The Analysis of Bovine Rhodopsin Cycling in Drosophila

Ciara C. Reyes, Valparaiso University, Dr. Alex Kiselev, University of Notre Dame, Dr. Joseph O’Tousa*, University of Notre Dame

Rhodopsins of both Drosophila and vertebrate photoreceptor cells are light sensor molecules that

are central to the process of vision. The rhodopsin molecule is converted to metarhodopsin by a

photon of light to activate the light response. Metarhodopsin is then rapidly phosphorylated by

rhodopsin kinase and bound by arrestin to terminate the light response. To complete the

Drosophila rhodopsin cycle, a photon of light converts metarhodopsin back to rhodopsin,

thereby releasing arrestin and allowing the RdgC rhodopsin phosphatase to dephosphorylate

rhodopsin. In mutants lacking RdgC, phosphorylated rhodopsin accumulates in the photoreceptor

and triggers retinal degeneration. Previous studies on rdgC mutants have investigated its action

the native Drosophila rhodopsin (Rh1). The goal of the study reported here is to investigate the

behavior of a vertebrate rhodopsin (Rho) when expressed in Drosophila photoreceptors. The

study made use of a Drosophila transgenic strain expressing the vertebrate Rho. In addition, two

transgenic strains carrying chimeric rhodopsins in which the C-terminal tails, the site of

phosphorylation, are exchanged between Drosophila and the vertebrate rhodopsin. Genetic

crosses were used to generate these transgenic strains in rdgC+ and rdgC mutant backgrounds.

Protein blots were used to test for the presence of rdgC protein in these strains, and pseudopupil

assays were used to look for retinal degeneration triggered by the presence of the rdgC mutation.

The fly strains generated from this experiment will be exposed to increasing increments of light.

Rhodopsin concentration and phosphorylation status, as well progression of retinal degeneration,

will then be monitored to understand the behavior of the vertebrate rhodopsins in Drosophila

photoreceptors.

Eric Hawley, Abhishek Tripathy, Rory Carmichael, Scott Emrich, and Kristin Hager

DePauw University and Dept. of Biological Sciences and Computer Sciences, University of

Notre Dame

Toxoplasma gondii is a protozoan parasite that, along with its apicomplexan relatives such as

Plasmodium and Theileria, poses a significant global health hazard. Apicomplexans are named

as such because they contain a number of unique secretory organelles including a plastid like

complex called the apicoplast. Toxoplasma gondii not only contains a traditional endoplasmic

reticulum retention deficiency (ERD) receptor, but also possesses a novel group of ERD2 related

receptors called plastid associated ERD-like proteins (PERLs). In T. gondii, the PERLs are

structurally similar to TgERD2, which functions in retrograde transport of proteins from the

Golgi back to the ER.  Yet, the PERLs do not appear to bind classical ERD-ligands with the

same efficacy. We hypothesized that the PERLs recognize a related but slightly different motif

that occurs in proteins bound for the apicoplast. Thus far our hypothesis has been supported by

both a phylogenetic analysis of three protein families and the use of profile hidden Markov

models. We have identified C-terminal motifs common across apicomplexa and rare among

metazoa using these techniques. As a secondary project we also investigated gene expansion

across the three strains of T. gondii. We hypothesized that gene expansion is negatively

correlated to virulence. We have found statistically significant differences in the gene expansion

rates between the three strains. Although the rates of genome wide expansion do not directly

correlate with virulence we have broken the data down into gene families and we believe our

hypothesis will hold true for gene families important for survival and infection.

Analysis of M2(H37A) Cytotoxic Activity as a Tool for Drosophila Cell Ablation Technology

Donna M. Cerabona, Victoria K. Lam, and Robert A. Schulz

Dept. of Biological Sciences, University of Notre Dame

Cell-specific ablation is a useful method to study cell function and cell-cell interdependency by

selectively destroying a small population of cells while leaving the rest of the tissues

undisturbed.  Many genes and cell signaling pathways studied in Drosophila are conserved in

humans, thus making Drosophila an ideal model organism for developmental studies.  A novel

cell ablation system using the M2(H37A) toxic viral protein has been created for use in

Drosophila.  In order to utilize this protein for future studies in developmental biology, its

function must be completely characterized and compared with current ablation technology.  This

study analyzed the timing, specificity and mechanism of M2(H37A)-induced cell death.  The

GAL4-UAS system was used to localize toxic protein expression to specific cell populations. 

M2(H37A)-induced cell death demonstrated specificity when driven in lamellocytes by MsnF9

GAL4.  HAND GAL4 localized toxic protein activity to the heart cells, where ablation occurred

by late 2nd instar of Drosophila larval development.  For each experiment, comparative studies

of this novel system were performed using reaper (UAS rpr), a suicide gene, which causes cell-

specific ablation through apoptosis when expressed.  Characterization of this novel system will

be useful in implementing this tool in future research to map Drosophila developmental

signaling pathways.

Ribozyme Driven Inhibition of The Hepatitis C Virus Replication Mechanism in Mammalian cells

Rafael Ramirez, Mark Fraser, David Yang, and Malcolm J. Fraser Jr.

Universidad del Sagrado Corazon and Dept. of Biological Sciences, University of Notre Dame

Hepatitis C Virus is the major causative agent of chronic hepatitis worldwide, constituting a

threat to public health. Hepatitis C Virus is a single stranded, positive, small and enveloped virus

belonging to the family of the Flaviviridae. Targeting the HCV genome with a ribozyme based

Group I Intron at the IRES region can provide inhibition of viral replication for ten of the 163

quasispecies of the virus (Ryu et al, 2003). Our research is developing a strategy based of the

functionality of a ribozyme based Group I Intron to bind and splice at a specific site in the 5

Untranslated region of the HCV genome that is conserved among all 163 quasispecies in order to

inactivate the viral replicating mechanism. The Introns will be tested in Hep2 cultured liver cells

that express a firefly luciferase reporter RNA containing the viral target sequence, and infectious

viral RNA. Once the introns are proven effective against the target RNA, the intron will be

coupled with an apoptosi  s-inducing tBax gene so that successful splicing produces an apoptotic

gene product, killing the cell. Expected results include the demonstration that the actual

ribozyme mechanism works for all 163 quasispecies by inhibiting the replication of viral RNA

through induced apoptosis via cell cytotoxicity. If successful results are demonstrated, research

would move on to testing the introns in transformed Hep2 cells expressing the Anti-HCV Intron,

and then move into humanized immunocompromised mouse models. 

Unraveling signaling pathways during epithelial glandular disruption

Andrew Warren, James Clancy, and Crislyn D'Souza-Schorey

John Hopkins University and Dept. of Biological Sciences, University of Notre Dame

Glandular epithelial organs are comprised of monolayers of adherent cells that enclose hollow

lumens. Many epithelial cancers are associated with disruption of normal epithelial glandular

architecture. The ability of epithelial cells to form cysts and tubules and adopt tissue-like

conformations when grown in a three-dimensional (3D) extracellular matrix has made 3D cell

culture a powerful approach to investigate the molecular basis of epithelial glandular

development as well as to identify molecular events that cause the disruption of these structures.

Here, using 3D epithelial cell cultures we show that constitutive activation of the protein, ARF6,

leads to marked disruption of epithelial cyst organization resulting in multiple lumens, luminal

filling and breakdown of cadherin-based cell-to-cell contacts, all of which are structural

perturbations observed in both early stage and invasive epithelial cancers. Pharmacological

inhibition of the MAP-kinase pathway, Phospholipase D (PLD) signaling and receptor

endocytosis all reverted the phenotype generated by constitutively active ARF6, while the PI3-

kinase inhibitor LY294002 had no effect.  These findings implicate MAP kinases, PLD and

receptor endocytosis in the generation of pathological cyst phenotypes downstream of ARF6.

Finally, we hypothesize that Rab5 activation is also affected in ARF6-GTP cysts.  To this end, a

pull-down assay to measure Rab5-GTP levels is being developed.  Cumulatively, these studies

contribute towards understanding the cellular mechanisms involved in epithelial glandular

disruption.  

Live-cell imaging of the delayed recruitment of LAMP1 in Leishmania infected murine macrophages

Melissa Harintho, Wibke Ballhorn, and Mary Ann McDowell

Dept. of Biological Sciences, University of Notre Dame

Leishmania are obligate intracellular parasites that infect and replicate inside hosts macrophages.

The manifestation of leishmaniasis is a direct result of the Leishmania species a person is

infected with, with the clinical forms ranging from self-healing cutaneous lesions at the site of

the sand fly bite to the potentially lethal dissemination of parasites to visceral organs such as the

liver, spleen, and bone marrow. A major question in the study of leishmaniasis is how

Leishmania engage macrophage receptors to delay phagosome maturation and avoid

proinflammatory responses and oxidative burst. Fixation and immunofluorescence of the

infection of bone-marrow derived macrophages from knockout mice revealed that MR, CR3, and

FcR receptors are necessary for Leishmania to delay phagosome maturation. However, previous

methods did not allow for the observation of receptor dimerization and cooperation, and were

restricted to the observation of single, static events. To develop a live-cell imaging technique to

visualize how Leishmania delay phagosome maturation and increase survival inside host

macrophages, we created a line of RAW 264.7 cells stably transfected with YFP-LAMP1.

LAMP1 is a visible indicator of the completion of the phagosome maturation process because it

distinguishes lysosomes containing digestive enzymes and cytotoxic chemicals from earlier

endosomes. Using real-time fluorescence imaging of phagosomes carrying Leishmania major

and L. donovani, we determined that parasite-infested phagosomes follow a uniform sequence of

LAMP-1 recruitment. Phagosome maturation in cells infected with Leishmania was delayed

compared with phagosomes harboring latex beads. 

Generation of a Cell Line to Investigate HD Pathogenesis

Samuel Lopez-Nieves, Andrew Warren, Joseph P. Krivda, and Cryslyn D’Souza-Schorey

University of Puerto Rico-Ponce and Dept. of Biological Sciences, University of Notre Dame

Huntington’s disease (HD) is a neurodegenerative, progressive and inherited disorder that affects

the physical and emotional life of a person.  The part of the brain most affected by this disease is

the striatum, composed by the putamen and caudate nucleus. An important protein in Huntington

Disease and striatal neurons is BDNF. These regions need brain derived neutrophic factor

(BDNF) for differentiation, proliferation and survival. Examination of patients with HD has

shown that a loss of BDNF correlates with the manifestation of the disease. The goal of this

project was to create a striatal neuronal cell line, ST14A stably expressing GFP-BDNF, with the

ultimate goal of assessing the trafficking and fate of BDNF in striatal neurons. Our efforts and

strategy to develop the cell line will be presented.  Further to examine the regulation of BDNF

trafficking along the endocytic pathway, we are generating recombinant virus expressing

dominantly activated and inhibitory forms of the Rab5 GTPase, which has been implicated in

early endosome fusion. 

The Role of the Vsx2 Transcription Factor In Zebrafish Retinal Regeneration

Jerry F. Shields, Travis J. Bailey, and David R. Hyde

Dept. of Biological Sciences, University of Notre Dame

The zebrafish (Danio rerio) is an organism with the ability to regenerate its retina, but the

specific genes involved in this process still need much study.  The gene vsx2, also known as

chx10, is a putative signal during retinal regeneration due to its requirement during retinal

development in Müller Glial cells, which are the retinal cells that differentiate to cause

regeneration.  Animals without vsx2 experience reduced proliferation of Müller Glial cells

during retinal development, resulting in microphthalmia (smallness of the eye).  As the role of

vsx2 in retinal development has been previously characterized, the goal of this study is to

determine the role of vsx2 in retinal regeneration.  Transgenic Vsx2 zebrafish, with the GFP

gene inserted after the Vsx2 promoter (Tg(chx10:EGFP) zebrafish) were given a Vsx2

morpholino to knock down Vsx2 expression.  Following light-induced degeneration of

Tg(chx10:EGFP) zebrafish retinas, fish with Vsx2 morpholino injections showed  reduced

Müller Glial cell division and proliferation during retinal regeneration as compared to

contralateral eyes.  This data suggests that during retinal regeneration, Vsx2 is required for full

regenerative potential.

Characterization of the Opsin 9 Protein in the Aedes aegypti mosquito

Michelle Stein, Michelle Whaley, and Joseph O'Tousa

Dept. of Biological Sciences, University of Notre Dame

Mosquitoes serve as vectors of many deadly human diseases, including malaria, dengue fever,

and yellow fever.  Mosquito behaviors, such as mating, oviposition and identification of food

sources, are largely dependent upon vision. However, the mosquito visual system has largely

remained uncharacterized.  My study proposes to characterize the opsin protein Op 9 in Aedes

aegypti by identifying express patterns across the retina as well as within the photoreceptor cells

of the compound eye. Using existing impure antibodies against the c-terminus end of the Op 9

protein, a series of western blots were completed to verify that the antibodies effectively

identified a protein of the correct size of approximately 35 kDa.  pET 32 Plasmids containing the

Op 9 insert were transformed and expression of the fusion protein was induced using IPTG. The

fusion protein was collected and purified using a His-Bind column. Op 9 was coupled to an

affinity chromatography column so that purified rabbit anti-op9 antibody could be generated.

The recovery of purified antibody was confirmed by a Coomaisse-stained gel showing protein

bands consistent with the antibody size. The purified antibody will be used in

immunohistochemistry experiments to determine the location of Op 9 expression in the

ommatidial units, likely in photoreceptor cells R1-6, as well as patterned Op 9 expression across

the retina. Cross-species comparison of expression will yield insight into disparate mosquito

ecologies.  

Testing routes and rates of pathogen transmission using agent based modeling

Noraliz Miranda, Kelly Lane, Agustin Fuentes, and Hope Hollocher

University of Puerto Rico-Aguadilla and Depts. of Biological Sciences and Anthropology,

University of Notre Dame

Infectious diseases are a common concern to our society and because of this; we developed an

agent-based model, LiNK, with a GIS interface that enables us to predict patterns of pathogen

transmission. We focused on parasite transmission dynamics among Balinese macaque (Macaca

fascicularis) populations located in a unique system of temples on the island of Bali, Indonesia.

By tracking macaque movement, the model allows us to input a variety of pathogen parameters

such as: virulence, infectivity, infectiousness, latency period and clearance time, which can help

us understand how different parasites will spread across the island. We hypothesized that

changing these pathogen parameters to higher levels will increase the number of infected

macaques. Also, we expected that a bigger temple population size will increase the amount of

infections. We tested our hypothesis by initiating infections in different temples across the island

taking into consideration population size and landscape surrounding them. This allowed us to

change pathogen parameters and observe the effect that each of them had on route and rate of

disease transmission. Our results confirmed that landscape type played a significant role in

infection success and that larger population sizes increased infections. Also, results showed that

virulence and infectivity increased the amount of infected macaques significantly; meanwhile

infectiousness didn’t cause any significant changes, it was clearly demonstrated that pathogen

parameter levels have a great impact in disease transmission

Multiple Invasion and Red Blood Cell Selectivity in Plasmodium falciparum

Walter J. Morales-Borrero, Mark Wacker, and Michael Ferdig

University of Puerto Rico-Ponce and Dept. of Biological Sciences, University of Notre Dame

Malaria, disease caused by protozoans parasites from the genus Plasmodium, has caused over a

million deaths worldwide. These parasites, once inside the bloodstream, rely on invading red

blood cells to survive and reproduce. Furthermore, more than one parasite often invades a single

red blood cell. It is known that at a higher parasitemia, the severity of the disease increases. The

objective of this research is to correlate the percentage of parasitemia with the number of

multiple invasions within blood cultures. We did this by growing different strains of Plasmodium

falciparum, the parasite that causes the most virulent form of Malaria, in 20mL flasks with

complete media until reaching the wanted parasitemia. After this, we generated three 5mL

cultures and waited until the next day to calculate the ending parasitemia and the ratio of single

invaded cells to multiple invaded cells. Also, we used the same method to analyze mixed

cultures. The results showed a decrease in multiple invasions in the mixed cultures at higher

parasitemias. On the other hand, the single parasite cultures showed an increase in multiple

invasions as parasitemia increased. We believe that competition between parasites may play a

crucial role. However, at the present time we do not fully understand the mechanisms behind

these events. 

Role of ID2 (Inhibitor of DNA binding 2) in the Mammalian Circadian Clock

Shanik J. Fernando, Sarah M. Ward, Tim Y. Hou, and Giles E. Duffield

Dept. of Biological Sciences, University of Notre Dame

Circadian rhythms are an essential component of life, which regulate many physiological,

biochemical, and behavioral processes. These 24 hr rhythms are driven by the master clock

located in the suprachiasmatic nucleus in the hypothalamus of the brain. The SCN receives

photic input via the retinohyopthalmic tract, and acts to signal peripheral oscillators throughout

the body and modulate physiological responses. These responses are due in part to the intrinsic

rhythms in each tissue, for example as much as 10% of expressed genes in the heart is under

local clock control. Disorders of circadian timing have been linked to metabolic and

cardiovascular disease, and therefore understanding the molecular basis and physiology of the

biological clock is critical to the development of new medical diagnoses and treatments. This

study examined the transcriptional inhibitor ID2 (Inhibitor of DNA binding 2) as a potential

modulator of the mammalian circadian clock. The current mode l suggests that Id2 is a novel

clock component which modulates INPUT, master pacemaker, and OUTPUT of the clock.

Interactions between ID2 and CLOCK/BMAL1 were demonstrated using the GAL4 /VP-16

mammalian two-hybrid interaction system. The identification of the Helix-Loop-Helix (HLH)

region as the relevant binding region was performed by in-vitro transient transfection with over

expressed proteins. It has been shown that ID2 operates rhythmically at both the transcript and

protein levels in the mammalian heart. As such qRT-PCR will be used to determine if ID2

regulates gene expression within the cardiac system. This would help to demonstrate that ID2 is

involved in modulating the biological clock.

Mueller Glia Cells Interaction with Apoptotic Ganglion Cells after Ouabain Injection

Joshua Oberley, Travis Bailey, and David Hyde

Pensacola Christian College and Dept. of Biological Sciences, University of Notre Dame

The structure of the eyes of a zebrafish is very similar to that of a human’s eye which makes the

zebrafish eye useful to study the eye of a human.   Unlike humans, the zebrafish eye has an

ability to regenerate cells by using distinct cells called Mueller glia cells.   Mueller glia cells

regenerate all the neurons of the retina including specific cells, such as the photoreceptor cells

after light damage or ganglion cells after ouabain poisoning. A subset of Mueller glial cells

respond to retinal damage and express Proliferating cell nuclear antigen (PCNA).  Responder

Mueller glia correlates with those in contact with dying photoreceptors.  These studies attempt

characterize a potential signal, that of engulfment, to potentially responder Mueller glial cells.

I used ouabain and injected different concentrations of ouabain and incubated for different times

before fixing the eye to determine optimal conditions.  I injected ouabain in a transgenic line,

Tg(isl3:EGFP).  Isl3 is a transcription factor important in the differentiation of ganglion cells,

thus the reporter gene Enhanced Green Fluorescence Protein is expressed in ganglion cells.

Terminal transferase dUTP nicked-end labeling (TUNEL) was used to detect apoptotic ganglion

cell debris.  Thus, if ganglion cells are engulfed by Mueller glia, EGFP would likely be detected

in the body of Muller glia cells colocalized with TUNEL labeling.

Long range nuclear gene flow through male dispersal has influenced the evolutionary history of

long-tailed macaques (Macaca fascicularis) on the island of Bali, Indonesia

Andrew Gloss, Kelly Lane, Agustin Fuentes, and Hope Hollocher

Depts. of Biological Sciences and Anthropology, University of Notre Dame

The long-tailed macaque (Macaca fascicularis) is one of the most successful non-human primate

species radiations, with a wide range extending throughout Southeast Asia.  Gene flow between

geographically separate regions has influenced the evolutionary history of this species,

particularly in Indonesia, where land bridges have connected the Malay Peninsula of continental

Asia and the islands to the south as recently as 10,000 years ago.  Male-mediated gene flow,

consistent with males dispersing between social groups and females remaining at their birth site,

is reflected in the presence of two Y chromosomal haplotypes in Java, the northernmost

Indonesian island – one originating in continental Asia and the other found only in the

neighboring islands.  Here, two Y chromosomal regions were sequenced from macaques on the

Indonesian island of Bali, which once temporarily formed the southeast tip of a large, continuous

landmass united with continental Asia during periods of low sea level.  Sequences were

compared with the corresponding published dataset for Southeast Asia.  Both the continental and

insular Y chromosomal haplotypes were detected, revealing that nuclear gene flow from

continental Asia progressed at least 1,600 km from the Asian mainland, more than 1,100 km than

previously known.  Additionally, the insular haplotype present on Bali clustered phylogenetically

with the insular haplotype present in the neighboring island of Java, suggesting recent genetic

exchange between the islands.  Both haplotypes were widely distributed throughout Bali, which

suggests that male dispersal events have connected the island’s spatially distinct populations over

a large timescale. 

Generating transgenic strains for the analysis of plasmatocyte differentiation and tumor suppressors

Clifford Thomas and Robert Schulz

Dept. of Biological Sciences, University of Notre Dame

Drosophila melanogaster has emerged as an important model organism in the analysis of

hematopoiesis. Recent studies have shown, much like the constitutive activation mutation of the

Janus Kinase Hopsctoch gene, the loss-of-function mutation in the Friend of GATA (FOG)

protein U-Shaped (USH) results in leukemia-like hyperplasia of hemocytes. USH is one protein

in a class of FOG proteins that are evolutionarily conserved multi-type zinc finger cofactors that

interact with the N-terminal zinc finger of GATA transcription factors. Experiments have shown

that USH acts on a GATA factor named Serpent (SRP). Ush loss-of-function means unregulated

SRP, which leads to over-proliferation of plasmatocytes, the phagocytic cells circulating the

hemolymph system, and lamellocytes, a larger neutralizing cell only circulating in the presence

of cellular damage, parasitic wasp infection, or cancer. In this study, crosses were made using the

USH loss-of-function allele and cell specific promoters coupled to GAL4. After three

generations, chromosomal crossovers can link the genes of interest on one chromosome. This

was tested using phenotypic screening. These recombinant strains will later be crossed with a

UAS RNAi strain to link associated genes to the cancer response. Further crosses were also

generated to test the differentiation of plasmatocytes, suspected in becoming lamellocytes.