Alumni Dissertations and Theses

 
 

Alumni Dissertations and Theses

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  • Role of Toll-NF-kB Signaling in Inflammation and Immune Homeostasis in Drosophila melanogaster

    Author:
    Indira Paddibhatla
    Year of Dissertation:
    2011
    Program:
    Biology
    Advisor:
    Shubha Govind
    Abstract:

    Inflammation is defined as a localized reaction in response to injury or tissue destruction. It serves to contain or sequester the causative agent. Fundamentally important to human health, inflammation and its aberrant regulation underlie many diseases including cancer, diabetes and heart disease. Like humans, fruit flies respond to infection by coordinating complex defense responses. Parasitic wasps are natural enemies of Drosophila and attack larvae or pupae. They inject one or more eggs, each approximately 100 mm in size, into the hemocoel of the fly larva. Oviposition results either in the development of the wasp larva that gradually eats the Drosophila host tissue, or the host's blood cells encapsulate the wasp egg. In the latter scenario, circulating host blood cells surround the wasp egg and inhibit its development. In addition to the activation and aggregation of the host blood cells in response to wasp attack, infected larvae also activate gene expression in the fat body. A number of antimicrobial peptides and other immune-related proteins are secreted into the hemolymph. The humoral arm is activated within the first couple of hours of infection, although the significance of this activation is not understood. It is also not clear how the encapsulation reaction is so tightly controlled or how it is terminated. Previous studies from our laboratory have focused on the role of Toll-NF-kB signaling in hematopoiesis. Larvae deficient in either IkB/cactus or the SUMO-conjugating enzyme, Ubc9, exhibit hematopoietic defects (overproliferation and abnormal differentiation of the blood cells) accompanied with aggregation and microtumor formation. These mutants also express antimicrobial peptides such as Drosomycin from the fat body even in the absence of infection. Given that both the humoral and cellular immune systems of these mutants are hyperactive, we hypothesized that changes induced after wasp infection represent acute inflammation, and these effects become chronic in cactus or Ubc9 mutants. Studies in the first chapter explore the cellular and molecular parallels between wasp-induced gene expression and encapsulation versus constitutive gene expression and encapsulation (in microtumors) of Ubc9 mutants. We show that several core components (including SPE, Toll, and cactus) of the Toll-Dorsal pathway are activated in each case. However, while gene expression after wasp infection shows acute phase profile (activation followed by downregulation), expression in mutants remains high. We show that the cytokine Spätzle (the Toll ligand) is expressed in the immune cells, hemocytes and the fat body. Spätzle protein levels are higher after wasp infection and in Ubc9 mutants. Misexpression of either SPE or Spz protein in immune tissues of wild type larvae leads to blood cell proliferation, differentiation, infiltration of the fat body tissue and melanized microtumors. We propose a pro-inflammaotry role for SPE/Spätzle whose downregulation is essential for limiting acute inflammation. Accordingly, loss of spz suppresses Ubc9- defects and loss of SPE in the immune cells fails to encapsulate the parasitic egg. In contrast to the pro-inflammatory role of SPE/Spz, SUMO conjugating enzyme, Ubc9, and IkB homologue, Cactus, are anti-inflammatory agents. RNAi knockdown of Ubc9, or even Uba2/Aos1 subunits of SUMO activating enzymes leads to systemic chronic inflammation. Affected animals activate Drosomycin in the fat body in the absence of infection. Blood cells infiltrate the fat body and these changes are accompanied with microtumor development. Extensive staining of fat body and blood cells reveals that while cactus transcription and protein levels increase after infection, Ubc9 mutants cannot sustain normal cytoplasmic levels of Cactus protein. These studies demonstrate the existence of a sumoylation dependent Ubc9/IkB modulated immune homeostasis mechanism that balances the pro- and anti-inflammatory factors in the hosts. This study on Drosophila inflammatory responses complements the existing mammalian models for cancer inflammation. Knowledge gained from our system should be highly relevant in developing novel strategies for medical and agricultural pest control.

  • The Sex of the Cell Dictates its Response

    Author:
    Carlos Penaloza
    Year of Dissertation:
    2014
    Program:
    Biology
    Advisor:
    Zahra Zakeri
    Abstract:

    Male and female differences in frequency of occurrence in disease have perplexed scientists for some time. This in part derives from limitations in the systems in which one can evaluate sex differences. At the organismal level, differences can be hidden by a myriad of extensive and complex factors. Additional limitations exist since most biomedical studies are performed almost exclusively on male subjects, as the female hormonal milieu is intrinsically more variable and too troublesome for routine inclusion in research protocols. Research documenting sex differences continues to grow, and while most researchers suggests that sex hormones are at the core of these differences, more evidence is suggesting other innate biological factors are at play. To address these limitations, we asked whether differences could be induced and assessed in a controlled cellular system. We then asked whether any aspect of sex dimorphism could be attributed to chromosomal rather than hormonal differences; and finally established a role for differential DNA methylation in sex differences. We found that male and female cells responded differently to cell death inducers as measured by cell viability. We then evaluated gene expression between the sexes, and found that many genes were differentially expressed in vivo. These differences persisted in our cultures, affording us the ability to further characterize these sex differences. Furthermore, we found that cell death induction led to dimorphic gene expression; at instances having opposite effects on cells, where one sex is repressed, and the other is induced. We then evaluated DNA methylation to characterize the differential gene regulation. Blocking DNA methylation globally, using 5-Aza-2-deoxycytidine, led to a loss of gene expression differences between the sexes and found that differences in methylation patterns correlated directly with differences in gene expression. Here we provided a model system, whereby we can test for individual differences resulting in sex dimorphisms at the cellular level. It is critical to continue to expand our knowledge in this area, as this work can be extrapolated and applied to other instances where differences are being measured between the sexes; providing more tools to better characterize those conditions.

  • A Systematic Revision of North American Tolypella A. Braun (Charophyceae, Charophyta)

    Author:
    William Pérez
    Year of Dissertation:
    2014
    Program:
    Biology
    Advisor:
    Kenneth Karol
    Abstract:

    Abstract A SYSTEMATIC REVISION OF NORTH AMERICAN TOLYPELLA A. BR. (CHAROPHYCEAE, CHAROPHYTA) by William Pérez Advisor: Dr. Kenneth G. Karol Charophyta comprises the algal classes Mesostigmatophyceae, Chlorokybophyceae, Klebsormidiophyceae, Coleochaetophyceae and Zygnematophyceae and the land plants. However, the precise phylogenetic position of these algal classes with respect to land plants is unresolved as are the phylogenetic relationships among genera in Charophyceae (Characeae). Characeae contains two tribes with six genera: tribe Chareae (Chara, Lamprothamnium, Lychnothamnus and Nitellopsis) and tribe Nitelleae (Nitella and Tolypella). Tolypella was considered the third most species-rich genus but, in the most comprehensive taxonomic treatment of Characeae, 16 Tolypella species were consolidated into two species, T. nidifica and T. intricata in sections Rothia and Tolypella, respectively. It was further suggested that Tolypella might be a derived group within the closely related genus Nitella. Currently, there are no comprehensive molecular phylogenetic studies of Tolypella. In this investigation into North American Tolypella, sequence data from the plastid genes atpB, psbC and rbcL and the nuclear ribosomal operon 18S-ITS1-5.8S-ITS2-28S were assembled for a broad range of charophytic algae and land plants in order to address Tolypella species diversity and resolve their relationship to other genera in Characeae. In addition, mitochondrial and plastid genomes were completely sequenced for three Tolypella species for a comparative analysis of genome architecture and gene content and for inclusion into a multi-gene phylogeny of the Charophyta. Phylogenetic analyses of the plastid and nuclear gene data showed that Nitelleae is paraphyletic with Nitella sister to Chareae. Genus Tolypella and Tolypella section Tolypella were recovered as monophyletic. Tolypella section Rothia was weakly to moderately supported as monophyletic or weakly supported as paraphyletic in several analyses. Several lineages of the traditionally recognized Tolypella species in addition to a new species, T. ramosissima sp. nov., were identified, which suggests greater species diversity in Tolypella than currently recognized. Comparative analyses of mitochondrial and plastid genomes of three Tolypella species, T. canadensis, T. glomerata and T. intricata, showed that gene content, gene order and genome architecture are relatively conserved, however, T. glomerata showed a complete loss of the Ndh 1-complex genes. Phylogenetic analyses also recovered a monophyletic Tolypella and sections Rothia and Tolypella but showed conflicting results regarding the sister taxon to land plants with either Charophyceae or Zygnematophyceae as their closest living relatives.

  • AUDITORY PROCESSING OF COMPLEX TONES IN NEWBORN INFANTS

    Author:
    HA PHAN
    Year of Dissertation:
    2010
    Program:
    Biology
    Advisor:
    BERNARD KARMEL
    Abstract:

    Pitch extraction from complex acoustic stimuli during neonatal period in infants was examined in this study. It is one of the most fundamental auditory processes. Pitch processing is crucial for speech intonation and musical melody perception. Preferences of newborns for "motherese" (or infant directed speech), which has different pitch characteristics compared to adult directed speech, suggest an early pitch information processing capability in newborns. Although auditory functional development in the auditory system at early ages draws much attention of researchers from different disciplines, the number of studies during the neonatal period is limited. We first asked how pitch information processing develops. It was hypothesized that newborns already possess ability to process pitch information early during perinatal period. Second, we hypothesized that the extraction of pitch information in acoustic stimuli depended on the integrity of the auditory pathway. Brain insult in the perinatal period has been shown highly likely to affect subcortical structures, including the auditory brainstem and midbrain, through different mechanisms. We reasoned that if there were delays in or problems with pitch processing in brain-injured or premature neonates when compared to healthy or premature infants of equal gestation, then, in the absence of peripheral disturbances, evidence for CNS mediation of the development of pitch processing could be argued. Two types of auditory brainstem responses were studied: (1) auditory brainstem evoked responses (ABRs) to click stimuli, and (2) the frequency following response (FFR) to complex tones were utilized to study the above questions in N=128 premature and term infants assigned to the Neonatal Intensive Care Unit (NICU) at birth, all at varying risk for a CNS injury (mean gestational age at birth=34±3.6 weeks). In summary, we confirmed that auditory pitch information processing of complex sound was present in newborn infants as early as 32 weeks gestation. Neonatal FFR studies provided positive evidence of responses to the pitch-related information both in the envelope-related frequency as well as in the difference tone and stimulus component-related frequencies. Similar responses at younger and older ages at test indicated that this capability remains stable across age during the first month of life. Moreover, NICU infants with evidence of a structural perinatal brain injury showed impairment in this type of auditory processing.

  • A Phylogenetic Revision of the Medicinal Leeches of the World (Hirudinidae, Macrobdellidae, Praobdellidae)

    Author:
    Anna Phillips
    Year of Dissertation:
    2011
    Program:
    Biology
    Advisor:
    Mark Siddall
    Abstract:

    The term "medicinal leech" refers to more species than just Hirudo medicinalis, the preferred species for bloodletting in 19th century Europe. In the past, freshwater, leeches with similar morphological characterstics to H. medicinalis have been divided into two families: the bloodfeeding Hirudinidae and the non-bloodfeeding Haemopidae. With a broader taxon sampling than in previous analyses, an analysis of multiple nuclear (18S rDNA and 28S rDNA) and mitochondrial (12S rDNA and cytochrome c oxidase I) gene fragments found Hirudinidae not to be monophyletic, instead placing in two independently originated lineages separated by the two families of terrestrial leeches, Haemadipsidae and Xerobdellidae. Members of Haemopidae were scattered among members of Hirudinidae of both lineages, thus rendering Haemopidae polyphyletic. The lineage containing H. medicinalis retained the name Hirudinidae, while the other lineage was shown to consist of three families: Macrobdellidae (North and South American bloodfeeders), Semiscolecidae (South American non-bloodfeeders), and Praobdellidae (a biogeographically diverse clade of species that feed primarily from mammalian mucous membranes). With the familial relationships within these two lineages established, attention was given to revising the intra-familial and generic relationships. Two new genera and two new species resulted from these investigations: Tyrannobdella rex n. gen. n. sp., a leech found feeding inside the nasal passages of humans placed within Praobdellidae, Mesobdella lineata was re-described as Parapraobdella lineata n. gen. within Praobdellidae, and Hirudinaria bpling n. sp. placed within the Hirudinidae. Endosymbiotic bacteria from the digestive tracts of members of the Hirudinidae, Macrobdellidae, and Praobdellidae were detected and determined to be Aeromonas species as well as an unculturable Bacteroidetes. The Aeromonas isolates did not show a predictable association based on the phylogeny of the leech hosts or geography, while the Bacteroidetes isolates did show a correlation with leech taxonomy. An analysis with the most thorough taxon sampling to date of the families of Hirudiniformes and Erpobdelliformes was performed. Gastrostomobdellidae, a group of macrophagous leeches hypothesized to be similar to Erpobdelliformes or Hirudiniformes, was strongly supported within Erpobdelliformes. The establishment of the relationships provides a basis for further systematic study, as well as a investigations into the evolution of these charismatic worms.

  • Transcriptome de novo assembly, clustering, and annotation of novel transcripts

    Author:
    Fatemeh Pooyaei Mehr
    Year of Dissertation:
    2013
    Program:
    Biology
    Advisor:
    David Gruber
    Abstract:

    Recent advances in Next Generation Sequencing (NGS) have allowed for unparalleled access to genetic information for organisms in both the functional and phylogenetic realms of biology. Analysis of the RNA transcripts of cells of organisms using Next Generation Sequencing (called RNA-seq) has opened doors for unique insights into the genomic complexity of organisms and has provided researchers with invaluable tools for analysis of function of gene products and phylogenetic relatedness. Application of this method has moved beyond model organisms. It has provided a lot of potentials, in ecological research and comparative transcriptomics, in non-model organisms. This thesis presents an overview on existing applications of RNA-seq in non-model organisms. Furthermore, it presents a new clustering design on handling the data, which led to identification of twelve new fluorescent protein isoforms in corals. In addition, de novo assembly and annotation of the data from polychaete Hermodice carunculata made possible the identification of one new phylogenetic marker and eight bioluminescent protein isoforms. Also, twelve new bilirubin-induced fluorescent proteins were identified from false moray eel Kaupichthys hyproroides. This approach can be applied on any other data.

  • The tree that held up the forest: Shihuahuaco (Dipteryx spp.) and the Chinese timber trade

    Author:
    Louis Putzel
    Year of Dissertation:
    2010
    Program:
    Biology
    Advisor:
    Christine Padoch
    Abstract:

    ABSTRACT Extending over an area of more than 5 million km2, the forests of Amazonia are a repository of 25% of terrestrial biodiversity and play a vital role in regulating regional and global ecological systems. In addition to the direct damages caused by logging, timber extraction entails important collateral impacts on forests, including facilitating access for conversion of land to agriculture and pastureland. Over 650,000 km2 (>12%) of the Amazon forest area is located in Peru. Unlike Brazil, where timber exports have declined in recent years, Peru's volume of annual timber exports increased 180% between 2004 and 2008. Mexico and the United States were long Peru's largest importers of timber. By 2006, however, China's imports had surpassed those of the US. Although only a relatively small percentage of China's total timber imports originate in Peru, since 2000, there has been an exponential increase in the wood trade between the two countries. Using a multi-disciplinary approach combining forest ecology, economic botany, and political economy this research identifies the species which represents 80% of China's imports from Peru, i.e. shihuahuaco (Dipteryx spp.), and then follows the supply chains of that species along rectilinear transects in the Amazonian forest and back through the highly non-linear Peruvian industrial milling centers and export market. Forest ecology methods are employed to assess the effects of logging on the regeneration of shihuahuaco, and the potential for local people to mitigate those effects and accelerate the reestablishment of shihuahuaco stands. Analysis of the Peruvian export market, broken down by species, illustrates the rapid increase of exports of shihuahuaco following intentional promotion by conservation-oriented programs and the emergence of the Chinese market. Finally, a dynamic commodity network analysis uncovers the connections between Chinese shihuahuaco traders located within the Amazon forest region and shows how these connections are evolving in unexpected ways in response to efforts to regulate the global timber trade. Key findings include: 1.) In areas logged for shihuahuaco, regeneration will be greatly reduced by the removal of seed trees; however, since post-logging conditions and treatments by local residents favor the recruitment of saplings, there is an opportunity for recovery of the resource. 2.) In response to extraction of shihuahuaco, a number of smallholder farmers living within and around logging zones manage residuals, transplant residual seedlings into their agricultural fields, and collect seeds to germinate in nurseries. 3.) Exports of shihuahuaco to China have increased more than three-fold over the past 5 years, and now represent over 50% of Peru's timber exports. 4.) While Chinese shihuahuaco exporters, like the majority of Peruvian timber exporters, continue to be supplied by the informal market, new vertically integrated supply chains owned by Chinese processing companies have obtained international certification. This certification enables them to increase their global market share, but the ecological and social impacts in Amazonia are unknown.

  • Systematics and Historical Biogeography of the Lampropeltinine Snakes

    Author:
    Robert Pyron
    Year of Dissertation:
    2009
    Program:
    Biology
    Advisor:
    Frank Burbrink
    Abstract:

    Comparative studies in biology require phylogenetic hypotheses to make inferences about the processes which have shaped the evolutionary history of organisms. Thus, a complete phylogenetic estimate of a diverse group offers an excellent opportunity for examining the factors which have promoted the diversification of ecomorphological assemblages. Here, I detail such a study of the New World snake tribe Lampropeltini. The lampropeltinines comprise 31 currently recognized species, occurring from Canada to northern South America, inhabiting most major North American biomes, and exhibiting an unusual temperate peak in species richness. The Lampropeltini also exhibit an array of ecomorphological diversity, with adult sizes differing by an order of magnitude, specialization for both endothermic and ectothermic diets, and the evolution of putative Batesian mimicry of venomous coral and rattle snakes in several species. A new phylogeny inferred using multiple nuclear and mitochondrial genes allows for the stabilization of the taxonomy of the Lampropeltini. Subsequent analyses reveal that the extratropical increase in species richness is attributable to a combination of historical biogeographic factors related to the Tropical Conservatism Hypothesis (Wiens & Donoghue 2004), which are proposed as a general explanation for the in-situ evolution of biodiversity, dubbed the `Biogeographical Conservatism Hypothesis.' The ecomorphological diversification of the Lampropeltini occurred primarily along an axis of adult body size, with which is observed the correlated evolution of diet and color pattern. This pattern of correlated evolution of putatively unrelated characters suggests that body size may be a primary determinant of morphological diversification when multiple traits are linked to variables such as body size. Finally, examining the factors which drove lineage formation at the species level through a phylogeographic assessment of the transcontinentally distributed Common Kingsnake (Lampropeltis getula) reveals patterns of allopatric speciation due to both phylogenetic niche conservatism and niche divergence. This suggests that niche preferences are labile on short evolutionary timescales, and that speciation can occur simultaneously in both geographic and ecological dimensions. This phylogeographic estimate also allows for a systematic assessment of the taxonomy of the Common Kingsnake group, which is revised to include five species corresponding to the major phylogeographic lineages.

  • Comparative Modeling and Functional Characterization of Two Enzymes of the Cyclooxygenase Pathway in Drosophila melanogaster

    Author:
    Yan Qi
    Year of Dissertation:
    2014
    Program:
    Biology
    Advisor:
    Shaneen Singh
    Abstract:

    Eicosanoids are biologically active molecules oxygenated from twenty carbon polyunsaturated fatty acids. Natural eicosanoids exert potent biological effects in humans, and a great deal of pharmaceutical research has led to the discovery of compounds for selective inhibition of specific enzymes in eicosanoid biosynthesis. Coupled with different receptors, eicosanoids mediate various physiological and pathophysiological processes, including fever generation, pain response, vasoconstriction, vasodilation, platelet aggregation, platelet declumping, body temperature maintenance and sleep-wake cycle regulation. In mammals, the eicosanoid biosynthesis has three pathways: the cyclooxygenase (COX) pathway, the lipoxygenase (LOX) pathway and the epoxygenase pathway. The COX pathway synthesizes prostanoids, which are important signaling molecules in inflammation. Because of their central role in inflammatory disease and human health, COX enzymes continue to be a focus of intense research as new details emerge about their mechanism of action and their interactions with NSAIDs. To date, the majority of studies dealing with the COX pathway are centered on mammalian systems. Although the literature is rich in speculations that prostaglandins are central signaling molecules for mediating and coordinating insect cellular immunity, genes responsible for encoding COX or COX-like enzymes and other enzymes in the COX pathway have not been reported in insects. The value of Drosophila melanogaster as a model organism is well established, and the fundamental regulatory signaling mechanisms that regulate immunity at the cellular level in human and flies are conserved. Given the importance of eicosanoids in mammalian and insect immunity, this study was designed to identify and characterize the enzymes that mediate eicosanoid biosynthesis in D. melanogaster computationally. After a preliminary extensive search for putative D. melanogaster homologues for all enzymes in the COX pathway, we conducted a systematic, comprehensive, and detailed computational investigation for two enzymes, COX and prostaglandin E synthase (PGES) in an endeavor to model and characterize the possible candidates and identify those that possess all the requisite sequence and structural motifs to qualify as valid COX(s)/PGE synthase proteins. In this study, we report the presence of qualified D. melanogaster COX(s)/PGE synthase proteins, characterize their biophysical properties, and compare them with their mammalian counterparts. This study lays the groundwork for further exploration of these proteins and establishing their role in D. melanogaster inflammation and immunity, opening up avenues for addressing the use of this model organism in COX signaling and its crosstalk with other signaling pathways.

  • Characterization of the CYP97 and HYD carotene hydroxylase enzymes

    Author:
    Rena Quinlan
    Year of Dissertation:
    2012
    Program:
    Biology
    Advisor:
    Eleanore Wurtzel
    Abstract:

    Vitamin A deficiency is a serious and widespread public health issue in developing countries. Provitamin A carotenoids such as β-carotene have therefore recently attracted interest as important neutraceuticals. Due to the nutritional value of carotenoids there is currently considerable interest in developing rational strategies for metabolic engineering of crops for enhanced carotenoid content. Efforts to improve the provitamin A content of cereal endosperm in staple crops such as maize will require characterization of the carotene ring hydroxylases involved in controlling the conversion of provitamin A carotenes to non-provitamin A xanthophylls. Based on early modeling by (Cunningham and Gantt, 1998) the cytochrome P450 CYP97 and diiron HYD carotene hydroxylases were predicted to localize to chloroplast membranes to function in separate multi-enzyme complexes for the respective conversions of the provitamin A carotenes α- and β-carotene to the non-provitamin A xanthophylls lutein and zeaxanthin. To gain a better understanding of the respective roles of the CYP97 and HYD enzymes in these conversions, the activities and localization/interaction of these enzymes were examined using both in vitro and in vivo approaches. Escherichia coli functional complementation systems were used to assess rice P450 CYP97A4 (β-ring hydroxylase) and CYP97C2 (ε-ring hydroxylase) as well as maize diiron HYD3 and HYD4 (β-ring hydroxylases) activities and substrate specificities. Preliminary investigations examining CYP97 enzyme activity only via E. coli complementation showed that the CYP97A4 exhibits major activity toward β-rings to convert β-carotene to β-cryptoxanthin (pathway intermediate) as well as a low amount of zeaxanthin (pathway end-product). In addition, this enzyme exhibited minor activity toward the ε-rings of ε-ε-carotene to convert this substrate to a low amount of lactucaxanthin (pathway end-product). These studies also indicated that the CYP97C2 appeared to be exclusively active toward the ε-rings of ε-ε carotene, converting this substrate to a low amount of lactucaxanthin; no activity toward the β-rings of β-carotene was detected for this enzyme. Subsequent complementation studies tested both individual and combined CYP97/HYD enzyme activities in E. coli accumulating both α- and β-carotene substrates. These studies demonstrated that the CYP97A4 and CYP97C2 enzymes function optimally when expressed together in the conversion of their preferred substrate α-carotene to produce lutein. Cells engineered to produce α- and β-carotene and which co-expressed these enzymes generated almost 30% lutein (% total carotenoids); a roughly 10-fold higher amount of lutein relative to zeaxanthin was observed. In contrast, when expressed as individual enzymes the CYP97A4 and CYP97C2 showed suboptimal activity (ie., no lutein produced; only approx. 14%, and 1% of the intermediates zeinoxanthin and α-cryptoxanthin generated respectively) regardless of substrate choice. In these cells, the CYP97A4, when expressed alone, preferred the β-carotene substrate to the α-carotene substrate generating a low amount of zeaxanthin (the monohydroxylated intermediate β-cryptoxanthin accumulated); the CYP97A4 was only moderately active toward the α-carotene substrate as only the intermediate zeinoxanthin accumulated (no lutein was produced). When expressed as an individual enzyme, the CYP97C2 was minimally active toward both α- and β-carotene substrates to respectively generate barely detectable amounts of the intermediates α-cryptoxanthin and β-cryptoxanthin. HYD3 + CYP97C2 and HYD4 + CYP97C2 combinations were also tested using this complementation system. Both enzyme combinations were moderately active toward α-carotene, respectively producing low amounts of lutein; although cells co-expressing the HYD3 + CYP97C2 appeared to be somewhat more active toward α-carotene than β-carotene, generating a more than two-fold higher amount of lutein relative to zeaxanthin. When expressed as an individual enzyme, the HYD3 was preferentially active toward β-carotene to convert this substrate to a low amount of zeaxanthin (end-product); this enzyme was only moderately active toward the α-carotene substrate as only the intermediate zeinoxanthin accumulated. The HYD3 exhibited suboptimal activity in our complementation systems whether expressed alone or in combination with the CYP97C2. By contrast, the HYD4 functioned optimally when expressed as an individual enzyme. This enzyme was preferentially active toward β-carotene and efficiently converted this substrate to zeaxanthin. This work also examined CYP97/HYD protein localization and protein interaction. In vitro chloroplast import and in vivo GFP fusion assays confirmed that these enzymes are localized to chloroplasts. In addition, import assays were used to determine the suborganellar locations of these enzymes, and in vivo Bimolecular Fluorescence (BiFC) assays were performed to assess protein-protein interaction. Taken together, these studies demonstrated that the CYP97A4 and CYP97C2 enzymes are peripherally-associated to chloroplast membranes where they interact to form a heterodimer complex to function in the efficient conversion of α-carotene to lutein. It was expected that these enzymes, which functioned optimally together toward the α-carotene substrate in the E. coli complementation system, would interact and localize to the same location in the chloroplast membrane. These data also indicated that the HYD3 and HYD4 enzymes are integrally-bound to the chloroplast membrane where they interact to function in the conversion of β-carotene to zeaxanthin. These enzymes were expected to localize to the same suborganellar location since they were both preferentially active toward the same substrate (ie., β-carotene) in the complementation system. In addition, BiFC analysis indicated that HYD4 formed a homodimer complex.