<p> Present throughout all classes of life, antimicrobial peptides (AMPs) confer defense against bacteria, viruses, fungi, and insects. Identifying maize AMPs would provide breeders with a new defense resource. Here, the investigation of maize AMPs is reported. The distribution of AMPs within a panel of ten Mississippi maize inbred lines with varying resistance to Lepidoptera larvae feeding and Aspergillus infection is explored to characterize their observed resistances. Homology data-mining with two comprehensive AMP databases revealed 88 unique maize AMP protein sequences across 81 genes in the MaizeGDB B73 genome assembly. AMP-related polymorphic sites were identified using genomic primers. Analyses with qRT-PCR revealed 8 differentially expressed maize AMP genes. Computational 3D models of AMPs are limited, and models of these eight maize AMP genes were predicted. Two-dimensional electrophoresis gels were used to contrast protein profiles of inbred lines with varying resistance to identify regions related to AMPs and other defense-related protein. </p><p>

<p> DNA&rsquo;s programmable nature and ability to self-assemble provides a powerful tool for the construction of complex nanostructures. The initial goal of the field was to use DNA to construct a continuous 3D DNA periodic lattice or crystal. The ultimate aim of the lattice structure would be to act as scaffold for the strategic placement of guest molecules such as macromolecules for structure determination using X-ray. Since that initial vision, the incorporation of guest molecules in DNA nanostructures has expanded to other applications such as cellular imaging, light-harvesting and drug delivery. However, there are several limitations to utilizing DNA crystals for these types of applications. They require relatively high cation concentrations to crystallize and often have low thermal stability. Additionally, crystals generally take on only one shape, or morphology, which can limit their uses in applications. </p><p> Our laboratory studies a 13-mer DNA oligonucleotide that self-assembles into crystals upon the addition of magnesium. I demonstrated that by treating these DNA crystals with a chemical crosslinker and depositing polydopamine on the crystal surface, we increased the overall durability of the crystals. Additionally, we modulated the morphology of the crystal without changing the underlying framework by designing crystal habit modifiers based on the known crystal structure and were able to predictably control the morphology of the overall crystal. This enhanced durability has allowed us to begin testing new applications for DNA crystals. I have explored the incorporation of doxorubicin into the stabilized DNA crystals as a potential form of a new drug delivery device. Together, this work significantly advanced several key areas necessary to diversify the capability of DNA crystals for nanotechnological applications. </p><p>

<p> The acquisition of fossil fuels has many harmful effects on the environment. One of those effects is the release of toxic heavy metals, which may find its way to water reservoirs or ecosystems. Metallothionein (MT) is a metal binding protein that has potential to alleviate such metal pollutions by scavenging for these metals. It can also be used to collect scarce metals used in industrial practices. The results showed that MT is able to bind metals in an organic environment, and that MT may be induced to release metals with the addition of acid. It was also found that the amount of metal bound is proportional to the amount of MT used. Despite efforts to show that MT may be used in PUREX, results indicate that the protein is unlikely to be used for this purpose. </p><p>

abstract: ABSTRACT The catalytic chaperone of Rubisco is AAA+ protein Rubisco activase (Rca), which hydrolyzes ATP and thus undergoes conformational change, helping in reactivating Rubisco. Rca reactivates Rubisco plausibly by removing its C- terminal tail from the opening of its active site thus releasing the inhibitor, a sugar phosphate molecule. Rubisco and Rca are regulated by the stromal environment, which includes the ATP/ADP ratio, Mg2+ concentration, redox potential etc. Here the mechanistic regulation of tobacco β-Rca was studied using steady state enzyme kinetics in terms of product inhibition, Mg2+ activation, cooperativity and asymmetry. A continuous Pi measurement assay was developed, and using this assay catalytic parameters were obtained, such as kcat 20.6 ± 6.5 min-1 ( n = 9) and KM 0.113 ± 0.033 mM (n = 4). A Mg2+ induced increase of substrate affinity in Rca was observed, where the KM changes from 0.452 mM to 0.069 mM, with the changing of free Mg2+ concentration from 0.1 mM to 10 mM. Fitting the catalytic efficiency as a function free Mg2+ concentration by use of a binding model gave a Hill coefficient of 2.2, which indicates a secondary magnesium binding site on the enzyme. A 8.4 fold increase of catalytic efficiency with increasing magnesium from 0.1 mM to 6.5 mM suggests a significant Mg2+ induced regulation of Rca. Moderate product inhibition was observed in inhibition study (Ki = 0. 063 ± 0.018 mM). A positive cooperativity (nH = 2.1) in ATP hydrolysis between two subunits was observed in the presence of 0.132 mM ADP, but not in the absence of ADP. This indicated the presence of two different classes of subunits, suggesting an asymmetric model for the enzyme. Inhibited Rubisco (ER) up to 20 μM concentration did not affect ATPase activity, in line with previous reports. The concentration dependent correlation of Rca activity (tobacco β-Rca) and oligomerization (cotton β-Rca) suggested that the dimer maybe the most active oligomeric species. A nucleotide induced thermal stabilization of Rca was observed, where ADP is more stabilizing than ATP in the absence of Mg2+. Mg2+ has a small destabilizing effect alone and in presence of the ADP, but a stabilizing effect in presence of ATP. The ligand induced thermal stability was similar for cotton and tobacco β-Rca. / Dissertation/Thesis / Doctoral Dissertation Biochemistry 2015

abstract: The linear chromosomes ends in eukaryotes are protected by telomeres, a nucleoprotein structure that contains telomeric DNA with repetitive sequence and associated proteins. Telomerase is an RNA-dependent DNA polymerase that adds telomeric DNA repeats to the 3'-ends of chromosomes to offset the loss of terminal DNA repeats during DNA replication. It consists of two core components: a telomerase reverse transcriptase (TERT) and a telomerase RNA (TR). Telomerase uses a short sequence in its integral RNA component as template to add multiple DNA repeats in a processive manner. However, it remains unclear how the telomerase utilizes the short RNA template accurately and efficiently during DNA repeat synthesis. As previously reported human telomerase nucleotide synthesis arrests upon reaching the end of its RNA template by a unique template-embedded pause signal. In this study, I demonstrate pause signal remains active following template regeneration and inhibits the intrinsic processivity and rate of telomerase repeat addition. Furthermore, I have found that the human telomerase catalytic cycle comprises a crucial and slow incorporation of the first nucleotide after template translocation. This slow nucleotide incorporation step drastically limits repeat addition processivity and rate, which is alleviated with elevated concentrations of dGTP. Additionally, molecular mechanism of the disease mutants on telomerase specific motif T, K570N, have been explored. Finally, I studied how telomerase selective inhibitor BIBR 1532 reduce telomerase repeat addition processivity by function assay. Together, these results shed new light on telomerase catalytic cycle and the importance of telomerase for biomedicine. / Dissertation/Thesis / Doctoral Dissertation Biochemistry 2018

Alfa-mannosidaasit ovat ryhmä entsyymejä jotka osallistuvat glykoproteiinien rakennukseen ja kierrätykseen leikkaamalla mannosidaasien välisiä alfa-sidoksia hydrolyysillä. Alfa-mannosidoosi on sairaus joka johtuu lysosomaalisen alfa-mannosidaasin mutaatioista. Sairauden vakavuus riippuu mutaation tyypistä ja sijainnista. Jos entsyymin toiminta on vain vähän hidastunut, oireet voivat olla lievät ja helposti hallittavissa, kun taas vakava mutaatio voi johtaa aikaiseen kuolemaan. Oireisiin kuuluvat kehitysvammaisuus, kuulo- ja luusto-ongelmat ja immuunikato. Oireet johtuvat kertyvien sokeriketjujen laajentamista lysosomeista, jotka vievät tilaa muilta soluelimiltä ja vaikeuttavat solun toimintaa. Parannusta sairauteen ei ole, mutta sitä voidaan helpottaa joko oirekohtaisilla hoidoilla tai entsyymikorvaushoidolla.

Makuaistin tehtävä on antaa informaatiota ravinnon laadusta jo ennen se nauttimista, ja auttaa välttämään mahdollisesti haitallisia tai myrkyllisiä aineita. Viidestä perusmausta makea on yksi tärkeimmistä ravinnon valintaan vaikuttavista tekijöistä. Yleisen käsityksen mukaan mieltymys makeaan on kehittynyt alun perin tunnistamaan runsasenergisiä ja ravinteikkaita ruoka-aineita, jonka vuoksi makean aistimista pidetään yleisesti ottaen miellyttävänä kokemuksena eläimillä ja ihmisillä. Koska yhä useampien makeutusaineiden käyttö on liitetty sairauksiin kuten diabetekseen, on nykyään tunnetuille pienimolekyylisille makeutusaineille kehiteltävä muita vaihtoehtoja. Jo 70-luvulla löydetyistä kasviperäisistä makeista proteiineista voisi olla seuraavaksi suureksi trendiksi elintarviketeollisuudessa, ihmisten suosiessa yhä luonnollisempia elintarvikkeita. Tässä tutkielmassa käyn lävitse yleisesti makeareseptorin rakenteen ja toiminnan, esittelen tähän mennessä löydetyt makeat proteiinit ja mitä niiden toimintamekanismista makeareseptorissa tiedetään. Lopuksi kerron vielä, miten luonnossa esiintyviä makeita proteiineja on kehitelty paremmin elintarviketeollisuuden käyttöön soveltuviksi, jotta niitä voitaisiin käyttää makeutusaineena tulevaisuudessa.

Tässä tutkielmassa tarkastellaan bakteriofagien käyttöä bakteeri-infektioiden hoidossa, erityisesti faagiterapian muodossa. Bakteriofagien kyky tappaa myös antibioottiresistenttejä bakteereita tekee faagiterapiasta hyvin merkittävän vaihtoehdon bakteeri-infektioiden hoitomenetelmänä. Bakteriofageilla voidaan sekä täydentää että korvata antibioottien käyttöä monissa erilaisissa tilanteissa.

HI-viruksen aiheuttama AIDS on ollut 1980-luvulta asti globaalisti merkittävä uhka terveydelle. Vielä nykyäänkin AIDS-liitännäisiin sairauksiin kuolee vuosittain tuhansia ihmisiä, eikä täydellistä parannuskeinoa ole onnistuttu löytämään. Uusi CRISPR/Cas9-geenieditointitekniikka voisi olla tähän ratkaisu, sillä sen soveltuvuutta HIV-1-infektioiden hoidossa on tutkittu ahkerasti positiivisin tuloksin. CRISPR/Cas9 on alunperin bakteerisoluissa adaptiivisen immuunijärjestelmän tavoin toimiva tekniikka, joka on viime aikoina yleistynyt geeniteknologian parissa nopeasti. Tekniikkana CRISPR/Cas9 on edullinen, helposti muokattavissa oleva ja pienen kokonsa ansiosta monikäyttöinen, mikä tekee siitä hyvin suositun työkalun tulevaisuuden geeniterapioita kehiteltäessä.

abstract: Spinal muscular atrophy (SMA) is a neurodegenerative disease that results in the loss of lower body muscle function. SMA is the second leading genetic cause of death in infants and arises from the loss of the Survival of Motor Neuron (SMN) protein. SMN is produced by two genes, smn1 and smn2, that are identical with the exception of a C to T conversion in exon 7 of the smn2 gene. SMA patients lacking the smn1 gene, rely on smn2 for production of SMN. Due to an alternative splicing event, smn2 primarily encodes a non-functional SMN lacking exon 7 (SMN D7) as well as a low amount of functional full-length SMN (SMN WT). SMN WT is ubiquitously expressed in all cell types, and it remains unclear how low levels of SMN WT in motor neurons lead to motor neuron degradation and SMA. SMN and its associated proteins, Gemin2-8 and Unrip, make up a large dynamic complex that functions to assemble ribonucleoproteins. The aim of this project was to characterize the interactions of the core SMN-Gemin2 complex, and to identify differences between SMN WT and SMN D7. SMN and Gemin2 proteins were expressed, purified and characterized via size exclusion chromatography. A stable N-terminal deleted Gemin2 protein (N45-G2) was characterized. The SMN WT expression system was optimized resulting in a 10-fold increase of protein expression. Lastly, the oligomeric states of SMN and SMN bound to Gemin2 were determined. SMN WT formed a mixture of oligomeric states, while SMN D7 did not. Both SMN WT and D7 bound to Gemin2 with a one-to-one ratio forming a heterodimer and several higher-order oligomeric states. The SMN WT-Gemin2 complex favored high molecular weight oligomers whereas the SMN D7-Gemin2 complex formed low molecular weight oligomers. These results indicate that the SMA mutant protein, SMN D7, was still able to associate with Gemin2, but was not able to form higher-order oligomeric complexes. The observed multiple oligomerization states of SMN and SMN bound to Gemin2 may play a crucial role in regulating one or several functions of the SMN protein. The inability of SMN D7 to form higher-order oligomers may inhibit or alter those functions leading to the SMA disease phenotype. / Dissertation/Thesis / Ph.D. Chemistry 2012