Pseudomonas aeruginosa and Candida albicans have shown antagonistic relationships, both in vivo and in vitro. The interaction between P. aeruginosa and C. albicans is one of the many prokaryotic-eukaryotic interactions existing in nature and needs more research due to their complex pathogenicity in humans. In this work, we have studied growth dynamics of Candida in a mixed population of Pseudomonas and Candida, and their receptor and ligand specificities, both from an ecological and a molecular point of view. Initially, growth, viability, and morphogenesis of Candida were studied in the presence of Pseudomonas and the conditioned medium of Pseudomonas using two Candida strains, namely CAF2 and tup1 mutant. The killing effect of Pseudomonas was more pronounced on the hyphal form of Candida. However, growth of Candida was inhibited by Pseudomonas irrespective of its morphological form. The conditioned medium had no effect on the growth rate of Candida. Nevertheless, it completely inhibited the germination of Candida. The attachment of Pseudomonas to Candida was studied using different strains of both, and with Pseudomonas cells harvested at different stages of its growth. The percent attachment varied with the age of the textit Pseudomonas culture. A lecB mutant of Pseudomonas showed a two-fold reduction in attachment compared to the wild type PAK strain. Carbohydrate inhibition studies confirmed that LecB is not directly involved in the attachment mechanism, but indirectly involves through regulating the expression of other proteins required for attachment. A genomic DNA library of Pseudomonas PAO1 was screened for clones that had acquired the ability to attach to C. albicans. A clone was isolated with a small increase in attachment and was subjected to genetic analysis. It contained nucleotides 458565 to 475917 of the Pseudomonas genome including some genes of the Pil-Chp gene cluster. Seven transposon mutants that represent mutations in ChpA,B,C,D,E operon and three other ORFs were selected, and their attachment ability was tested. All seven mutants showed a reduction in attachment indicating that this was a non specific effect, which could be attributed to the in vitro manipulation of the bacteria. We conclude that the attachment of Pseudomonas to Candida is multi-factorial.

The cadA gene in Dictyostelium encodes a unique Ca2+-dependent cell adhesion molecule DdCAD-1. It is synthesized as a soluble protein in the cytoplasm and then transported to the plasma membrane by contractile vacuoles. The solution structures of Ca2+-free and Ca2+-bound DdCAD-1 reveals that it contains two β-sandwich domains, belonging to the βγ-crystallin and immunoglobulin fold classes, respectively. Whereas the N-terminal domain has a major role in homophilic binding, the C-terminal domain tethers the protein to the cell membrane. Although hydrophobic interactions constitute the major force for adhesion, electrostatic interactions may act as a ‘switch’ to regulate the homophilic binding by a change in electrostatic potential caused by the binding of Ca2+ to the three binding sites. To further investigate DdCAD-1 transport, DdCAD-1-GFP fusion proteins were expressed in cadA-null cells. Time-lapse microscopy revealed that DdCAD-1 was imported by invagination of the contractile vacuole membrane. The N-terminal, C-terminal domains, and two of the three Ca2+-binding site mutant forms of DdCAD-1 failed to enter the contractile vacuole, suggesting that Ca2+-binding and the integrity of DdCAD-1 are required for import. Indeed, proteins with altered conformation failed to enter the contractile vacuole, indicating that the import signal is integrated in the three-dimensional structure of DdCAD-1. Finally, we describe how the cadA gene acts as a single-gene green beard. In chimera experiments, cells expressing DdCAD-1 were more likely to form fruiting bodies than cadA-null cells on soil plates. Here cadA behaved as a single gene green beard. However, cadA exhibited anti-green beard behaviour on non-nutrient agar plates. Wild-type cells differentiated mostly into prestalk cells and eventually died, whereas the cadA-null cells survived as spores. DdCAD-1 was enriched in cell-cell contact regions of anterior cells, while it was mostly localized in the cytoplasm of posterior cells. The presence of DdCAD-1 on the cell surface of prestalk cells is crucial for cell sorting, which in turn explain the anti-green beard effect observed in chimeras containing cadA+ and cadA- cells. These observations demonstrate that DdCAD-1 plays a direct role in cell sorting through differential cell-cell adhesion which results from the differential distribution of DdCAD-1.

The cadA gene in Dictyostelium encodes a unique Ca2+-dependent cell adhesion molecule DdCAD-1. It is synthesized as a soluble protein in the cytoplasm and then transported to the plasma membrane by contractile vacuoles. The solution structures of Ca2+-free and Ca2+-bound DdCAD-1 reveals that it contains two β-sandwich domains, belonging to the βγ-crystallin and immunoglobulin fold classes, respectively. Whereas the N-terminal domain has a major role in homophilic binding, the C-terminal domain tethers the protein to the cell membrane. Although hydrophobic interactions constitute the major force for adhesion, electrostatic interactions may act as a ‘switch’ to regulate the homophilic binding by a change in electrostatic potential caused by the binding of Ca2+ to the three binding sites. To further investigate DdCAD-1 transport, DdCAD-1-GFP fusion proteins were expressed in cadA-null cells. Time-lapse microscopy revealed that DdCAD-1 was imported by invagination of the contractile vacuole membrane. The N-terminal, C-terminal domains, and two of the three Ca2+-binding site mutant forms of DdCAD-1 failed to enter the contractile vacuole, suggesting that Ca2+-binding and the integrity of DdCAD-1 are required for import. Indeed, proteins with altered conformation failed to enter the contractile vacuole, indicating that the import signal is integrated in the three-dimensional structure of DdCAD-1. Finally, we describe how the cadA gene acts as a single-gene green beard. In chimera experiments, cells expressing DdCAD-1 were more likely to form fruiting bodies than cadA-null cells on soil plates. Here cadA behaved as a single gene green beard. However, cadA exhibited anti-green beard behaviour on non-nutrient agar plates. Wild-type cells differentiated mostly into prestalk cells and eventually died, whereas the cadA-null cells survived as spores. DdCAD-1 was enriched in cell-cell contact regions of anterior cells, while it was mostly localized in the cytoplasm of posterior cells. The presence of DdCAD-1 on the cell surface of prestalk cells is crucial for cell sorting, which in turn explain the anti-green beard effect observed in chimeras containing cadA+ and cadA- cells. These observations demonstrate that DdCAD-1 plays a direct role in cell sorting through differential cell-cell adhesion which results from the differential distribution of DdCAD-1.

In teleosts, the Yolk Syncytial Layer (YSL) is functionally similar to the anterior visceral endoderm found in mice and is required for morphogenesis of the overlying blastoderm. The YSL undergoes dramatic reorganization during early development through processes that mirror the morphogenetic movements of the blastoderm. The YSL and YSL nuclei (YSN) undergo epiboly, and during convergence and extension movements of the blastoderm, the YSN underneath the animal cap also converge and extend underneath the axial hypoblast. Our work with pollywog ( pwg ) maternal-effect mutants highlights the delicate control of the YSL during yolk morphogenesis, and provides novel insight into understanding which tissues of the embryo are affected by loss of a cohesive YSL. I found that pollywog encodes the zebrafish mitogen activated protein kinase kinase kinase 4 ( map3k4 ) gene and that it acts upstream of p38a MAPK in the YSL. I show that this pathway acts in the YSL along with a mixer gene family member, mix-type homeobox gene 1 ( mxtx1 ), to non-autonomously coordinate extracellular matrix deposition and morphogenetic movements in the overlying blastoderm. Our data describes an early and novel role for Map3k4, p38a and Mxtxl activity that is required for proper morphogenesis of the YSL and the blastoderm. In embryos lacking maternal Map3k4, the YSL undergoes a rapid and catastrophic retraction and the YSN lose their normal distribution around the yolk. The prechordal plate of pwg mutant embryos deflect laterally or plunge into the yolk, and the overall animalward extension of the prechordal plate is diminished. I also show that the anterior neural plate of pwg mutant embryos fail to converge dorsally to the same extent as in wild embryos. These data show that the p38 MAPK pathway is essential for maintaining normal yolk cell equilibrium during early development and that without proper cues from the YSL, the blastoderm cannot complete its morphogenetic movements. Incuded in this thesis is work highlighting the alpha-actinin gene family in zebrafish. alpha-actinins are actin microfilament crosslinking proteins. Vertebrate actinins fall into two classes: the broadly-expressed actinins 1 and 4 ( actn1 and actn4 ) and muscle-specific actinins, actn2 and actn3 . Members of this family have numerous roles, including regulation of cell adhesion, cell differentiation, directed cell motility, intracellular signaling and stabilization of f-actin at the sarcomeric Z-line in muscle. Here I identify five zebrafish actinin genes including two paralogs of ACTN3 . I describe the temporal and spatial expression patterns of these genes through embryonic development. All zebrafish actinin genes have unique expression profiles, indicating specialization of each gene. In particular the muscle actinins display preferential expression in different domains of axial, pharyngeal and cranial musculature. There is no identified avian actn3 and approximately 16% of humans are null for ACTN3 . Duplication of actn3 in the zebrafish indicates that variation in actn3 expression may promote physiological diversity in muscle function among vertebrates.

<p>The gonad is a unique primordial organ that retains the ability to adopt one of two morphological fates through much of mammalian embryonic development. Previous work in our lab found that dimorphic vascular remodeling was one of the earliest steps during sex-specific morphogenesis. In particular, vessels in XY gonads display highly ordered behavior that coincides with testis cord formation. It was unknown how the vasculature may influence testis cord morphogenesis and, if so, how this was mechanistically related to sex determination. The work in this thesis addresses a single over-arching hypothesis: Male-specific vascular remodeling is required for testis morphogenesis and orchestrates differentiation of the XY gonad. </p><p>To address this question we have modified and developed techniques that allow us to isolate aspects of vascular behavior, gene expression, and endothelial influence on surrounding cells. In particular, the application of live imaging was instrumental to understanding the behavior of various gonadal cell-types in relation to remodeling vessels. It is difficult to grasp the complexity of an organ without understanding the dynamics of its constituents. A critical aim of my work was to identify specific inhibitors of the vasculature that do not affect the early stages of sex determination. Combining inhibitors, live imaging, cell sorting, qRT-PCR, mouse models, and whole organ culture has led to a far richer understanding of how the vasculature behaves and the cell-types that mediate its influence on organ morphogenesis. The beauty of our system is that we do not have to settle for a snapshot of the fate of cells in vivo, but can document their journeys and their acquaintances along the way. </p><p>Vascular migration is required for testis cord morphogenesis. Specific inhibitors revealed that in the absence of vessels, testis cords do not form. The work below shows that vessels establish a feedback loop with mesenchymal cells that results in both endothelial migration and subsequent mesenchymal proliferation. Interstitial control of testis morphogenesis is a new model within the field. The mechanisms regulating this process include Vegf mediated vascular remodeling, Pdgf induced proliferation, and Wnt repression of coordinated endothelial-mesenchymal dynamics. Our work also suggests that vascular patterning underlies testis patterning and, again, is mediated by signals within the interstitial space not within testis cords themselves. </p><p>A final aspect of my work has been focused on how vessels continue to influence morphology of the testis and the fate of surrounding cells. Jennifer Brennan, a graduate student in our lab, previously showed that loss of Pdgfr&#945; antagonizes cord formation and development of male-specific lineages. The mechanisms and cell-types related to this defect were not clear. I began to reanalyze Pdgfr&#945; mutants after finding remarkable similarity to gonads after vascular inhibition. This work is providing data suggesting that vessels are not simply responsible for testis morphology but also for the fate of specialized cells within the testis. On the whole, this thesis describes specific roles for endothelial cells during gonad development and mechanisms by which they are regulated.</p> / Dissertation

The work presented in the thesis was carried with the aim to understand how Widerborst (Wdb) regulate planar cell polarity in Drosophila wing. In search of proteins interacting with Wdb I carried a Yeast Two Hybrid screen and identified a protein, bitesize, with tandem C2 domains in its C terminus interacting with Wdb. Wdb also interacts with btsz genetically and removal of one copy each of Wdb and btsz enhances the truncated hair phenotype observed in Wdb EMS mutants and btsz P element insertion mutants. There are at least three predicted isoforms of bitesize and loss of the btsz-II isoform is lethal. Clonal analysis of a btsz mutant, btszJ5-2, which removes the btsz II isoform resulted in truncated wing hair outgrowth. On the other hand over expression of a myc-btsz-II construct resulted in hair duplication phenotype. However, over expression of the GFP-CT is sufficient to give wing hair duplication phenotype and this hair duplication phenotype is stronger than that caused by myc-btsz-II over expression. The Myc tagged btsz-II protein shows apical localization. Though most of the protein is confined to cytoplasm, btsz-II also marks the plasma membrane. The GFP-CT construct marks the plasma membrane strongly and is enriched in the apical region. The over expression of CT domain is sufficient to give hair duplication phenotype and the strong difference observed in the localization pattern of full length btsz-II protein and GFP-CT together suggest that regulation of membrane localization of btsz through its CT region is important to regulate hair morphogenesis. As the loss of function (truncated wing hair) and gain of function (hair duplication) both affect the process of hair morphogenesis, it can be said that btsz is a positive regulator of hair morphogenesis. Since no defect in cortical polarization of Fmi was observed in cells lacking btsz-II, btsz is required for establishment of cortical domains. However with the present study it remains unknown how exactly the C2 domains might regulate hair morphogenesis and whether Wdb targets btsz for dephophorylation to PP2A catalytic subunit.

Linseed flax studies in vitro were carried out at the Laboratory of Genetic – Biotechnological, Department of Crop Science and Animal Husbandry of Lithuanian University of Agriculture in 2002-2005, photomorphogenetic processes were investigated at the Laboratory of Plant Physiology of the Lithuanian Institute of Horticulture in 2004-2005. Optimal conditions for the dedifferentiation of linseed flax stem segments and hypocotyls cells and those of secondary differentiation in vitro were ascertained. Light technology of a solid body was used for investigation and an optimal combination of light parameters has been selected, promoting photomorphogenesis in vitro. Factors determining androgenesis and gynogenesis processes in vitro were evaluated and potential androgenic and gynogenic genotypes for the development of initial selection material were selected.

Study aim is to study endogenic and exogenic factors preconditioning morphogenesis of linseed flax in vitro. Linseed flax studies in vitro were carried out at the Laboratory of Genetic – Biotechnological of Lithuanian University of Agriculture in 2004-2006. Factors which influence morphogenetic capability of linseed flax cultivars ‘Lirina’, ‘Barbara’ and ‘Szaphir’ in tissue culture were investigated. Two different types of explants – stem segments and hypocotyls – of the three genotypes were cultivated in nutrient media differing in their macro and micro salts concentration, vitamins and different levels of growth regulators. Our results showed that intensity of morphogenesis was affected not only by exogenous growth regulators, but also by the type of explant and genotype. Hypocotyls-derived callus gave the better results than that stem-segment-derived callus on all tested media. Four-weeks-old callus showed the maximum shoot regeneration frequency. The best morphogenetic capabilities were demonstrated for cultivar ‘Szaphir’. The largest number of shoots was produced by all the cultivars on MSB5 nutrient medium supplemented with cytokinin 2iP (2.0 mg 1-1). The master’s final work comprises 50 pages, including 3 tables, 10 figures and 66 literature references.

Magistrantūros studijų baigiamajame darbe pateikiami žaliosios šerytės morfogenezės indukcija in vitro tyrimų duomenys. Darbo metodai: vertintas kaliaus susidarymo dažnis (%), ūglių susidarymo dažnis (%), ūglių skaičius iš eksplanto (vnt.) bei šaknų formavimosi dažnis (%). Darbo objektas: Setaria viridis L. Darbo rezultatai. Tyrimų rezultatai atskleidė skirtingą kaliaus, ūglių bei šaknų formavimosi dažnį, dėl skirtingų egzogeninių augimo reguliatorių derinio ir jų kiekio maitinamojoje terpėje. Žaliosios šerytės dediferenciacijos indukcija intensyviau vyko terpėse, papildytose didesniu auksinų nei citokininų kiekiu. Žaliosios šerytės kaliaus formavimosi dažnis varijavo priklausomai nuo egzogeninių augimo reguliatorių derinio indukcijos terpėje. Augimo reguliatorių derinys 2,0 mg l-1 2,4-dichlorfenoksiacto rūgšties + 0,4 mg l-1 6-furfurylamino purino skatino intensyvesnę dediferenciaciją in vitro kultūroje. Antrinė diferenciacija intensyviausiai vyko maitinamojoje terpėje, papildytoje 0,4 mg l-1 α naftilacto rūgšties ir 2,0 mg l-1 N6-(2-izopentil) adenino. Daugiausiai ūglių iš eksplanto susiformavo maitinamojoje terpėje, papildytoje 0,3 mg l-1 NAR + 2,0 mg l-1 2iP. Žaliosios šerytės rizogenezės procesą in vitro skatino augimo reguliatorių deriniai 2,0 mg l-1 2,4D + 0,4 mg l-1 KIN, 0,2 mg l-1 NAR + 2,0 mg l-1 2iP ir 0,4 mg l-1 NAR + 2,0 mg l-1 BAP. / The master work presents the results of increasing of green millet induction of morphogenesis in vitro studies. Method of the work - evaluated callus formation frequency (%), shoot formation rate (%), number of shoots per explant (units) and root formation frequency (%). Object of the work - of Setaria viridis L. The results of work. The results of research showed differences in callus, shoot and root formation frequency of different exogenous growth regulator combination and quantity of nutrient medium. Setaria viridis L dedifferentiation induction intensively was in medium, supplemented by more quantity of auxin than cytokinin. Setaria viridis L callus formation frequency varied depending on the exogenous growth regulators combination of induction medium. Combination of growth regulators for 2.0 mg l-1 2,4-dichlorophenoxy acetic acid + 0.4 mg l-1 6-purine furfuryl amine stimulated more intensive dedifferentiation in vitro culture. Secondary differentiation was most intensive in nutrient medium supplemented with 0.4 mg l-1 α naphthyl acetic acid and 2.0 mg l-1 N6-(2-isopentyl) adenine. The most shoots from the explant were formed in a nutrient medium supplemented with 0.3 mg l-1 NAA + 2.0 mg l-1 2iP. Setaria viridis L the process of rhizogenesis in vitro stimulated the growth regulators combinations 2.0 mg l-1 2,4 D + 0.4 mg l-1 KIN and 0.2 mg l-1 NAA + 2.0 mg l-1 2iP and 0, 4 mg l-1 NAA + 2.0 mg l-1 BAP.

The ability of Candida albicans to change morphology plays several roles in its virulence and as a human commensal. The yeast-to-hyphae transition is tightly regulated by several sets of activating and repressing pathways. The DNA-binding proteins Rfg1p, Nrg1p and the global repressor Tup1p are part of the repressors found to regulate this morphogenesis. Knowledge of these repressors is based on extrapolations from homology to S. cerevisiae and from expression studies of mutants in inducing conditions, all of which are indirect means of determining a protein's function. We proposed a genome-wide location study of the Nrg1 and Rfg1 transcription factors to obtain direct data to identify their in vivo targets. Our results suggest different avenues for Nrg1p function and a regulation behaviour diverging from the previously suggested model: Nrg1p acts not only as a repressor but also as a transcription activator. Furthermore it regulates its target genes through binding in their coding regions instead binding to the expected regulatory elements on promoters.