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AUDITORY PROCESSING OF COMPLEX TONES IN NEWBORN INFANTS
Year of Dissertation:
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)
Year of Dissertation:
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.
Evolution of bat-trypanosome associations and the origins of Chagas Disease
Year of Dissertation:
Trypanosoma cruzi is a genetically diverse parasite that causes Chagas disease, one of the most important zoonoses in the Americas. This generalist parasite of mammals belongs to a clade mostly comprised of bat parasites, the T. cruzi clade. The origins (i.e., biogeographic history and evolution of hosts associations) of this parasite are far from being understood, and the main areas that need further study are: species limits within T. cruzi sensu lato, further studies on the diversity of T. cruzi clade members and their hosts, and research on adaptations of the hosts to trypanosome infections. In this dissertation I explore these research areas in five core chapters (Ch.2 to Ch.6), and these are the main results per chapter: ch.2) Proposed the recognition of Tcbat as a major diagnostic typing unit of T. cruzi. Ch.3) Found high genetic diversity in the bat exclusive lineage T. c. marinkellei than in the other subdivisions of T. cruzi. Also, reported Tcbat and T. c. marinkellei for Ecuador. Ch.4) Determined that the number of putative species in the T. cruzi clade is underestimated, and more strikingly, that the bat Artibeus jamaicensis is the vertebrate host with the highest number of trypanosomes (5 species) detected in a single locality. Ch.5) Results indicate that T. cruzi sensu lato is actually comprised of three species - the generalist T. cruzi, and two parasites of bats, Trypanosoma marinkellei and Trypanosoma sp. nov.; that there is a previously undetected high diversity of T. cruzi relatives associated with bats, and use of bats as hosts has evolved several times and in three different clades of parasites. Ch.6) Tested the hypothesis that immune genes in bats evolve under stronger positive selection than in other mammals because of the high diversity of pathogens associated with bats--including trypanosomes of the T. cruzi clade. I used the gene TLR2, as a candidate gene. These results help to understanding better the diversity of the T. cruzi clade, and the importance of bats on the origins of T. cruzi sensu strict and Chagas disease.
Transcriptome de novo assembly, clustering, and annotation of novel transcripts
Fatemeh Pooyaei Mehr
Year of Dissertation:
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
Year of Dissertation:
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
Year of Dissertation:
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
Year of Dissertation:
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
Year of Dissertation:
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.
Neural Targets of Electric Field Stimulation
Year of Dissertation:
Clinical application of electric fields to the brain are promising non-invasive approaches for the treatment of psychiatric, neurological, and pain disorders. Low-magnitude electric fields, which do not cause neuronal firing but only create changes to the voltage necessary for inducing neuronal firing by approximately 1%, are known to have substantial behavioral effects and therapeutic outcomes. Transcranial direct current stimulation is one electric stimulation modality that induces such low-magnitude electric fields. Electric fields of magnitude sufficient to directly trigger neuronal firing may be induced by transcranial electric stimulation and transcranial magnetic stimulation. Many therapeutic advances have been made using these techniques, and they create new experimental approaches to increase our knowledge of how the brain works. Still, the fundamental mechanisms as to how these electric fields may affect neuronal elements of the brain are not fully understood. Chapter 1 of this thesis creates a mechanistic model describing how an electric field of any small magnitude may still have an effect on neuronal processing by changes in spike timing. This mechanistic framework has implications for the effects of endogenous, brain-generated electric fields as well as electric stimulation modalities. The second chapter of this thesis develops a model of how cortical neuronal morphology and cell type may be used to predict changes in polarization and firing caused by electric fields. Both of these studies employed techniques to record from single cells in the brain, and all models described have been experimentally verified through these techniques.
Mechanisms of Vocal Learning in Songbirds: how song syllable structure is learned
Year of Dissertation:
When learning to perform continuous actions one needs to cope with conflicting motor requirements: while some parts of the action may require exploratory variability to find motor states that can efficiently produce a desired outcome, other parts may require consolidation if they are already close to the desired goal. A possible solution to this problem is partitioning the action into segments that could be controlled independently, but it is not known if exploratory variability can be locally regulated during the sensory-motor learning of a continuous action. This dissertation explores song learning in songbirds as a model of learning to perform continuous actions. In the first set of experiments we manipulated song learning in zebra finches (Taeniopygia guttata) to experimentally control different requirements for vocal exploration in different parts of the song. We first trained birds to perform a one-syllable song, and once they mastered it we added a new syllable to the song model. Remarkably, when practicing the modified song, birds rapidly alternated between high and low acoustic variability to confine vocal exploration to the newly added syllable. Analysis of exploratory variability within syllables revealed that acoustic variability changed independently across song elements that were only milliseconds apart. The variability of each song element decreased as it approached the target, correlating with momentary local error and less so with the global error (the mean of local errors across the syllable). We concluded that vocal error is computed locally in sub-syllabic time scales and that song elements can be learned and consolidated independently. However, our evidence for partitions singing behavior into segments that may be learned independently, also indicate that those segments are not developmentally stable. For example, we observed that the appearance of distinct syllable types usually precedes the appearance of distinct sub-syllabic song elements, suggesting that the time scales at which syllables are learned might get shorter over development, perhaps in a hierarchical manner, from the learning of course temporal structure to the learning of fine temporal structure. To test this hypothesis we studied song learning at the articulatory level by measuring the control of respiratory pressure during song learning. We found that during early song learning, respiratory pressure patterns were coarse, and fine temporal structure was later added to that pre-existent coarse structure. This effect was not observed in socially isolated birds that developed their song without learning a tutored model. We therefore propose that the learning of continuous singing action is achieved by first partitioning the song into coarse segments, and then to finer units, where exploratory variability in each unit is dynamically gated until a local match to the song model is achieved.