energetic cost. An analytical model was built to further explore functional and structural aspects in optimizing energy efficiency. The model supported that energy efficiency optimization requires high PAZ. However, terminals with low PAZ were better able to sustain neurotransmitter release. We suggest that tension between energy efficiency and stamina sets PAZ and thus determines synaptic strength. To test the hypothesis that nerve terminals regulate PAZ rather than N to maintain synaptic strength, we induced sustained synaptic homeostasis at the nerve terminals. Ca2+ imaging revealed that terminals of the MN innervating only one muscle fiber utilized greater Ca2+ influx to achieve compensatory neurotransmitter release. In contrast, morphological measurements revealed that terminals of the MN inner vating multiple postsynaptic targets utilized an increase in N to achieve compensatory neurotransmitter release, but this only occurred at the terminal of the affected postsynaptic target. In conclusion, this dissertation provides several novel insights into a prominent question in neuroscience: how is synaptic strength established and maintained. The work indicates that tension exists between energy efficiency and stamina in neurotransmitter release likely influences PAZ. Furthermore, PAZ and N are rebalanced differently between terminals during synaptic homeostasis.