ETD Collection

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Author: search.filters.author.Alexandre, Kabamba Bankoledi

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    Sensitivity of HIV-1 subtype C viruses to Griffithsin, cyanovirin-N and scytovirin: potential HIV-1 microbicides
    (2013-05-07) Alexandre, Kabamba Bankoledi
    The majority of HIV-1 infections around the world occur via sexual intercourse and women, especially in developing countries, are disproportionately affected. Recently a number of strategies have been proposed to control the spread of HIV, among these the use of microbicides to prevent the sexual transmission of the virus. A clinical trial of 1% tenofovir gel that conferred up to 39% protection provided a proof-of-concept that an anti-HIV microbicide is feasible. Various other compounds, acting at different stages of HIV-1 life cycle, are also being investigated as potential microbicides. These include the lectins Griffithsin (GRFT), cyanovirin-N (CV-N) and scytovirin (SVN). GRFT was isolated from the red algae griffithsia sp. while CV-N and SVN were isolated from the blue green alga Nostoc ellipsosporum and the cyanobacterium Scytonema varium, respectively. These lectins bind mannose-rich glycans found on the surface of HIV-1 envelope and act as entry inhibitors. Although HIV-1 subtype C is the main cause of infections around the world, almost all studies conducted with GRFT, CV-N and SVN are based on subtype B viruses. The Chapter Two sought to establish the neutralization sensitivity of HIV-1 subtype C viruses to the three lectins, using both a cell line and primary cells, and compared this sensitivity to subtype B. This Chapter also examined mannose-rich glycans on HIV-1 that are involved in GRFT, CV-N and SVN binding. The conclusion from this study was that the neutralization of subtype C viruses by these lectins is similar to subtype B and that the 234 and 295 mannose-rich glycans were involved in their interaction with the virus. In general these data supported further studies on the use of GRFT, CV-N and SVN for prevention of HIV-1 subtype C sexual transmission. In Chapter Three, the ability of GRFT to expose the CD4 binding site (CD4bs) on HIV-1 gp120 is explored. I found that this exposure resulted in the enhancement of HIV-1 binding to plates coated with anti-CD4bs antibodies b12 and b6 or the CD4 receptor mimetic CD4-IgG2. This lectin also synergized with b12 and HIVpositive plasma containing antibodies to the CD4bs to neutralize the virus. Furthermore, the glycan at position 386, which shields the CD4bs, was shown to be involved in both GRFT enhancement of HIV-1 binding to b12 and b6 and in the synergistic interaction between the lectin and these antibodies. The importance of this study is that it investigated in details the effect of GRFT binding on HIV-1 envelope and also suggests this lectin can be used in combination with anti-HIV-1 antibodies to synergistically enhance the anti-viral activity. In Chapter Four I investigated GRFT, CV-N and SVN inhibition of the virus binding to the DC-SIGN receptor and their inhibition of the DCSIGN transfer of HIV-1 to target cells. These lectins only moderately inhibited the virus binding to the receptor while they potently inhibited its transfer to target cells. However, the inhibition of transfer was stronger when the virus bound the lectins after binding the DC-SIGN receptor compared to when it bound the lectins prior to binding the receptor. These three lectins can, therefore, inhibit the sexual transmission of HIV-1 since the DCSIGN- mediated transfer of the virus to susceptible cells is pivotal to this mode of transmission. Chapter Five is an investigation of the ability of HIV-1 subtype C to escape GRFT, CV-N and SVN, which involved growing the virus under escalating concentrations of these compounds. This was to know how this virus behaves under conditions of continuous exposure to the lectins. I found that HIV-1 subtype C became increasingly resistant to the lectins and viral envelope sequence analysis showed that this was associated with the deletion of mannose-rich glycans on gp120. Furthermore, of the 11 potential mannose-rich glycosylation sites on gp120 seven (230, 234, 241, 289, 339, 392 and 448) were involved in GRFT, CV-N and SVN resistance. Thus, the conclusion was that although these three lectins are potent inhibitors of HIV-1 infection, the virus is also able to escape their neutralization by deleting mannose-rich glycans on its envelope. However, the fact that escape to these lectins involved multiple deglycosylation and was only partial suggests that HIV-1 subtype C escape from GRFT, CV-N and SVN in a microbicide formulation may not be an easy process. We discuss the implications of these findings in Chapter Six and suggest future studies that could complement data presented in this thesis. Overall our data show that GRFT, CV-N and SVN can prevent the sexual transmission of both free and DC-SIGN associated HIV-1 particles and supports further development of these lectins as microbicides against HIV-1.
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    The effect of metformin-induced AMPK activation on adipogenesis and HIV replication
    (2008-04-08T13:51:39Z) Alexandre, Kabamba Bankoledi
    ABSTRACT Metformin is the most common drug used against type 2 diabetes mellitus. However, it was only recently shown, in human and rat hepatocytes, that metformin-like 5-aminoimidazole-4-carboximide ribonucleoside (AICAR), acts via activation of the AMP-activated protein kinase (AMPK), an enzyme that plays a central role in lipid metabolism. Although it is well known that metformin is used in the treatment of type 2 diabetes and results in significant fat loss, no study has investigated the effects of this drug on adipocytes. In this report I studied the effects of metformin on the formation of fat deposits in mouse 3T3-L1 preadipocytes, as well as its effects on the activation of AMPK in these cells. Our results suggested that metformin significantly inhibits the transformation of pre-adipocytes into adipocytes. This is achieved via the inhibition of intracellular lipid accumulation during adipogenesis. In addition to its inhibition of intracellular lipid accumulation, metformin induced a significant increase in the phosphorylation of AMPK. It has been shown that AMPK activation with AICAR results in the inhibition of the nuclear factor-κB (NF-κB) induced gene expression. Since NF-κB is the key nuclear factor used by HIV-1 during the initiation of its gene transcription, I investigated the possibility of inhibiting HIV-1 replication in U1 cells with metformin and AICAR. I observed that AICAR and metformin inhibit HIV-1 replication in U1 cells. This inhibition wasparalleled by the accumulation of NF-κB in the cytoplasm of AICAR and metformin treated cells, and at the same time by a significant decrease in the concentration of this nuclear factor in the nucleus of these cells. However, I failed to observe any phosphorylation of AMPK by metformin and AICAR in U1 cells. In conclusion, metformin inhibits adipogenesis in mouse adipocytes and this inhibition is likely to take place via the activation of AMPK. AICAR and metformin have inhibitory properties against HIV-1 replication. However, this inhibition does not seem to be by the activation of AMPK.