Resilin is a disordered elastomeric protein and can be found in specialized regions of insect cuticles. Its protein sequence, functions and dynamic mechanical properties vary substantially across the species. Resilin can operate across the frequency range from 5 Hz for locomotion to 13 kHz for sound production. To understand the functions of different exons of resilin, we synthesize recombinant resilin-like hydrogels from different exons, and investigate the water content and dynamic mechanical properties, along with estimating surface energies relevant for adhesion. The recombinant resilin-like hydrogel has 80wt% water and does not show any sign of tack even though it satisfies the Dahlquist criterion. Finally, doubly shifted dynamic moduli master curves are developed by applying the time-temperature concentration superposition principle (TTCSP), and compared to results obtained with natural resilin from locusts, dragonflies and cockroaches. The resulting master curves show that the synthetic resilin undergoes a prominent transition, though the responsible mechanism is unclear. Possible explanations for the significant increase in modulus include the formation of intramolecular hydrogen bonds, altered structural organization, or passing through a glass transition, all of which have been reported in the literature for polymeric materials. Results show that in nature, resilin operates at a much lower frequency than this glass transition frequency at room temperature. Moreover, recombinant resilins from different clones have comparable resilience with natural resilin, though the modulus is around 1.5 decades lower. Results from the clones with and without chitin binding domains (ChBD) indicate that the transition for the clone without ChBD occurs at lower frequencies than for those with the ChBD, perhaps due to the disordered nature of the clone without ChBD. Atomistic molecular modeling is applied on the repetitive motifs of resilin and different elastomeric proteins to better understand the relationship between elastomeric behavior and amino acid sequences. Results show that the motifs form a favorable bent conformation, likely enabled by glycine's lack of steric hindrance and held in place through intramolecular hydrogen bonds. During Steered Molecular Dynamic (SMD) pulling of these motifs, the hydrogen bonds break and they reform again when the peptides are released to move freely, returning to similar bent conformations. The transition seen in the master curves of recombinant resilins might be due to either these intramolecular hydrogen bonds or to glass transition behavior, though evidence indicates that the transition probably due to the glass transition. What we learned from the synthesized recombinant resilin and simulating the repetitive motifs of resilin may be applicable to the biology and mechanics of other elastomeric biomaterials, and may provide deeper understanding of their unique properties. / Ph. D.

Vetores lentivirais são ferramentas fundamentais para modificação celular. Sua utilização ganhou destaque devido à capacidade desses em integrar ao genoma de células que estão ou não em divisão. Grande parte dos vetores desenvolvidos são derivados do genoma do Vírus da Imunodeficiência Humana (HIV-1), portanto, modificações foram necessárias a fim de evitar a formação de Partículas Competentes em Replicação (RCLs) e garantir uma utilização segura. Com as modificações, foram produzidos os vetores lentivirais de terceira geração utilizados atualmente. Esses vetores podem ser usados para expressão constitutiva de genes, produção de proteínas recombinantes, produção de animais transgênicos e terapia gênica. Com isso, torna-se necessário o desenvolvimento de vetores lentivirais para aplicação em pesquisa básica e ensaios clínicos. Dessa forma, o presente estudo teve por objetivo a construção de vetores de expressão lentivirais aplicáveis à: 1- expressão constitutiva de genes de interesse e 2-vetores com promotores específicos para expressão de proteínas em megacariócitos. Esse trabalho descreve a construção desses vetores, sua importância e discute suas possíveis aplicações. As sequências selecionadas para produção dos vetores foram: os genes Runx1C e VkorC1 e os promotores proPF4 e proITGA2b. Todas as sequências encontram-se clonadas em vetor de clonagem e estoques de bactérias com esses vetores congeladas em glicerol foram confeccionados. Para a confecção dos vetores lentivirais, o gene Runx1C foi subclonado no vetor lentiviral base p1054-CIGWS sob controle do promotor forte CMV, enquanto o promotor proITGA2b foi subclonado no vetor base p1054-FVIII, em substituição ao promotor CMV, de forma a controlar a expressão de FVIII. Os dois vetores produzidos apresentam ainda o gene para proteína verde GFP precedida do sítio de ligação do ribossomo IRES, com expressão controlada pelo mesmo promotor interno do vetor. O trabalho possibilitou, portanto, a produção de dois vetores lentivirais bi-cistrônicos: p1054-Runx1C e pL-proITGA2b-FVIII. A construção p1054-Runx1C ainda não foi sequenciada, mas foi confirmada por restrição enzimática e apresenta potencial para aplicação em estudos de diferenciação hematopoética. Já a construção pL-proITGA2b-FVIII foi sequenciada, porém sem confirmação da região de ligação do proITGA2b ao vetor. Reações de PCR e de restrição enzimática confirmaram a ligação e sequenciamento mostrou 67% de similaridade entre a região sequenciada e o promotor ITGA2b depositado no banco de dados. Análise funcional foi realizada através da transfecção desse vetor em células HEK-293T. As células transfectadas apresentaram expressão positiva para GFP e secreção de FVIII no sobrenadante celular, evidenciando que o promotor proITGA2b clonado no vetor encontra-se ativo. Esse vetor apresenta potencial para aplicação em terapia gênica para hemofilias, pois apresenta expressão do fator de coagulação direcionado a megacariócitos e plaquetas, células que estão diretamente relacionadas ao processo de coagulação, representando grandes veículos para secreção desses fatores. Ainda, os dois vetores lentivirais gerados apresentam segurança e eficiência elevadas, pois são vetores de terceira geração auto-inativantes (SIN) e apresentam elementos regulatórios que melhoram o transporte e integração do DNA ao genoma hospedeiro. / Lentiviral vectors are fundamental tools for cell modification that gained prominence due to their ability to integrate the genome of non-dividing cells. Most of developed lentiviral vectors are derived from the genome of Human Immunodeficiency Virus (HIV-1), so modifications were necessary in order to avoid the formation of Competent Replication Particles (RCLs) and ensure safer operations. The modifications led to development of third generation lentiviral vectors currently used. These vectors can be used for constitutive gene expression, production of recombinant protein, production of transgenic animals and gene therapy. It\'s evident the need to develop lentiviral vectors for application in basic research and clinical trials. Thus this study aimed to construct lentiviral expression vectors applicable to: 1- constitutive expression of genes of interest and 2-vectors with specific promoters for expression of proteins in megakaryocytes and platelets. This paper describes the construction of these vectors, their importance and discuss their possible applications. Sequences were selected for production of the vectors: genes Runx1C and VkorC1 and proPF4 and proITGA2b promoters. All four sequences are cloned into cloning vectors and stocks of bacteria with these vectors frozen in glycerol were prepared. Lentiviral vectors were engineered from subcloning the sequence Runx1C into the basic lentiviral vector p1054- CIGWS under control of the strong CMV promoter, and from subcloning proITGA2b promoter into p1054-FVIII basic vector, replacing the CMV promoter in order to control the expression of FVIII. Both vectors exhibit the green fluorescence protein GFP gene preceded by a ribosome binding site IRES under control of vector\'s internal promoter. Therefore, this work resulted in the production of two bi-cistronic lentiviral vectors: p1054-Runx1C and pLproITGA2b-FVIII. The p1054-Runx1C construction has not yet been sequenced, but it was confirmed by digestion and has potential for use in hematopoietic differentiation studies. Though, pL-proITGA2b-FVIII construct was sequenced, but the technique didn\'t allow to confirm the binding region between proITGA2b and the vector. Although PCR reaction and digestion confirmed the construction. Sequence analysis showed 67% similarity between the sequenced region and ITGA2b promoter deposited in the database. Functional analysis was performed by transfection of this vector in HEK-293T cells. The transfected cells showed positive expression of GFP and FVIII secretion in cell supernatant, indicating that the proITGA2b promoter cloned into the vector is active. This vector has potential usage in gene therapy for hemophilia, since it can be used to express coagulation factors in megakaryocytes and platelets and these cells are directly related to the clotting process, representing great vehicles for secretion of these factors. Even more, the two lentiviral vectors generated have higher safety and efficiency, as they are self-inactivating (SIN) third-generation vectors and have regulatory elements that enhance transport and integration of DNA into the host genome.

Vetores lentivirais são ferramentas fundamentais para modificação celular. Sua utilização ganhou destaque devido à capacidade desses em integrar ao genoma de células que estão ou não em divisão. Grande parte dos vetores desenvolvidos são derivados do genoma do Vírus da Imunodeficiência Humana (HIV-1), portanto, modificações foram necessárias a fim de evitar a formação de Partículas Competentes em Replicação (RCLs) e garantir uma utilização segura. Com as modificações, foram produzidos os vetores lentivirais de terceira geração utilizados atualmente. Esses vetores podem ser usados para expressão constitutiva de genes, produção de proteínas recombinantes, produção de animais transgênicos e terapia gênica. Com isso, torna-se necessário o desenvolvimento de vetores lentivirais para aplicação em pesquisa básica e ensaios clínicos. Dessa forma, o presente estudo teve por objetivo a construção de vetores de expressão lentivirais aplicáveis à: 1- expressão constitutiva de genes de interesse e 2-vetores com promotores específicos para expressão de proteínas em megacariócitos. Esse trabalho descreve a construção desses vetores, sua importância e discute suas possíveis aplicações. As sequências selecionadas para produção dos vetores foram: os genes Runx1C e VkorC1 e os promotores proPF4 e proITGA2b. Todas as sequências encontram-se clonadas em vetor de clonagem e estoques de bactérias com esses vetores congeladas em glicerol foram confeccionados. Para a confecção dos vetores lentivirais, o gene Runx1C foi subclonado no vetor lentiviral base p1054-CIGWS sob controle do promotor forte CMV, enquanto o promotor proITGA2b foi subclonado no vetor base p1054-FVIII, em substituição ao promotor CMV, de forma a controlar a expressão de FVIII. Os dois vetores produzidos apresentam ainda o gene para proteína verde GFP precedida do sítio de ligação do ribossomo IRES, com expressão controlada pelo mesmo promotor interno do vetor. O trabalho possibilitou, portanto, a produção de dois vetores lentivirais bi-cistrônicos: p1054-Runx1C e pL-proITGA2b-FVIII. A construção p1054-Runx1C ainda não foi sequenciada, mas foi confirmada por restrição enzimática e apresenta potencial para aplicação em estudos de diferenciação hematopoética. Já a construção pL-proITGA2b-FVIII foi sequenciada, porém sem confirmação da região de ligação do proITGA2b ao vetor. Reações de PCR e de restrição enzimática confirmaram a ligação e sequenciamento mostrou 67% de similaridade entre a região sequenciada e o promotor ITGA2b depositado no banco de dados. Análise funcional foi realizada através da transfecção desse vetor em células HEK-293T. As células transfectadas apresentaram expressão positiva para GFP e secreção de FVIII no sobrenadante celular, evidenciando que o promotor proITGA2b clonado no vetor encontra-se ativo. Esse vetor apresenta potencial para aplicação em terapia gênica para hemofilias, pois apresenta expressão do fator de coagulação direcionado a megacariócitos e plaquetas, células que estão diretamente relacionadas ao processo de coagulação, representando grandes veículos para secreção desses fatores. Ainda, os dois vetores lentivirais gerados apresentam segurança e eficiência elevadas, pois são vetores de terceira geração auto-inativantes (SIN) e apresentam elementos regulatórios que melhoram o transporte e integração do DNA ao genoma hospedeiro. / Lentiviral vectors are fundamental tools for cell modification that gained prominence due to their ability to integrate the genome of non-dividing cells. Most of developed lentiviral vectors are derived from the genome of Human Immunodeficiency Virus (HIV-1), so modifications were necessary in order to avoid the formation of Competent Replication Particles (RCLs) and ensure safer operations. The modifications led to development of third generation lentiviral vectors currently used. These vectors can be used for constitutive gene expression, production of recombinant protein, production of transgenic animals and gene therapy. It\'s evident the need to develop lentiviral vectors for application in basic research and clinical trials. Thus this study aimed to construct lentiviral expression vectors applicable to: 1- constitutive expression of genes of interest and 2-vectors with specific promoters for expression of proteins in megakaryocytes and platelets. This paper describes the construction of these vectors, their importance and discuss their possible applications. Sequences were selected for production of the vectors: genes Runx1C and VkorC1 and proPF4 and proITGA2b promoters. All four sequences are cloned into cloning vectors and stocks of bacteria with these vectors frozen in glycerol were prepared. Lentiviral vectors were engineered from subcloning the sequence Runx1C into the basic lentiviral vector p1054- CIGWS under control of the strong CMV promoter, and from subcloning proITGA2b promoter into p1054-FVIII basic vector, replacing the CMV promoter in order to control the expression of FVIII. Both vectors exhibit the green fluorescence protein GFP gene preceded by a ribosome binding site IRES under control of vector\'s internal promoter. Therefore, this work resulted in the production of two bi-cistronic lentiviral vectors: p1054-Runx1C and pLproITGA2b-FVIII. The p1054-Runx1C construction has not yet been sequenced, but it was confirmed by digestion and has potential for use in hematopoietic differentiation studies. Though, pL-proITGA2b-FVIII construct was sequenced, but the technique didn\'t allow to confirm the binding region between proITGA2b and the vector. Although PCR reaction and digestion confirmed the construction. Sequence analysis showed 67% similarity between the sequenced region and ITGA2b promoter deposited in the database. Functional analysis was performed by transfection of this vector in HEK-293T cells. The transfected cells showed positive expression of GFP and FVIII secretion in cell supernatant, indicating that the proITGA2b promoter cloned into the vector is active. This vector has potential usage in gene therapy for hemophilia, since it can be used to express coagulation factors in megakaryocytes and platelets and these cells are directly related to the clotting process, representing great vehicles for secretion of these factors. Even more, the two lentiviral vectors generated have higher safety and efficiency, as they are self-inactivating (SIN) third-generation vectors and have regulatory elements that enhance transport and integration of DNA into the host genome.

Resumo: O mal-do-fio (ou queima-do-fio) é uma doença causada pelo fungo Basidiomiceto Ceratobasidium sp., que afeta diversas plantas frutíferas nativas ou cultivadas. A doença ocorre com mais freqüência em zonas de alta precipitação e temperaturas elevadas, típicas de regiões de florestas tropicais como a Amazônia e a Mata Atlântica. Em São Paulo, recentemente detectou-se a ocorrência do mal-do-fio, em caquizeiro na região de Mogi das Cruzes. Essa doença pode- se tornar importante com a expansão do cultivo de fruteiras no Estado. A maioria das pesquisas sobre o patossistema focalizou a epidemiologia e o controle do fungo. Entretanto a etiologia do patógeno ainda não está totalmente definida, especialmente para populações do fungo infectando caqui e chá no Estado de São Paulo. Há também pouca informação sobre a divergência genética entre populações do patógeno de hospedeiros distintos como caqui e chá. O primeiro objetivo deste trabalho foi determinar o posicionamento filogenético global de populações de Ceratobasidium sp. do caqui e do chá, em relação a espécies de Ceratobasidium sp. descritas no mundo. Foram analisadas seqüências de DNA da região ITS-5.8S do rDNA, inferindo-se a história dos alelos ou haplótipos deste lócus, por meio de métodos filogenéticos, cladísticos e coalescentes. Observou-se que uso de C. noxium é apropriado para denominar espécies associadas ao mal-do- fio em caqui e chá, apesar de C. noxium do caqui e do chá constituírem populações filogeneticamente independentes, as quais denominamos de Grupo Diospyrus e Grupo Camellia. Este estudo trouxe uma contribuição importante para a compreensão das relações filogenéticas e biologia de populações de C. noxium em caqui e chá. Uma vez esclarecidas as questões filogenéticas, o segundo objetivo deste trabalho foi testar a patogenicidade cruzada de... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The white-thread blight is a disease caused by the Basidiomycete fungus Ceratobasidium sp. that affects several native or cropped tree fruits. This disease frequently occurs in zones of high precipitation and high temperatures typical of the tropical forest regions such as the Amazon and the Atlantic Forest. In São Paulo, the occurrence of the white-thread blight was detected only recently on kaki orchards closer to Mogi das Cruzes. That disease can become important with the expansion of the fruit trees cropping areas in the State. Most of the researches about the pathosystem has focused on the epidemiology and control of fungus. However the etiology of the pathogen is not totally defined yet, especially for the fungus populations infecting kaki and tea in São Paulo State. There is also little information available about the biological and genetic divergence between pathogen populations from distinct hosts, such as kaki and tea. The first objective of this research was to determine the global phylogenetic placement of populations of Ceratobasidium from kaki and tea, considering the species of Ceratobasidium described throughout the world. Sequences of the ITS-5.8S region of the rDNA were analyzed, inferring the alleles or haplotypes history for this locus, by phylogenetics, cladistisc and coalescent methods. We observed that the use of C. noxium is appropriate to denominate the fungus species associate with the white-thread blight on kaki and tea, despite the fact that C. noxium from kaki and tea constitutes phylogenetically independent populations, which we denominate Diospyrus and Camellia groups. This study brought an important contribution for the understanding of the phylogenetics and population biology of C. noxium infecting kaki and tea. Once the phylogenetics subjects have been cleared, the second objective of this work was to test the cross-pathogenicity... (Complete abstract, access electronic address below) / Orientador: Paulo Cezar Ceresini / Coorientador: Alcebíades Ribeiro Campos / Banca: Adriana Zanin Kronka / Banca: Cézar Junior Bueno / Mestre

Plants are constantly exposed to pathogens and various environmental stresses, such as cold, salinity and drought. Plants normally respond rapidly to these biotic and abiotic stresses. Efficient perception of biotic and abiotic stresses and cell programmed signaling mechanisms for appropriate responses are important for growth and survival of plants. Calcium is an important second messenger in signaling pathways that respond to environmental stresses, pathogen attack as well as hormonal stimuli (For review, see DeFalco et al., 2010; Reddy and Reddy, 2004; Sanders et al., 2002). The transient increase of cytosolic free calcium concentration has been shown in a variety of external signals (Reddy, 2001), which in turn triggers many signal transduction pathways leading to a variety of cellular responses (Bush, 1995). Any calcium mediated signal transduction process involves generation of signal-specific calcium signature in the cytosol (Scrase-Field and Knight, 2003). These changes in cytosolic calcium level or ‘calcium signatures’ are sensed by the specific group of proteins called the ‘calcium sensors’. Different calcium sensors recognize specific calcium signatures and transduce them into downstream effects, including altered protein phosphorylation and gene expression patterns. In plants the protein kinases are a large and differentiated group of calcium sensors. After analyzing 1264 protein kinase sequences, a superfamily of protein kinase called CDPK/SnRK family of protein kinase were defined (Hrabak et al., 2003). CDPK/SnRK family of protein kinases encompasses five subfamilies viz., calcium-dependant protein kinases, (CPKs), calcium/calmodulin dependant protein kinases (CCaMKs), calmodulin-dependant protein kinases (CaMKs), CPKrelated kinases (CRKs), and SnF1 related kinase 3 (SnRK3) and are regulated by calcium directly or indirectly. Among these, in plants, calcium-dependant protein kinases (CPKs) are predominant calcium sensors, which are shown to be involved in myriads of physiological responses. They are Ser/Thr family of protein kinases typically made up of five domains with an Nterminal variable domain followed by catalytic protein kinase domain, an autoinhibitory/ junction domain, a regulatory calmodulin-like domain (CaMLD) and a Cterminal domain of variable length. The CPKs are unique due to the presence of CaMLD which couples the calcium sensor directly to its responder (kinase domain). Although CPKs are highly conserved, there are several features that distinguish different members of the plant CDPK family. In an attempt to investigate the role of a CPK isoform, in the present work we bring out the results and inferences on isolation and characterization of a novel cDNA encoding a calcium-dependant protein kinase isoform ScCPK1 from Swainsonacanescens, a pharmaceutically important Australian herb known to produce an anticancer drug, swainsonine. Initially, we have cloned an 800 bp partial CPK cDNA from S. canescens by reverse transcription polymerase chain reaction (RTPCR) using degenerate oligonucleotide primers designed based on conserved regions of the other known CPKs. A 2.1 kb full length CPK was obtained using 5` and 3` RACE which was designated as ScCPK1. An open reading frame (ORF) of 1659 bp was detected that encodes a protein of 552 amino acids with a calculated molecular mass of 61.8 kDa. Comparison of the deduced amino acid sequence of ScCPK1 with sequences of other CPKs revealed the highest identity (>90%) to Glycine max and Vigna radiate CPKs. As described for other CPKs, ScCPK1 has a long variable domain (88 aa), an auto-inhibitory domain (31 aa) and a C-terminal calmodulin domain (145 aa) containing four EF-hand calcium binding motifs, which is found in many CPKs. Phylogenetic tree analysis showed that ScCPK1 was closely related to StCPK4 , CmCPK1 and CmCPK2. The entire coding region of ScCPK1 was cloned into pRSETA expression vector and expressed as fusion protein in E.coli. The recombinant ScCPK1 protein was purified to homogeneity by NiNTA affinity chromatography. The recombinant purified ScCPK1 was catalytically active in a calcium-dependent manner. The recombinant ScCPK1 phosphorylated itself and histone IIIS as substrate only in the presence of Ca2+. Phosphoaminoacid analyses showed that ScCPK1 phosphorylates serine and threonine residues of histone IIIS and its autophosphorylation also occurs on serine and threonine residues. ScCPK1 has a pH and temperature optima of 7.5 and 37 °C, respectively. It showed high affinity to histone III-S with a Km of 4.8 µM and had a Vmax of 4.700 pmoles of γ32P incorporation/min/mg at saturating substrate concentrations. The ScCPK1 is ~100fold active and showed 10fold higher affinities to histone III-S than CaCPK1 and CaCPK2, CPKs which were characterized from Cicer arietinum previously in our laboratory (Prakash and Jayabaskaran, 2006). From literature it is known that many CPKs are activated or inhibited by metal ions. (PutnamEvans etal., 1990; Anil and Rao, 2001). The influence of Na+ and Mg2+on the in vitro substrate phosphorylation activity of the recombinant ScCPK1 was tested in this work. Addition of NaCl strongly inhibited ScCPK1 activity. The inhibition of substrate phosphorylation activity by salt implies ionic interactions between the positively charged substrate and the enzyme’s active site. The optimum concentration of Mg2+ for ScCPK1 substrate phosphorylation activity was found to be 810 mM, similar to CaCPK1 and CaCPK2 (Prakash and Jayabaskaran, 2006). However, the activity was inhibited above 10 mM Mg2+suggesting the disruption of ionic interactions between the enzyme and the substrate. The kinase and autophosphorylation activities of the recombinant ScCPK1 were calmodulin independent and sensitive to CaM antagonists’ calmidazolium and W7 (N(6aminohexyl)5chloronaphthalene sulphonate). This indicates that the activation was supported by calmodulin-like domain, which is typical of CPK family. Farmer and Choi (1999), showed that DcCPK1 activity was inhibited by polyamines vizspermine and spermidine, and polylysine. We found that substrate phosphorylation activity of ScCPK1 was inhibited by polyLLysine with an IC50 of 8 M but not the polyamines, spermine and spermidine. An interesting aspect that makes CPKs attractive for research is their functional similarity to mammalian PKCs. There are no structural PKC analogues found in plant genomic data. Similar to PKCs, CPKs are regulated by intracellular Ca2+ signals. There is also experimental evidence that some of the CPKs are additionally activated by phospholipids (Farmer and Choi, 1999; Szczegielniak etal., 2000). We investigated the effects of lipid molecules on the activity of ScCPK1. Phosphorylation of histone IIIS by ScCPK1 was stimulated by phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol in the presence of Ca2+, where as lysophosphatidylcholine, phosphatidylcholine and phosphatidic acid did not increase the enzyme activity. Our data that shows interaction of ScCPK1 with phospholipids supports the idea that this protein kinase could be associated with the membrane. The work from Farmer and Choi (1999), with DcCPK1 suggested that some of the PKClike activities observed in plants may be attributed to CPKs. They also demonstrated that DcCPK1 phosphorylated PKC pseudosubstrate peptide and also was sensitive to staurosporine inhibition. However, the protein kinase inhibitor, staurosporine inhibited the substrate phosphorylation activity of ScCPK1 completely with an IC50 value of 700 nM invitro. But PKC inhibitor PMA was less effective, inhibiting the substrate phosphorylation activity of ScCPK1 to a maximum of 50%, but at a very high concentration (200 nM). Our data suggests that ScCPK1 may not have any features attributable to PKC. We investigated subcellular localization of the ScCPK1. To gain a better understanding of the subcellular localization of the ScCPK1, we generated GFP fusion protein of ScCPK1 and transiently expressed it in Agrobacterium-mediated transformed tobacco BY2 cells. Analysis of the GFP expression patterns in transformed tobacco BY2 cells revealed ScCPK1 localization in the plasma membrane of the transformed tobacco BY2 cells despite lacking consensus myristoylation and palmitoylation motifs (as per in silico analyses). Taking together, our data have demonstrated that ScCPK1 is a Ser/Thr protein kinase and its sub-cellular localization studies revealed that it is localized to plasma membrane. We propose that ScCPK1 is a key component of one or more signaling pathways and plays vital roles in plant development, responses to environmental stimuli and/ or in secondary metabolite biosynthetic gene expression. The involvement of the ScCPK1 as a component of signaling pathways warrants further studies.

Indiana University-Purdue University Indianapolis (IUPUI) / The development of new methods to carry out gene transfer has many benefits to several fields, such as gene therapy, agriculture and animal health. The newly established lentiviral vector systems further increase the efficiency of gene transfer dramatically. Some studies have shown that lentiviral vector systems enhance efficiency over 10-fold higher than traditional pronuclear injection. However, the timing for lentiviral vector integration to occur remains unclear. Integrating in different stages of embryogenesis might lead to different integration patterns between tissues. Moreover, in our previous study we found that the vector copy number in transgenic sheep varied, some having one or more copies per cells while other animals having less than one copy per cell suggesting mosaicism. Here I hypothesized that injection of a lentiviral vector into a single cell embryo can lead to integration very early in embryogenesis but can also occur after several cell divisions. In this study, we focus on investigating integration sites in tissues developing from different germ layers as well as extraembryonic tissues to determine when integration occurs. In addition, we are also interested in insertional mutagenesis caused by viral sequence integration in or near gene regions. We utilize linear amplification-mediated polymerase chain reaction (LAM-PCR) and next- generation sequencing (NGS) technology to determine possible integration sites. In this study, we found the evidence based on a series of experiments to support my hypothesis, suggesting that integration event also happens after several cell divisions. For insertional mutagenesis analysis, the closest genes can be found according to integration sites, but they are likely too far away from the integration sites to be influenced. A well-annotated sheep genome database is needed for insertional mutagenesis analysis.

MSc (Microbiology) / Department of Microbiology / See the attached abstract below

Indiana University-Purdue University Indianapolis (IUPUI) / The phototropin photoreceptors are important regulators of plant growth and development and can therefore affect the photosynthetic activity of plants. Phototropin1 and Phototropin2 are versatile protein kinases that become activated when exposed to blue light. Their photobiological actions are best understood in the model plant Arabidopsis thaliana, where they are known to trigger several responses to blue light, one of which is phototropism, the bending of plant organs towards light. Additionally, phot1 and phot2 drive stomatal opening, chloroplast arrangement in leaf cells, leaf expansion, and leaf orientation. The phot1-specific response is rapid inhibition of hypocotyl growth, leaf positioning and mRNA stability whereas phot2 mediates the chloroplast avoidance response to high light. These responses impact a plant’s ability to capture light for photosynthesis, therefore the phototropins play important roles in optimizing a plant’s photosynthetic activity. Soybean (Glycine max) is a very important crop plant in Indiana known for its nutritional versatility and is also utilized for biodiesel production.In spite of soybean being a key crop, there is currently no information about the functionality of soybean phototropins. Also, being a legume, soybean has many structural and functional features that are not present in Arabidopsis. Interestingly, PsPHOT1A (a photoreceptor from garden pea) was found to be a functional phototropin as it was able to complement the phot1 mutation in Arabidopsis. The roles of these proteins in soybean will be elucidated based on the hypothesis that soybean phototropins play essential roles in regulating photosynthetic activity as do the Arabidopsis phototropins. To date, five soybean phototropins, 3 PHOT1s and 2 PHOT2s, are believed to exist. These GmPHOT protein coding regions were amplified by RT-PCR and cloned into pCR8/TOPO or pENTR-D/TOPO vectors via TOPO cloning to utilize Gateway cloning technology to create plant transformation constructs subsequently. The cloned GmPHOT cDNAs from each of the 5 GmPHOTs were sequenced and compared to the GmPHOT sequences from the Phytozome database to assess the accuracy of the gene models. The gene models of all the GmPHOTs were found to be accurate except that of GmPHOT1B-2. The high level of sequence identity between the GmPHOTs and AtPHOTs and the conservation of LOV domains and catalytic domains indicate structural resemblance between them. This suggests that soybean phototropins should encode active photoreceptors. The cloned protein coding regions from soybean were then recombined into a plant expression vector via Gateway technology,which were then used for transformation of Agrobacterium tumefaciens. These plant expression constructs will be utilized in the future to determine the functionality of soybean phototropins in Arabidopsis.