The human preprotachykinin-A gene promoter has been highly conserved and can drive human-like marker gene expression in the adult mouse CNS.

Peer reviewed

The human preprotachykinin-A gene promoter has been highly conserved and can drive human-like marker gene expression in the adult mouse CNS.

Peer reviewed

Molecular and functional studies of human immunodeficiency virus type 1 accessory protein

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Investigating the developmental and behavioral consequences of maternal immune activation on affected offspring

Maternal infection during pregnancy increases the risk of several neuropsychiatric disorders later in life, many of which have a component of dopaminergic (DA) dysfunction, including schizophrenia, autism spectrum disorders (ASD), and attention deficit hyperactivity disorder (ADHD). The majority of DA neurons are found in the adult midbrain; as such the midbrain is a key region of interest regarding these disorders. The literature is conflicting regarding the behavioral alterations following maternal immune activation (MIA) exposure, and the cellular and molecular consequences of MIA on the developing midbrain remain to be fully elucidated. Thus, this thesis aimed to establish the consequences of acute and mild MIA on offspring dopamine-related behaviors, as well as the associated cellular and molecular disturbances of MIA on offspring midbrains. We utilized a rat model of MIA using low dose (50μg/kg, I.P.) of LPS administered at different gestational ages. Our first study indicated that MIA at later gestational ages significantly increased pro-inflammatory IL-1β expression, and reduced HSD11B2 expression in the placenta, which is an important regulator of fetal development. In utero LPS exposure at later gestational ages also impaired the growth of neurons from affected offspring. This study identified key gestational stages during which MIA resulted in differential effects. We utilized these time points in subsequent studies, the next of which investigated neurobehavioral outcomes following MIA. Our results from that study showed that motor differences occurred in juvenile offspring following MIA at E16 only, and these differences were compensated for in adolescence. Then, there was a decline in motor behavior capabilities in adulthood, again only for animals exposed to MIA on E16 (and not E12). Furthermore, our results also demonstrated adolescent and adult offspring that were exposed to MIA at E12 had diminished responses to amphetamine in reward seeking behaviors. In our final study, we aimed to investigate the molecular and cellular changes following MIA which might explain these behavioral alterations. This final study showed a differential inflammatory response in fetal midbrains depending on gestational age of exposure as well as differential developmental alterations. For example, LPS exposure at E16 resulted in decreased VM neurosphere size after 7DIV and this was associated with an increased susceptibility to neurotoxic effects of pro-inflammatory cytokines for VM neurospheres and VM DA neurons treated in culture. In utero LPS exposure at E16 also reduced DA neuron count of fetal VM, measured by TH staining. However, there were no differences in DA neuron number in juvenile, adolescent, or adult offspring. Similarly, LPS exposure did not alter cell number or morphology of glial cells in the midbrains of affected offspring. In conclusion, this thesis indicated later rat pregnancy (E16) as vulnerable time for MIA to affect the development of the nigrostriatal pathway and subsequent behavioral outcomes, possibly implicating a role for MIA in increased risk for disorders associated with motor behavior, like PD. These effects may be mediated through alterations in the placenta and altered inflammatory mediators in the offspring brain. This thesis has also shown that MIA in earlier rat pregnancy (E12) results in altered mesocorticolimbic function, and in particular MIA on E12 resulted in a differential response to amphetamine in affected offspring, which may implicate a role for MIA in increasing the risk for disorders associated with this pathway, including drug tolerance and addiction.

Molecular and functional studies of human immunodeficiency virus type 1 accessory protein

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Molecular and isotopic compositions of hydrocarbons at DSDP Site 76-533

In an investigation of gas hydrates in deep ocean sediments, gas samples from Deep Sea Drilling Project Site 533 on the Blake Outer Ridge in the northwest Atlantic were obtained for molecular and isotopic analyses. Gas samples were collected from the first successful deployment of a pressure core barrel (PCB) in a hydrate region. The pressure decline curves from two of the four PCB retrievals at in situ pressures suggested the presence of small amounts of gas hydrates. Compositional and isotopic measurements of gases from several points along the pressure decline curve indicated that (1) biogenic methane (d13C = -68 per mil; C1/C2 = 5000) was the dominant gas (>90%); (2) little fractionation in the C1/C2 ratio or the C carbon isotopic composition occurred as gas hydrates decomposed during pressure decline experiments; (3) the percent of C3, i-C4, and CO2 degassed increased as the pressure declined, indicating that these molecules may help stabilize the hydrate structure; (4) excess nitrogen was present during initial degassing; and (5) C1/C2 ratios and isotopic ratios of C gases were similar to those obtained from conventional core sampling. The PCB gas also contained trace amounts of saturated, acyclic, cyclic, and aromatic C5-C14 hydrocarbons, as well as alkenes and tetrahydrothiophenes. Gas from a decomposed specimen of gas hydrate had similar molecular and isotopic ratios to the PCB gas (d13C of -68 per mil for methane and a C1/C2 ratio of about 6000). Regular trends in the d13C of methane (about -95 to -60 per mil) and C1/C2 ratios (about 25000 to 2000) were observed with depth. Capillary gas chromatography (GC) and total scanning fluorescence measurements of extracted organic material were characteristic of hydrocarbons dominated by a marine source, though significant amounts of perylene were also present.

On the microstructure, growth pattern and original porosity of belemnite rostra: insights from calcitic Jurassic belemnites

Calcitic belemnite rostra are usually employed to perform paleoenvironmental studies based on geochemical data. However, several questions, such as their original porosity and microstructure, remain open, despite they are essential to make accurate interpretations based on geochemical analyses.This paper revisits and enlightens some of these questions. Petrographic data demonstrate that calcite crystals of the rostrum solidum of belemnites grow from spherulites that successively develop along the apical line, resulting in a “regular spherulithic prismatic” microstructure. Radially arranged calcite crystals emerge and diverge from the spherulites: towards the apex, crystals grow until a new spherulite is formed; towards the external walls of the rostrum, the crystals become progressively bigger and prismatic. Adjacent crystals slightly vary in their c-axis orientation, resulting in undulose extinction. Concentric growth layering develops at different scales and is superimposed and traversed by a radial pattern, which results in the micro-fibrous texture that is observed in the calcite crystals in the rostra.Petrographic data demonstrate that single calcite crystals in the rostra have a composite nature, which strongly suggests that the belemnite rostra were originally porous. Single crystals consistently comprise two distinct zones or sectors in optical continuity: 1) the inner zone is fluorescent, has relatively low optical relief under transmitted light (TL) microscopy, a dark-grey color under backscatter electron microscopy (BSEM), a commonly triangular shape, a “patchy” appearance and relatively high Mg and Na contents; 2) the outer sector is non-fluorescent, has relatively high optical relief under TL, a light-grey color under BSEM and low Mg and Na contents. The inner and fluorescent sectors are interpreted to have formed first as a product of biologically controlled mineralization during belemnite skeletal growth and the non-fluorescent outer sectors as overgrowths of the former, filling the intra- and inter-crystalline porosity. This question has important implications for making paleoenvironmental and/or paleoclimatic interpretations based on geochemical analyses of belemnite rostra.Finally, the petrographic features of composite calcite crystals in the rostra also suggest the non-classical crystallization of belemnite rostra, as previously suggested by other authors.

Mechanical properties and cellular response of novel electrospun nanofibers for ligament tissue engineering: Effects of orientation and geometry

Electrospun nanofibers are a promising material for ligamentous tissue engineering, however weak mechanical properties of fibers to date have limited their clinical usage. The goal of this work was to modify electrospun nanofibers to create a robust structure that mimics the complex hierarchy of native tendons and ligaments. The scaffolds that were fabricated in this study consisted of either random or aligned nanofibers in flat sheets or rolled nanofiber bundles that mimic the size scale of fascicle units in primarily tensile load bearing soft musculoskeletal tissues. Altering nanofiber orientation and geometry significantly affected mechanical properties; most notably aligned nanofiber sheets had the greatest modulus; 125% higher than that of random nanofiber sheets; and 45% higher than aligned nanofiber bundles. Modifying aligned nanofiber sheets to form aligned nanofiber bundles also resulted in approximately 107% higher yield stresses and 140% higher yield strains. The mechanical properties of aligned nanofiber bundles were in the range of the mechanical properties of the native ACL: modulus=158±32MPa, yield stress=57±23MPa and yield strain=0.38±0.08. Adipose derived stem cells cultured on all surfaces remained viable and proliferated extensively over a 7 day culture period and cells elongated on nanofiber bundles. The results of the study suggest that aligned nanofiber bundles may be useful for ligament and tendon tissue engineering based on their mechanical properties and ability to support cell adhesion, proliferation, and elongation.

Fasciola hepatica surface tegument: glycoproteins at the interface of parasite and host

Fasciola hepatica, commonly known as liver fluke, is a trematode which causes Fasciolosis in ruminants and humans. The outer tegumental coat of F. hepatica (FhTeg) is a complex metabolically active biological matrix that is continually exposed to the host immune system and therefore makes a good vaccine target. F. hepatica tegumental coat is highly glycosylated and helminth-derived immunogenic oligosaccharide motifs and glycoproteins are currently being investigated as novel vaccine candidates. This report presents the first systematic characterisation of FhTeg glycosylation using lectin microarrays to characterise carbohydrates motifs present, and lectin histochemistry to localize these on the F. hepatica tegument. We discovered that FhTeg glycoproteins are predominantly oligomannose oligosaccharides that are expressed on the spines, suckers and tegumental coat of F. hepatica and lectin blot analysis confirmed the abundance of N- glycosylated proteins. While some oligosaccharides are widely distributed on the fluke surface other subsets are restricted to distinct anatomical regions. We selectively enriched for FhTeg mannosylated glycoprotein subsets using lectin affinity chromatography and identified 369 proteins by mass spectrometric analysis. Among these proteins are a number of potential vaccine candidates with known immune modulatory properties including proteases, protease inhibitors, paramyosin, Venom Allergen-like II, Enolase and two proteins, nardilysin and TRIL, that have not been previously associated with F. hepatica Furthermore, we provide a comprehensive insight regarding the putative glycosylation of FhTeg components which could highlight the importance of further studies examining glycoconjugates in host-parasite interactions in the context of F. hepatica infection and the development of an effective vaccine.

Molecular and Functional Characterization of Antimicrobial Peptides in Crustaceans

Most living organisms are constantly exposed to potentially harmful pathogens. It is the immune system of the organism that enables it to survive in an environment loaded with dangerous pathogenic microorganisms. The innate immunity provides organisms with a rapid and non-specific first line of defense against pathogens. It includes physical barriers such as skin and mucous membranes and chemical barriers including the high acidity of gastric juice, and specialized soluble molecules that possess antimicrobial activity. One of the well-known innate immune defense mechanisms is the production of antimicrobial substances by specific cells or tissues of the organisms. Antimicrobial peptides (AMPs) are such natural substances that

Molecular and Functional Characterization of Histone Derived Antimicrobial Peptides from Marine Organisms

Antimicrobial peptides (AMPs) are gene encoded, small sized, generally cationic, amphiphathic peptides characterized by antimicrobial activity against bacteria, fungi, viruses and other pathogens. They are a major component of the innate immune defense system of almost all living organisms, ranging from bacteria to humans and represent the first line of defense against the invading microbial pathogens (Boman, 1995; Zasloff, 2002). Antimicrobial peptides represent a heterogeneous group displaying multiple modes of action that are determined by the sequence and concentration of peptides. Their remarkable specificity for prokaryotes with low toxicity for eukaryotic cells has favored their investigation and exploitation as new antibiotics

Age, growth, tagging of tilapia spp. and reproductive biology of siluroid catfishes in Lake Victoria

This report encompasses the time period 1 September 1973 through 31 July 1975. The research Officer's tour of duty officially terminated 30 September 1975, consisting of a regular 2-year tour of duty with three month extension of contract. During this time research into age and growth of several tropical species of fishes was conducted. In addition a closely related tagging program was initiated in order to determine both growth and movements of fishes. Lastly, some effort was directed towards aspects of the basic biology of several of the siluroid catfishes. This report delineates the relative effort into and success of the various research projects; presents results of research not previously offered, summarizes findings, and makes recommendations for future research endeavour. Two annual reports along with quarterly reports up to 30 June 1975 have been produced and are on file both at Headquarters in Jinja and the Kisumu Sub-station. These only will be referred to in some instances and built upon in others. Opinions offered, conclusions drawn and recommendation given within this report are solely those of the research officer employed in an official capacity for E.A.F.F.R.O.

Molecular and biological characterisation of orthobunyaviruses

Orthobunyaviruses are the largest genus within the Bunyaviridae family, with over 170 named viruses classified into 18 serogroups (Elliott and Blakqori, 2001; Plyusnin et al., 2012). Orthobunyaviruses are transmitted by arthropods and have a tripartite negative sense RNA genome, which encodes 4 structural proteins and 2 non-structural proteins. The non-structural protein NSs is the primary virulence factor of orthobunyaviruses and potent antagonist of the type I interferon (IFN) response. However, sequencing studies have identified pathogenic viruses that lack the NSs protein (Mohamed et al., 2009; Gauci et al., 2010). The work presented in this thesis describes the molecular and biological characterisation of divergent orthobunyaviruses. Data on plaque morphology, growth kinetics, protein profiles, sensitivity to IFN and activation of the type I IFN system are presented for viruses in the Anopheles A, Anopheles B, Capim, Gamboa, Guama, Minatitlan, Nyando, Tete and Turlock serogroups. These are complemented with complete genome sequencing and phylogenetic analysis. Low activation of IFN by Tete serogroup viruses, which naturally lack an NSs protein, was also further investigated by the development of a reverse genetics system for Batama virus (BMAV). Recombinant viruses with mutations in the virus nucleocapsid protein amino terminus showed higher activation of type I IFN in vitro and data suggests that low levels of IFN are due to lower activation rather than active antagonism. The anti-orthobunyavirus activity of IFN-stimulated genes IFI44, IFITMs and human and ovine BST2 were also studied, revealing that activity varies not only within the orthobunyavirus genus and virus serogroups but also within virus species. Furthermore, there was evidence of active antagonism of the type I IFN response and ISGs by non-NSs viruses. In summary, the results show that pathogenicity in man and antagonism of the type I IFN response in vitro cannot be predicted by the presence, or absence, of an NSs ORF. They also highlight problems in orthobunyavirus classification with discordance between classical antigen based data and phylogenetic analysis.

Analysis of radiation induced DNA damage by LC-MS/MS in isolated and cellular DNA

Abstract: It is well established that ionizing radiation induces a variety of damage in DNA by direct effects that are mediated by one-electron oxidation and indirect effects that are mediated by the reaction of water radiolysis products, e.g., hydroxyl radicals (•OH). In cellular DNA, direct and indirect effects appear to have about an equal effect toward DNA damage. We have shown that ϒ-(gamma) ray irradiation of aqueous solutions of DNA, during which •OH is the major damaging ROS can lead to the formation several lesions. On the other hand, the methylation and oxidative demethylation of cytosine in CpG dinucleotides plays a critical role in the gene regulation. The C5 position of cytosine in CG dinucleotides is frequently methylated by DNA methyl transferees (DNMTs) and constitutes 4-5% of the total cytosine. Here, my PhD research work focuses on the analysis of oxidative base modifications of model compounds of methylated and non methylated oligonucleotides, isolated DNA (calf-thymus DNA) and F98 cultured cell by gamma radiation. In addition, we identified a series of modifications of the 2-deoxyribose moiety of DNA arising from the exposure of isolated and cellular DNA to ionizing radiation. We also studied one electron oxidation of cellular DNA in cultured human HeLa cells initiated by intense nanosecond 266 nm laser pulse irradiation, which produces cross-links between guanine and thymine bases (G*-T*). To achieve these goals, we developed several methods based on mass spectrometry to analyze base modifications in isolated DNA and cellular DNA.