Kirchman, Paul A.

Lithium (Li) is used in many commercial products. With the recent development of a
potent new lithium battery suitable for hybrid and plug-in electric cars, demand for
lithium should soar. Seawater has low Li ion concentrations, and has not been considered
a profitable commercial source of this element. For my thesis work, I attempted to
develop a mutant strain of the marine bacteria Photobacterium damselae subsp. damselae
capable of sequestering lithium from seawater. I expose the bacteria to shortwave ultra
violet (UV) light with the intent of obtaining a lithium dependent mutant. I was not
successful at isolating such a mutant. However, I noted differences in colony size,
between colonies grown in lithium media and sodium media, after UV treatment. Based
on other research, the bacterial recovery methods of lithium and other metals from
seawater remains a plausible option.

Oxidative stress occurs when reactive oxygen species (ROS), such as superoxide anion (O2' —), hydroxyl radical (HO.), and hydrogen peroxide (H2O2), build up to
detrimental levels in the cell, and effects of oxidative stress are associated with aging.
Mitochondria are the main site of oxidative stress, most likely due to the high level of
ROS produced during respiration. Mitochondrial aconitase is an enzyme involved in
respiration; due to the presence of an iron-sulfur cluster in the active site, it is a target for ROS. A plasmid containing the aconitase gene to be inserted into Saccharomyces
cerevisiae was designed, and will be used in the future for the pop-in pop-out method;
mitochondrial aconitase will be subjected to point mutations, replacing several leucine
and isoleucine amino acid residues with methionine residues near the active site and on
the surface of the enzyme. The amino acid methionine has a sulfur group, which also acts as a target for ROS and could prevent inactivation of the iron-sulfur cluster of aconitase.

Oxidative stress from reactive oxygen species (ROS) is hypothesized to be a primary cause of aging and aging-related diseases. For my thesis research, my advisor and I examined adaptation to oxidative stress in the red-eared slider, Trachemys scripta elegans, a turtle that goes long periods with low oxygen during hibernation, but then quickly re-acclimates to higher oxygen levels upon surfacing. This dramatic ischemia/reperfusion event should result in ROS formation, but the turtle appears to reduce oxidative stress. We aimed to isolate the gene for methionine sulfoxide reductase B3, a protein that alleviates oxidative stress by reconverting oxygen damaged methionine-R-sulfoxide to the amino acid methionine. We designed degenerate primers to amplify a conserved region of the MSRB gene to isolate a partial DNA sequence. We then used 5’ RACE PCR and vectorette library PCR to isolate much of the coding region of the gene from T. scripta.

T-lymphocytes develop from bone marrow derived hematopoietic stem cells (HSCs) and
mature in the thymus, where they participate in reciprocal signaling with thymic stromal
cells. The thymic developmental stages are well characterized, but only a few intrathymic
signals that influence the development of T-lymphocytes have been identified. Previous
microarray experiments revealed interleukin-17A (IL-17A) and its receptor (IL-17RA) as
a possible stromal-lymphoid signal. In this study, an IL-17RA-/- knockout was used to
determine whether the IL-17RA gene has a role in T-lymphocyte maturation. We made
competitive bone marrow chimeras and analyzed the percentage of donor wildtype and
mutant HSCs present in the bone marrow, and compared it to the percentage of a
particular blood cell type that developed from these donor HSCs. We found that IL-17RA
influences the maturation of T-lymphocytes, but does not affect the development of other
immune cells such as B-lymphocytes, macrophages, and granulocytes.

Previously isolated mutants of the superoxide dismutase 2 gene (SOD2), selected
for increased activity in E. coli, were transformed into yeast cells in order to observe their
effects on aging. Polymerase chain reaction and DNA sequencing were used to confirm
that the yeast incorporated the mutant form of the gene. The strains with mutant forms of
the SOD2 gene were observed to have decreased growth rates compared to the unmutated
strain. Lifespan analyses were then conducted in order to see if yeast with
mutant versions of SOD2 had lifespans that differed significantly from those with the unmutated
forms of the gene. The mutant forms of SOD2 had no significant effect on the
lifespan of yeast.

Reactive oxygen species (ROS) are molecular oxygen-derived molecules
that are exceedingly reactive, often generated as free radical bi-products of
mitochondrial respiration, which cause oxidative stress that leads to aging. To
avoid the generation of ROS, aerobic organisms have antioxidant defense
mechanisms that use enzymes such as Superoxide Dismutase (SOD) to convert
the superoxide radicals into hydrogen peroxide and oxygen. Manganesecontaining
SOD (Mn-SOD), a product of the SOD2 gene is found in all eukaryotic
organisms in the mitochondrial matrix, including T. scripta (red-eared slider
turtle). Extraordinarily, T. scripta can live without oxygen for long periods of
time without experiencing adverse effects when oxygen is again available. The
purpose of our experiment is to clone the T. scripta SOD2 gene, and test its
activity. A partial fragment of the gene has been isolated by screening the cDNA
library, and we are currently working on finding the remaining sequence.