If I re-upload a list, will it remember which users unsubscribed?
The only time the system will remember if someone previously unsubscribed would be if the recipient had chosen to be added to your master unsubscribe list. In general, you should avoid re-uploading contact lists. If you keep sending to the same hard-bounced email addresses you are uploading into new lists, your email deliverability will suffer.
Ideally, you should use the same lists each time you send, and simply add new contacts to the lists.