11/1/14

I guess I was tired yesterday because I was fantasizing and dreaming the whole night. Yesterday, he came over to my apartment in the end and take a look at some of the things…

Sining was talking about inhibitory factors…I guess for him it’s totally what Miguel said he wanted an open relationship…so I guess at this point, I feel that Miguel knew something, maybe…

anyway…I dont’ know

nothing happened I guess…

Today he stopped by to take the cloth..which later he also bought, so…

There’s really not much link left…I also trash the other bag of clothes….

He asked how long I chatted with Sining last night about him..haha…I don’t know…

Initially I really thought I’m going to tell him let’s just date for couple months, and set up rules to see each other twice a week… I also wanted to take picture with him for every date and that will be a farewell gift of some sort to me…

think about myself…I don’t know…I got attached easily with people..

anyway, there is no reason for him to come over anymore..
I kept those condoms in the end, but I won’t need them.

IF I date someone else again, they will have to get their own I guess…

I just feel rather awkward…anyway…study some tonight, and tomorrow morning, go to lab and work on the cells… then need to organize the notes…

I feel scared seeing people moving forward…

btw…I’m surprised that even Anthony send out the email for BGSO thing, and even I was thinking about going, in the end…no one went…

true that when things happen, and when you have the chance, you should grab it… I don’t know…I feel really complciated.

today, I dressed up for lab…
nothing in particular, actually is more like I want to cheer myself up..
today, we had lunch together with Andre, but again we avoid each other…
there’s nothing we talked about…

it used to be so sweet that someone would say that I looked good..you are sexy..whatever, but..

I guess those only come out when you are into someone.

I’m going to go home to cook instant noodles with vege..so sad..lonely life.

and none of the experiments worked…
and I plated 4X more cells that I supposed to…so bad

somehows…it annoys me

Miguel and Anthony are talking about Andre from time to time…which i guess on one hand I wanted to know, but on the other hand also makes me feel a bit annoyed.

Mostly because, why don’t I know about this?

And figuring out the why, or knowing about the why complicates my feelings.

Miguel said, he didn’t go anywhere but visited some friends only one night.
And he cooked last night, curry? I don’t know what he cooked…

we were also chatting a bit last night too, he would say, maybe he will join my at library.. then he will say, maybe, then probably not because of experiment.

He can tell me that he already went home, but won’t you just say, I’ll cook tonight.

I don’t know what I am, who I am and what I want…

Sining is interpreting his sentence, after the new apartment, but… ya, when she pointed out, maybe I have a smile, but when thinking more, I guess. there is nothing to it.

I have no idea where he apply to live, what he did etc…

every bits about life is not minor, every thing can be interesting, if you can, and if I’m interested in you. but not at all the otherwise.

I’m even nervous about the dinner, even if it will happen…
what are we going to talk about? I don’t know..
Why am I so lack of things to talk about with people, and why people lost interest in me gradually.

I was nervous, very nervous to see him again… And here it is today..
He arrived yesterday…O haven’t talked to him for quite a while, or we talked, but short responses…all those reminded me about Zheng… a replicate of zheng.. just that this is shorter…this seems cooler, this seems trying a bit more to stay as friend…

but I don’t think I can do it..

I was hoping, oh maybe he will come in together with Miguel to the lab, we’ll hug then… but that didn’t happen..
Then I thought maybe I’ll see him at the busstop…that didn’t happen…
finally, I saw him at the reception, and at that moment, maybe air freeze, and I just want to escape from him…
He saw him, nice enough, he gave me a hug… a hug that can actually makes me cry…I did said, I miss you. even though it’s something that I hope, or I warn myself not to say.

We chatted during lunch, talked about project, other than that, I don’t know what to say..
we chatted, then he walked away… he turned back, but I pretended to be cool, and move on..
we chatted, I sat down next to him, he moved away…and that moment, I think…maybe I should give up and quit…

we walked on the beach…and the sand hurted my leg… Andreas was the one supporting me from behind, I thought, it’s him…it’s not…
then the sand hurted my leg, I screamed a little, I thought he turned around, but that’s Anthony…
I walked away, I thought someone who scream…I hope it’s him, but it’s Miguel…

Your own memory is something that drag you down and kills you.

A year ago, roughly, in here, I left some good memories…but those don’t last.

I wasn’t happen about that facebook picture in particular.. but should I care…and no one explain either.

when he said, see you tomorrow…I thought he would really come out just for me…

there are little things between other people, that I never know…and I afraid to know…

There are somethings that left at home, and they are like scars in my heart…

I’m afraid with anyone, and honestly…maybe I only want to be alone.

Yesterday…Sining bought people over without acknowledging me in advance, and I was mad, real mad, and I ignored her.. that was impolite …. but I just want to be alone.. all the sudden… I’m really lonely…but seeing others, I feel worse.

Once upon a time, I was walking over into the darkroom developing a film…and seems like I get the same feeling today..

Things takes time to heal….. especially for a lonely person like me.

labeling of specific enzyme classes in a covalent fashion.

three different elements with partially opposing,
but complementary roles; the ubiquitin conjugating cascade, the
deubiquitylating enzymes and the proteasome complex (Fig. 1).

>> The alkylation of nucleophilic residues (Cys, Ser, Thr) present in protease active sites
by reactive electrophiles has been a very successful realization of this
method. The

>> deconjugating enzymes
that usually contain strong nucleophiles at their active sites
when compared to the respective ligases/transferases.

>> probes bearing a C-terminal
electrophile (Fig. 2).

>>A complex proteome such as a cell or tissue lysate is labeled by
incubation with ubiquitin probe, followed by SDS-PAGE separation
and immunoblotting against the epitope tag genetically encoded
in the probe construct. Thereby a read-out, which is dependent
on DUB abundance and activity, is obtained.

>> competition assays
between an inhibitor candidate compound and the activitybased
probe can be performed in cell lysates. In this type of assay
format the competition between the small molecule inhibitor and
the probe leads to a reduced labeling profile for the activity-based
probe.

>>specificity of inhibition,

The proteasome complex, composed of at least
three different proteolytically active subunits β1, β2 and β5, degrades
the majority of soluble proteins within the cell following
polyubiquitylation [4]. The UPS not only enables the targeted destruction
of proteins no longer required by the cell, but also enables
the selective deactivation of signaling molecules, removal of pathogenic
proteins and production of peptides for presentation to the
immune system. The

>> is highly conserved
among eukaryotes (Kerscher et al., 2006). Ub adopts a globular
structure in which all seven lysine residues and the N terminus
are solvent exposed and can therefore form linkages with the
C terminus of another Ub to form poly-Ub chains

>>The Ub linkage type and the chain length encode the
fate of a ubiquitinated substrate.

DUB labelling

>> profile activity of DUBs in cell extract
>> DUB remove covalently bound ubiquiin moieties from protein substrates, thereby controlling many cellular functions. Alterations in these processes contribute to pathologies
>> post-translational modification of priteins by ubiquitin is a reversible process
>> ubiquitylation is carried out by a cascade of E1, E2, E3 ubiquitinating enzmes
>> apart from mono- and milto-ub, future ubiquitlation diversity is achieved by the assembly of poly ub chains through ispeptide bond formation between C-terminal glycine and one of the seven K residues of Ub.

>> modification of target proteins by poly-Ub chans linked through K48 serves as degratoin signals..
shuttles the Ubed protein through a series of Ub-binding proteins to the proteasome for degraation
>> K63 –> regulation of signaling evermts.

>> all 7 lysine residues on Ub can be used to create poly-Ub chains
>> activity based probes
>>Electrophilic probes based
on the ubiquitin scaffold, such as Ub-vinyl methylester (VME)
or Ub-vinyl sulfone (VS) act as Michael acceptors towards the
catalytic thiol of the DUB active site, which enables their covalent
capture and activity-based protein profiling (ABPP), a concept
successfully applied to other proteases.[9]

Profiling ubiquitin linkage specificities of DUB with branched ubiquitin isopeptide probes

branched Ub isopeptide activity-based probes (UIPPs) –> to profile ubiquitin (Ub) linkage specificity of DUBs in cell extracts